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dissertation entitled

DEVELOPMENT OF NEW TOOLS FOR THE APPLICATION OF
BIOTECHNOLOGY TO AGRICULTURAL IMPROVEMENT AND
ASSESSING RISKS OF BIOTECHNOLOGY AND ITS PRODUCTS

presented by

MERIDITH AYN COOK

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has been accepted towards fulfillment
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Ph.D. degree in Plant Biology

 

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DEVELOPMENT OF NEW TOOLS FOR THE APPLICATION OF
BIOTECHNOLOGY TO AGRICULTURAL IMPROVEMENT AND ASSESSING
RISKS OF BIOTECHNOLOGY AND ITS PRODUCTS

By

Meridith Ayn Cook

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Plant Biology

2008

ABSTRACT
DEVELOPMENT OF NEW TOOLS FOR THE APPLICATION OF
BIOTECHNOLOGY TO AGRICULTURAL IMPROVEMENT AND ASSESSING
RISKS OF BIOTECHNOLOGY AND ITS PRODUCTS
By

Meridith Ayn Cook

The genetic modification of plants through biotechnology, in conjunction with
traditional plant breeding, will serve as the basis for future plant improvements for both
food and energy. As we employ this relatively new technology, there are numerous issues
to consider. In addition to identifying new areas where biotechnology can provide for
agricultural improvement, the expansion of biotechnology through development of new
methods tailored to specific crops is critical. Simultaneously, the potential risks of
biotechnology must be evaluated.

In this thesis I describe the development of a novel transformation system for dry
bean (Phaseolus vulgaris L.), an important staple crop in many parts of the world. This
unique plant transformation method, referred to as electro-transforrnation, uses an
electrical current to drive DNA into the apical meristem of a seedling. Electro-
transformation was successful at transforming P. vulgaris as evidenced by the presence in
the T1 generation of the bar gene for resistance to the herbicide glufosinate ammonium
and the germin gene which encodes an oxalate oxidase. ElectrO-transformation will
hopefully be applicable to other legumes and agriculturally-important species.

Cherry, an important Michigan crop, is susceptible to numerous plant viruses.

One significant cherry viral pathogen is prunus necrotic ringspot ilarvirus (PNRSV) and

infection with this virus results in decreased fruit quality and yield. Here we characterize
PNRSV isolates in Michigan. Our results indicate that PNRSV is prevalent in Michigan
cherry orchards. Phylogenetic analysis of the coat protein gene nucleotide sequences
demonstrates that Michigan PNRSV isolates have high sequence similarity to worldwide
PNRSV isolates, and that there was likely more than one introduction of the virus into
Michigan. Since there is no source of natural PNRSV resistance in cherry, we propose a
novel resistance strategy based on silencing, which is described in the Appendix.

While numerous applications of plant biotechnology are foreseen, each presents
potential risks which must be addressed. Virus-resistant transgenic plants (VRTPs)
transcribing plant viral transgenes pose a unique potential environmental risk because
recombination between the transgenic transcript and a challenging virus could produce a
novel virus. Here we evaluate RNA recombination activity in transgenic Nicotiana
benthamiana transcribing a portion of the cowpea chlorotic mottle virus (CCMV) coat
protein gene inoculated with a CCMV coat protein mutant. We found evidence of five
recombination events. Our results suggest the presence of a recombination hotspot in
CCMV RNA 3. This study demonstrates that there is baseline recombination activity in
inoculated plants expressing a viral transgene. Our results contribute to knowledge in the

broader area of RNA recombination and VRTPs.

C0pyright by
MERIDITH AYN COOK
2008

ACKNOWLEDGEMENTS

I would like to thank my guidance committee members Dr. Richard Allison, Dr.
Rebecca Grumet, Dr. Sheng Yang He, and Dr. Suzanne Thiem for all of their help and
support during my time at MSU. I especially thank my major professor Dr. Richard
Allison for all of his support, guidance, and encouragement. Graduate school has been a
pivotal time in my development as a scientist and as a person and I am grateful for his
mentorship during this time. I would like to express my gratitude to all the former Allison
Lab members for their assistance, particularly Katie Knauf, Julia Rabe, and Jennifer
Cirino. I also appreciate the help of Ken Sink with cherry tissue collection, Kelly Mann
with cherry RNA extractions, and Heather Adams and Grant Godden with PNRSV
phylogenetic analyses. I appreciate the cooperation and generosity of the Michigan cherry
growers who allowed collection of cherry tissue from their orchards. I would like to
thank all of my colleagues and friends in the department and building. It has been a
pleasure being part of the plant sciences program at MSU and I am thankful for having
the opportunity to be a part of this community. Thanks to all of the Plant Biology
departmental staff for their patience and assistance throughout my graduate student years.
I would like to honor the memory of Dr. Ann Greene, whose previous work inspired the
bromovirus recombination experiments. Many thanks to my family for all of their
incredible love and support, especially my husband Fred and daughters Jordan and

Breaenna.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
INTRODUCTION .................................................................................... 1
CHAPTER 1
DEVELOPMENT OF A NOVEL PLANT TRANSFORMATION SYSTEM FOR
PHASEOLUS VULGARIS .......................................................................... 13
Introduction ................................................................................. 13
Materials and Methods ..................................................................... 24
Results ....................................................................................... 31
Discussion ................................................................................... 38
CHAPTER 2
CHARACTERIZATION OF PRUNUS NECROTIC RINGSPOT ILARVIRUS (PNRSV)
ISOLATES IN MICHIGAN ....................................................................... 46
Introduction ................................................................................. 46
Materials and Methods ..................................................................... 53
Results ....................................................................................... 58
Discussion ................................................................................... 75
CHAPTER 3
RNA RECOMBINATION IN BROMOVIRUS COAT PROTEIN DELETION
MUTANTS .......................................................................................... 78
Introduction ................................................................................. 78
Materials and Methods ..................................................................... 86
Results ....................................................................................... 93
Discussion ................................................................................... 99
CONCLUSIONS AND PERSPECTIVES ..................................................... 108
APPENDIX
DEVELOPMENT OF A RESISTANCE STRATEGY FOR PRUNUS NECROTIC
RINGSPOT ILARVIRUS (PNRSV) IN CHERRY ............................................ 113
Introduction ................................................................................ 1 13
Materials and Methods ................................................................... 116
Summary and Status ..................................................................... 118
REFERENCES .................................................................................... 120

vi

LIST OF TABLES

Table 1.1. Treatments of P. vulgaris apical meristems prior to electro-transforrnation, and
results of analyses on T1 generation ...................................................... 31

Table 2.1. Incidence of PNRSV infection in cherry trees sampled from Michigan
orchards ...................................................................................... 58

Table 2.2. Origins of cloned Michigan PNRSV isolates ....................................... 59

Table 2.3. PNRSV isolates included in the present study with origins and accession
numbers ...................................................................................... 60

Table 3.1. Results of analyses of clones isolated from wild-type or transgenic plants
inoculated with AG3 mutant or wild-type CCMV inoculum ........................ 93

vii

LIST OF FIGURES

Figure 1.1. HindIII-SspI fragment from pBKSbar/gf-2.8 used to transform P. vulgaris
seedlings .................................................................................... 24

Figure 1.2. Depiction of electro-transformation setup ......................................... 26

Figure 1.3. PCR amplification of the bar gene from P. vulgaris total genomic DNA from

T1 plants ..................................................................................... 33
Figure 1.4. PCR amplification of the germin gene from P. vulgaris total genomic DNA

from T1 plants .............................................................................. 34
Figure 1.5. PCR amplification of the actin gene from P. vulgaris total genomic DNA

from T1 plants ............................................................................... 35
Figure 1.6. Southern blots of DNA hybridized with a radiolabeled bar gene probe ....... 36

Figure 1.7. Southern blots of DNA hybridized with a radiolabeled germin gene probe...37

Figure 1.8. Expected inheritance pattern of a transgene following integration into

meristematic tissue and subsequent selection .......................................... 41
Figure 2.1. Depiction of the ilarvirus genome ................................................... 48
Figure 2.2. Map of counties in the lower peninsula of Michigan ............................. 54
Figure 2.3. RT-PCR products of PNRSV isolates from Michigan cherry trees ............. 59

Figure 2.4. Phylogenetic relationships among coat protein gene nucleotide sequences of
new and previously-described PNRSV isolates...........................................65

Figure 2.5. Alignment of the N-terminus of the coat protein of representative PNRSV
isolates ....................................................................................... 67

Figure 2.6. Alignment of the amino acid sequence of the movement protein of

representative PNRSV isolates ........................................................... 70
Figure 3.1. Depiction of CCMV sequences used in this study ................................ 86
Figure 3.2. PCR amplification products from CCMV constructs ............................. 95

Figure 3.3. Depiction of sequences present in recombinants characterized in this study..96

viii

Figure 3.4. Map of recombination sites in characterized recombinants ...................... 97
Figure 3.5. Predicted hairpin structure within CCMV RNA 3 .......................................... 98

Figure A1. Intron-containing hairpin construct designed to induce silencing of
PNRSV ............................................................................................. 118

ix

INTRODUCTION

Conventional plant breeding is responsible for modem-day agriculture. Numerous
beneficial traits, including disease resistance and insect tolerance, have been integrated
into cultivated crop species via traditional breeding. Despite these successes,
conventional breeding has both challenges and limitations. If agriculture is to keep pace
with the 21st century’s demand for both food and energy, then conventional agriculture
must be used in conjunction with genetic engineering which has the capacity to move
traits beyond their natural species boundaries. To date Ihe most notable genetically
engineered varieties include cotton, soybean, and maize, and improvements to numerous
other species are in the experimental phase. As we approach large-scale employment of
this relatively new technology, there are numerous issues to consider. Perhaps most
important are the risks, real or perceived, of biotechnology to consumers and the
environment. It is critical that we ensure the safety of genetically modified plants and
their products. In addition, as biotechnology expands to include additional plant species,
there will be a role for alternative transformation technologies and creative uses of
existing techniques.

There are a number of reasons why agriculture must become more productive in
the future. First, the world population of 6.6 billion is predicted to rise to over 9 million
by 2050 (US. Census Bureau, 2007). A major limiting factor to continued population
growth will be food and energy; both are directly related to sustained increases in
agricultural productivity. Additionally, the land base suitable for crop cultivation is

diminishing (Khush, 1999). Cropland availability is decreasing due to desertification,

salination, and urbanization. Desertification refers to the degradation of land due to
climatic factors and human activities. Salination of land occurs when salts accumulate to
phytotoxic levels as a result of fertilization and irrigation and evaporation. Urbanization
involves the conversion of cropland to housing, recreation, and supporting facilities.
These processes continue to decrease land available for cultivation; therefore even
maintaining the current food supply will require increasing food productivity.

The availability of fresh water is another challenge to agriculture. As the world
population increases there will be increased demands on this limited resource. This will
inherently pose problems, as the predicted increase in agricultural productivity will
necessitate adequate water.

The dietary demands of the growing population will also result in a significant
impact on the world’s grain resources. Much of the world’s population desires meat in
addition to grain as part of a daily diet. Approximately 7 kg of grain is required to
produce one kg of beef. To produce one kg of pork roughly 4 kg of grain is needed, and
about 2 kg of grain is required for one kg of poultry (Brown, 1997). As a result,
significantly more grain will be needed to produce meat for the predicted population.

The advent of biofuels will place increased pressure on the world’s agricultural
resources as well. The new commitment of cropland to biofuel production will result in
the conversion of food crops to those used as precursors for ethanol or biodiesel. This
reallocation of agricultural products and land from food to biofuels will place severe
pressure on our current agricultural productivity. Significant improvements in yield
through both conventional breeding and genetic engineering will be required to meet

these demands.

Conventional breeding has thus far improved the yield and quality of crops
worldwide. The Green Revolution contributed to modern day agriculture through the
development of high-yielding hybrids. In the 19603, dwarf rice and wheat varieties were
introduced which had greater yield, produced more edible biomass, and were more
responsive to fertilizer (Khush, 1999). Also, photoperiod-insensitive rice was developed
which could be planted any time of year and had a shorter maturation time than existing
varieties. Agriculture during the Green Revolution resulted in greater grain production
and converted several importing countries to exporters. The average rice yield per area
increased 71% between 1966 and 1995. As a result, total rice production doubled from
1966 to 1990. Likewise, total worldwide grain production doubled between 1966 and
1995 (847 million tons to 1680 million tons) (Khush, 1999). Despite the significant
contributions of conventional breeding to the world’s agricultural yield, the predicted
increase in demand for food will be difficult to meet with conventionally-bred crops
alone.

In addition to meeting the caloric needs of the world’s population, there is also a
need to provide complete nutrition. The number of people undernourished according to
2000-2002 statistics was estimated at 852 million (FAO, 2004). Malnutrition is
widespread in developing countries and has severe consequences. Common, although
easily preventable, deficiencies include folic acid, iron, and vitamin A. Vitamin A
deficiency has a profound effect on the immune system, and deficiencies in developing
countries result in the deaths of approximately 1 million children per year. Maternal
folate deficiency is responsible for more than 200,000 severe cases of birth defects per

year worldwide and severe anemia caused by lack of iron results in the death of

approximately 60,000 women annually during pregnancy and childbirth (Micronutrient
Initiative & UNICEF, 2004). Consequently, in addition to ensuring adequate caloric
resources, a healthy population will require complete nutrition.

Despite the significant contributions of conventional breeding to world
agriculture, there are a number of limitations to traditional breeding approaches.
Conventional breeding has had limited success in producing crops with tolerance to
abiotic stresses such as drought, salinity, and low temperature. One possible reason is that
abiotic stress resistance is likely a complex trait affected by multiple genes (Garg et al.,
2002). Often, breeders need to turn to wild relatives of domesticated crop species to find
a desirable trait. However, there can be drawbacks of these wide crosses including
transmission of undesirable chromosomes and sterility as a result of unfavorable genetic
interactions. In some cases, the existence of fertilization baniers (pre- and post-) further
limits conventional breeding (Jauhar, 2006). More often the desired trait is not available
in the natural gene pool of a particular crop. Even if there is donor material for a desirable
trait, it may take up to 10 years to obtain the one trait of interest in a suitable genetic
background.

Another obstacle presented by conventional breeding is that a species must be
sexually propagated to be a potential candidate for integration of a trait from a donor
plant via conventional means. This is difficult for some species, including banana which
is sexually sterile (Manshardt, 2004). Biotechnology will therefore be a useful approach
for improvement of sterile, vegetatively propagated crops.

Conventional breeding is particularly limiting in perennial woody fruit crops.

These limitations are due to a variety of reason, including: extended juvenile phase of

woody crops, self-incompatibility, single seed per fruit, polyploidy, and lack of
information on inheritance of desirable traits (Krishna & Singh, 2007). Therefore, the
potential contributions of biotechnology to improvement of fruit crops are extensive.

The future of agriculture will need to include alternative methods of crop
improvement. One such alternative is genetic engineering, which involves inserting a
gene of interest into the genome of a target plant species. One benefit of genetic
engineering is that the gene pool available for crop improvement becomes limitless.
Genes isolated from a variety of species, even different kingdoms, can be used to modify
a target crop. Thus, breeding obstacles that are present in conventional breeding are
reduced or eliminated. Transformation techniques are well established for model plant
species, such as tobacco and Arabidopsis. Despite the transformation successes of some
plant species, there are difficulties in transforming some economically important crops.
For example, some large-seeded legumes, such as dry bean, are recalcitrant to
transformation. As a result, there is a need to expand the available technology for genetic
modification of crop species.

Potential contributions of biotechnology to agricultural improvement are
seemingly endless. Despite past efforts in crop improvement, up to 30% of crops are lost
due to pests (Lenne, 2000). An example of an agriculturally significant disease is maize
streak geminivirus (MSV), which can reduce maize yield by 70% (Bosque-Perez et al.,
1998). Yield losses due to pests in cassava, an important staple in sub-Saharan Africa,
can range from 20 to 80% (Lenne, 2000). A relevant example in which conventional
breeding actually exacerbated loss from disease is that of the Southern corn leaf blight

epidemic (Tatum, 1971). High-yielding corn hybrid varieties displayed increased

susceptibility to the fungus Helminthosporium maydz's, the causative agent of corn leaf
blight, and in the 1970’s many Southern states experienced 50% or more yield loss of
corn due to the disease. Abiotic stresses, such as drought, high salinity, and extreme
temperatures, also pose agricultural challenges and may cause complete crop failure.

Micronutrient malnutrition is a significant cause of human health problems in
developing nations, and many important food staples, such as rice, have micronutrient
deficiencies (Toenniessen, 2002). Efforts are underway to biofortify important dietary
staples, such as rice and cassava, and projects are at various stages of development
(Sautter et al., 2006).

A number of crops developed through biotechnology have been commercialized.
In fact, in 2005, 222 million acres worldwide were planted with genetically modified
crops (Sankula, 2006). Members of the cry gene family, encoding Bacillus thuringiensis
Bt proteins, are used to provide resistance to a variety of insect species. These Monsanto
products include YieldGard® corn, BollGard® cotton, and NewLeaf® potato. In addition
to Colorado beetle resistance, NewLeaf® Y provides resistance to the potyvirus potato
virus Y (PVY). Considering the wide range and effects of plant pathogens, there are
numerous opportunities for pathogen-resistant crops.

Herbicide resistance is a desirable trait in a transgenic crop for weed control. The
resistant crop can be sprayed with a relatively benign herbicide, such as Roundup®
(glyphosate), and weed competition can be eliminated. Use of the more toxic agricultural
herbicides correlates with health problems of agricultural workers and likely contributes
negatively to the health of consumers and the environment. The use of a broad-spectrum

herbicide, such as Roundup®, which is relatively harmless to mammals and the

environment, in conjunction with herbicide-resistant plants has many potential benefits.
A number of approved herbicide—resistant crops are commercially planted, including
Monsanto’s Roundup Ready® corn, soy, and cotton.

Transgenic papaya has had notable success in Hawaii, where the viral pathogen
papaya ringspot potyvirus (PRSV) had a disastrous effect on papaya agriculture. Papaya
expressing the coat protein gene of PRSV displays resistance to the virus, and papaya
yields have increased immensely since transgenic papaya was introduced (Gonsalves,
1998). Another commercial success is virus resistance in summer squash. Yellow
crookneck squash line ZW-20 which contains the coat protein genes for zucchini yellow
mosaic potyvirus (ZYMV) and watermelon mosaic potyvirus 2 (WMV2), and line CZW-
3 containing the coat protein genes of ZYMV, WMV2, and cucumber mosaic
cucumovirus (CMV), are resistant to the viruses from which the genes were derived.
Transgenic lines display increased fruit yield and quality upon challenge with the viruses
when compared to non-transgenic squash (Fuchs & Gonsalves, 1995; Fuchs et al., 1998).

An important issue to consider in the application of biotechnology to crop
improvement is that of intellectual property rights. Of the US agricultural biotechnology
patents granted from 1982 to 2001, approximately 75% of them are owned by private
entities (Graff et al., 2003). The major transformation systems currently used are patented
and/or licensed by private corporations. Syngenta holds the patent for Agrobacterium
binary vector transformation, DuPont has the license for the helium particle gun
transformation approach, and Agracetus (currently Monsanto) holds the electric discharge

particle gun patent (Pray & Naseem, 2005).

Many of the frequently-used marker genes and regulatory sequences are patented
as well. Monsanto holds the patent on the widely-used cauliflower mosaic caulimovirus
(CaMV) 358 promoter, as well as one of the most commonly-used marker genes, the
kanamycin resistance gene nptII. In addition, Monsanto holds a patent for any antibiotic
resistance gene used under the control of a plant promoter (Pray & Naseem, 2005). These
are only a few examples of the wide range of patented material relating to plant
biotechnology. Because most marker genes and promoters characterized to date are
patented, and many patents are held by private corporations that charge a licensing fee for
use, it is difficult to develop a product that can be used in countries where it is not
economically feasible to pay the large royalty costs. To contribute to agricultural
improvement in countries where it is needed most, alternative methods of transformation
as well as alternative marker genes and regulatory sequences with limited patent
restrictions are desirable.

One relevant example of the challenges intellectual property present in
agricultural improvement is seen in the instance of Golden Rice, rice genetically
modified to contain beta-carotene biosynthesis genes (Ye et al., 2000). The development
of Golden Rice involved 70 intellectual property and technical property rights from 32
different companies and universities (Potrykus, 2001). This posed an obstacle to making
the nutritionally-enhanced rice available to developing countries. Fortunately, in the case
of Golden Rice, an agreement was arranged between all the patent-holders and the
developers of Golden Rice and this extremely beneficial crop can now be used for
humanitarian purposes with no royalties, providing the income gained by a grower is less

than $10,000 (Potrykus, 2001). However, this emphasizes the need to consider any

potential patent violations before embarking on development of any technology for
agricultural improvement. Because of the many complications posed by intellectual
property rights, which can be a frustrating deterrent to researchers, advancement in
agricultural technology may be hindered (Pray & Naseem, 2005).

New technologies which enable beneficial traits to be added to agriculturally-
important plant species while avoiding obstructions caused by intellectual property issues
have vast potential to benefit society. In Chapter 1 we describe a novel transformation
system for dry bean, an important staple crop in many parts of the world. Previously,
there was no reliable transformation method for dry bean. While designed and tested with
the improvement of dry bean in mind, this novel transformation system also has potential
applications for the modification of other species. Although the university has patented
this transformation system, the licensing agreement includes a clause enabling the use of
the system for crop improvement in developing countries.

Most of the agricultural area in the US is dedicated to the cultivation of major
crops, which include com, soybean, wheat, and cotton. As a result, these crops have
typically been the focus of crop improvement via biotechnology. Because of the high
profit potential of major crops, corporate interests tend to focus mainly on genetic
improvement of these crops. As described above, Monsanto has focused much of its crop
improvement efforts on these major crops.

While major crops play significant roles in the agricultural economy, minor crops
such as pulses, fruits, and vegetables must also be considered as candidates for
improvement. Unfortunately, research funding for minor crops is limited. However,

several committed researchers have developed useful transgenic crops. The success of

transgenic papaya, described previously, is one noteworthy illustration of the success of
biotechnology in a minor crop. The cultivation and subsequent success of this transgenic
papaya has much to do with Monsanto’s decision to forgo intellectual property rights on
this genetically modified crop. This has made it economically feasible for farmers in
Hawaii to grow the crop without the financial burden of licensing costs.

