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This is to certify that the

dissertation entitled

PRODUCTION, MOLECULAR EVALUATION AND DISEASE RESPONSE
OF TRANSGENIC POTATO PLANTS (SOLANUM TUBEROSUM L.)
EXPRESSING A FUNGAL GLUCOSE OXIDASE GENE

 

presented by
Kimberly J. Felcher

has been accepted towards fulfillment
of the requirements for

 

Ph.D. degree in Plant Breeding and Genetics

 

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Date 4/ 4/00

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PRODUCTION, MOLECULAR EVALUATIONAND DISEASE RESPONSE OF
TRANSGENIC POTATO PLANTS (SOLANUM TUBEROSUM L.) EXPRESSING A
FUNGAL GLUCOSE OXIDASE GENE

By

Kimberly J. Felcher

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics
Department of Crop and Soil Sciences

2000

ABSTRACT
PRODUCTION, MOLECULAR EVALUATION AND DISEASE RESPONSE OF
TRANSGENIC POTATO PLANTS (SOLANUM TUBEROSUM L.) EXPRESSING A
FUNGAL GLUCOSE OXIDASE GENE
By

Kimberly J. Felcher

Late blight, (Phytophthora infestans (Mont) De Bary), is one of the most
devastating diseases of potato worldwide and the development of cultivars resistant to
this disease is an important objective for potato breeding programs. Current emphasis is
on the utilization of horizontal resistance rather than vertical resistance conferred by
specific R (resistance) genes. This research was undertaken to determine if enhanced
resistance to late blight could be obtained by combining natural resistance and engineered
resistance conferred by the fungal transgene glucose oxidase. Late blight susceptible
cultivars 'Atlantic', 'Snowden' and 'Spunta' and partially resistant cultivars 'Zarevo' and
'Libertas' were transformed with the glucose oxidase gene under the control of the Gelvin
Super Promoter. Copy number ranged from 1 to 8 and glucose oxidase enzyme activity
ranged from O to 93.96 Units/mg plant tissue * 105. In agronomic trials, 44% of the
transgenic lines tested were determined to be "off-type" compared to appropriate non-
transgenic controls based on plant and/or tuber phenotypic characteristics. Because
several off-type lines did not express the glucose oxidase protein, this phenomenon can
not be attributed to the glucose oxidase transgene.

Lines expressing the glucose oxidase transgene were evaluated for reaction to P.
infestans in grth chambers and inoculated field studies. Results from growth chamber

trials suggested a small effect in 'Spunta'-derived transgenic lines but high coefficient of

variation (CV) values prevented the identification of any significant differences between
the transgenic lines and their respective non-transgenic controls. Field studies revealed
no significant effect of the glucose oxidase transgene in either late blight susceptible or
partially late blight resistant cultivars.

Transgenic lines were also evaluated for response to Erwinia. carotovora ssp.
carotovora (soft rot) and F usarium sambucinum (dry rot). Following inoculation, disease
progression was determined by measuring the diameter and depth of lesions over time for
F usarium dry rot and by measuring lesion diameters over time for Erwinia sofi rot.

The glucose oxidase gene had a minimal effect on resistance of the cultivars to
F usarium dry rot and Erwim’a soft rot although differences between lines were difficult to
detect due to high error variation. Lines SGO-l and 860—2 were the only ones
significantly different from the non-transgenic control for response to F usarium dry rot
and lines SGO-9, SGO-12, LGO-l and LGO-3 were the only lines significantly different
from the non-transgenic controls for response to Erwinia soft rot. There did not appear to
be any correlation between resistance to F usarium and resistance to Erwim'a among the
transgenic lines.

These results indicate that the glucose oxidase gene alone or in combination with
natural host plant resistance will not produce completely late blight resistant cultivars nor
will it produce high levels of resistance to Fusarium dry rot or Erwinia soft rot.
However, potato cultivars with moderate levels of disease resistance may be useful in
combination with reduced input chemical control programs. This potential benefit would
need to be weighed against the time and cost required for the development of transgenic

potato cultivars and the potential for off-type tubers and plants.