Another success story of improvement of a minor crop using biotechnology is
seen in the development of a plum line resistant to plum pox potyvirus (PPV). PPV
causes devastating yield losses in fruit trees, particularly in European countries. A
transgenic plum line, ‘HoneySweet,’ containing the PPV coat protein gene, is resistant to
PPV. The resistance of ‘HoneySweet’ has been tested in the field for 10 years and PPV
resistance has remained durable and stable (Scorza & Ravelonandro, 2006).
‘HoneySweet’ is currently undergoing the approval process for commercialization in the
US (Bliss, 2007) and it will play a promising role in preventing the establishment of PPV
on this continent.

Cherry is another important minor crop, particularly in Michigan. A number of
pathogens negatively impact the cherry industry. One notable pathogen of cherry is
prunus necrotic ringspot ilarvirus (PNRSV) and infection with this virus can result in
decreased fruit yield. There is no reliable source of PNRSV resistance in commercial
cherry varieties. As a result, we have turned to biotechnology as a means of increasing
disease resistance in this valuable crop. In Chapter 2 we characterize PNRSV isolates in
Michigan, and recommend a resistance strategy for this minor crop in the Appendix.

While there are many possible benefits to the application of biotechnology to

agriculture, the potential risks of this new technology need to be assessed. Possible risks

10

include introduction of allergenicity factors into otherwise non-allergenic products,
negative effects on the environment and non-target organisms, and movement of the
transgene into non-transgenic crops and/or wild relatives. Safety of transgenic crops must
be thoroughly evaluated prior to commercialization.

There are risks unique to virus-resistant transgenic plants (VRTPs) carrying a
viral transgene (Tepfer, 2001). If the transgene is translated and the viral protein product
is present in the transgenic plant, there may be risks associated with infection by a
challenging virus. The transgene product may complement the challenging virus,
potentially increasing the infectivity of the virus. Even if the transgene is not translated, a
challenging virus may acquire all or part of the transgene genetic material via RNA
recombination. The resulting chimeric virus may cause different symptoms and/or have a
different, and perhaps expanded, host range than that of the parent virus. In order to better
understand the interaction of a challenging virus with VRTPs, we used a model system of
a challenging mutant bromovirus inoculated onto plants containing a bromovirus
transgene. The results presented in Chapter 3 will help assess the contribution of a
transgene to viral evolution. This will help us further understand recombination, which
can contribute to our understanding of the risks associated with VRTPs.

Society will face many challenges in the future with the continued increase in
population and demand for energy. One of our most significant challenges is how to
provide adequate food for this growing population. With obstacles to agriculture such as
space constraints, and biotic and abiotic stresses, we will need to have more options to

complement conventional breeding for agricultural improvement. While biotechnology

11

offers many potential contributions to crop improvement, we must also wisely evaluate

its products to ensure safety, success, and sustainability for the future.

12

CHAPTER 1

DEVELOPMENT OF A NOVEL PLANT TRANSFORMATION SYSTEM FOR
PHASE 0L US VUL GARIS

INTRODUCTION

Phaseolus vulgaris (L.), common bean, is an important crop worldwide. The
seeds of P. vulgaris provide a valuable dietary staple, particularly in developing
countries. With approximately 25% of calories from protein (Michigan Bean
Commission, 2006), dry beans serve as a significant protein source. Dry beans also
contain high levels of folic acid, which is crucial to the prevention of many congenital
brain and spinal cord malformations, and iron, which is critical to the prevention of iron-
deficiency anemia. In developing countries, where malnutrition is more prevalent, dry
beans are an economically-feasible source of dietary protein and minerals.

In addition to its dietary importance, P. vulgaris plays a significant role in the
nitrogen cycle of the agricultural ecosystem. The symbiotic relationship of P. vulgaris
with the nitrogen-fixing bacterium Rhizobium etli results in the accumulation of nitrogen
in dry bean fields. As nitrogen is often a limiting factor in agriculture production, dry
bean is an important rotational crop.

Afiica has the largest dry bean production area, 13.3 million Ha, followed by
Latin America (6.4 million Ha) and the United States (642,000 Ha) (FAO, 2005). In
2005, Africans consumed 4.4 million tons, Latin Americans consumed 5.3 million tons,
while the United States consumed nearly one million tons (FAO, 2005).

Both biotic and abiotic factors limit dry bean production. The causal agent of

white mold, the fungus Sclerotinia sclerotiorum, can result in crop losses of up to 100%

13

(Schwartz et al., 2005). Disease reports indicate that angular leaf spot, caused by the
fungal pathogen Phaeoisariopsis griseola, can result in up to 80% yield loss (de Jesus
Junior et al., 2001). Another fungal disease, anthracnose, a consequence of infection by
Colletotrichum lindemuthianum, has lead to complete crop losses (Schwartz et al., 2005).
Bean common mosaic necrosis potyvirus (BCMNV) can reduce yield up to 80%, and, as
a seed-home virus, can negatively impact seed quality (Schwartz et al., 2005). Bean
golden yellow mosaic geminivirus (BGYMV) has been shown to cause yield reductions
ranging from 20-50% (Morales & Anderson, 2001). Common bacterial blight of beans,
caused by the bacterium Xanthomonas campestris pv. phaseoli, can result in yield losses
of 30% (Wallen & Galway, 1977).

In addition, abiotic stress can decrease dry bean yields. P. vulgaris is highly
sensitive to drought conditions, and drought stress can reduce yield by 50% (Teran &
Singh, 2002). Insect damage can also drastically reduce crop yields. Diminishing the
impact of diseases and drought on dry beans by developing resistant lines could result in
a significant contribution to the world’s food supply.

Various international projects exist for dry bean improvement. The Institute of
Plant Biotechnology for Developing Countries (IPBO) is centered at Ghent University,
Belgium and has a number of P. vulgaris improvement projects. The Center for Tropical
Agriculture (CIAT) is also focused on dry bean improvement (CIAT, 2006). At Michigan
State University, the Bean/Cowpea Collaborative Research Support Program (CRSP)
supports efforts in bean and cowpea improvement. Dry bean improvement projects

supported by these agencies include the development of varieties that are resistant or

14

tolerant of both biotic and abiotic stresses, as well as the nutritional enhancement of bean
varieties.

Conventional breeding has traditionally been used as a means to improve crop
varieties. An important limitation of conventional breeding is the time required to move a
trait from a source isolate to a commercial variety. Crossbreeding and selection processes
often require many generations to complete. A more significant limitation of
conventional breeding is that desirable traits may not be available in sexually compatible
species. Consequently, some desirable goals cannot be achieved through conventional
breeding.

Genetic engineering provides an optional method for moving desirable traits to a
crop species in a comparatively short period of time. Genes of interest, such as those that
confer disease resistance, can be isolated from an unrelated organism and transformed
into an agriculturally important crop variety. Once the gene is inserted into a particular
crop, conventional breeding can be used to transfer the trait to related varieties. Thus,
transformation can be used independently and in conjunction with traditional breeding for
crop improvement.

Transformation of large-seeded legumes, such as P. vulgaris, has considerable
potential as a tool for crop improvement. Prospective improvements include the
introduction of genes for drought tolerance, disease and insect resistance, nutritional
enhancement, and cold tolerance. Examples of genes which have been successfully
introduced into crop plants include members of the cry gene family that provide
resistance to a range of insects, the bacterial EPSPS gene encoding resistance to the

herbicide Roundup® (N ida et al., 1996), the papaya ringspot potyvirus (PRSV) coat

15

protein gene which is effective in conferring PRSV resistance (Fitch et al., 1992;
Gonsalves, 1998), and beta-carotene biosynthesis genes utilized to create the
nutritionally-enhanced Golden Rice (Ye et al., 2000). Alternatively, transformation can
be used to induce gene silencing, which can turn off the expression of a selected gene.
For example, phytic acid, a product of both legume and cereal crops, is considered an
anti-nutrient because it inhibits absorption of important minerals in humans. Gene
silencing has been used to decrease the phytic acid content of soybean seeds (Nunes et
al., 2006) and maize (Shi et al., 2007). Similarly, other genes could be turned off to
obtain desired characteristics.

Currently, the most commonly used methods for plant transformation include
Agrobacterium-mediated transformation, biolistic methods, and protoplast
transformation. A grobacterium-mediated transformation takes advantage of the capability
of Agrobacterium tumefaciens to insert DNA into a host’s chromosome. In this case, a
defined region of the bacterial plasmid is added to a plant chromosome during infection;
this area can carry selected genes and expression control sequences. Biolistic methods,
such as particle bombardment, utilize high-speed gold or tungsten particles coated with
foreign DNA to insert genes and controlling sequences into plant cells, where the DNA is
incorporated into the chromosomes by an, as yet, undefined natural mechanism.
Protoplast transformation involves removal of the plant cell wall by digestion with fungal
cellulases and subjection of cells to chemical and/or electrical treatments to introduce
transgene DNA. Common to each of these techniques is the selection of single

transformed cells, their exclusive cultivation, and regeneration into whole plants.

16

A significant obstacle to P. vulgaris transformation is the difficulty of
regenerating P. vulgaris whole plants from callus cultures. Although there are several
reports of P. vulgaris regeneration (Mohamed et al., 1993; Zambre et al., 1998; Ahmed et
al., 2002; Veltcheva & Svetleva, 2005), these results have proven difficult to reproduce,
especially in cultivated bean varieties. Therefore, a transformation method for dry bean
which does not require regeneration of plants via tissue culture is highly desirable.

Phaseolus acutifolius (A. Gray), commonly known as tepary bean, has been
successfiilly transformed by Agrobacterium (Dillen etal., 1997; Zambre et al., 2005).
Dillen et al. (1997) discusses the possible use of P. acutz’folius as a “bridging” species to
introduce genes of interest into P. vulgaris. Although P. acutifolius and P. vulgaris can
be successfully crossed with one another, fertility of the hybrids is low, and numerous
backcrosses are required to obtain stable and desirable transforrnants (Andradf-Aguilar &
Jackson, 1988; Haghighi and Ascher, 1988). While this cumbersome approach may
produce P. vulgaris varieties containing a transgene, it would be more efficient and
effective to directly transform P. vulgaris with genes of interest rather than using P.
acutifolius as an intermediate species.

There have been several reports of successful P. vulgaris transformation.
Although A. tumefaciens has been shown to infect P. vulgaris (Lippincott & Heberlein,
1965; Karakaya & Ozcan, 2001), and transgene expression has been attained in infected
tissues (McClean et al., 1991), whole transformed plants have not been produced. A
related A grobacterium species, A. rhizogenes, induces hairy roots in P. vulgaris species.
Several reports describe using A. rhizogenes to obtain transgene expression in P. vulgaris

roots (Estrada-Navarrete et al., 2006; Estrada-Navarrete et al., 2007).

17

The most successful transformation approach for P. vulgaris to date involves
particle bombardment of axillary meristems. Studies emanating from two laboratories
have demonstrated successful transformation, with inheritance of the transgene into
subsequent generations (Russell et al., 1993; Aragao et al., 1996; Aragao et al., 1999;
Aragao et al., 2002; Bonfim etal., 2007). Transformation efficiencies ranged from 0.03%
(Russell et al., 1993) to 0.9% (Aragao et al., 1996).

Although particle bombardment is a successful technique for transformation of P.
vulgaris, albeit at low efficiencies, and A grobacterz'um shows potential for success, the
patents/licenses for these transformation methods are owned by private corporations. The
Agrobacterium binary vector transformation patent is held by Syngenta. Cornell holds the
patent for helium particle gun transformation, with DuPont owning the license, while
Agracetus (currently owned by Monsanto) holds the electric discharge particle gun patent
(Pray & Naseem, 2005). Patenting and exclusive licensing to major corporations has
limited the use of these important techniques to research and has in turn stymied the use
of these methods for crop improvement by those not affiliated with these corporations.

Considering the challenges to P. vulgaris transformation, there remains a
substantial need for a reliable dry bean transformation system. As the potential benefits of
improved dry bean varieties are significant, especially for developing countries, the
development of a reliable and accessible transformation method for P. vulgaris would be
valuable. Such a method would enable the improvement of bean lines through the
addition of disease resistance, stress tolerance, and nutritional enhancement. Therefore,
the novel dry bean transformation method described in this study has the potential to play

a role in bean improvement worldwide.

18

DNA is a negatively-charged molecule and travels from the negative electrode
toward the positive electrode when exposed to an electrical current in an electrophoresis
gel. In the same respect, a mild electrical current could be used to direct DNA into a
plant, with the plant tissue, rather than an agarose gel, acting as the matrix for DNA
passage. A similar approach was used to inoculate plants with virus in 1980, when Polson
and von Wechmar used a mild direct current to introduce charged virus particles into
plant leaves; viral infection resulted.

The use of an electrical current to drive transgene DNA into plant tissue has been
demonstrated previously. Electrophoresis was used successfully to introduce transgenes
to orchid embryos (Griesbach & Hammond, 1993; Griesbach, 1994) and barley seedlings
(Ahokas, 1989). However, transgene inheritance by subsequent sexually-produced
generations was not demonstrated, and the possibility of transient expression from the
introduced DNA was not ruled out. If a plant transformation system is to be
commercially relevant, it must result in the stable integration of the transgene DNA into
the chromosome of cells that are involved in forming the progeny of the plant.

To encourage stable transformation and sexual inheritance of a transgene, the
approach described in this thesis targets the meristematic area of a seedling. Among the
dividing meristematic cells are those destined for meiosis in the flower of the adult plant.
If these cells are transformed, then progeny plants will inherit the transgene. A
subsequent challenge of this transformation approach is the selection and. identification of
transformed plants. Since a goal of this transformation system is to avoid tissue culture
and single cell selection and regeneration, selection of whole transformed plants is

required.

19

Herbicide resistance provides a selectable characteristic that can be used with
whole plants. An additional advantage is that selection pressure can be applied within
greenhouse confinement or, with appropriate approvals, in field studies. The selectable
marker used in this study is the bar gene (Thompson et al., 1987), which encodes
resistance to the herbicide glufosinate ammonium, also known as bialaphos, Basta®, and
Liberty®. The bar gene, originally isolated from the bacterium Streptomyces
hygroscopicus, encodes phosphinothricin acetyl transferase (PAT), which inactivates
glufosinate ammonium. Use of the bar gene as a selectable marker in transformation
provides an economical screen for putative transforrnants.

In addition to the bar gene, this study includes the germin gene, originally
isolated from wheat. The germin gene encodes an oxalate oxidase (OxO) (Lane et al.,
1993). Evidence suggests that OxO provides resistance to the fungus Sclerotinia
sclerotiorum, the pathogen that causes white mold, by oxidizing oxalic acid, a
pathogenicity factor produced by the pathogen (Donaldson et al., 2001; Hu et al., 2003).

Theoretically, there are numerous barriers that should prevent the movement of
DNA into a cell and into a chromosome. In the transformation approach described here,
the response of the charged DNA molecule to the electrical gradient established within a
seedling will move DNA into the vicinity of mitotic activity. By targeting actively
dividing cells, the formidable cell wall barrier of mature cells is avoided. This leaves the
movement of DNA across the plasma and nuclear membranes and the incorporation of
the DNA into the genome. There is evidence that natural mechanisms exist for the uptake
and incorporation of DNA. One significant illustration of DNA uptake and genome

incorporation is seen in particle gun transformation, mentioned above. A variety of

20

studies describe passage of oligonucleotides through eukaryotic cell membranes. Hanss et
a1. (1998) demonstrate the movement of radiolabeled oligos through the cell membrane.
Foreign DNA has been found to stimulate immune responses in murine cells, suggesting
that the DNA traveled through the membranes of the cells (Branda et al., 1993; Branda et
al., 1996; Halpem et al., 1996). Schablik and Szabo (1981) describe the uptake and
incorporation of foreign DNA into Neurospora crassa cells. Since the actual mechanisms
of DNA uptake have not been identified, we are unable to determine if they are functional
in meristematic cells. In contrast to Agrobacterz’um’s enzyme-mediated DNA
recombination, other transformation methods rely on natural eukaryotic DNA
recombination methods for the incorporation of transgene DNA into genomic DNA. The
transformation method described in this study also relies on natural recombination
activity. However, it may be possible to enhance natural recombination efficiencies.
Stressful conditions can induce genomic rearrangements in plants. In 1984,
Barbara McClintock reflected on the “importance of stress in insti gating genome
modifications by mobilizing cell mechanisms that can restructure genomes.” Pathogen
infection, including viral infection, is one form of biotic stress that has been demonstrated
to trigger genomic rearrangements. Lucht et a1. (2002) observed somatic recombination
in Arabidopsis following infection by an oomycete pathogen. Viral infection was shown
by Kovalchuk et a1. (2003) to induce DNA rearrangements in tobacco plants. Mottinger
et al. (1984) described mutations in the maize genome following Barley stripe mosaic
virus infection. Abiotic stress, such as radiation (McClintock, 1984; Ries etal., 2000),

can also induce genomic changes. If plants respond to stressfirl conditions by rearranging

21

their genetic material, perhaps stress could enhance uptake of foreign DNA, and thus
could be utilized in a transformation system to increase transformation efficiency.

Previous experiments in this laboratory established electrical parameters that
moved DNA from an external source to the meristematic area. DNA stained with
ethidium bromide was applied to the apical meristem of bean seedlings and subjected to
an electrical current. The apical meristem was sliced lengthwise and observed under
ultraviolet light to determine movement of the transgene DNA into the plant tissue. These
studies concluded that 15 minutes was adequate to move the DNA into the optimal
position of the apical meristem (unpublished results). Additionally, several stress
pretreatment conditions were tested, including heat treatment and application of plant
growth hormones such as 2,4-D, kinetin, NAA, and BAP. Lipofectin® (Invitrogen,
Carlsbad, CA), a transfection reagent, was also evaluated as a pretreatment in an attempt
to increase uptake of transgene DNA. No transformation events were identified with
these pretreatments (unpublished results). This lead to the search for other stress factors
that may assist transgene recombination events.

Homoserine lactones are well-characterized signaling compounds of gram-
negative bacteria (Fuqua et al., 2001; Williams etal., 2007) and play significant roles in
bacterial-host infection and symbiosis. In addition to their function as bacterial quorum-
sensing signals, homoserine lactones also induce responses in eukaryotes. Plants respond
to homoserine lactones in a variety of ways, including activating defense and stress
responses (Bauer & Mathesius, 2004; Mathesius et al., 2003; Schuhegger et al., 2006).

Since stress responses can include genome reorganization, homoserine lactone may

22

contribute to initiation of recombination events in plants. Homoserine lactone treatments
were evaluated in this study in an attempt to increase transformation efficiency.

The economic importance of P. vulgaris, the limitation of sexually-compatible
genetic resources, and the lack of a suitable plant transformation system justify P.
vulgaris as a target for the development of a novel transformation system.
Morphologically P. vulgaris is ideal because of its large and accessible meristematic
dome which cases the mechanical manipulation of the target area. This study
demonstrates preliminary success in transformation of P. vulgaris by a novel method
referred to hereafter as electro-transformation. While the technique was developed using

P. vulgaris, it should find applications in other agriculturally important species.

23

MATERIALS AND METHODS
Plasmid Construction

The plasmid pRD400/3SS-gf-2.8 (Donaldson et al., 2001) was digested with
EcoRI and Hindlll, and the 1.7 kb fragment containing the cauliflower mosaic
caulimovirus (CaMV) 35S enhanced promoter, the 0.9 kb SphI fragment from the germin
gene gf-2.8 (accession number M63223), and a CaMV 35$ polyadenylation signal was
isolated. This fragment was ligated into the EcoRI and Hindlll sites of pCAMBIA3300
(CAMBIA, Canberra, Australia), preceding the bar gene which encodes resistance to
glufosinate ammonium. The resulting plasmid was digested with Hindlll and Sspl, and
the fragment containing both the bar and germin genes plus their controlling sequences
was ligated into the pBluescriptKSII(-) plasmid (Stratagene, La J olla, CA). The final
plasmid product is referred to as pBKSbar/gf-Z 8. Prior to electro-transformation,
pBKSbar/gf-2.8 was digested with HindIII and Sspl to release a 4.8 kb fragment
containing both the bar and germin genes and their controlling sequences (Figure 1.1). In
most cases the desired transformation fragment was not isolated from the 2.1 kb bacterial

plasmid backbone prior to transformation.

left
Hindlll CaMV 353 germin polyA CaMV 358 bar polyA border Sspl

arr-mg“ .. CWT—w _. «unriva- we q. . w . . .— ,
‘ -' . . --.r
‘_ :ilr':1-.:'.’.5"j:' 1‘5) ' ’I-‘- J :5 L113... é. 'o':"-." f. "4" - ,

 

 

 

 

Figure 1.1. Hindlll-Sspl fragment from pBKSbar/gf-2.8 used to transform P. vulgaris
seedlings. CaMV 35S indicates the enhanced 358 promoter from cauliflower mosaic
caulimovirus. The germin gene encodes a putative factor involved in resistance to white
mold. The bar gene provides resistance to the herbicide glufosinate ammonium. PolyA
refers to the termination signal from cauliflower mosaic virus. Left border refers to the
Agrobacterium left border sequence from pCAMBIA3300.

24

Electra-Transformation

Phaseolus vulgaris, cultivar T9905 from Hyland Seeds (Blenheim, Ontario,
Canada), was used for electro-transformation. To seeds were germinated in Baccto High

Porosity Professional Planting Mix (Michigan Peat Company, Houston, TX) and 5- to 8-
day-old seedlings were removed and their roots were rinsed with distilled water. The
apical meristem was exposed by teasing leaf primordia away from the meristematic dome
with a dissecting needle. If the experiment required a pretreatment, it was incorporated
into a 1.5 ul solution of IX TAE buffer and injected into the tip of the meristem with a
30G1/2 needle (see Table 1.1). Pretreatrnents included varying concentrations of DL-
homoserine lactone (ICN Biomedicals Inc., Costa Mesa, CA), ranging from 1 mM to 100
mM. Thirty plants were transformed for each treatment.

Figure 1.2 illustrates the electro-transforrnation setup. For the application of DNA
to the apical meristem, a mixture of 49 pl of 1% SeaPlaque low melting point agarose
(Hoefer Scientific Instruments, San Francisco, CA) in 1X TAE buffer and 1 ul of 10
ng/ul DNA was drawn into a 200 u] pipet tip. Each yellow pipet tip had been modified by
removing 2 mm from its terminus to increase the diameter of the pore. Following
solidification of the agarose, the head space remaining in the tip was filled with 1X TAE.
Each seedling was supported within a 50 ml polypropylene vial filled with 1X TAE, and
the pipet tip containing the DNA-agarose mixture was lowered gently upon the seedling’s
apical meristem. A platinum positive electrode was placed within the vial containing the
root of the bean seedling, and the negative electrode was placed within the buffer
reservoir of the yellow tip containing the DNA-agarose mixture. With the pipette tip in

contact with the plant meristem, the system was subjected to 125 V and 0.15 mA for 15

25

min of direct current followed by 30 sec of alternating current (Figure 1.2). These
electrical conditions were previously established by visualizing the migration of ethidium

bromide stained DNA within the apical meristem.

   
 
 

agarose
K

bean
seedfing

cotyledons

 

Figure 1.2. Depiction of electro-Iransformation setup. The seedling was placed into a
conical tube filled with buffer. The pipet tip containing the DNA/agarose mixture
(stippled area) was placed onto the seedling meristem and filled with buffer. “+” indicates
the positive electrode, an “-“ indicates the negative electrode. Dotted line designates
surface of buffer.

26

Following each electro-transformation attempt, the bean seedling was rinsed with
distilled water and potted into soil. Plants were tented with plastic wrap and maintained
in a laboratory growth room for 24-48 hr at 22 °C without supplemental lighting,
followed by transfer to a transgenic plant approved greenhouse. Each plant was labeled
individually and grown to maturity. Seeds were harvested and preserved for screening.
Herbicide Screen

Prior to greenhouse screening for herbicide resistance, kill curves were
established for glufosinate ammonium (Liberty®, Bayer CropScience, Research Triangle
Park, NC) using non-transformed P. vulgaris plants, T9905, at the secondary leaf growth
stage. Herbicide concentrations ranging from 50-300 mg/l were tested. Herbicide was
applied with a spray bottle and both the top and bottom leaf surfaces were saturated. Kill

curves were re-established periodically to account for seasonal variations and to help

avoid escapes. Glufosinate ammonium was applied to T1 plants at the secondary-leaf

growth stage. Surviving plants were sprayed an additional time, and T1 resistant plants

were identified 1-2 weeks following the herbicide application. DNA was extracted from
seedlings resistant to the herbicide and analyzed for the presence of marker genes via

polymerase chain reaction (PCR) and Southern blot analysis. Resistant plants were grown
to maturity and the T2 seeds were collected. Plants were labeled in the following manner:

the letter designation refers to the treatment, and the first number refers to the plant

transformation event, while subsequent numbers refer to progeny plants. For example,
B20 refers to plant 20 of Treatment B (To), while 820-3 refers to the third T1 progeny

plant derived from B20.

27

DNA Extraction

Total DNA was isolated from plants displaying resistance to glufosinate
ammonium. One hundred to three hundred mg of expanding leaf tissue was removed
from each plant using forceps, frozen in liquid nitrogen, and ground in an Eppendorf tube
using a mini pestle. DNA was extracted from the pulverized tissue using Plant DNAzol
Reagent (Invitrogen, Carlsbad, CA), following manufacturer’s protocol with the
following modifications: initial incubation with DNAzol Reagent was extended to 15
min, RNase A was added during extraction to ensure RNA degradation, and the initial
centrifugation step was performed at 4 °C. Following extraction, each DNA pellet was
resuspended in 30 pl of TE buffer and incubated at 65 °C for 10 min to aid in DNA
resuspension. DNA was separated on a 1% agarose gel, stained with ethidium bromide,
and the concentration was estimated based on known DNA size marker concentrations.
Polymerase Chain Reaction (PCR)

PCR reactions contained the following components: 200 uM dNTPs, 1.5 mM

MgC12, 1X PCR Buffer, 0.2 uM each of forward and reverse primers, 1 unit of T aq DNA

Polymerase (Invitrogen, Carlsbad, CA; Promega, Madison, WI), and approximately 20
ng of genomic DNA. The forward primer for the bar gene was 5’-
GGTCTGCACCATCGTCAACC-3’ and reverse primer was 5’-
GAAGTCCAGCTGCCAGAAAC—3’ (Aragao et al., 2002), which provides for the
amplification of a 448-bp fragment. The forward primer for the germin gene gf-2.8 was
5’-AACCAGTGCCATAGACACTCTCCA-3’ and reverse primer was 5’-

TTCCCACGAGAAGCTCACCTTTCA-3’. The forward primer for the actin gene, which

28

was used as a PCR control, was 5’-GGACGAGGCTCAATCGAAGA-3’ and reverse
primer was 5’-ACTGACACCGTCTCCGGAGT-3’ (Hamada et al., 2002), which
amplified an approximately 900-bp fragment. Amplification conditions were as follows:
94 °C for 2 min, followed by 30 cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C,
with a final extension step at 72 °C for 5 min.

Southern Analyses

Total genomic DNA (10-15 ug) fiom herbicide-resistant plants was digested with
35 units of HindIII (Invitrogen, Carlsbad, CA) overnight. Solutions for agarose gel
manipulations and post-hybridization membrane washes were as in Vallejos et a1. (1992),
which describes Southern analyses of P. vulgaris DNA. Digested samples were loaded
using a bromophenol blue/ glycerol loading dye onto 1% agarose gels containing 1X
modified TAE buffer (100 mM Tris-acetate, 1 mM EDTA, pH 8.1), and electrophoresed
at approximately 47 V at 4 0C. Gels were stained with ethidium bromide and visualized
under UV light. Gels were depurinated with 0.25 M HCl for 10 min, followed by
denaturation in 0.4 M NaOH, 1.5 M NaCl for 30 min. DNA was transferred from agarose
gels to Nytran nylon membrane (Schleicher & Schuell, Keene, NH) in alkaline transfer
buffer (20 mM NaOH, 0.6 M NaCl) overnight. Following transfer, membranes were
washed briefly in 2X SSPE, air-dried, and UV crosslinked.

The DNA templates for bar, germin, and actin gene probes were synthesized
using PCR. Amplification conditions and primers were the same as those described in the
previous section. The plasmid pBKSbar/gf-2.8 served as the amplification template for
bar and germin, while wild-type P. vulgaris genomic DNA was used as the template for

the actin control. The amplification products from the PCR reactions were gel isolated

29

using Qiaquick Gel Isolation Kit (Qiagen, Valencia, CA) and used as the template for
random prime labeled probes. Radiolabelled probes were created using a Random Prime
Labeling Kit (Invitrogen, Carlsbad, CA), incorporating a-32P-labeled dCTP
(PerkinElmer, Waltham, MA). Membranes were prehybridized in UltraHyb solution
(Ambion, Austin, TX) for 30 min to 1 hr at 42 °C, followed by hybridization at 42 °C
with the radiolabelled probe in UltraHyb solution overnight. Blots were washed briefly
with 2X SSPE, 0.1% SDS, followed by two or three 15 min washes with 2X SSPE, O. 1%
SDS and one or two washes with 0.5X SSPE, 0.1% SDS. Hybridizing fragments were

visualized by autoradiography.

30

RESULTS
Each treatment included thirty P. vulgaris seedlings. Seven different homoserine

lactone pre-treatments were evaluated, for a total of 2 1 0 treated seedlings (Table 1.1). A
total of915 seeds were collected from the To generation and planted for herbicide
screening. Glufosinate ammonium concentrations between 150 and 200 mg/l, as

determined by a control application prior to T1 testing, were sprayed onto T1 plants at the

second leaf growth stage. Ninety-two T1 seedlings were resistant to glufosinate

ammonium and were evaluated further with molecular tests.

Table 1.1. Treatments of P. vulgaris apical meristems prior to electro—transformation, and
results of analyses on T1 generation.

Homoserine # of T1 8 # of T1s # 0f T1s # 0f T15

 

Series #T l nt . . . . ' ' f r with
. . Lactone 1 p a s h rb d t f r posrtive o

DGSIQDaUO" - analyzed e .'c' e posr we 0 germin positive
000090" 31'0" resistant bar PCR PCR Southern

control 0 mM 255 O 0 0 0

A 1 mM 306 31 12 2 1

B 5 mM 183 33 11 1 0

C 15 mM 75 1 O 0 O

D 30 mM 137 15 9 2 0

E 50 mM 67 O O 0 0

F 75 mlvl 116 11 4 1 O

G 100 mM 31 1 1 1 O

 

Genomic DNA extraction from P. vulgaris was challenging, with previous
approaches yielding very little DNA. Extraction from P. vulgaris leaf tissue using
DNAzol was optimized for these experiments. Protocol modifications described in the
previous section allowed for the effective isolation of genomic P. vulgaris DNA suitable

for PCR, restriction enzyme digestion, and Southern blot analyses.

31

Of the 92 plants displaying herbicide resistance, 37 plants provided PCR products
with primers specific to the bar gene (Figure 1.3). Seven plants had PCR amplification
products with germin-specific primers (Figure 1.4). In each case, the PCR-amplified
fragment co-migrated with the PCR band derived from the original transformation
plasmid. All plants showed positive amplification with actin-specific primers (Figure
1.5).

Southern blot analysis with the bar gene (Figure 1.6) and germin gene (Figure
1.7) probes showed hybridization to genomic DNA from one T1 plant, A25-4.
All T2 progeny, a total of approximately 870 plants, from T1 plants showing a

positive bar PCR were analyzed by PCR for the presence of the bar gene. None of the

plants from the T2 generation showed PCR amplification of the bar gene (data not

shown).

32

A27-4
A27-8
A28-1
A30—1
31 3-2

‘7 v . '7
2 2 2 2
‘

 

828-4
026-1

 

026-3
029-1
029-3
029-4
029-5
030-2
F-3
F-4
F-5
F-7

G-2
water
T9905
plasmid

 

Figure 1.3. PCR amplification of the bar gene from P. vulgaris total genomic DNA from
T1 plants. Ladder = lkb Plus DNA Ladder (Invitrogen, Carlsbad, CA). T9905 =
untransformed P. vulgaris control. Plasmid = pBKSbar/gf—Z. 8. Water = no DNA negative
control. Gel images were combined to provide continuity of the figure. Positive and
negative controls were included in each original gel. Band size is indicated on the left-
hand side of each gel.

33

A27-4
A27-8
A28—1
A30-1
BI 3-2
B1 4-1
B14-3

' 'Ladder
A2-
A2-4
A2-5
A25-3
A25-4
A27-2

   
 

 

400 bp —
300 bp —
S ‘7 ‘7 9.1 ‘7 ‘l .9 «.9 N. '7 9.1 <2 <2 v
13 In C) 0) co (0 CO CD w to (.0 (D C) O)
(U ‘- \- u- N N N N N N N N N N
.J m m m m m m (D m D D C) D D
400 bp —
300 bp — ' '
5 to 9
If) N L. E
'U I c— O Q)
8 81’ 8 <2 ‘I «2 N. on 8 a E
_l D O u. u. L LL (9 I— 3 o.
400 bp —
300 bp —

Figure 1.4. PCR amplification of the germin gene from P. vulgaris total genomic DNA
from T1 plants. Ladder = lkb Plus DNA Ladder (Invitrogen, Carlsbad, CA). T9905 =
untransformed P. vulgaris control. Plasmid = pBKSbar/gf—2.8. Water = no DNA negative
control. Gel images were combined to provide continuity of the figure. Positive and
negative controls were included in each original gel. Band size is indicated on the left-
hand side of each gel.

34

E 4 .0 ,2 n- o? ‘i 9.1 ‘l 09 F. v 9.1
‘o ‘T . . . co Lo LO ix ix ix oo o to
re N N N N N N N N N g N co -—
_l < < < < < < < < < < < m
850 bp — -
650 bp '—

 

Ladder
814-1
314-3
315-1
819-1
819-2
828-1
828-4
828-5
828-6
828—7
D10-2
026-1
026-2

8501’“ .... flue-”CUM.-

650 bp —

 

 

Figure 1.5. PCR amplification of the actin gene from P. vulgaris total genomic DNA
from T1 plants. Ladder = lkb Plus DNA Ladder (Invitrogen, Carlsbad, CA). T9905 =
untransformed P. vulgaris control. Water = no DNA negative control. Gel images were
combined to provide continuity of the figure. Positive and negative controls were
included in each original gel. Band size is indicated on the left-hand side of each gel.

35

A)

 

‘ 'A27-8 T9905
12.0 -
8.0 —

5.0 -
4.0 —

3.0 -

Molecular Size (Kb)

2.0 -

 

3)

Molecular Size (Kb)

 

Figure 1.6. Southern blots of DNA hybridized with a radiolabeled bar gene probe. A) P.
vulgaris total genomic DNA from several T1 plants. T9905 = untransformed P. vulgaris
control. Film was exposed to blot for 2 days. B) pBKSbar/gf-ZB digested with HindIH
and Sspl (positive control for bar probe). Film was exposed to blot for 30 min.

36

Molecular Size (Kb)

A)

\ v

       
       

 

029-3 F-7 ‘D_26-2 .D15-1 A25-4 A27-8 T9905

12.0—

8.0 -

5.0 -
4.0 -

3.0 -

2.0 -

3)

Molecular Size (Kb)

 

Figure 1.7. Southern blots of DNA hybridized with a radiolabeled germin gene probe. A)
P. vulgaris total genomic DNA from several T1 plants. T9905 = untransformed P.
vulgaris control. Film was exposed to blot for 4 days. B) pBKSbar/gf-2.8 digested with
HindIII and Sspl (positive control for germin probe). Film was exposed to blot for 20
mm.

37

DISCUSSION

One T1 plant, A25-4, showed phenotypic evidence for the bar gene, plus both

PCR and Southern genotypic evidence for the two distinct marker genes. PCR amplified
bands derived from A25-4 were the same size as the band amplified from the plasmid
control. These five lines of evidence demonstrate that electro-transformation is a
functional method for Phaseolus vulgaris transformation. There does not appear to be a
significant correlation between homoserine lactone concentration and number of plants
positive for the presence of the marker genes (Table 1.1). Rather, it appears the presence
of homoserine lactone is sufficient for transformation. Since one plant out of 915 plants
analyzed was confirmed to be transformed, we could conclude that the efficiency of the
electro-transformation method is approximately 0.1%. Reported transformation
efficiencies of P. vulgaris range from 0.03% to 0.9%. Consequently, the transformation
efficiency of electro-transformation is roughly comparable to previously-described dry
bean transformation methods.

Several questions arise from the results of this study: 1) Why did the majority of
herbicide-resistant plants lack the bar gene when analyzed via PCR? 2) Why did PCR
analysis result in amplification of the germin gene from only seven of the 37 plants

positive for bar PCR? 3) Why did Southern analysis show the presence of the bar and
germin genes in only one of the PCR positive plants? 4) Why were all of the T2
generation plants from bar-positive parents negative for the bar PCR?

Although 92 T1 plants survived herbicide spraying, only 37 of the surviving

plants showed amplification of the bar gene. Previous transformation experiments using

the bar gene have reported up to 90% escape of screening with glufosinate ammonium

38

(Becker et al., 1994; Nehra et al., 1994; Ortiz et al., 1996). Therefore, our observation of
approximately 60% escape may not be unreasonable in the context of previously-
described herbicide screening experiments. Herbicide concentration kill curves were re-
established periodically in an attempt to apply an appropriate concentration for 100% kill

of non-resistant plants, but by design these concentrations had to be established at least

one week prior to T1 testing. Therefore, the herbicide concentration may not have been

appropriate for the greenhouse conditions on the days T1 plants were screened. Further,

extra high herbicide concentrations were avoided to help prevent overwhelming a
potential transforrnant.

Concerning the unequal PCR amplification of bar and germin genes, the bar gene
was originally isolated from Streptomyces hygroscopicus, a soil bacterium. If even a trace
amount of soil were adhered to the plant leaves from which DNA was isolated, and the
soil contained S. hygroscopicus, then the bar PCR product could have been amplified
from the bacterial genome rather than from a plant transgene. However, if this is the case,
we would expect to see false positives from the control T9905 plants as well. Since we do
not understand the method of DNA integration into the chromosome, there is a possibility
that the bar region of the intended insert was preferentially inserted or maintained within
the chromosome. These may in part explain the lack of germin gene amplification from
the majority of the plants showing a positive bar PCR result.

An important point to note is that the germin PCR amplification appears to be
much less efficient than the bar PCR. Although the PCR analyses in this study were not
quantitative, it is obvious from observing numerous gels that the amplification product

from the bar PCR is consistently much more intense than the germin amplification

39

product. This may be an indication that the germin-specific primers were not as efficient
as the bar primers due to DNA modifications or structural modifications of the
chromosomal DNA. As a result, it may have been more difficult for the polymerase to
amplify the germin gene from genomic DNA. However, modification of the PCR
amplification conditions did not reveal additional positive samples.

An alternative explanation for bar false positives is that a homolog to the bar
gene exists in P. vulgaris. However, if this were the case, false positive bands of an

alternative size would be predicted in T9905 control plants; none were observed.

Following selection of the T1 generation, we expected that the transgene would be
inherited in a Mendelian fashion in subsequent generations. Figure 1.8 depicts the
anticipated inheritance pattern. The T0 generation should only have the transgene
integrated into one or a few meristematic cells, some of which are destined to become
germ cells. The T0 plant is therefore chimeric, as only some cells will contain the

transgene. Assuming a single insertion into a pre-meiotic cell, that cell would produce

two gametes containing the transgene and two without the transgene. Upon selfing of the
To plant, 50% of the seeds resulting from fertilization of the gametes from the original
cell should be heterozygous for the transgene. These transgenic seeds (T1 generation)

should not be chimeric; all cells within the plants should contain a copy of the transgene.
As a result, these plants would display resistance to the herbicide, and DNA from any

tissue would show amplification of the bar gene. If the bar gene functions dominately,

selfing should provide for a 3:1 phenotype in the T2 generation, with 75% of plants

carrying the transgene. Of the transgenic T2 plants, one-third will be homozygous and

40

two-thirds will be heterozygous for the transgene. Our screens should identify both the

heterozygous and homozygous plants.

 

1 herbicide
selection

9 = transgene

T1 @
// \\

o o o o
25 /o 25 /o 25 /o 25 /o 75% Of T2

T 2 generation is
transgenic

Figure 1.8. Expected inheritance pattern of a transgene following integration into
meristematic tissue and subsequent selection. P. vulgaris is self-pollinating.

To avoid any inadvertent loss of transformed plants, T2 plants originating from T1
bar PCR positive plants were not screened with herbicide. Rather, we relied on the more
sensitive and less destructive PCR test for T1 transforrnants. Out of the 870 T2 plants

tested, we did not find any to be positive for the bar gene PCR screen. This observation

differs significantly fi'om the expected inheritance pattern. One possible explanation for

the absence of the transgene in the T2 generation is that the transgene was somehow

eliminated.

41

There are numerous examples in the literature of transgene loss and/or
inactivation. The examples comprise a variety of different plant species, including

legumes, as well as different transformation approaches.

Transgene loss in the R1 generation was observed in both P. vulgaris and soybean
(Glycine max L.) transformed via particle bombardment (Romano et al., 2005). The

transgenes were maintained during vegetative propagation of the R0 plant lines, but were

lost upon meiosis and were consequently absent in the R1 generation. Interestingly, upon

analysis of the transgene integration sites within the genome, both dry bean and soybean.
had lost transgenes that had been integrated into rRNA encoding regions. The authors
provide several hypotheses to explain transgene elimination: intrachromosomal
recombination, activation of a genomic defense system, or stress caused by tissue culture
conditions.

Upon transformation of soybean via particle bombardment, Choffnes et al. (2001)
initially observed Mendelian inheritance of the transgene, but the integration pattern was

not stably inherited by subsequent generations. Rearrangements of the transgene locus
were Observed in both the T1 and T2 generations. Inter- or intrachromosomal

recombination was thought to be responsible for the observed transgene rearrangements.
Another large-seeded legume, guar (Cyamopsis tetragonoloba (L.) Taub), showed
transgene loss in many of the transformed lines produced by Agrobacterium-mediated
transformation (J oersbo et al., 1999). Nearly half of the original primary transformants
produced progeny lacking the transgene. Transgene elimination was confirmed both by

phenotypic and genotypic analyses.

42

Examples exist in other plant groups, including both herbaceous and woody
species. Risseeuw et al. (1997) report deletions of all or part of a transgenic locus in
Nicotiana spp. transformed using Agrobacterium and discuss the possible role of tissue
culture in the observed transgene instability. Transgene elimination has also been
observed in wheat (T riticum aestivum L.) transformed by particle bombardment (Altpeter
et al., 1996). Srivastava et a1. (1996) also noted rearrangements in transgene sequences in
previously stably-transformed wheat lines. In addition, the transgene was methylated in
most lines. Aspen (Populus tremuloides Michx) transformed using Agrobacterium
displayed phenotypic reversion to wild-type, and transgene loss was confirmed by
molecular analyses (F ladung, 1999). The original transgene locus showed unusual
organization, containing a partial inverted repeat, which may have contributed to
transgene elimination.

Transgene loss is not limited to sexually-produced offspring. Elimination of
transgenes has been observed in banana plants produced by vegetative propagation
(Matsumoto et al., 2007). Therefore, meiosis is not necessarily a prerequisite for
transgene loss.

There are a number of potential reasons for transgene elimination from one
generation to the next. During meiosis, specific conformation and pairing of the
chromosomes are required, and the presence of extra transgenic DNA that has no allelic
meiotic partner may affect this process. The foreign DNA may be eliminated to ensure
proper progression of meiosis. Alternatively, due to their foreign nature, transgene
integration sites may be targeted by host nucleases for degradation (Kumpatla et al.,

1998).

43

Research on the mammalian genome has demonstrated preferential integration of
transgene DNA into certain regions, suggesting the existence of possible recombination
“hotspots” in the genome (Merrihew et al., 1996). These target insertion regions were
found to be unstable. Although this study involved mammals, there are likely similar
genetic mechanisms in all eukaryotes, thus these observations may be applicable to
plants.