ACKNOWLEDGMENTS

Wow! After five years of hard work I've finally finished my Ph.D.! However,
saying, "I've" finished is horribly inaccurate due to the many people who have
contributed their time, knowledge and emotional support. I would like to thank my
committee members Dr. Jim Kelly, Dr. Ken Sink and Dr. Ray Hammerschmidt for
lending their expertise to the completion of this research. My major professor, Dr. Dave
Douches, deserves a special thanks for his input on the design and execution of my
research and for hours of conversation and debate on my research, various plant breeding
topics and things totally unrelated to science. I would also like to thank the "potato
crew", Dilson Bisognin, Joe Coombs, Chris Long, Anne Lund, Beth Leary and Kelly
Zarka for all their help and for the fun and friendship we experienced together. Special
thanks to Kelly for putting up with my mishaps in "her" lab. I also need to thank Dr.
Willie Kirk and the late blight crew for their help both in the lab and in the field. Last but
not least, thanks to my husband Rob for his support, encouragement, humor (he told
everyone that I was getting my Ph.D. in light bright) and for not proposing until after I
passed my comprehensive exams! Thanks again to you all for your invaluable part in this

chapter of my life.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ vi
LIST OF FIGURES .............................................................................................. vii
CHAPTER I ............................................................................................................. 1
General introduction .................................................................................... 1
Literature cited ........................................................................................... 16
CHAPTER II .......................................................................................................... 21
Abstract ...................................................................................................... 21
Introduction ................................................................................................ 23
Materials and Methods ............................................................................... 31
Cloning of the glucose oxidase gene ............................................. 31
Transformation of potato cultivars ................................................. 31

Molecular characterization of transgenic potato lines ................... 33

Glucose oxidase enzyme activity assay .......................... 36

Late blight screening of transgenic potato lines ............................ 37

Results ........................................................................................................ 40
Transformation and molecular analysis ......................................... 4O

Foliar late blight screening ............................................................. 48

Tuber late blight evaluation ........................................................... 50

Enzyme activity assay .................................................................... 52

Discussion .................................................................................................. 53
Literature cited ........................................................................................... 59
CHAPTER III ........................................................................................................ 65
Abstract ...................................................................................................... 65
Introduction ................................................................................................ 67
Materials and Methods ............................................................................... 73
Cloning of the glucose oxidase gene ............................................. 73
Transformation of potato cultivars ................................................. 73

F usarium dry rot screening of trasngenic potato lines ................... 75

Erwim'a soft rot screening of transgenic potato lines ..................... 76

Results ........................................................................................................ 78

F usarium dry rot ............................................................................ 78

Erwinia soft rot .............................................................................. 82

Discussion .................................................................................................. 88
Literature Cited .......................................................................................... 94

LIST OF TABLES

TABLE 1.1 Chloroplast DNA (ct-DNA) types and ploidy levels of wild and cultivated
potato species .................................................................................................................... 4

TABLE 2.1. Molecular analyses, field and greenhouse late blight experiments and tuber
phenotype of ‘Spunta' - derived (SGO) lines containing the glucose oxidase gene ......... 41

TABLE 2.2. Molecular analyses, field and greenhouse late blight experiments and tuber
phenotype of 'Atlantic' - derived (AGO) lines containing the glucose oxidase gene ...... 42

TABLE 2.3. Molecular analyses, field and greenhouse late blight experiments and tuber
phenotype of 'Libertas' - derived (LGO) lines containing the glucose oxidase gene ...... 43

TABLE 2.4. Tuber late blight evaluations for 1998 and 1999 ..................................... 51

TABLE 3.1. Molecular analyses, F usarium dry rot evaluations and tuber phenotype of
'Spunta' - derived (SGO) lines containing the glucose oxidase gene ............................... 79

TABLE 3.2. Molecular analyses, F usarium dry rot evaluations and tuber phenotype of
'Atlantic' - derived (AGO) lines containing the glucose oxidase gene ............................ 80

TABLE 3.3. Molecular analyses, F usarium dry rot evaluations and tuber phenotype of
'Libertas' - derived (LGO) lines containing the glucose oxidase gene ............................ 81

TABLE 3.4. Molecular analyses, Erwinia soft rot evaluations and tuber phenotype of
'Spunta' - derived (SGO) lines containing the glucose oxidase gene ............................... 83

TABLE 3.5 Molecular analyses, Erwim'a soft rot evaluations and tuber phenotype of
'Atlantic' - derived (AGO) lines containing the glucose oxidase gene ............................ 84

TABLE 3.6. Molecular analyses, Erwinia soft rot evaluations and tuber phenotype of
'Libertas' - derived (LGO) lines containing the glucose oxidase gene ............................ 85

vi

LIST OF FIGURES

FIGURE 1.1. Evolutionary relationships of cultivated potatoes and their ploidy levels

(adapted from Hawkes, 1990) .............................................................................................. 3
FIGURE 1.2. The relationship of five potato chloroplast DNA (ct-DNA) types based on

restriction fragment length polymorphisms (adapted form Hosaka, 1986) ......................... 4
FIGURE 1.3. Diagram of the life cycle of Phytophthora infestans (Agrios, 1988) ........... 8

FIGURE 2.1. Hypothetical model showing the role of H202 and NO in the induction of
gene expression and cell death during plant-pathogen interactions. H202, salicylic acid,
and cell death are linked in a self-amplifying process, termed the oxidative cell death
(0CD) cycle. The 0CD cycle is reiterated in distal leaves, leading to SAR. Cell death in
infected leaves is initiated through several routes, including, besides the 0CD cycle,
ethylene- and NO-dependent pathways. Broken arrows marked by a plus indicate
putative agonistic effects of salicylic acid and ethylene on H202 mediated cell death
(VanCamp et al., 1998) ...................................................................................................... 30

FIGURE 2.2. Diagram of the pSPUDll vector containing the glucose oxidase gene
from Aspergillus niger under the control of the Gelvin "super promoter" [ P(ocs)3 mas].
Restriction sites used for Southern blotting are shown. ..................................................... 32

FIGURE 2.3. Southern blot of BamI-II digested DNA verifying insertion of the glucose
oxidase gene (A) and Xbal digested DNA to estimate transgene copy number (B). ........ 44

FIGURE 2.4. Northern blot of transgenic potato lines containing the glucose oxidase
gene hybridized with the glucose oxidase probe (A) and a potato 18S ribosomal probe

(B). ..................................................................................................................................... 44

FIGURE 2.5. Western blot showing leaf protein hybridized to glucose oxidase
polyclonal antibody ............................................................................................................ 46

FIGURE 2.6. Photograph showing the off-type tuber morphology of several transgenic
lines containing the glucose oxidase gene. ........................................................................ 47

FIGURE 3.1. Photograph of tubers inoculated with F usarium sambucinum showing
internal (A) and external (B) symptoms of infection ......................................................... 68

FIGURE 3.2. Diagram of the pSPUDll vector containing the glucose oxidase gene

fi'om Aspergillus niger under the control of the Gelvin "super promoter" [ P(ocs)3 mas].
Restriction sites used for Southern blotting are shown ...................................................... 74

vii

FIGURE 3.3. Photograph of 'Libertas' and LGO lines inoculated with Erwim'a
carotovora ssp. carotovora. All LGO lines shown had lesion diameters significantly less
than the 'Libertas' control (p<0.05) .................................................................................... 86

FIGURE 3.4. Photograph of 'Atlantic' and AGO-4 tubers inoculated with Erwim'a
carotovora ssp. carotovora. Lesion diameter of AGO-4 was significantly less than that
of the non-transgenic 'Atlantic' control (p<0.05). .............................................................. 87

FIGURE 3.5. Photograph showing the off-type tuber morphology of several transgenic
lines containing the glucose oxidase gene. ........................................................................ 91

viii

Chapter I

GENERAL INTRODUCTION

The cultivated potato, Solanum tuberosum, has had an interesting history ranging
fi'om its role in the Irish potato famine of the 1840’s, to its current importance in the fast-
food industry. Though it was slow to be accepted as a food crop when introduced into
Europe from South America, the potato has become one of the most important food crops
in the world. Although it is enjoyed in the United States for its taste and versatility, in
other parts of the world the potato is desired for its high nutrient value for humans and for
livestock. Ranking fifth in area among crop plants grown for human consumption and
fourth in yield, the potato is superior to many crop plants in protein production per unit
area and time, energy production and the presence of essential amino acids (Ross, 1986).