An alternative explanation of our observed lack of confirmed transgenic plants in
the T2 generation may be that the transgene insertions resulted in gamete disruption

and/or embryo lethality. Non-Mendelian segregation has been observed in transformed
Arabidopsis thaliana and found to be the result of T-DNA insertion into genes essential
for gametophytic function (F eldmann et al., 1997; Howden et al., 1998). Because of these
gametophytic disruptions, transgenic progeny were observed at a lower frequency than

expected.

Despite the lack of positive T2 plants, it is important to emphasize the evidence
supporting the initial success of the electro-transformation method. One transformation
event was successful as demonstrated by a single T1 plant containing the bar gene,

confirmed by three different lines of evidence, plus PCR and Southern evidence for the
co-transgene, germin. The phenotypic evidence of herbicide resistance compounded with
the molecular confirmation by both PCR and Southern analyses give us confidence that
this novel plant transformation method is indeed possible.

This study demonstrates the development of a novel plant genetic modification
system referred to as electro-transformation. Phaseolus vulgaris, dry bean, was selected

for the development of a novel transformation system because: 1) it is a crop species with

44

worldwide nutritional importance, 2) a critical need exists for both disease resistance and
abiotic stress tolerance, and 3) current transformation methods, including particle
bombardment and Agrobacterium, have proven unsuitable for this species. With the
proof-of-concept in hand, further research will focus on increasing the transformation
efficiency. Other P. vulgaris genotypes will be evaluated to avoid any transgene stability
issues that may be peculiar to T9905. In addition, use of smaller DNA constructs may
increase transgene incorporation. Short repeated flanking regions derived from bean
genomic DNA sequences may stimulate homologous recombination between the
transgene construct and bean DNA and thus increase integration of the transgene.

We anticipate that electro-transformation will facilitate the genetic improvement
of dry bean and eventually contribute to the nutrition of individuals who rely on this
species as a calorie and protein source. In addition to contributing to the improvement of
dry bean agriculture, we predict that electro-transforrnation will be applicable to not only
other large-seeded legumes, such as cowpea and soybean, but to other important crop

species.

45

CHAPTER 2

CHARACTERIZATION OF PRUNUS NECROTIC RINGSPOT ILARVIRUS
(PNRSV) ISOLATES IN MICHIGAN

INTRODUCTION

Cherry, both sweet (Prunus avium L.) and tart (Prunus cerasus L.), is an
agriculturally important crop in Michigan, contributing significantly to the state’s
economy. In 2005, Michigan produced 208 million pounds of tart cherries, and accounted
for 77% of total US. tart cherry production. Michigan produced 27,000 tons of sweet
cherry in 2005, 11% of the US. total. Washington State is also a significant contributor
to the US. cherry industry and in 2005 produced 16.5 million pounds of tart cherries (6%
of US. total) and 138,000 tons of sweet cherries (55% of US. total) (USDA, 2006).

A number of bacterial, fungal, and viral pathogens infect cherry, and disease
symptoms can impact fruit quality and yield. Prunus necrotic ringspot ilarvirus (PNRSV)
infects sweet and tart cherry, and both visual and antigenic (ELISA) analyses have shown
the presence of this virus in Michigan cherry orchards (Hamilton, 1987; Monissey,
1988). Although PNRSV infection is not lethal to agriculturally important cherry
varieties, it significantly impacts crop yield and quality. Lewis (1951) observed up to a
41% yield loss in tart cherry trees showing ring spot symptoms compared to
asymptomatic trees. Morrissey (1988) described the effect of PNRSV infection on tart
cherry tree decline in Michigan. Decline orchards were defined as orchards composed of
trees with characteristics such as trunk damage, blindwood, and low vigor. There was a

positive relationship between orchard decline and PNRSV infection rate within these

46

decline orchards (Morrissey, 198 8). However, economic losses to cherry due to PNRSV
have not been quantified in Michigan.

In addition to infecting cherry trees, PNRSV infects a variety of other stone fruit
species, including peach, almond, and plum. A study of PNRSV infection of peach in
New Zealand estimated a yield loss of 18% in infected plants (Wood et al., 1997). This
study also quantified the spread of PNRSV in a peach orchard. The authors reported a
60% infection rate of PNRSV in an initially virus-free planting block after 7 years,
indicating the virus had spread efficiently from nearby peach trees within the same
orchard. These results emphasize the magnitude of PNRSV spread throughout an
orchard.

PNRSV is also a concern in many European countries. The European Union
sponsored the “Ilarvirus Ringtest” in 1998 in an attempt to better characterize PNRSV in
European orchards (Hammond, 2003). The goals of the Ringtest included expanding the
number of PNRSV nucleotide sequences available, in addition to assessing and
improving diagnostic procedures for ilarviruses in woody crops. Numerous other groups
have published characterizations of PNRSV in Europe (Aparicio et al,. 1999; Glasa et al.,
2002; Herranz et al., 2005; Spiegel et al., 2004; Vaskova et al., 2000), resulting in the
availability of a large number of partial PNRSV sequences in GenBank.

PNRSV (ICTV # 00.010.002.016) is a member of the Ilarvirus genus in the
family Bromoviridae. Ilarviruses are icosahedral viruses and their genome consists of
three single-stranded positive-sense RNA molecules (Figure 2.1). RNA 1 encodes the la
protein, involved in replication. RNA 2 encodes the 2a protein, also part of the replicase,

and the 2b protein, a putative silencing suppressor which is expressed from subgenomic

47

(sg) RNA 4A. RNA 3 encodes the 3a protein, which functions as the movement protein,

and the 3b protein, which is the coat protein and is expressed from ngNA 4.

RNA 1 1a
RNA 2 2a

ngNA 4A 2b

RNA 3 movement coat

ngNA 4 coat

Figure 2.1. Depiction of the ilarvirus genome. RNAs l and 2 encode components of the
replicase. ngNA 4A is derived from RNA 2, and encodes the 2b protein gene. RNA 3
encodes the movement protein gene, and the coat protein gene which is expressed from
ngN A 4.

Symptoms of PNRSV infection, defined as the pathotype, can range from
symptomless to severe, depending on the particular virus strain (isolate). Infected trees
may show symptoms such as chlorosis or necrotic rings on leaves. The more severe
infections, referred to as the rugose pathotype, manifest as wrinkled, deformed leaves.
PNRSV infection can result in delayed fruit set.

PNRSV is spread via pollen and seed, and through mechanical inoculation,
including pruning and grafting. Because PNRSV is spread through pollen, bee movement
throughout an orchard can transmit the virus from tree to tree (George & Davidson, 1963;

George & Davidson, 1964). Transporting bees from an infected orchard to an uninfected

orchard can also transmit the viral infection. In the late 19703, beehives from Washington

48

State were loaned to stone fruit orchards in California for early spring pollination and,
upon return to Washington, PNRSV antigens were detected in stored bee pollen (Howell
& Mink, 1988). The natural movement of bee pollinators is the primary mechanism by
which PNRSV is spread within an orchard and between adjacent orchards, while the
commercial movement of hives contributes to long-distance spread. Seed transmission is
not likely a significant contributor to PNRSV spread, as seed planting is not a
predominant method of modem-day cherry propagation. In addition, PNRSV
transmission via seed is relatively low, ranging from 5-14% (Maeso Tozzi et al., 1995).
Vegetative propagation of cherry and other hosts and transportation of cherry rootstock
among nurseries have contributed to worldwide distribution of PNRSV. Although
recently implemented phytosanitary measures and virus-free certification programs
(Cembali et al., 2003; Rowhani et al., 2005) have likely reduced the spread of PNRSV,
orchards with established infection continue to provide a local source of the virus. Since
the virus is non-lethal, many growers do not recognize its presence or its contribution to
yield loss.

To create a strategy to decrease the impact of PNRSV, it is essential to thoroughly
characterize the 'virus and understand its diversity and evolution. A number of different
approaches have been attempted to understand the relationship between PNRSV isolates
around the world. Serological analyses of PNRSV using polyclonal antisera (Mink,
1987), have identified three distinct serotypes, CH3, CH9, and CH30, each named after
the isolate from which the antibody for the serotype was obtained. However, no
relationship between the pathotypes and serotypes was established, suggesting symptoms

are not directly correlated with capsid epitopes. Other attempts have been made to

49

demonstrate a relationship between pathotype and serotype. Nucleic acid hybridization
(Crosslin et al., 1992) and assessment of viral particle biophysical characteristics
(Crosslin & Mink, 1992) were unsuccessful at correlating pathotype and serotype. In
contrast, evaluation of electrophoretic mobility of PNRSV virions (Ong & Mink, 1989)
proved useful in distinguishing among some, but not all, isolates.

More recent approaches have been more successful at categorizing PNRSV
isolates. Hammond and Crosslin (1998) observed correlations between nucleotide/protein
sequences, serology, and pathotypes. Hammond et a1. (1999) described the use of
multiplex polymerase chain reaction to differentiate among PNRSV isolates, and
phylogenetic analyses have been useful in understanding the relationships among PNRSV
isolates (Aparicio & Pallas, 2002; Glasa etal., 2002; Hammond, 2003; Hammond &
Crosslin, 1998; Vaskova et al., 2000). Comparison of 3a proteins of PNRSV showed
homology among different isolates (greater than 90% similarity) even though the isolates
were extracted from different hosts (cherry and peach). Scott et al. (1998) observed high
homology among coat protein sequences of different isolates, with only four to ten
differences in amino acids of coat proteins.

When Hammond and Crosslin (1998) compared the nucleotide sequences of RNA
3 of various US isolates, they found that the CH9 serotype grouped together. Within this
CH9 serotype, mild isolates grouped together and could be distinguished from rugose
isolates, which also grouped together. Similarly, the CH30 serotypes were distinct. These
relationships were reflected in the amino acid sequences of both the movement and coat

proteins.

50

Several authors have classified PNRSV isolates into three groups based on
phylogenetic analyses: PV32 (also known as Group I), PV96 (Group II), and PE5 (Group
III) (Aparicio & Pallas, 2002; Glasa ct al., 2002; Hammond, 2003; Vaskova et al., 2000).
Some authors describe an additional PNRSV group, referred to as CH30 (Glasa et al.,
2002). The PV32 group is named for representative isolate PV32 from the US (Sanchez-
Nevarro & Pallas, 1997), PV96 is named for a German isolate PV-0096 (Guo et al.,
1995), and PE5 is named for US isolate PE5 (Hammond & Crosslin, 1995). Regarding
the serotype classifications of Washington isolates by Hammond & Crosslin (1998),
groups PV32 and PV96 contain isolates of the CH9 serotype, while group PE5 contains
isolates of the CH30 serotype in addition to one CH9 serotype isolate (Hammond, 2003).
Upon comparison of the nucleotide sequence of part of the coat protein coding region,
Hammond (2003) noted a correlation between sequence and pathotype. Group PV32
consisted of mainly severe isolates, group PV96 contained isolates with mostly a mild
pathotype, and group PE5 consisted of isolates with varying symptomology. Hammond
& Crosslin (1998) also discussed the relationship between isolate sequence and symptom
severity. The authors noted that the area of most sequence variation in the movement
protein amino acid sequence lies within the carboxy-terrninus of the protein, with amino
acid substitutions correlating with symptom severity. The area of highest sequence
divergence in the coat protein is the amino-terminus of the protein, with variation again
showing correlation to pathotype.

Numerous sequences of PNRSV isolates are available in GenBank. Most are of
isolates obtained from European and Middle-Eastem countries. Of the sequences derived

from United States isolates, most are from Washington State. Only a single isolate

51

sequence from Michigan is available in GenBank (Scott et al., 1998). Given the economic
importance of cherry to the Michigan economy, this study focuses on identifying PNRSV
in Michigan orchards, and characterizing Michigan isolates of this agriculturally-
important pathogen by sequencing Michigan PNRSV isolates and comparing them to
previously-sequenced isolates from other regions of the world.

Results of this current study add to the existing PNRSV sequence database and
suggest the prevalence of PNRSV in Michigan cherry orchards. Characterization of the
nucleotide sequences of Michigan isolates contributes to the understanding of the
evolutionary relationship of PNRSV isolates and has inspired continuing research on a

virus resistance strategy for perennial plants (see Appendix).

52

MATERIALS AND METHODS
Sample Collection and RNA Isolation

In cooperation with Michigan State University Extension agents and Michigan
cherry growers, leaves were gathered in September 2003 from 59 Michigan sweet and
sour cherry trees representing fifteen different commercial orchards across Michigan. Of
the 59 cherry samples, 25 were collected from southwest Michigan (Berrien County) and
34 were collected from northwest Michigan (Leelanau County) (Figure 2.2). Since the
intentions were to identify variation in virus isolates, trees showing potential PNRSV
symptoms were sampled purposely. Sample designation consists of two letters, referring
to the orchard from which the sample was collected, followed by a number, which refers
to a particular tree within the orchard.

For each sample, total RNA was isolated from approximately 70 mg of leaf tissue
using the RN easy Plant Mini Kit (Qiagen, Valencia, CA). Manufacturer’s instructions
were followed, including the optional steps of heating the sample in Buffer RLT for 3
min at 56 °C and an additional centrifugation following the addition of wash buffer. The

column membrane was eluted twice with 30 ul of RNase-flee water.

53

     

    

Cheboyga

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

Presque
Isle
Leelanau 03990 Montmor-- Alpena
ency
Kalkaska Crawford Oscoda Alcona
‘ Benzie
- Roscom- O emaw Iosco
Manisteejwexford Missaukee mon 9
Arenac
Mason Lake Osceola Clare Gladwin
Huron
Bay
Oceana Mecosta Isabella Midland
Newago Tusoola Sanllac
Saginaw
Muskegon Montcalm Gratiot
Lapeer '
Kent Shia- Genesee St. Clair
Ottawa lonia Clinton
wassee
Macom
Oakland
Allegan Barry Eaton lngham .Livingston
Van iKalamazooi Calhoun Jackson Washtenaw
Buren
Berrien Cass Jossel‘p h Branch Hillsdale Lenawee

 

 

 

 

 

 

Figure 2.2. Map of counties in the lower peninsula of Michigan. Stars indicate the
counties from which cherry samples originated. The shaded regions represent counties

with commercial cherry production.

54

Reverse Transcription Polymerase Chain Reaction

Primers used for amplification of the RNA 3 segment of PNRSV were modified
from Hammond and Crosslin (1998). The amplified region of RNA 3 was an
approximately 1.64 kb fragment containing the entire movement protein gene, the
intergenic region, and the majority of the coat protein gene. This region was chosen in
order to compare Michigan isolate sequences with those from Washington State, as this
was the same region analyzed for most of the Washington isolates. Forward primer
(RA352), homologous to nucleotides 112-131 of PE5 (accession number L38823;
Hammond & Crosslin, 1995) RNA 3 is as follows: 5’ —
afiatgzggaGTGGGTTTAGAGATTGTIGG — 3’, the non-underlined lowercase letters
denote the added 5’ tail, underlined lowercase letters indicate the added XbaI restriction
site, and uppercase letters represent the PNRSV sequence. Reverse primer (RA353) is
complementary to nucleotides 1757-1738 of PE5 RNA 3: 5’ —
atttaaa_agc_t_tCATCGACCAGCAAGACATCA — 3’, with the same notation except the
underlined lowercase letters indicate the added HindIII restriction site. The restriction
enzymes HindIII and XbaI were selected for incorporation into the primers because
sequence analysis of the region of interest in PE5 RNA 3 showed the absence of both
HindIII and XbaI restriction sites, and these two sites are present in the cloning vector
used.

A 10 pl aliquot of total RNA was used as template for the reverse transcription
(RT) reaction. SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) was used
to synthesize the first-strand cDNA, following manufacturer’s protocol. The sample was

RNaseH-treated for 20 min at 37 °C following the RT reaction. A 2 pl aliquot of the RT

55

reaction served as template in the polymerase chain reaction (PCR) with Herculase
Enhanced DNA Polymerase (Stratagene, La Jolla, CA). Amplification conditions were as
follows: 95 °C for 2 min, followed by 10 cycles of 30 sec at 95 °C, 30 sec at 58 °C, 1 min
40 sec at 72 °C, then 20 cycles of 30 sec at 95 °C, 30 sec at 58 °C, 1 min 40 sec plus 10
sec per cycle at 72 °C, with a final extension step at 72 °C for 10 min.

Cloning and Sequencing

PCR amplification products were purified using Qiaquick Gel Isolation Kit
(Qiagen, Valencia, CA) and digested with XbaI and HindIII (Invitrogen, Carlsbad, CA).
Digestion products were examined on a 0.6% agarose, 1X TBE gel and gel-isolated using
Qiaquick Gel Isolation Kit. Following gel isolation, restricted PCR products were ligated
using T4 DNA Ligase (Invitrogen, Carlsbad, CA) into the pBluescript plasmid
(Stratagene, La Jolla, CA) digested with XbaI and HindIII.

Eight PNRSV clones in pBluescript were sequenced at the Michigan State
University Macromolecular Structure, Sequencing, and Synthesis Facility using ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Each clone was
sequenced at least two times to ensure accuracy of sequencing data.

Phylogenetic Analyses

Nucleotide sequences of the coat protein gene of Michigan PNRSV isolates were
aligned with other PNRSV isolates available in GenBank using ClustalX (Thompson et
al., 1997). The coat protein gene was chosen for comparison in order to include the
maximum number of isolates in the analysis, as the movement protein gene sequence is
not available for some PNRSV sequences. Gaps, due to availability of only truncated coat

protein gene sequences for many isolates, were treated as missing data by the analysis

56

program. The sequence of apple mosaic ilarvirus (ApMV) was used as the outgroup. A
heuristic search in PAUP" version 4.0b10 (Swofford, 2002), in which starting trees were
generated using a random addition sequence (10 replicates), was used to generate
maximum parsimony trees. MaxTrees was set to 500. Five-hundred bootstrap replicates
were generated. One neighbor-joining tree, with topology identical to one of 500 equally-
parsimonious trees, was generated, visualized in TREEVIEW (Page, 1996), and bootstrap
values were added in Adobe Illustrator CS (Adobe, San Jose, CA, USA). In addition,
amino acid sequences of the movement protein and coat protein were aligned using

ClustalX.

57

RESULTS
Table 2.1 shows the incidence of PNRSV infection in Michigan cherry orchards

sampled during this study. Of the 59 trees sampled, 26 (44%) showed an RT-PCR
product of 1.64 kb, indicating PNRSV infection. Of the samples showing positive
PNRSV infection results, 11 were from southwest Michigan (Berrien County) and 15
were from northwest Michigan (Leelanau County). Eighty percent of orchards sampled
from northwest Michigan, and 70% of orchards from southwest Michigan, contained
PNRSV-infected trees.

Table 2.1. Incidence of PNRSV infection in cherry trees sampled from Michigan
orchards.

 

 

Leelanau Berrien
County County Total
(Northwest) (Southwest)

# trees sampled 34 25 59

# orchards sampled 5 7 12

# PNRSV-Infected trees 15 11 26
°/o PNRSV-Infected trees 44% 44% 44%

# orchards containing 4 5 9

PNRSV-Infected trees

% orchards containing 80% 70% 75%

PNRSV-infected trees

Because of the limited quality and quantity of leaf material and low viral titers
due to fall isolation, most samples did not yield enough PCR product to continue with
successful restriction digests and cloning. Of the 26 PNRSV positive samples, eight were
successfully cloned into pBluescript and sequenced. The RT-PCR products of the eight

cloned isolates are shown in Figure 2.3.

58

Table 2.2 lists the geographical source of the Michigan PNRSV isolates cloned

and sequenced in this study, including the Michigan county in which isolation occurred,

as well as cherry species and cultivar. Table 2.3 lists all the PNRSV isolates included in

the phylogenetic comparisons, including isolate origin, accession number, and reference

(if available).

Approximately 1,610 nucleotides were sequenced for Michigan PNRSV isolates.

Nucleotide differences between Michigan isolates within the sequenced region ranged

from 7 nucleotides to 97 nucleotides. Thus, nucleotide sequence similarities of Michigan

isolates ranged from 94% similarity to over 99% similarity.

2000 bp —
1650 bp —

   
 

A81 SW2 RW3 CB7 GF5 GF6 GF7 DA9

. . ;_-‘«?‘. b .5 . — I
.' . 1.. ‘ e .

Figure 2.3. RT-PCR products of PNRSV isolates from Michigan cherry trees. M = lkb+
DNA Ladder (Invitrogen, Carlsbad, CA), neg = cherry not infected with PNRSV.

Table 2.2. Origins of cloned Michigan PNRSV isolates.

 

 

Isolate Location Cherry Species

Designation

AB1 Berrien County Prunus avium (sweet), cv. N/A

CB7 Leelanau County Prunus cerasus (tart), cv. Montmorency
DA9 Leelanau County P. cerasus, cv. Montmorency

GF5 Leelanau County P. cerasus, cv. Montmorency

GF6 Leelanau County P. cerasus, cv. Montmorency

GF7 Leelanau County P. cerasus, cv. Montmorency

RW3 Berrien County P. cerasus, cv. Montmorency

SW2 Berrien County P. cerasus, cv. Montmorency

 

59

Table 2.3. PNRSV isolates included in the present study with origins and accession
numbers.

 

 

Isolate Group Accession

Designation Origi_n Homology" Number Reference

AB1 Michigan, USA (PV96) EF495167 this study

CB7 Michigan, USA (PV32) EF495168 this study

DA9 Michigan, USA (PV32) EF495169 this study

GF5 Michigan, USA (PV32) EF495170 this study

GF6 Michigan, USA (PV96) EF495171 this study

GF7 Michigan, USA (PV32) EF495172 this study

RW3 Michigan, USA (PV32) EF495173 this study

SW2 Michigan, USA (PV32) EF495174 this study

SW6 Michigan, USA N/A AF013287 Scott et al., 1998

Prune California, USA PV96 AF013286 Scott et al., 1998

Mission California, USA PV96 AF013285 Scott et al., 1998

CH3 Washington, USA PE5 AF465235 Hammond, 2003

CH9 Washington, USA PV32 AF034992 Hammond & Crosslin, 1998
CH19 WA (Washington) PV32 AF465236 Hammond, 2003

CH30 Washington, USA PE5 AF034994 Hammond & Crosslin, 1998
CH38 Washington, USA PV32 AF034991 Hammond & Crosslin, 1998
CH39 Washington, USA PV96 AF034990 Hammond & Crosslin, 1998
CH57 Washington, USA PV32 AF034993 Hammond & Crosslin, 1998
CH61 Washington, USA PV96 AF034989 Hammond & Crosslin, 1998
CH71 Washington, USA PE5 AF034995 Hammond & Crosslin, 1998
NRSV Hop1 USA N/A AF465234 Hammond, 2003

PE5 USA PE5 L38823 Hammond & Crosslin, 1995
leAI.unk1 Albania PV32 AJ133211 Aparicio et al., 1999

1/13 Czech Republic PV32 AF 1701 56 Vaskova et al., 2000

4/8 Czech Republic PV96 AF170165 Vaskova et al., 2000

6/54 Czech Republic PV96 AF170160 Vaskova et al., 2000

7/20 Czech Republic PV96 AF170164 Vaskova et al., 2000

21/1 Czech Republic PV32 AF170157 Vaskova et al., 2000

Na hrbu Czech Republic PV96 AF170170 Vaskova et al., 2000
Nahnuta Czech Republic PV96 AF170169 Vaskova et al., 2000
PS7/5a Czech Republic PV96 AF170166 Vaskova et al., 2000
PS7/11 Czech Republic PV96 AF170161 Vaskova et al., 2000
PS7/12 Czech Republic PV96 AF 170162 Vaskova et al., 2000
PS12/16 Czech Republic PV32 AF170158 Vaskova et al., 2000
PS14/22 Czech Republic PV32 AF170159 Vaskova et al., 2000

Sss Czech Republic PV96 AF170168 Vaskova et al., 2000

UH1 Czech Republic PV96 AF170167 Vaskova et al., 2000

 

 

60

 

 

Table 2.3 (cont’d).

 

Isolate
Designation
UN
Valticka
Ring13
Rin923
Rin924
Rin925
Rin926
Ring15
Ring16
Ring1?
Ring18

AI Kochav
Apr 755-256
Ring1 1

PI 505—6
Almlt.cor1
Almlt.pre1
Aprlt.caf1
Aprlt.nap1
Aprlt.try1
Chrlt.bla1
Chrlt.lam1
Chrlt.mrs1
Pchlt.may1
Pchlt.mry1
lelt.clf1
lelt.mrb1
Ring1
RingZ
Ring3
RingG
Ring1 0
RingZ1
Rin922
Jordan

I-23 Poland
NRSizO

Origin

Czech Republic
Czech Republic
France
France
France
France
France
Germany
Germany
Germany
Germany
Israel
Israel
Israel
Israel
Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

Italy

N/A (Jordan)
Poland
Poland

Group
Homology‘
PV96
PV96
PV32
PV96
PV96
PV32
PV32
PV96
PV96
PV96
PV96
(PV96)
(PV96)
PV96
(PV96)
PV96
PV96
PV96
PV96
PV32
PE5
PV96
PV96
PV96
PE5
PV96
PV32
PV96
PV96
PV32
PV96
PV32
PV96
PV96
(PV32)
(PV32)
PE5

 

 

NRSiz1

Poland

Accession
Number

Reference

 

AF1 70163
AF1 701 71
AF465220
AF465227
AF465228
AF465229
AF465230
AF465221
AF465222
AF465223
AF465224
AY434715
AY434716
AF465219
AY434714
AJ133204
AJ133202
AJ1 33199
AJ133200
AJ133201
AJ1 3321 0
AJ133203
AJ133209
AJ133205
AJ133207
AJ1 3321 2
AJ1 3321 3
AF465214
AF465215
AF465216
AF465217
AF465218
AF465225
AF465226
AY463362
00003584
AF33261 1

 

PV96

61

AF332612

 

Vaskova et al., 2000
Vaskova et al., 2000
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Spiegel et al., 2004
Spiegel et al., 2004
Hammond, 2003
Spiegel et al., 2004
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Apan'cio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Aparicio et al., 1999
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
Hammond, 2003
N/A

N/A

N/A

N/A

Table 2.3 (cont’d).

 

 

 

 

 

 

Isolate Group Accesslon

Deslgfltion Origin Homology* Number Reference
NRSiz2 Poland PV32 AF332613 N/A

NRSIZS Poland PV32 AF332614 N/A

NRSiz6 Poland PV96 AF 33261 5 N/A

NRSiz7 Poland PV96 AF332616 N/A

NRSi28 Poland PV96 AF 33261 7 N/A

NRSizQ Poland PV96 AF 33261 8 N/A

Rin928 Poland PV96 AF465232 Hammond, 2003
Rin929 Poland PV96 AF465233 Hammond, 2003
GG Slovakia PV96 AY037788 Glasa et al., 2002
KU Slovakia PV96 AY037791 Glasa et al., 2002
NT Slovakia PV96 AY037790 Glasa et al., 2002
YUG Slovakia PV96 AY037789 Glasa et al., 2002
thSp.mur1 Spain PV96 AJ133208 Aparicio et al., 1999
PctTu.unk1 Tunisia PV32 AJ133206 Aparicio et al., 1999
Beijing China ( PV96) DQ300178 N/A

Yunnan China (PV32) AY684271 N/A

E260 India PV32 AJ61 9958 N/A

Pal India (PV32) AJ9691 10 N/A

Rin927 N/A PV96 AF465231 Hammond, 2003
ApMV N/A outgroup NC003480 Shiel et al., 1995

 

MA = Not Available

* parentheses in this column indicate group homology based on phylogenic analysis from this

study

62

Figure 2.4 displays one of the most parsimonious trees showing the phylogenetic
relationships among nucleotide sequences of the PNRSV coat protein gene, with a tree
length of 772, consistency index of 0.661 , and retention index of 0.822. Of 685 total
characters, 293 were constant, 216 were parsimony-uninfonnative and 176 characters
were parsimony-informative.

The largest clade of the phylogenetic comparison shown in Figure 2.4 separates
into two distinct groups, supported by high bootstrap values (above 90). The topology of
the clade is concordant with previous studies which classify isolates into groups labeled

PV32 and PV96 (see below).

63

Figure 2.4. Phylogenetic relationships among coat protein gene nucleotide sequences of
new and previously-described PNRSV isolates. Shown in this figure is one of 500 most
parsimonious trees, with a tree length of 772. The consistency index (CI) equals 0.661
and retention index (RI) equals 0.822. Bootstrap support values above 50 are shown at
their respective branches. Branch length is indicated at the bottom of the figure. Michigan
sequences are designated by asterisks. Hatched lines on the outgroup branch indicate
truncated branch length shown.

64

53

 

100
92
100

 

 

 

SW6 *

— 5 changes

Figure 2.4

68 NRSIZO

i—C:
DA9* L—-— CH30
'___sw2*

'_ 63 CB7* RW3 *
Jordan

     
   
 

l-23 Polang
- unnan
Rin926 Ring3
PImIt.mrb1
NRSIZZ CH9

 

1’13 PS14/22
s 6

F

124% Chrlt.lam1
. Aprlt.caf1

gRS'ze thSp.mur1
6’54 NRSi26
SSS UH1

  
   
  
   
 
  
  
 

N iii?

a r u .
NRSiz7

Nahnuta Rlngzz

Rm” Valticka
_Chrlt.mrs1
Prune R'”929
R'"92 PS7/12
PS7’" PImIthf1
Rin921 .

Rin928
Rings Ring15
NRSIZQ

Ring27 Almlt.cor1

Almlt.pre1
PI 505-6

Pchlt.may1
K

- 7/20
4/8

UN U
53 NT

NRSiz1
Aprlt.nap1

 

 

' Apr 755-256
{figmgfl Ring18
ng16
AI Kochav
NRSizS

NRSV HL

CH39

II
I,

65

CH3
CH71

PImAI.unk1 Aprlt.try1

 

90 Chrlt.bla1
Pchlt.mry1

PE5

PE5

PV32

PV96

ApMV

The majority of isolates from Michigan appear most closely related to isolates
previously classified into the PV32 group. The PV32 group includes the isolates Ring3,
CH19, and RinglO. In our coat protein tree, Michigan isolates CB7, DA9, SW2, GF5,
RW3, and GF 7 cluster together in the same clade as the PV32 isolates. The PV32 group
has been previously described by other authors, and our groupings appear to be consistent
with these previous analyses (Aparicio & Pallas, 2002; Hammond, 2003; Glasa et al.,
2002; Codoner et al., 2006).

Michigan isolates grouping together with PV32 isolates have similar
polymorphisms in the coat protein, including a 2 amino acid insert at position 42 that is
lacking in other isolates (Figure 2.5). This polymorphism has previously been described
as characteristic of many isolates falling into the PV32 group (Codoner et al., 2006).

According to Figure 2.4, two Michigan isolates, AB1 and GF6, appear most
closely related to isolates previously classified into the PV96 group, or Group II. This
group forms the largest clade of our phylogenetic comparison and includes many
European isolates, Washington isolates CH61 and CH39, and California isolates Prune
and Mission. The results of our phylogenetic tree appear to be consistent with other
authors’ analyses in regards to the PV96 grouping of previously-described isolates

(Aparicio & Pallas, 2002; Hammond, 2003; Glasa et al., 2002; Codoner et al., 2006).

66

Figure 2.5. Alignment of the N-terminus of the coat protein of representative PNRSV
isolates. Asterisks indicate Michigan isolates.

l 50
GF5* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAANNPSRSRNPNRVSS
CH19 ------------------------- LVPLRARQRAANNPSRSRNPNRVSS
DA9* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAANNPNRNRNPNRVSS
CB7* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAANNPNRNRNPNRVSS
SW2* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRVANNPNRNRNPNRVSS
RW3* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAANNPNRNRNPNRVSS
GF7* MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAASNPNRNRNPNRVSS
CH9 MVCRFCNHTHAGGCRSCKKCHPNDALVPLRAQQRVANNPNRERNPNRVSS
CH57 MVCRFCNHTHAGGCRSCKKCHPNDALVPLRAQQRVANNPNR--NPNRVSS
CH38 MVCRFCNHTHASGCRSCKKCHPNDALVPLRAQQRVVNTPNR--NPNRVSS
Prune MVCRICNHTHAGGCRSCKKCHPNGALVPLRAQQRAANNPNR--NPNRASS
Mission MVCRICNHTHAGGCRSCKKCHPNGALVPLRAQQRAANNPNR--NPNRASS
GF6* MVCRICNHTHAGGCRSCKKCHPNGALVPLRAQQRAANNPNR--NPNRASS
ABl* MVCRICNHTHAGGCRSCKKCHPNGALVPLRAQQRAANNPNR--NPNRASS
CH61 MVCRICNHTHASGCRSCKKCHPNGALVPLRAQQRDANNPNR--NPNRVSS
CH39 MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRDANNPNR--NPNRASS
SW6* MVCRICNHTHAGGCRSCKKCHPNNALVPLRAQQRAANNPNR--NPNRVSS
CH3 ------------------------- LVPLRAQQRAANNPNR--NPNRVSS
CH71 MVCRICNHTHAGGCRSCKKCHPNDALVPLRAQQRAVNNPNR--NPNRVSS
CH30 MVCRICNHTHASGCRSCKKCHPNDALVPLRAQQRAANNPNR--NPNRVSS
51 100
GF5* GVGPAIRPQPVVKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEAVKYLSI
CH19 GVGPAIRPQPVVKTTWTVRGPNVPPRIPKGYVAHSHREVTTTEAVKYLSI
DA9* GVGPAIRPQPVVKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEAVKYLSI
CB7* GVGPAIRPQPVVKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEAVKYLSI
SW2* GVGPAIRPQPVAKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEAVKYLSI
RW3* GIGPAIRPQPVVKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEAVKYLSI
GF7* GIGPAVRPQPVVKTTWTVRGPNVPPRIPKGYVAHNHREVTTTEVVKYLSI
CH9 GIGPMVRPQPVAKTTWTVRGPNVHPRIPKGYVAHSHREVTTTEAVKYLSI
CH57 GIGPTVRPQPVAKTTWTVRGPNVHPRIPKGYVAHNHREVTTTEAEKYLSI
CH38 GIGPTVRPQPVAKTTWTVRGPNVPPRIPKGYVAHSHREVTTTEAVKYLSI
Prune GTGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVTTTEAVKYLSI
Mission GYRTVVRPQPVVKTIWTVRGPNVPPRIPKGFVAHNHREVTTTEAVKYLSI
GF6* GTGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVTTTETVKYLSI
ABl* GTGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVMTTEAVKYLSI
CH61 GTGPMVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVMTTEAVKYLSI
CH39 GTGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVTTTEAVKYLSI
SW6* GIGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNHREVTTAEAVKYLSI
CH3 AAGRDARSKPVVKTTWTVRGPNAPPRIPKGFVAHSHREVTTTEVVKYLSI
CH71 AAGRDARSKPVVKTTWTVRGPNVPPRVPKGFVAHSHREVTPNEVVKYLSI
CH30 AAGPDARSKPVVKTTWTVRGPNVPPRVPKGFVAHSHREVTTTEVVKYLSI

67

Figure 2.5 (cont’d).

101 150
GF5* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
CH19 DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
DA9* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
CB7* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
SW2* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
RW3* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVNQPDGPNALS
GF7* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
CH9 DFTTTLPQLMGQNLTLLTVIVRMNSVSSNGWIGMVEDYKVDQPDGPNALS
CH57 DFTTTLPQLMGQNLTLLTVIVRMNSVSSNGWIGMVEDYKVDQPDGPNALS
CH38 DFTTTLPQLMGQNLTLLTVIVRMNSVSSNGWIGMVEDYKVDQPDGPNALS
Prune DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVERPDGPNALS
Mission DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVEQPDGPNALS
GF6* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVEQPDGPNALS
ABl* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVEQPDGPNALS
CH61 DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVEQPDGPNALS
CH39 DFTTTLPQLMGQNLTQLTVIVRMNSMSSNGWIGMVEDYKVDQPDGPNALS
SW6* DFTTTLPQLMGQNLTLLTVIVRMNSMSSNGWIGMVEDYKVNQPDGPNALS
CH3 DFTTTFPQLMGQNLTLLTVINRMNSMSSNGWIGMVEDYRVDNPEGPNALS
CH71 DFTTTFPQLMGQNLTLLTVINRMNSMSSNGWIGMVEDYRVDNPDGPNALS
CH30 DFTTTLPQLMGQNLTLLTVINRMNSMSSNGWIGMVEDYRVDNPDGPNALS
151 200
GF5* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH19 RKGFLKDQPRGWQFEPPSDLDFDTFAR -----------------------
DA9* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CB7* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
SW2* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
RW3* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
GF7* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH9 RKGFLKDQPRGWOFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH57 RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVAAGPKVLVRDL
CH38 RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIKFKTEVPAGAKVLVRDL
Prune RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
Mission RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
GF6* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
ABl* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH61 RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH39 RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
SW6* RKGFLKDQPRGWQFEPPSDLDFDTFARTHRVVIEFKTEVPAGAKVLVRDL
CH3 RKGFLKDQPRGWQFEPPSDLDFDTFAK -----------------------
CH71 RKGFLKDQPRGWQFEPPSDLDFDTFAKTHRIVIEFKTEVPVGAKVLVRDL
CH30 RKGFLKDQPRGWQFEPPSDLDFDTFAKTHRIVIEFKTEVPVGAKVLVRDL

68

Figure 2.5 (cont’d).

201 226
GF5* YVVVSDLPRVQIPT ------------
CH19 --------------------------
DA9* YVVVSDLPRVQIP -------------
CB7* YVVVSDLPRVQIPT ------------
SW2* YVVVSDLPRVQIPT ------------
RW3* YVVVSDLPRVQIPT ------------
GF7* YVVVSDLPRVQIPT ------------
CH9 YVVVSNLPRVQIPTDVLLVDE -----
CH57 YVVVSDLPRVRIPTDVLLVDE -----
CH38 YVVVSDLPRVQIPTDVLLVDE -----
Prune YVVVSDLPRVQIPTDVLLVDEDLLEI
Mission YVVVSDLPRVQIPTDVLLVDEDLLEI
GF6* YVVVSDLPRVQIPT ------------
ABl YVVVSDLPRVQIPT ------------
CH61 YVVVSDLPRVQIPTDVLLVDE -----
CH39 YVVVSDLPRVQIPTDVLLVDE -----
SW6* YVVVSDLPRVQIPTDVLLVDEDLLEI
CH3 --------------------------
CH71 YVVVSDLPRVQIPTDVLLVDE -----
CH30 YVVVSDLPRVQIPTDVLLVDE -----

Alignment of the movement protein validates the grouping of A81 and GF6 into
PV96 and the remaining Michigan isolates into PV32 (Figure 2.6). AB1 and GF6 show
similar polymorphisms in the amino acid sequence of the movement protein as other
PV96 isolates, such as Washington isolates CH61 and CH39. For example, AB1 and GF6
both contain valine at position 60 like other PV96 isolates, whereas the other Michigan
isolates contain isoleucine, consistent with other PV32 isolates. Also, AB1 and GF6
contain valine at position 253 and threonine at position 261 , as do other PV96 isolates,

while PV32 isolates contain isoleucine at 253 and leucine at 261.

69

Figure 2.6. Alignment of the amino acid sequence of the movement protein of
representative PNRSV isolates. Asterisks indicate Michigan isolates.

1 50
ABl* MADVSKNPSTSDFSVVECSMDEMGQISEDLHKLMLSDEMRALPTKGCHIL
CH61 MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMRALPTKGCHIL
CH39 MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
Chrlt.lam1 MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGRHVL
GF6* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
Prune MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
Mission MADVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
SW6* MADVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMRALPTKGCHIL
CB7* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
GF5* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
CH38 MAGVSKNPSTSDFSVVECSMDEMCQISEDLHKLMLSDEMKALPTKGCHIL
CH57 MAGVSKNPSTSDFSVVECSMDEMCQISEDLHKLMLSDEMKALPTKGCHIL
CH9 MAGVSKNPSTSDFSVVECSMDEMCQISEDLHKLMLSDEMKALPTKGCHIL
DA9* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMKALPTKGCHIL
GF7* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMRALPTKGCHIL
SW2* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMRALPTKGCHIL
RW3* MAGVSKNPSTSDFSVVECSMDEMSQISEDLHKLMLSDEMRALPTKGCHIL
CH30 MAGVSKNPSTSDFSVVECSMDEMSQISEDLHNLMLSDEVRHLPTKGCHIL
CH71 MADVSKNPSTSDFSVVECSMDEMSQISEDLHNLMLSDEMRHLPTKGCHIL

Chrlt.bla1 MAGVSKNPSTSDFSVVECSMDEMSQISEDLHNLMLSDEMRSLPTKGCHIL

51 100
ABl* HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
CH61 HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
CH39 HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
Chrlt.lam1 HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
GF6* HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
Prune HLVNLPKSNVLRLASKEQKGFLSRQADRVKNKIYRCVGRIFLVYVPIIQA
Mission HLVNLPKSNVLRLASKEQKGFLSRQADKVKNKIYRCVGRIFLVYVPIIQA
SW6* HLVNLPKSKVLRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
CB7* HLVNLPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
GF5* HLVNLPKSNILRLASKEQKGFLSRQADKVKNKIYRCVGRVFLVYVPIIQA
CH38 HLVNFPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
CH57 HLVNFPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
CH9* HLVNFPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
DA9* HLVNLPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
GF7* HLVNLPKSNILRLASKBQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
SW2* HLVNLPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
RW3* HLVNLPKSNILRLASKEQKGFLSRQADKVKKKVYRCVGRVFLVYVPIIQA
CH30 HLVNLPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA
CH71 HLANLPKSNILRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA

Chrlt.bla1 HLVNLPKSNVLRLASKEQKGFLSRQADKVKKKIYRCVGRVFLVYVPIIQA

70

iii

 

Figure 2.6 (cont’d).

101 150
ABl* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
CH61 TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
CH39 TTSGLITLKLQNSYTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
Chrlt.lam1 TASGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
GF6* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
Prune TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
Mission TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
SW6* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVVMDRWGRSLVDSAELNLL
CB7* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
GF5* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
CH38 TTLGLITLKLQNSDTGEISDVVTDVAANRAFVIMDRWGRSLVESADLNLL
CH57 TTSGLITLKLQNSDTGEISDVVTDVAANRAFVIMDRWGRSLVESADLNLL
CH9 TTSGLITLKLQNSDTGEISDVVTDVAANRAFVIMDRWGRSLVESADLNLL
DA9* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
GF7* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
SW2* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
RW3* TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
CH30 TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL
CH71 TTSGLITLKLQNSDTGEISDVVTDVEANRAFVIMDRWGRSLVESADLNLL

Chrlt.bla1 TTSGLITLKLQNSDTGEVSDVVTDVEANRAFGIMDRWGRSLVESADLNLL

151 200
AB1* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH61 YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH39 YSISCPDVRPGARVGEMMAFWDERMSRQQTYSEKGNPILFPIAETKPSKY
Chrlt.lam1 YSISCPDVRPGARVGETMVFWDERMSRQQTYLEKGNPILFPIAETKPSKY
GF6* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
Prune HFISCPEVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
Mission YYISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNLILFPIAETKPSKY
SW6* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CB7* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
GF5* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH38 YSISCPDVRPGARVGDMMVFWDERMSRQQTYLEKGNPILFPITETKPSKY
CH57 YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH9 YSISCSDVRPGARVGEMMAFWDERMSRQQTYLEKGNPFLFPIAETKPSKY
DA9* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
GF7* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
SW2* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
RW3* YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH30 YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY
CH71 YSISCPDVRPGARVGEMMAFWDERMSRQQTYLEKGNPILFPIAETKPSKY

Chrlt.bla1 YSISCPDVRPGARVGEMMVFWDERMSRQQTYLEKGNPILFPIAETKPSKY

71

 

Figure 2.6 (cont’d).