Although the origin of the cultivated potato is not certain, archaeological evidence
and the current distribution of primitive cultivated potatoes and wild species suggests that
the potato was first domesticated in northern Bolivia near Lake Titicaca. It then spread
rapidly throughout the high Andes of Bolivia and Peru where other staple crops such as
maize did not thrive (Hawkes, 1992). In addition to the cultivated potato, there are many
wild potato species that range in ploidy level fi'om diploid (2n=2x=24) to hexaploid
(2n=6x=72) with most species functioning as autopolyploids and only a few as
allopolyploids. These species occupy a large geographical area ranging from the
southwestern United States, through Mexico and Central America and throughout many
South American countries. It is from these wild species that the cultivated potato was
derived. Although the exact progenitor of cultivated potato is unknown, it is generally

assumed to be a diploid species whose natural distribution area coincides with the

proposed region of domestication (high Andes from central Peru southwards to central
Bolivia) (Hawkes, 1990). The wild species S. leptophyes is found in the appropriate area
and has been proposed as similar or identical to the progenitor species for cultivated
diploid potato (Hawkes, 1990) (Figure 1.1). Other species such as S. canasense have
been proposed as the progenitor species for diploid cultivated potato. Based on
chloroplast genome type (ct-genome), Hosaka (1986) suggest that S. canasense is one of
the most probable ancestral species as it has the C type ct-genome which is ancestral to
the S type ct-genome common in cultivated diploid species (Table 1.1; Figure 1.2).

Although many of the cultivated potatoes and wild species are short-day adapted,
selection from S. tuberosum ssp. andigena both in Europe and Chile produced the long-
day adapted S. tuberosum ssp. tuberosum which is cultivated in many areas of the world.
Although it is generally agreed that the cultivated potato arrived in Spain around 1570
and in England around 1590 (Hawkes, 1992) the nature and source of the early European
potato has been disputed. Some botanists believed that the European potato must have
been imported from Chile already adapted to long day lengths (S. tuberosum ssp.
tuberosum) (Hawkes, 1992). However, several pieces of information refute this assertion
leading both Salaman (1946) and Hawkes (1967) to suggest that the European potato was
derived from Andean sources of the short day adapted S. tuberosum ssp. andigena. First
of all, herbarium specimens and descriptions from the late 16‘h and early seventeenth
centuries more closely resemble subspecies andigena than subspecies tuberosum
(Hawkes, 1967). Secondly, the trip from Chile to Europe would have taken too long for
potato tubers to survive thus it is more likely that the first shipments came from Peru or

Colombia (Salaman 1946). In addition, the conversion of andigena to tuberosum has

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Table 1.1 Chloroplast DNA (ct-DNA) types and ploidy levels of wild and
cultivated potato species.

 

 

 

 

 

 

 

 

Species Chloroplast DNA type Ploidy

S. leptophyes W 2x

S. sparsipilum W 2x

S. acaule C 4x

S. canasense C 2x

S. stenotomum S 2x

S. tuberosum ssp. andigena A 4x

S. tuberosum ssp. tuberosum T 4x

S. chaucha S 3x

S. curtilobum S 5x

S. phureja S 2x

S. juzegczukii C 3x

_ W — S' acaule S. stenotomum
—_’ S' canasense" —_+ S. phureja
S. Ieptophyes S. Juzepczuku S. c hauc ha
5' sparszpzlum S. curtilobum

 

 

 

 

 

 

T _ A _*
—_" S. tuberosum

S tuberosum ssp. andigena
’ ssp. tuberosum

 

 

 

 

 

 

 

 

Figure 1.2. The relationship of five potato chloroplast DNA (ct-DNA) types based on
restriction fragment length polymorphisms (adapted from Hosaka, 1986).