201 250
ABl* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
CH61 LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
CH39 LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
Chrlt.lam1 LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
GF6* LNDKKVLMSMVRSRILAGTEGCDIAPENIBVKRLGDNRKVLTIQPKAPIV
Prune FNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
Mission LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
SW6* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPVV
CB7* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
GF5* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
CH38 LNDKKVLMSMVRSRILAGTEGCDIAPETIEVKRLGDNRKVLTIQPKAPIV
CH57 LNDKKVLMSMVRSRILAGTEGCDIAPETIEVKRLGDNRKVLTIQPKAPIV
CH9 LNDKKVLMSMVRSRTLAGTEGCDIAPETIEVKRLGDNRKVLTIQPKAPIV
DA9* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
GF7* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
SW2* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPTV
RW3* LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKAPIV
CH30 LSDKKVLMSMVRSRILAGTEGCDIVPENIEVKRLGDNRRVLTIQPKVPVI
CH71 LSDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRRVLTIQPRAPVI

Chrlt.bla1 LNDKKVLMSMVRSRILAGTEGCDIAPENIEVKRLGDNRKVLTIQPKTPII

 

251 285
ABl* EEVKDE-DEPTGSNG-ENHMEEKTVTVKVGSSGNA
CH61 EEVKDE-DEPTGSNG-ENHMEEKTVTVKVGSSGSA
CH39 EEVKDE-DEPTGSNG-GNHMEEKTVTVKVGSSGSA
Chrlt.lam1 EEVKNE-DEPTGSNG-GNHMEEKTVTVEVGSSGSA
GF6* BEVKDE-DEPTGSNG-ENHMEEKTVTVKVGSSGSA
Prune EEVTEK-DEPTGSNG-ENHMEEKTVTVKVGSFGSA
Mission EEVKEK-DEPTGSNG—ESHMEEKTVTVKVGSSGNA
SW6* EBIKDE-VEPVGSNG-ENHMEEKTVTVKVGSSGSA
CB7* EBIKDD-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
GF5* EBIKDD-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
CH38 EEIKDD-VEPLGSNG-GNHMEEKTVTVKVGSSGSA
CH57 EEIKDD-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
CH9 EEIKND-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
DA9* EEIKND-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
GF7* EEIKDD-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
SW2* EEIKDD-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
RW3* EEVKND-VEPLGSNG-ENHMEEKTVTVKVGSSGSA
CH30 EELKDEEIEPIGSIGTENHMEEKVVTTKVGGTGSA
CH71 EELKDEEIEPIGSIGTENHMEEKVVTTKVGGTGSA

ChrIt.bla1 EELKND-GEPIGSSSTBKHMEEKVVAVEVGRTGSA

72

As depicted in Figure 2.4, the Michigan isolate SW6 described by Scott et a1.
(1998) does not appear to group closely with the PV32, PV96, nor PE5 isolates.
Hammond (2003) also found that SW6 does not group with any of the classifications,
however, this observation differs from the analyses of Aparicio and Pallas (2002) and
Glasa et a1. (2002) which group SW6 with PV96. Alignment of the amino acid sequence
of the coat protein (Figure 2.5) shows that SW6 lacks the 2 amino acid insert at position
42 that is characteristic of isolates from the PV32 group. In addition, at amino acid
position 81, SW6 has a phenylalanine as do isolates of the PV96 group, whereas isolates
grouping as PV32 contain a tyrosine at this position. In the movement protein alignment
(Figure 2.6), SW6 contains valine at amino acid position 60 as do other PV96 isolates, as
opposed to isoleucine in the PV32 isolates. However, at amino acid position 253, SW6
contains isoleucine as do most PV32 isolates, instead of the valine seen in the PV96
isolates. Interestingly, SW6 is unique from either the PV32 or PV96 group at position
261, containing valine rather than leucine, as in PV32 isolates, or threonine, like PV96
isolates. One possible explanation for the unique nature of the SW6 sequence is that the
isolate may be a product of recombination between two different isolates, one from the
PV32 group, and one from the PV96 group.

Figure 2.4 shows Washington isolates CH3, CH71, and CH30 grouping together
with isolates Chrlt.bla1, Pchlt.mry1, PE5, and NRSizO. These seven isolates appear to be
significantly different than any of the other isolates analyzed in that they group into a
distinct cluster. Other authors also show these isolates to form a unique cluster, referred
to as the PE5 group (Aparicio & Pallas, 2002; Hammond, 2003; Vaskova et al., 2000).

None of the Michigan isolates groups with these Washington isolates into the PE5 group.

73

 

Overall, high homology was observed among all PNRSV isolates analyzed, with
no apparent correlation among isolate sequence or geographic location. These results are
consistent with previous studies concluding that there is no significant relationship
between PNRSV sequence and geographic origin (Hammond, 2003; Scott et al., 1998).
Among the Michigan isolates, geographical origin within the state does not appear to
correlate with sequence. AB1 and GF6 group most closely with each other according to
sequence, however, AB1 was isolated from Berrien County in the southwest corner of
Michigan, and GF 6 was isolated from Leelanau County in the northwest comer of
Michigan’s lower peninsula. The remaining Michigan isolates, some of which were
isolated from Berrien County and others from Leelanau County, group closely with each
other. Interestingly, although all the “GF” samples were gathered from the same orchard,
this geographical proximity is not reflected in sequence similarity. GF5 and GF 7 isolates
group together according to sequence, and GF6 groups separately from them. These
sequence differences within Michigan suggest separate introductions into the state, and

even within orchards.

74

DISCUSSION

Sour cherry originated in the Middle East and then disseminated to Europe,
followed by North America. The source of sweet cherry was Asia Minor, and it also
dispersed to Europe then North America (Rieger, 2006). Analyses of the sequences of
PNRSV reveal that there is no correlation between geographic origin and isolate
sequence. All PNRSV isolates show high sequence similarity, suggesting time and
geographical spread have not resulted in high sequence divergence.

Despite high sequence similarity, PNRSV isolates can be grouped into three main
groups based on sequence analysis: PV32, PV96, and PBS. There does not appear to be
any correlation between grouping and the geographic source of members of these groups.
We would predict some degree of sequence difference to be proportional to distance from
cherry origin if there were a single introduction of PNRSV to the new location. However,
European, Asian, Middle Eastern, and US isolates are represented throughout the
phylogenetic groupings, and do not group according to region. One explanation for this
phenomenon could be that the orchards from which similar isolates were derived shared
either plant or pollination material directly with each other.

Our results demonstrate that PNRSV is prevalent in Michigan orchards, with 75%
of orchards sampled containing PNRSV-infected trees. In addition, it appears that
PNRSV incidence in Michigan is relatively consistent between the northwest and
southwest regions of Michigan. In order to more thoroughly understand the status of
PNRSV in Michigan cherry orchards, it would be beneficial to analyze trees from other
orchards throughout the commercial cherry growing regions of Michigan. Nonetheless,

our results emphasize the incidence of PNRSV in Michigan. Because PNRSV can

75

decrease fruit quality and yield, this virus can negatively impact the cherry industry and
PNRSV-resistant cherry can therefore benefit cherry growers. The Appendix of this
thesis addresses a PNRSV resistance strategy for cherry.

If there were a single PNRSV introduction into Michigan, we would expect
Michigan isolates to group closely together upon sequence comparison. However,
isolates GF6 and AB1 group closely with each other and with other US and European
isolates in the PV96 group while the remaining Michigan isolates cluster into the PV32
group. This would suggest that there were multiple PNRSV introductions into the state.
Sources of PNRSV into the state were most likely virus-infected rootstocks which were
imported into Michigan cherry orchards.

Multiple introductions may even have occurred within a single orchard. For
example, GF5, GF6, and GF7 were all isolated from the same orchard; however GF5 and
GF7 group together based on sequence, while GF6 fits into a separate group. The
similarity between GF5 and GF7 suggests the virus may have been spread via pollen
between these two trees. The differences seen between GF5 and GF 7, and the more
unique isolate GF6 may be a result of separate rootstock introductions into the GF
orchard. Alternatively, these isolates may have evolved independently.

Isolate SW6 (Scott et al., 1998) displayed some polymorphisms characteristic of
the PV32 group and others characteristic of the PV96 group. This could be a result of
either a recombination event between isolates of each group, or could be indicative of a
strain that had existed longer in Michigan and thus may have had a longer time period to

evolve and develop these polymorphisms. Analysis of isolates from PNRSV-infected

76

trees located in the same area as SW6 may shed light on how this isolate may have
evolved.

Another explanation for the lack of correlation between geographic location and
sequence could be that some or all of the polymorphisms are random, perhaps because
that particular part of the genome is “flexible” and the slight changes do not interfere
with the viral life cycle. For instance, groups PV96 and PE5 lack a two amino acid insert
at position 42 of the coat protein (Codoner et al., 2006) that is present in most isolates of
the PV32 group. Presence or absence of these two amino acids may not significantly
affect the pathogenicity of PNRSV and as a result a deletion in that particular region of
the coat protein may be readily tolerated.

PNRSV can result in crop yield loss, particularly in conjunction with other
Prunus viruses. Therefore, development of a resistance strategy against PNRSV would
benefit crop growers. We have characterized a number of different Michigan PNRSV
isolates and have used the sequence information derived from our study to initiate
development of a novel resistance strategy to PNRSV in cherry using biotechnology,

described in the Appendix.

77

CHAPTER 3

RNA RECOMBINATION IN BROMOVIRUS COAT PROTEIN DELETION
MUTANTS

INTRODUCTION

Plant viruses present a major threat to agriculture and both crop yield and quality
can be significantly impacted by viral diseases. For example, epidemics of rice tungro
virus have caused rice yield losses of up to 50% (Muralidharan et al., 2003), which
equated to an annual economic loss of $1.5 billion in Southeast Asia (Hull, 2002).
Tomato spotted wilt tospovirus causes annual losses of $1 billion (Prins & Goldbach,
1998). Cucumber mosaic cucumovirus (CMV) with its broad host range, including many
economically important plants, causes crop losses worldwide. Since there are no methods
of eliminating virus from infected plants, prevention of infection and resistance are the
only available means of decreasing losses due to viruses.

Traditional approaches of controlling viral diseases in crops include phytosanitary
measures such as virus-free seed or transplants, chemical control of insect vectors, and
the utilization of natural sources of virus resistance. Although conventional sources of
resistance would appear to be the most reliable approach to virus control, there are
limited sources of natural virus resistance genes for cultivated crop species. In addition,
resistance to plant viruses can be controlled by several genes and may therefore
complicate introgression of the resistance trait into cultivated varieties. As a result, even
when R genes are available, moving a resistance gene to a specific variety may take many
years to accomplish, require a significant financial input, and present the subsequent

challenge of assuring resistance durability (Lecoq et al., 2004). Incorporation of a

78

resistance locus into a background of interest through conventional breeding may also
have undesired effects through genetic drag, as unwanted genetic material may
accompany the desired resistance genes which may provide undesirable traits in the
resulting resistant variety.

In light of the significant crop losses due to viruses and limitations of attaining
resistance through conventional breeding, biotechnology can play an important role in
engineering viral resistance in crops. The concept of pathogen-derived resistance (PDR)
was introduced over three decades ago when Powel Abel ct a1. (1986) demonstrated
successful resistance to tobacco mosaic tobamovirus (TMV) in tobacco (Nicotiana
tabacum L.) expressing the TMV coat protein gene. Since this revolutionary study, there
have been a vast number of successful attempts to attain virus resistance by transforming
plants with various viral sequences, and numerous virus-resistant transgenic plants
(VRTPS) have resulted.

Several crops transcribing transgenic viral sequences have been commercialized,
including: two lines of yellow crookneck squash, one resistant to zucchini yellow mosaic
potyvirus (ZYMV) and watermelon mosaic potyvirus 2 (WMV2), and one resistant to
ZYMV, WMV2, and CMV; papaya containing the papaya ringspot potyvirus (PRSV)
coat protein gene resistant to PRSV; and potato resistant to potato virus Y (PVY). These
virus-resistant crops are contributing to the fi'uit and vegetable industry by providing
disease resistance. PRSV was poised to devastate the Hawaiian papaya industry until
PRSV-resistant papaya became available, which has allowed the papaya industry to
flourish once again (Gonsalves, 1998). As mixed virus infections can be common in the

field, resistance to multiple viruses can be accomplished rather easily through genetic

79

engineering. For instance, crookneck squash resistant to ZYMV, WMV2, and CMV
showed a 50-fold increase in marketable fruit yield compared to non-transgenic squash
when challenged by these three viruses (Fuchs et al., 1998).

Despite the potential contributions of biotechnology to virus disease resistance,
the safety of the resulting products must be thoroughly evaluated before employing the
technology on a large scale. Potential risks of VRTPs include heterologous encapsidation
and recombination events involving the transcript of the viral transgene.

Heterologous encapsidation may occur when a transgenic coat protein
encapsidates the genome of a challenging virus. If the challenging virus is not able to
systemically infect the transgenic plant by itself, the coat protein from the transgenic
plant may complement the challenging virus’s inadequacy. This may impart the ability of
the challenging virus to move systemically throughout the plant if it was not able to
before (Kaplan et al., 1995). In addition, heterologous encapsidation may allow a
challenging virus to be transmitted by a different insect vector (Lecoq et al., 1993) and be
introduced to plant species that it does not normally encounter.

Although heterologous encapsidation may only temporarily complement a
challenging virus, RNA recombination could result in a more permanent modification
and host range expansion. Recombination is a proven RNA virus evolutionary
mechanism (Nagy & Simon, 1997; Roossinck, 1997) and is thought to occur through
template switching which involves substitution of RNA templates during virus
replication. This mechanism, which is referred to as “copy choice,” was demonstrated as
the mode of recombination in poliovirus, a positive-sense single-stranded RNA virus

(Kirkegaard & Baltimore, 1986). The copy choice mechanism of RNA recombination

80

involves a viral replication complex reading a single-stranded RNA template pausing
during RNA synthesis, dissociating from the original template, binding to an alternate
RNA template in close proximity, and continuing RNA synthesis on the new template
(Lai, 1992). The result is a chimeric RNA product which reflects parts of at least two
distinct RNA templates.

There are several RNA features which may encourage recombination activity.
A/U-rich areas appear to characterize hotspots for recombination because these areas may
result in pausing or slippage of the RNA-dependent RNA polymerase (RdRP) and
switching of the polymerase onto a different RNA template. Recombination may also
occur more frequently in areas with complex secondary structure as these regions may
also encourage the RdRP to pause and switch templates.

Homologous recombination refers to recombination which occurs between two
areas with high sequence similarity. The RdRP switches from one region on the template
molecule to another region with a homologous sequence, either on a different molecule or
another part of the same molecule. In contrast, heterologous recombination occurs
between two areas of different sequence. As described above, structural characteristics of
the RNA molecule contribute to both heterologous and homologous recombination.

Recombination in plant viruses was first demonstrated in the tripartite brome
mosaic bromovirus (BMV) (Bujarski & Kaesberg, 1986). This study showed that an
attenuated BMV RNA 3 with a deletion near the 3’ end of RNA 3 was restored during
infection through homologous recombination with co-inoculating BMV genomic RNAs l

and 2 which have similar 3’ terminal sequences.

81

Evidence of recombination has been observed in the nucleotide sequences of
numerous plant viruses, including members of Bromoviridae (Allison et al., 1989; Bonnet
et al., 2005), Comoviridae (Le Gall et al., 1995; Vigne et al., 2005), Potyviridae (Moreno
et al., 2004; Chare & Holmes, 2006), and Potexviridae (Sherpa et al., 2007). In addition
to observations of natural recombination between plant viruses, there is evidence of
recombination between viral RNA and host RNA. Mayo and J olly (1991) demonstrated
that the nucleotide sequence of the 5’ region of the potato leafroll luteovirus (PLRV) is
nearly identical to a transcribed sequence from the tobacco chloroplast genome,
suggesting that recombination occurred between viral RNA and host RNA during PLRV
evolution. Analysis of the turnip mosaic potyvirus (TuMV) genome also suggests
recombination with a plant chloroplast RNA. The 3’ non-translated region of one TuMV
isolate is homologous to a chloroplast ribosomal protein gene (Sano et al., 1992).

Co-infecting viruses within the same plant are also able to recombine with each
other. Recombination was observed between two cucumoviruses, tomato aspermy virus
(TAV) and CMV, in tobacco plants (Aaziz & Tepfer, 1999). These recombination events
occurred between wild-type viruses in non-transforrned plants and thus arose under
minimal selection pressure.

Viral RNA recombination is considered a potential risk of VRTPs because the
RNA transcript from a plant viral transgene is available within the cytoplasm for
recombination with a challenging virus. The use of a constitutive promoter for viral
transgene expression ensures the availability of the transgenic transcript in each cell to a
challenging virus and distinguishes the possibility of recombination in VRTPs from

recombination opportunities during most mixed infections, where the two viruses are

82

introduced at different times and complete active replication in the absence of the other
virus. Such situations would occur when viruses are introduced independently and/or at
different times. It is difficult to predict the effects that an acquired sequence may have on
the pathogenicity and host range of the challenging virus, although it seems obvious that
most recombination events will be non-functional and have no selective advantage.
However, there is the possibility that a new chimeric virus will be more pathogenic
and/or have an increased or altered host range.

Greene and Allison (1994) observed recombination between a movement-
defective coat protein deletion mutant bromovirus and the RNA transcript from a
transgenic plant containing the missing portion of the bromovirus coat protein gene
sequence. In these experiments the 3’ one-third of the coat protein gene of cowpea
chlorotic mottle bromovirus (CCMV) was deleted and this mutant RNA 3 molecule was
used as part of a tripartite inoculum onto transgenic Nicotiana benthamiana plants
transcribing the 3’ two-thirds of the CCMV coat protein gene plus the complete 3’
untranslated region (UTR) of CCMV RNA 3. Although the RNA 3 coat protein deletion
mutant was unable to move systemically, it contained the CCMV replication recognition
sequence and therefore served as a template for the replication complex encoded by co-
inoculating RNAS 1 and 2. The observed recombination events between the transgenic
transcript and the replicating RNA 3 resulted in the mutant CCMV gaining the ability to
move systemically throughout the plant.

Movement was also restored in a movement-defective plum pox potyvirus (PPV)
coat protein mutant upon recombination. When transgenic N. benthamiana plants

containing the PPV coat protein gene were inoculated with the PPV mutant, the mutant

83

recombined and gained the ability to systemically infect its host (Varrelmann et al.,
2000).

One concern of recombination between a virus and a transgene is that the
pathogenicity and host range of the resulting recombinant may be distinct from those of
the parental virus. Using cauliflower mosaic caulimovirus (CaMV), a DNA virus that
replicates through an RNA intermediate, Schoelz and Wintermantel (1993) characterized
a recombinant that formed upon infection of transgenic Nicotiana bigelovii' that
transcribed a sequence fiom a different CaMV strain. The recombinant displayed
modified symptoms and a wider host range when compared to the parental wild-type
virus.

Experiments by Borja et a1. (1999) also demonstrated increased pathogenicity of a
recombinant virus. A coat protein mutant of tomato bushy stunt tombusvirus (TBSV),
which elicited mild symptoms, was inoculated onto transgenic plants containing the
TBSV coat protein gene. Lethal symptoms occurred in approximately one-fifth of the
inoculated plants. The observed increased pathogenicity was a result of recombination of
the TBSV mutant with the transgene transcript. In this case, recombination occurred with
no obvious selection pressure, as the inoculation mutant was able to cause disease and
move systemically.

Although the experiments with TBSV showed recombination under non-selective
pressure, experiments with TMV demonstrated recombination only under selective
pressure (Adair & Kearney, 2000). When a movement-defective TMV coat protein
mutant was inoculated onto N. benthamiana plants expressing the TMV coat protein

gene, recombinants were recovered that restored TMV movement. However, when non-

84

movement defective TMV was inoculated onto the transgenic plants, no recombination
was observed.

The experiments of Greene and Allison (1994) described recombinants that
gained the ability to systemically infect the transgenic host upon restoration of the deleted
region of the coat protein gene through recombination with the transgene RNA. The
study described in this chapter uses the same coat protein mutant virus inoculum and the
transgenic lines described by Greene and Allison (1994). In contrast, this study analyzes
the recombination activity that occurs in the initially infected leaf where, theoretically,
there is no selection pressure for restored recombinants. While studies of this nature may
identify functional recombinants, it is designed to examine non-functional recombinants
in the absence of selection pressure and thus report on the baseline recombination activity
in an inoculated leaf. Understanding baseline recombination in a transgenic plant can

contribute to the understanding and evaluation of risks of viral recombination in VRTPs.

85

MATERIALS AND METHODS
Transgenic Plants and Viral Transcripts

Nicotiana benthamiana plants were transformed with the 3’ two-thirds of the
CCMV coat protein gene containing three silent marker mutations which introduced a
Not I restriction site, and the CCMV 3’ untranslated region (UTR) (Greene & Allison,
1994). Transgenic plants remained susceptible to CCMV infection as the transgene did
not provide resistance to CCMV. Wild-type CCMV plasmids pCClTPl , pCC2TP2, and
pCC3TP4 (referred to hereafter as TPl, TP2, and TP4) were derived from cDNAs of
CCMV RNA 1, RNA 2, and RNA 3, respectively (Allison et al., 1988). In vitro
transcripts from these RN As represent the complete viral genomic RNA and are
infectious. The CCMV deletion mutant pCC3AG3 (AG3) lacks 119 nucleotides from the

3’ end of the coat protein gene (Greene & Allison, 1994) (Figure 3.1).

RNA 1 (TP1) 1a
RNA 2 (TP2) 2a
—’ ‘—
RNA 3 (TP4) movement ' coat I/
RNA 3 deletion
mutant (AG3) m°V°m°nt “at
Notl
Ii

transgene coat

Figure 3.1. Depiction of CCMV sequences used in this study. RNA 1, RNA 2, and RNA
3 refer to the wild-type components of the CCMV genome. AG3 is the coat protein
deletion mutant of RNA 3. The transgene consists of the 3’ two-thirds of the coat protein

gene plus the 3’ UTR, with an introduced NotI marker site. Horizontal arrows indicate
the region amplified by RT-PCR.

86

Prior to viral transcript synthesis, plasmids TPl, TP2, TP4, and AG3 were
linearized with Xba I (New England Biolabs, Beverly, MA). Viral transcript reactions
(modified from Ahlquist et al., 1984) contained the following components: 10 pg
linearized plasmid DNA, 10 mM dithiothreitol, 1 mM rNTPs, 500 pM G(5’)ppp(5’)G
(Amersham, Piscataway, NJ), 200 U RNasin Ribonucleotide Inhibitor (Promega,
Madison, WI), 100 U T7 RNA polymerase (Roche, Indianapolis, IN), 1X polymerase
buffer (Roche, Indianapolis, IN), in a final volume of 500 pl. Transcript synthesis
reactions were incubated at 37 °C for 45 min after which an additional 1.9 pl of 20 mM
rGTP and 100 U T7 RNA polymerase were added to each reaction. Transcript synthesis
was continued at 37 °C for an additional 45 min. Synthesis was terminated by adding 10
U RNase-free DNase (Promega, Madison, WI) and incubating for 15 min at 37 °C. RNA
transcripts were purified and concentrated through several phenol/chloroform extractions
and sodium acetate/ethanol precipitation. RNA pellets were resuspended in 50 pl of
RNase-free water and quantitated by visualizing on an agarose gel stained with ethidium
bromide.

Viral Transcript Inoculation

Sixty transgenic N. benthamiana plants, representing six different transgenic plant
lines (lines 3-22, 3-51, 3-57, 3-63, 5-26, and 5-58) and one non-transgenic plant were
inoculated with TPl, TP2, and AG3 transcripts. One transgenic plant and one non-
transgenic plant were mock-inoculated with water. One transgenic plant and one non-
transgenic plant were inoculated with wild-type transcripts TPl , TP2, and TP4 and one
non-transgenic plant was inoculated with virion CCMV derived from previously infected

tissue. Viral transcript inoculations contained the following components for each leaf: 4

87

pl of each viral transcript (approximately 7 pg), 5 pl of 5 mg/ml bentonite, and 3 pl of
water. The most mature secondary leaf from each plant at the 5-leaf stage was dusted
with carborundum and the inoculum was rubbed gently across 25% of the leaf.
RNA Extraction and RT-PCR

Two weeks post-inoculation, total RNA was extracted from approximately 80 mg
of leaf tissue from the viral transcript inoculation site using TRIzol Reagent (Invitrogen,
Carlsbad, CA), following the manufacturer’s protocol. Each RNA pellet was resuspended
in 30 pl of RNase-free water. A 2 pl aliquot of each RNA extraction was used as the
template in a reverse transcription (RT) reaction, using SuperScript II Reverse
Transcriptase (Invitrogen, Carlsbad, CA), following the manufacturer’s protocol. The
reverse primer used in the RT reaction was: 5’ —
aaaaaaggat_chGGTCTCCTTAGAGATC — 3’. The non-underlined lowercase letters
denote the added 5’ tail, underlined lowercase letters indicate the added PvuI restriction
site (not utilized in these experiments), and uppercase letters represent the
complementary sequence of nucleotides 2173-2157 of CCMV RNA 3 (accession number
NC003542; Allison et al., 1989). The CCMV sequence of the reverse primer is also
100% homologous to the 3’ terminal sequences from CCMV RNA 1 and RNA 2 as the 3’
UTRs of all three CCMV genomic RNAS have an identical 37—nucleotide sequence at
their 3’ termini.

The product of the RT reaction was used as the template for amplification through
the polymerase chain reaction (PCR) using Herculase Enhanced DNA Polymerase
(Stratagene, La J olla, CA), following the manufacturer’s protocol. The forward primer

was: 5’ — aaataacgatcgGCAGTACGCACCTATGTATAAG — 3’, with the same notation

88

except the uppercase letters indicate the sequence homologous to nucleotides 856-877 of
CCMV RNA 3. The reverse primer was the same as that used in the RT reaction.
Amplification conditions were as follows: 95 °C for 2 min, followed by 10 cycles of 30
sec at 95 °C, 30 sec at 60 0C, l min 20 sec at 72 °C, then 20 cycles of 30 sec at 95 °C, 30
sec at 60 °C, 1 min 20 sec plus 10 sec per cycle at 72 °C, with a final extension step at 72
°C for 10 min.
Cloning of RT-PCR Products

A 3’-A overhang was added to the RT-PCR products by adding 6.4 pl of purified
RT-PCR product to a 10 pl reaction containing the following components: 1X buffer
(Promega, Madison, WI), 1.5 mM MgC12, 200 pM dATP, 5U T aq Polymerase in Storage
Buffer A (Promega, Madison, WI). Reactions were incubated at 70 °C for 20 min.
Following 3’-A overhang addition, 2 pl of each reaction were cloned into the pGEM-T
Easy Vector plasmid using pGEM-T Easy Vector System I (Promega, Madison, WI)
following manufacturer’s protocol and transformed into competent cells, Escherichia coli
strain DHSa. Plasmids with cDNA inserts were identified by blue-white screening of
bacterial colonies on agar plates containing 70 pg/ml of X-gal (Roche, Indianapolis, IN)
and 80 pM IPTG (Roche, Indianapolis, IN). White colonies were transferred to a gridded
agar plate.
Dot Blot and Colony Blot Analyses

To determine whether plants were systemically infected, attempts were made to
extract virus particles from upper leaves of inoculated plants two weeks post-inoculation.
Approximately 80 mg of leaf tissue was placed into 750 pl of virus isolation buffer (0.2

M sodium acetate, 10 mM ascorbic acid, 10 mM EDTA, pH 4.8 with glacial acetic acid).

89

Samples were held on ice for 30 to 60 min and then centrifuged for 5 min at 14,000 rpm
in a microcentrifuge. 3 pl of supernatant was dot-blotted onto Nytran nylon membrane
(Schleicher & Schuell, Keene, NH) and UV crosslinked.

To establish if the white colonies contained cloned CCMV sequence, colony blots
were probed with a radiolabeled CCMV probe. Nytran membranes were placed on
gridded white colonies and adhering samples were lifted for hybridization analyses. The
membranes were placed in a solution of 0.5 M NaOH, 1.5 M NaCl to lyse the bacterial
cells and denature the DNA. The DNA was neutralized with 1 M Tris-HCl (pH 8.0)
followed by an incubation with a solution of 0.1 M Tris HCl (pH 7.5), 2X SSC and UV
crosslinked to the membrane.

The DNA template for the CCMV probe was synthesized through PCR using
pCC3TP4. Amplification conditions and primers were the same as those described
previously. The amplification product was gel isolated using a Qiaquick Gel Isolation Kit
(Qiagen, Valencia, CA) and used as the template for synthesizing a random prime labeled
probe. The radiolabeled probe was created using a Random Prime Labeling Kit
(Invitrogen, Carlsbad, CA), incorporating a-3ZP-labeled dCTP (PerkinElmer, Waltham,
MA). Membranes were prehybridized at 40 °C for 1-2 hr in a solution of 45% formamide,
7% SDS, 0.3 M NaCl, 0.05 M NazHPO4-NaH2PO4 (pH 7), 1X Denhardt’s solution, and
100 pg/ml salmon sperm DNA. Following prehybridization, the radiolabeled CCMV
probe was added to the solution and hybridized overnight at 40 °C. Membranes were
washed twice in a solution of 2X SSC, 0.1% SDS and once in 0.1X SSC, 0.1% SDS, each

wash lasting 10-15 min. Probe hybridization was visualized by autoradiography.

90

Colonies hybridizing to the CCMV probe were further analyzed by PCR colony
screen and/or restriction digests of purified DNA.
PCR Colony Screen and Plasmid Restriction Digest

Bacterial colonies were transferred from the gridded plate to 50 pl of colony lysis
buffer (1% Triton X-100, 20 mM Tris pH 8, 2 mM EDTA) and heated at 95 °C for 5 min.
2 pl aliquots of the lysis reaction served as template for 25 pl PCR reactions. The PCR

reactions contained 1X PCR buffer (Invitrogen, Carlsbad, CA), 200 pM dNTPs, 1.5 mM
MgC12, 12.5 pmol each of forward and reverse primers (described above), and 1 unit of

T aq DNA Polymerase (Invitrogen, Carlsbad, CA). Thermocycler conditions were the
same as described above.

Colonies were propagated in liquid medium and plasmid DNA was extracted and
purified using the Wizard Plus SV Miniprep System (Promega, Madison, WI). Purified
plasmid DNA was restricted with either PstI which produced a single-cut product or
EcoRI which cleaved the clones in the multicloning site on either side of the insert to
remove the cloned CCMV sequence. Restriction digest products were separated on a
0.8% agarose gel and stained with ethidium bromide.

Size comparisons were made between restriction and PCR fragments derived
from the control plasmid AG3, which represented the inoculum, and those of putative
recombinants derived from inoculated plants. Samples differing in size fi'om the AG3
mutant samples were fiirther analyzed.

Recombinant Sequencing
Clones were sequenced at the Michigan State University Macromolecular

Structure, Sequencing, and Synthesis Facility using ABI PRISM 3100 Genetic Analyzer

91

(Applied Biosystems, Foster City, CA) with the SP6 and T7 promoter primers.
Sequenced recombinants were compared to known CCMV sequences using NCBI

BLAST (Altschul, et al. 1990).

92

RESULTS

To characterize the recombination events that occured in the transgenic plant cells
expressing the CCMV transgene that had been inoculated with the deletion mutant AG3,
total RNA was isolated from the inoculation site. The viral RNA encompassing the area
of deletion in AG3 was reverse transcribed into cDNA, PCR amplified, and cloned for
identification of RNA recombination activity within this area. Table 3.1 indicates the
number of clones analyzed and number of recombinants identified for each
inoculum/plant combination.

Table 3.1. Results of analyses of clones isolated from wild-type or transgenic plants
inoculated with AG3 mutant or wild-type CCMV inoculum.

 

 

CCMV # of # of %
Inoculum Plant clones recombinants recombinants
analyzed Identified recovered
mfifait wild-type 5 0 0%
mziit transgenic 1 100 5 0.5%
wild-type wild-type 9 0 0%
wild-type transgenic 7 0 0%

 

Five recombinants were identified which contained sequence differing from that
of the inoculum. PCR amplification fragments from the wild-type plasmid TP4, the coat
protein mutant AG3, and recovered recombinant plasmids are compared in Figure 3.2.
Figure 3.3 is a depiction of the recombinant molecules, showing sequence sources of
each recombinant. Recombinants 27.32 and 39.10 were isolated from plants representing
transformation line 5-26; recombinants 42.6 and 46.5 were from plants of transformation
line 3-22; and recombinant 80.5 was from plant transformation line 5-58. No

recombinants were recovered from the non-transgenic plant inoculated with AG3, and

93

only wild-type sequences were isolated from transgenic and non-transgenic plants
inoculated with the wild-type virus.

The majority of the RT-PCR products cloned from the transgenic plants
inoculated with AG3 mutant inoculum represented the original AG3 inoculum sequence.
Of approximately 1,100 clones analyzed, 5 recombinants were identified (Table 3.1).
Thus, more than 99% of the clones recovered from the transgenic plants were derived
from accurately replicated inoculum with RNA recombination events had affecting only
0.5% of the recovered RNAS.

Recombinant 27.32 reflects a deletion beginning at nucleotide 924 and includes
the loss of the 3’ end of the movement protein gene, the entire intercistronic region, the
entire coat gene region, and the 3’ UTR from RNA 3. The 3’ UTR sequence from this
recombinant is derived from CCMV RNA 1 (nt 3011-3171). Recombinant 27.32 is
simply the majority of the movement protein gene attached to the 3’ UTR of RNA 1.

The majority of the coat protein gene is deleted in recombinant 39.10. In addition,
163 nucleotides from the 5’ region of the 3’ UTR are deleted. The 693-nucleotide
deletion in recombinant 39.10 spans the region between nt 1405 and nt 2096.

Recombinant 42.6 is nearly identical to recombinant 27.32. The deletion of the
movement protein gene begins at nucleotide 923, and the intercistronic region, coat gene,
and the 3’ UTR from RNA 3 are absent. Like recombinant 27.32, recombinant 42.6
contains the 3’ UTR region from CCMV RNA 1. However, in addition to the 3’ UTR,
recombinant 42.6 contains the 3’ region of the 1a gene from RNA 1. Overall,
recombinant 42.6 contains the CCMV RNA 1 region spanning from nucleotide 2935 to

3171.

94

Recombinant 46.5 resulted from the deletion of both the intercistronic region and
the coat gene, as well as the 3’ end of the movement protein gene (beginning at nt 926).
In this case, the 3’ UTR is derived from CCMV RNA 2 (nt 2630-2765).

Recombinant 80.5 has a deletion of 327 nucleotides (nt1372-1687) from the coat
gene. On each side of the deleted region of the original AG3 inoculum sequence is a
homologous sequence of five nucleotides (UACAG). Following recombination, only one
copy of this five-nucleotide sequence wass preserved. This would appear to be an
example of homologous recombination, as the deletion occurred with a homologous
sequence on either side.

None of the characterized recombinants contained a restored coat protein gene,

consequently no systemic infection was observed in plants yielding recombinant RNA.

L TP4 AG3 27.32 39.10 42.6 46.5 80.5

_S

O
O

o
l

850 -

Molecular Size (bp)
(II
8
l

 

Figure 3.2. PCR amplification products from CCMV constructs. TP4 = wild-type
sequence; AG3 = coat protein deletion mutant; 27.32, 39.10, 42.6, 46.5, and 80.5 =
CCMV recombinants. L = lkb Plus DNA Ladder (Invitrogen, Carlsbad, CA).

95

RNA 1 w/W/WilifiiW/m

 

27.32 movement\ //////.
AG3 movement coat
AG3 movement coat

I/

39.10 movement

RNA 1 m///////////////////////19’///////////////////W

  

42,6 movement %( W.
AG3 movement coat
FUNPiZ 23““.-—-—-'l'fiéJ-——
46.5 movement
\
AG3 movement coat
AG3 movement coat

augucUACAG UACAGagacg

augucUACAGagacg

80,5 movement ‘ coat

7% RNA 1 I RNA2 RNA 3

Figure 3.3. Depiction of sequences present in recombinants characterized in this study.
Regions derived from each wild-type RNA molecule or coat protein mutant RNA
molecule are indicated.

96

2934 2943

gaaguauauc

2928 3911

RNA 1 1a
(71) (2947)(3171)
2628 2637
uuacaagcaa
2630
*
RNA2 2a
(110) (2536)(2774)
916 934
uaaacaauugaaaaauuau
926
934 1372
'k
922 1405 1687 2096
+ * 'k * * ‘—
RNA 3 movement coat
(239) (1147)(1360) (1932)(2173)

Figure 3.4. Map of recombination sites in characterized recombinants. Asterisks signify
positions on the CCMV genome where recombination occurred, with the nucleotide
position noted above each astrix. The region between the arrows indicates the sequence
amplified by RT-PCR. Numbers in parentheses designate the beginning and end positions
of open reading frames in the genome, as well as the 3’ terminal nucleotide position of
each genomic RNA molecule. Sequence is shown for A/U-rich regions at which
recombination occurred, with nucleotide positions labeled.

Figure 3.4 denotes the areas of the CCMV genome where recombination was
observed. Three recombinants (27.32, 42.6, and 46.5) showed recombination events that
occurred within a two-nucleotide span of nt 924 on the RNA 3 molecule. Each of these
recombinants contained a different sequence adjoined to this region of RNA 3. The
region surrounding nt 924 in the wild-type RNA 3 is A/U rich. In addition, preliminary

analysis of the secondary structure of the CCMV RNA 3 molecule using the software

97

RNAstructure Version 4.5 (Mathews et al., 2004) suggests the presence of a hairpin loop

structure in the area surrounding nt 924 of the RNA 3 molecule (Figure 3.5).

 

940 950
9110 U-A A’G ‘ti A A 3’
/ \ A -
A/A\AAU ueuu’ \eec’ \u—AAce’ \UCAAUCUG
i Ill Illl III I lllI IIIIIIII
A\ /UUA AthA\ /ccc\ ,cli\ /UUGC\A_A/AGIIJUGGAC
920 u 910 900
924

Figure 3.5. Predicted hairpin structure within CCMV RNA 3. Nucleotides 895 to 959 of
the plus strand of RNA 3 are shown. A similar structure is predicted in the corresponding
region of the RNA 3 minus strand.

98

DISCUSSION

RNA recombination events of a CCMV deletion mutant that occurred within
inoculated cells of transgenic plants were analyzed. Previous experiments involving a
similar deletion mutant and transgenic plants identified functional recombinants derived
through recombination between the inoculum and the transgene transcript (Greene &
Allison, 1994). This current study was intended to look at recombination activity in the
initially infected leaf in the absence of selection pressure for functional recombinants.
Towards this goal, five RNA 3 recombinants were recovered that reflect RNA
recombination activity only among the CCMV RNAS. None of the recovered
recombinants contained either host or transgenic RNA. In the absence of selection
pressure, the recovery of non-functional recombinants was anticipated. The chance of _
identifying a random recombination event that would restore the coat protein gene or
compensate its deletion is unlikely to be identified in the recombinants recovered from
the inoculated leaf. However, since none of the inoculated plants became systemically
infected it is unlikely that a restored recombinant virus was formed.

In addition, none of the recombinants acquired the plant transgene sequence.
However, chimeric RNA 3 molecules were identified and demonstrate that CCMV
recombination occurred in transgenic plants. All recombinants recovered had maintained
the original 3’ terminus or exchanged this region with a coinoculating RNA. This region
is critical to the replication of CCMV RNAS as it possesses the recognition site for the
viral replication complex. Thus by virtue of our RT-PCR primers, recovered

recombinants were likely amplified by the viral replication complex.

99

Among the recovered RNA 3 recombinants, both homologous as well as
heterologous recombination was evident. Homologous recombination is suspected in
recombinant 80.5. As shown in Figure 3.3, the region between the repeated UACAG
motif of AG3 was deleted. The deletion area of this recombinant is positioned directly
following the intergenic region, which is a region with extensive secondary structure. In
fact the secondary structure of the intergenic region results in binding preference of the
replicase to the subgenomic promoter within this area in BMV (Choi et al., 2004). The
subgenomic promoter, which is required for the synthesis of subgenomic RNA 4 and coat
protein expression, has been implicated in RNA recombination events in BMV
(Wierzchoslawski et al., 2003). A likely scenario is that the RdRP paused during
synthesis of the plus strand at or within the non-single-stranded RNA structure of the
subgenomic promoter and then resumed strand synthesis on the same or another similar
strand at the alternative UACAG sequence, skipping the region between the two UACAG
repeats and excluding this region in the resulting RNA product.

Most of the identified recombinants displayed heterologous recombination, where
there was no apparent sequence similarity at the recombination sites. Three distinct
recombinants, 27.32, 42.6, and 46.5, displayed recombination within a two-nucleotide
vicinity of nt 924 on the RNA 3 molecule. This region of RNA 3 is A/U-rich and also
may have a complex secondary structure. The RdRP might have an increased tendency to
pause during replication at this region due to its sequence and/or structural
characteristics. Upon pausing, there is a higher chance of template switching occurring.
Although recombinants 27.32, 42.6, and 46.5 contain different sequences attached to a

nearly identical region of AG3, they all contain 3’ UTRs derived from CCMV RNAS.

100

Recombinants 27.32 and 42.6 contain sequence from the 3’ end of the RNA 1 molecule,
while recombinant 46.5 contains sequence from the 3’ UTR of RNA 2. The UTRS of
CCMV all have extensive secondary structure resembling tRNA structures which enables
the replicase complex to recognize these regions as templates and to initiate minus-strand
synthesis (Ahlquist et al., 1981). A possible scenario to explain the formation of
recombinants 27.32, 42.6, and 46.5 could be that, during minus-strand synthesis, initiated
on RNA 1 in the case of recombinant 27.32 and 42.6, or RNA 2, in the case of
recombinant 46.5, the polymerase may have paused near the 5’ end of the 3’ UTRS due to
the secondary structures of these regions. After pausing, the replicase may have bound
near the hairpin-loop region around nt 924 of an RNA 3 molecule and resumed
replication on the new template. Alternatively, recombination may have occurred during
plus-strand synthesis if the replicase paused around nt 924 due to either the A/U-rich
sequence, secondary structure characteristics, or both, and after pausing bound to the 3’
UTR of either RNA 1 or RNA 2 and continued replication on the new template molecule.

Recombinant 39.10 may have been produced during minus-strand synthesis. A
possible scenario to explain the formation of this recombinant is that the RdRP paused
near the 5’ end of the 3’ UTR of RNA 3 and then switched to a different area of the RNA
3 molecule, possibly one closer to the intergenic region to which the polymerase may
have a higher affinity, and resumed RNA synthesis.

Our results suggest the presence of a recombination hotspot in CCMV RNA 3,
around nucleotide 924. Greene and Allison (1994) did not describe recombination events
occurring at this region in RNA 3, nor at the other regions where recombination was

observed in this study.

101

Although none of the characterized recombinants contained the deleted RNA 3
region or marker present in the transgene sequence, we cannot completely rule out a
potential contribution of the transgene transcript to recombination. The 3’ UTR sequence
of RNA 3 was present in the transgene transcript and may have affected recombination in
some way. The presence of the 3’ UTR in a transgene transcript was shown to increase
the rate of recombination (Greene & Allison, 1996). Because the 3’ UTR sequence of the
AG3 inoculum was identical to the 3’ UTR sequence in the transgene transcript it is
impossible to determine the origin of the 3’ UTR sequence of the recombinants.

The experimental design of this study used the non-functional nature of the viral
inoculum as a diagnostic tool since systemic infection would have indicated a functional
recombinant. Because replication of the coat protein mutant was limited to inoculated
cells, the concentration of the viral RN As may have increased beyond natural
concentrations because they were unable to move from the cells. An unusually high
concentration of replicating viral RNAS may influence recombination simply because of
increased template availability.

Another significant difference between the deletion mutant and the wild-type
inocula besides the inability of the deletion mutant to move out of the inoculation site is
that there is no functional coat protein produced in the deletion mutant. The absence of
the coat protein during replication may have a number of different implications for
recombination activity. For example, the coat protein may contribute to the template
specificity of the replication complex. If this component is missing there may be an
increase in recombination resulting from decreased specificity of the replicase. Also, the

completion of active viral replication may be controlled by a critical concentration of the

102

coat protein or the resulting virion. Thus the lack of the coat protein may sustain
recombination opportunities in plants inoculated with the deletion mutant.

In one sense, these experiments, which used a coat protein deletion mutant, are
not equivalent to a natural field inoculum and may have biased the recombinant recovery.
On the other hand, this may be an applicable scenario, since many viruses are unable to
systemically infect a host plant but remain replication competent within the initially
infected cells (Hull, 2002). As a result, these replicating viruses would come in contact
with a viral transgene expressed from a constitutive promoter and may be able to acquire
the new genetic material.

Even though results from these experiments may be an overestimate of
recombination events, recombinants were observed in 0.5% of the clones derived from
transgenic plants. If a recombinant virus is to be successful, it must be competitive with
the wild-type virus. Many of the initial recombination studies did not compare the fitness
of recombinant viruses to their parental viruses. It is important to evaluate the ability of
recombinants to compete with wild-type viruses in order to assess the potential risks of
VRTPS in a field environment.

Several laboratory experiments have evaluated the fitness of recombinant viruses.
When RNA 3 recombinants of CMV were co-inoculated with wild-type CMV, the wild-
type virus out-competed the recombinants (Pierrugues et al., 2007). Dietrich et al. (2007)
evaluated the fitness of a recombinant PPV strain. Although the recombinant virus
showed more severe symptoms than the wild-type virus, it was out-competed by the wild-
type virus. The CCMV recombinants described by Greene (1995) differed in regards to

symptom severity compared to wild-type CCMV, with some recombinants displaying

103

more severe symptoms. However, all these recombinants were out-competed by the wild-
type virus in mixed infections. These studies suggest that the majority of randomly
occurring recombinants may be less fit than the wild-type virus and will likely not be
dominant in a field environment.

The risk of recombination is only agriculturally relevant if recombination occurs
at a high enough frequency to significantly change the virus population, or if the
recombinants are fit enough that they compete with wild-type viruses. These instances
could result in a virus that poses an agricultural threat. To date, there have been no
reports from field studies of new recombinant viruses derived from recombination events
involving the transgenic transcript or influenced by its presence.

The population of grapevine fanleaf nepovirus (GFLV) isolates in a field of
grapes with rootstocks transgenic for the GFLV coat protein was analyzed for
recombinants (Vigne et al., 2004). This is an example of study performed in a field with
high disease pressure. No recombinants were detected in the transgenic field.
Recombinants were isolated from a nearby field containing only non-transgenic grapes.
The recombinants, however, did not show characteristics of the GFLV strain from which
the transgene was derived. The authors concluded that the transgenic plants did not aid in
recombination of GFLV.

Capote et a1. (2007) examined the effect of transgenic plum lines containing the
PPV coat protein gene on the population of PPV isolates in the field. The study found
that there was no significant difference between the molecular diversity of PPV isolates
derived from transgenic plum trees and those derived from non-transgenic trees. In

addition, no recombinant viruses were identified in the transgenic trees. This is one

104

example of a relatively long-terrn field study, as the plum trees had been in the field
exposed to the PPV population for 8 years at the time of analysis.

The contribution of virus-resistant transgenic squash to recombination and
molecular diversity of CMV in the field was analyzed (Lin et al., 2003). No
recombination was observed in isolates from transgenic squash, and no correlation
between CMV diversity and transgenic field status was found. It has been more than ten
years since the release of most of the commercially-grown VRTPS, and there have been
no observations to date of novel viruses emerging or epidemics resulting from the use of
these VRTPS (Fuchs and Gonsalves, 2007).

Although VRTPS expressing a viral gene from a constitutive promoter should
contain the viral transcript in the cytoplasm of each cell throughout the plant, the likely
compartmentalization of viral replication may prohibit the viral transgene transcripts
from coming into contact with a replicating virus. Most positive-sense RNA plant viruses
replicate in association with a membrane (Hull, 2002). For example, BMV replication
occurs in association with the endoplasmic reticulum while alfalfa mosaic alfamovirus
(AMV) replicates on the chloroplast outer membrane (Hull, 2002). Thus, there may be a
spatial separation between viral transgene transcript and replicating viral genomic RNA
which may decrease the probability of RNA recombination between the transgene and
viral RNA.

It is prudent to continue evaluations of the contribution of plants containing a
viral transgene to viral evolution. Although recombination between a transgene and

challenging virus has not been observed in the field so far, it still remains that viral

105

transgenes are likely available for recombination, and evaluations should continue to be
conducted and growers should remain vigilant.

Not only should recombination between a viral transgene and the virus from
which the sequence was derived be examined, but recombination between a transgene
and other viruses, particularly related viruses, should be evaluated. These analyses
inevitably pose logistical obstacles, and novel analysis methods will likely need to be
developed.

In an attempt to evaluate the fitness of recombinants containing genes of unrelated
plant viruses, Chung et al. (2007) developed hybrid viruses containing gene sequences
from potato virus Y (PVY). Tobacco rattle tobravirus (TRV) and potato virus X (PVX)
were engineered to contain genes from PVY. All three viruses were unrelated. The
recombinant viruses were all unstable, and loss of the PVY sequence was observed. This
study demonstrated that in this virus system, recombinant viruses with sequences from
unrelated viruses are not stable and are outcompeted by the wild-type virus.

Although recombination can be one mechanism for evolution of RNA viruses, it
appears it is a relatively rare event. Recombination between a viral transgene transcript
and an infecting virus has been demonstrated in a laboratory setting under high selection
pressure. However, emergence of recombinant viruses due to VRTPS in a field
environment has not been observed. The probability of a recombinant emerging as a
result of acquisition of genetic material from a transgene by a challenging virus is low,
and the ability of such a recombinant to compete with a wild-type virus is even lower.
VRTPS have the potential to benefit agriculture by decreasing crop losses due to viruses.

In order to wisely utilize this important resource, risks must continue to be assessed. Any

106

potential risks must be evaluated with the benefits of the use of VRTPS in mind, and if
the contribution to agricultural improvement clearly outweighs any possible risks, then

sensible use of biotechnology in attaining virus resistance seems appropriate.

107

CONCLUSIONS AND PERSPECTIVES

Significant pressures will be placed on the world’s agricultural resources as our
population continues to rise, energy demands turn to crop-based resources, and the land
base available for crop cultivation decreases. Conventional breeding has contributed
greatly to crop improvement. However, because of the time required for breeding
improvements, the limited gene pool available, and genetic drag resulting from trait
introgression, conventional breeding in and of itself may not be sufficient for crop
improvement demands of the future. Genetic engineering has a variety of advantages
including: an unlimited source of genes for improvement, a relatively short period of time
for development, and the ability to add a trait of interest to a desired variety without
changing the genetic background. Plant biotechnology will complement conventional
breeding and result in a synergistic contribution to agricultural improvement.

The goals of these studies were to contribute to knowledge in the area of plant
biotechnology in the following ways: 1) develop novel tools for biotechnology; 2)
identify an area where biotechnology has the potential to contribute to crop improvement;
and 3) assess risks of biotechnology products.

New techniques for plant biotechnology can help expand the number of candidate
plant species to modify as well as types of modifications possible. We demonstrated
successful transformation of Phaseolus vulgaris using electro-transformation, a unique
method for genetically engineering plants. Electro-transformation utilizes a mild
electrical current to drive transgene DNA into the meristematic tissue of a seedling. A
DNA construct containing two genes, the bar gene encoding phosphinothricin

acetyltransferase (PAT) for resistance to the herbicide glufosinate ammonium and the

108

germin gene encoding an oxalate oxidase (OxO), was transformed into P. vulgaris
seedlings. One plant line was verified to contain both the bar and germin genes. The
electro-transformation approach circumvents regeneration via tissue culture, one of the
key limiting steps of dry bean transformation. Additionally, electro-transfonnation avoids
the obstacles posed by existing corporate transformation patents. Because electro-
transforrnation can be used for humanitarian purposes free of charge, it is more accessible
to developing countries interested in dry bean genetic modification than current methods.
Dry bean is an important staple food in many cultures, and genetic engineering will
hopefully add desirable traits such as disease resistance, drought tolerance, and
nutritional enhancement.

Future experiments in electro-transforrnation may find ways to increase
transformation efficiency. Homoserine lactone appears to stimulate electro-
transforrnation, and perhaps other stress-inducing treatments, such as heat treatment,
combined with homoserine lactone prior to electro-transforrnation may increase the rate
of transformation.

Future studies should also evaluate electro-transforrnation in other plant species.
As research in the biofuels field expands, there will be a need for reliable transformation
techniques. Electro-transformation may play a role in modifying plants for biofuel
purposes. Lipid modification of plants will likely play a critical part in large-scale use of
biodiesels, and lignin and starch modifications could be significant in the bioethanol
field. Use of a variety of plant species, including those currently difficult to transform,
will likely be involved in this emerging field. Although electro-transformation has only

been demonstrated in dry bean thus far, we anticipate its success in transforming other

109

plant species. Electro-transformation will likely be most successful in the genetic
modification of large-seeded plants, because such plants are generally more tolerant to
seedling manipulations required during the protocol; however, the method could
potentially be modified for smaller-seeded plants.

To identify another area where biotechnology could contribute to agricultural
improvement, we examined a viral pathogen of cherry in Michigan. Cherry is an
important crop in Michigan as well as in other areas of the US. and the world, and
characterization of pathogens and development of resistance strategies can positively
impact the cherry industry. We characterized isolates of prunus necrotic ringspot ilarvirus
(PNRSV) from cherry trees in Michigan orchards. Total RNA was isolated from cherry
trees and cDNA was synthesized using PNRSV-specific primers. RT-PCR products were
cloned and sequenced, and a portion of the coat protein sequence from Michigan isolates
was compared to sequences of PNRSV isolates from other geographic locations. No
correlation was found between sequence and geographic origin. Michigan isolates did not
show more similarity to each other than to isolates from other origins suggesting that
there was more than one introduction of the virus into the state. Overall, high homology
was observed among worldwide isolates, and groupings from our analysis corroborated
those of other studies. Sequencing of the Michigan isolates has enabled development of a
novel resistance strategy for PNRSV in cherry using biotechnology, which is described in
the Appendix. Areas of sequence similarity among coat protein genes of various isolates
should enable the broad application of the silencing strategy and constructs.

The safety of biotechnology applications must be evaluated. Virus-resistant

transgenic plants (VRTPS) raise particular concerns in that they may contribute to the

110

evolution of plant viruses by making a viral transgene transcript available for
recombination and acquisition into the genomes of challenging viruses. A number of
laboratory studies conducted in the 1990’s have validated this risk potential by
demonstrating that movement-defective viruses, inoculated onto a transgenic plant
containing a viral transgene, recombined with the transgene transcript and gained the
ability to move systemically. The goal of our recombination study was to use a
previously-described bromovirus system to characterize recombination events and their
frequency.

Five recombinants were identified in Nicotiana benthamiana plants containing a
cowpea chlorotic mottle bromovirus (CCMV) coat protein transgene inoculated with a
CCMV coat protein mutant. Both homologous and non-homologous recombination was
observed, and a potential recombination hotspot on the RNA 3 molecule was identified.
None of the recombination events resulted in acquisition of the transgene transcript
sequence and none of the recombinants was able to systemically infect the host plant.

It seems logical that increased recombination would occur in a movement-
defective virus that was destabilized by a deletion. Recombination could be a “survival”
mechanism for the deficient virus. If a virus is unable to be successful in its environment
(i.e., unable to systemically infect a plant), then one strategy could be to acquire genetic
material which might increase the success of the virus. Recombination could be one such
approach. It would be interesting to study recombination of a virus in inoculated cells of
one of its non-host plants. Since viral replication can often occur in inoculated cells of

non-systemically infecting viruses, characterization of recombinants occurring in these

111

inoculated cells could give insight into recombination events that occur in a natural
setting.

Ultimately, as scientists and consumers we must ask ourselves: Which risks are
relevant? It is apparent that recombination between a viral transgene and a challenging
virus is possible, however the consequences of this may be negligible relative to the
global benefits of increased virus resistance.

Plant biotechnology is poised to play a critical role in ensuring that the predicted
demands for plant productivity are met. To realize its full potential, we must take
advantage of the creativity of the scientist, ensure the thorough testing of each product,
and gain the acceptance of the consumer. Thorough and thoughtful evaluation will ensure

that the application of plant biotechnology achieves its potential.

112

APPENDIX
DEVELOPMENT OF A RESISTANCE STRATEGY FOR PRUNUS NECROTIC
RINGSPOT ILARVIRUS (PNRSV) IN CHERRY
INTRODUCTION

The modification of plants through genetic engineering offers a reliable method
for plant improvement and numerous examples providing disease resistance, herbicide
resistance, and nutritional enhancement are available. Almost all of these examples are in
annuals, rather than perennials, since genetically modified (GM) annuals can be
developed much faster than perennials and the economic benefits due to their importance
and acreage are much greater. Thus the development of GM perennials has lagged behind
that of annuals.

Further, at this point in time there is a greater economic risk in the
commercialization of GM perennials than in annuals; this is because GM foods have not
been fully embraced by consumers. A GM orchard would require a significant investment
of both time and money prior to harvest of the first salable crop, and consumer
acceptance at the time of harvest is unpredictable. Hence, a method by which a perennial
species could reap the benefit of genetic engineering while the fi'uit crop remained
unmodified would be ideal. Our background with prunus necrotic ringspot virus
(PNRSV) positions us to test a novel hypothesis whereby the rootstock of a perennial is
genetically modified and the rootstock passes the benefit of that modification to the
unaltered scion. This appendix describes the initiation of these experiments.

Post-transcriptional gene silencing (PTGS) is a phenomenon by which a cellular

system is established that targets a specific RNA sequence for destruction (Baulcombe,

113

2004; Hamilton & Baulcombe, 1999; Voinnet & Baulcombe, 1997). PTGS is initiated by
a double-stranded RNA (dsRN A) which is cleaved into short dsRNAs by the enzyme
Dicer. Degradation products are then incorporated into a protein complex, RISC, which
recognizes and cleaves mRNAs that contain an homologous sequence. In this manner
mRNA with the same sequence as in the trigger dsRNA is degraded prior to translation.

RNA silencing in plants can provide virus resistance (Baulcombe, 1996; Ratcliff
et al., 1997; Voinnet, 2001; Waterhouse et al., 2001). If RNA of a replicating virus
triggers the silencing mechanism, then the plant can display resistance to that particular
virus. This silencing phenomenon can be initiated by a virus infection, or it can be
engineered prior to infection by transcription from a viral transgene. High levels of a
transgene transcript may trigger a plant RN A-dependent RN A polymerase (RdRP) to
synthesize the complementary RNA strand to form dsRNA. This dsRNA then enters the
silencing pathway resulting in degradation of transgene RNA as well as genomic RNA of
a challenging virus containing the same nucleotide sequence.

Virus resistance through RNA silencing has been demonstrated in plum trees.
Scorza et a1. (1994) developed transgenic plum lines containing the coat protein gene
from plum pox potyvirus (PPV). One plum line, CS, was resistant to PPV (Ravelandro et
al., 1997). This resistance was later shown to be silencing-based (Scorza et al., 2001; Hily
et al., 2005). Field resistance of the C5 line to PPV over a ten-year period has
demonstrated that silencing-based virus resistance in plum can be maintained (Hily et al.,
2004; Scorza & Ravelonandro, 2006). These studies suggest that durable PTGS-based

virus resistance in other perennial fruit trees is feasible.

114

Wesley et a1. (2001) describe enhanced silencing in transgenic annuals by using
an intron-containing hairpin construct. These authors developed a series of constructs
which contained a multicloning site adjacent to the enhanced 35S promoter, followed by
the orthophosphate dikinase (pdk) intron and an additional multicloning site. These
plasmid constructs, including pKANNIBAL, were designed to clone the sense and
antisense orientations of a specific nucleotide sequence and in planta transcripts would
fold into a dsRNA that would trigger the silencing system. There are numerous examples
of successful RNA silencing based on these constructs.

To address public concerns surrounding GM crops, it would be advantageous to
produce trees in which the fruit itself is not transgenic, and engineered virus resistance in
one part of a plant is shared with the remainder of the plant. One plausible approach to
achieve this goal is to graft a non-GM scion to a GM rootstock. Palauqui et al. (1997)
demonstrated in tobacco that a silencing signal is transmitted from a rootstock to a non-
silenced scion resulting in silencing in the scion. If the rootstock of a perennial, such as
cherry, were genetically engineered to express a silencing construct that targets PNRSV
and were grafted to a non-transgenic scion, then resistance may be conferred to the scion
without the actual presence of the transgene in the scion’s genomic DNA. As a result, the
tree may display PNRSV resistance, but the fruit would not be genetically modified.

Our goal is to test this hypothesis by producing cherry trees that are resistant to
PNRSV by creating transgenic rootstocks silenced for the PNRSV coat protein gene and
grafting non-transgenic scions to these silenced rootstocks. The use of grafting as part of

a virus resistance strategy is a logical approach which complements current pomological

115

practices. Scions of varieties exhibiting desired fi'uit qualities are typically grafted onto
rootstocks displaying characteristics such as dwarfing or root pathogen tolerance.
PNRSV infects a number of cultivated stone fruit species in addition to cherry,
including peach, plum, and almond. A successful silencing—based PNRSV resistance
strategy for cherry may serve as a model for virus resistance in other woody perennials.
Furthermore, the use of a GM rootstock with the migration of a silencing signal to an
unmodified scion may find alternative uses in disease resistance and crop improvement.
As described in Chapter 2, Michigan is the largest US producer of sour cherry and
our research suggests that PNRSV is slowly spreading within numerous Michigan
orchards. PNRSV-resistant cherry varieties would benefit Michigan cherry growers as
they would help combat this viral pathogen. Sequence derived from several Michigan
PNRSV isolates has enabled the identification of a highly conserved sequence within the
coat protein gene that is being tested for the induction of PNRSV resistance through RNA

silencing.

MATERIALS AND METHODS

Nucleotides 1-413 of the coat protein gene of Michigan PNRSV isolate CB7
(accession number EF495168) were amplified from pCB7 via polymerase chain reaction
using Platinum T aq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA). Primers
used to amplify this region are described below, depicted in 5’ to 3’ orientation.
Sequences are written as follows: the lower-case, non-underlined sequence at the 5’ end
of each primer denotes a non-viral added 5’ tail; the lower-case, underlined sequence

indicates an added restriction site; the upper-case sequence represents PNRSV sequence.

116

The first reaction contained primer set RA457 and RA458. The second reaction contained
primer set RA459 and RA460. Forward primer RA457 is as follows:
tttttcggggAATGGTTTGCCGAATTTGC. It adds an XhoI restriction site, underlined.
Reverse primer RA458 is: ttttttggta_ggTAGTCCTCCACCATCCCA and adds a KpnI
restriction site, underlined. Forward primer RA459,
tttttt’ggggAATGGTTTGCCGAATTTGC, adds an Xbal restriction site, underlined.
Reverse primer RA460, ttttttatggatTAGTCCTCCACCATCCCA, adds the underlined
ClaI site. The annealing temperature was 59 °C and the extension temperature was 68 °C.
Amplification products were purified using Qiaquick Gel Isolation Kit (Qiagen, Valencia,
CA).

The RA457/458 PCR product was digested with XhoI and KpnI (New England
Biolabs, Beverly, MA) and ligated into pKANNIBAL (Wesley et al., 2001; accession
number AJ311873), digested with XhoI and KpnI, using T4 DNA Ligase (Invitrogen,
Carlsbad, CA). The RA459/460 PCR product was digested with Xbal and ClaI (New
England Biolabs, Beverly, MA) and ligated into pKANNIBAL containing the
RA457/458 piece with T4 DNA Ligase. This plasmid, containing the CB7 coat protein
gene fragment in both the sense and antisense orientations, was called pKANsaPNRSV.
The region between the 35S promoter and the ocs terminator is shown in Figure Al.

pKANsaPNRSV was digested with NotI (New England Biolabs, Beverly, MA)
and ligated into pART27 (Gleave, 1992) digested with NotI. This binary plasmid was
called pART-PNRSV. The accuracy of pART-PNRSV was verified by restriction digest

analyses and sequencing at the Michigan State University Macromolecular Structure,

117

Sequencing, and Synthesis Facility using ABI PRISM 3100 Genetic Analyzer (Applied

Biosystems, Foster City, CA).

PNRSV .de PNRSV Ocs term

-v

. E“h353-_- .

t ' 24‘0'. ‘LQ‘T- I I I
Xhol Kpnl Clal Xbal

 

Figure A1. Intron-containing hairpin construct designed to induce silencing of PNRSV.
Arrows indicate orientation of sequence, with the forward arrow indicating sense
direction, and reverse arrow indicating antisense direction. Enh358 indicates the
enhanced 358 promoter from cauliflower mosaic virus. PNRSV refers to the 414-
nucleotide region of the PNRSV coat protein gene. Ocs term indicates the octopine
synthase terminator from Agrobacterium tumefaciens.

SUMMARY AND STATUS

Successful virus resistance in plants through gene silencing has been
demonstrated in many plant species, including both herbaceous and woody species.
Although there is a vast number of crop improvements possible through genetic
engineering, consumer concern regarding GM crops is not trivial and must be considered
prior to making the required investments for GM crop development and cultivation. One
approach to producing virus-resistant fi'uit trees and reaping the benefits associated with
disease resistance while addressing consumer concerns is to produce virus-resistant GM
rootstock and graft onto it a non-GM scion. With such an approach the fruit produced
will be non-GM.

The PNRSV silencing construct pART-PNRSV has been successfully
transformed into cherry rootstock ‘Golden’ in Michigan State University’s Plant

Transformation Facility and GM calli have been Obtained. This was verified by

118

kanamycin resistance and PCR analysis. The next step is to regenerate whole plants from
transformed callus material, determine if the transgene has triggered the silencing system,
and nurture these plants to a maturity where they can serve as a rootstock. Non-transgenic
cherry scions will be grafted to the PNRSV-resistant rootstocks and the resistance level
of the scions will be evaluated. If PNRSV resistance successfully traverses the graft
junction to non-transformed scions then the transgenic resistant rootstock lines will be
propagated and, following appropriate approvals, made available to cherry growers.

We are hopeful that this PNRSV resistance strategy will provide PNRSV
resistance, result in an overall increase Of cherry yield, be acceptable to consumers, and

be applicable to other perennial species.

 

119

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