been accomplished by Simmonds (1966) in only a few generations of selection. Thus, it
would be quite possible to develop tuberosum from imported andigena in the years
between the first introduction to Europe (circa 1570) and the description of the European
tuberosum by Linnaeus (circa 1750). Lastly, the early European potato was regarded as a
botanical curiosity until the mid-eighteenth century due to poor yields and late crops
indicating adverse photoperiodic response (Hawkes, 1992). Hosaka and Hanneman
(1988) provided further evidence supporting andigena as the first European potato.
These authors evaluated the ct-genome of hybrids from “Myatt’s Ashleaf’, which is one
of the relics of the first European potato, and determined that these hybrids had the A
type ct-genome found in ssp. andigena but not in ssp. tuberosum. Based on their ct-
genome determinations, Hosaka and Hanneman (1988) proposed that the original
European potato was ssp. andigena that was adapted to long day lengths. These potatoes
prevailed in Europe until the mid 1800’s when they were mostly destroyed by disease and
were replaced by the American cultivars derived from “Rough Purple Chili” which was
selected from Chilean ssp. tuberosum (Salaman, 1949). Therefore, current European and
American potatoes carry the T type ct-genome found only in ssp. tuberosum (Hosaka and
Hanneman, 1988).

In addition to its poor initial performance the potato was not well accepted in
Europe for several other reasons. First of all, the attributes of known plants with similar
characteristics were applied to the potato. Thus, the undesirable flatulence and lust
inciting qualities of sweet potato and the toxicity of black nightshade were associated
with potatoes (Salaman, 1949). In addition, many doctors and herbalists attributed

leprosy, consumption, rickets and many other diseases to the consumption of potatoes

(Salaman, 1949). Lastly, the potato was the first edible plant in Europe to be grown from
tubers and have the edible portion grown on underground stems. Therefore, the potato
was immediately under suspicion simply because it was an oddity (Salaman, 1949). The
potato was mostly disfavored and was slow to be accepted in parts of England, France
and Russia. However, in Ireland, the potato was more readily accepted for several
reasons (Salaman, 1949). The Irish climate and soil were generally well suited to
growing potatoes and the crop required limited time, energy and skill to produce. The
potato was also easily incorporated into the traditional diet and preparation techniques.
Perhaps more importantly, potatoes could be stored through the winter to feed both
family and livestock after all the surplus of grain had been used to pay rent.

A large population dependent on the potato for sustenance, the economic and
political environment of Ireland in the mid-1800’s, and the arrival of the potato late blight
pathogen, Phytophthora infestans mont. DeBary, lead to the Irish Potato famine in 1845
and 1846. An outbreak of late blight in England was reported in August of 1845 and by
the end of September the disease had spread through Poland, Germany, Belgium, France
and Ireland (Salaman, 1949). Although early varieties escaped infection most of the Irish
potato crop was a late variety called ‘Lumper’ which either rotted in the field or later in
storage. The disease reappeared early in the season of 1846, spread rapidly across Ireland
and destroyed most of the crop (Salaman, 1949). With little aide from the ruling English
government, the result in Ireland from 1845-1852 was the death of around one million
peOple who relied on potatoes for food and the emigration of another million and a half

people fleeing from poverty and starvation (Kinealy, 1996).

Late blight continues to be a devastating disease of potatoes and much research
has been done to understand the causal pathogen (P. infestans) and to develop methods of
control. Not a true fungus, P. infestans belongs to the Oomycete class and is classified as
a “water mold” due to its adaptation for thriving where there is fi'ee water and cool
temperatures. The life cycle of P. infestans can be divided into a highly efficient asexual
life cycle that can be repeated in 5-7 days under ideal conditions (Kirk, 1996) and a
sexual cycle (Figure 1.3). The asexual cycle is carried out via sporangia that can fruit
quickly on leaf and stem tissues. Sporangia are non-motile and rely on wind and rain for
dispersal but can undergo indirect germination to produce mobile zoospores (Lacy and
Hammerschmidt, 1995). Tuber infection occurs when zoospores or sporangia swim or
are washed into the soil and enter the tubers through lenticels or cracks or when healthy
tubers physically contact diseased foliage at harvest. P. infestans is heterothallic with
two known mating types, A1 and A2. If mycelia of different mating types interact, sexual
propagation can occur through the formation of oospores. Sexual reproduction in this
pathogen is of great importance for two reasons. First, oospores have been shown to
survive freezing and thawing, long-term storage, and passage through the digestive
system of snails (Umaerus and Umaerus, 1994). Thus, these spores may survive in soils
separate from the host plant for long periods of time and may serve as an inoculum
source for newly planted potatoes. Secondly, sexual reproduction implies genetic
recombination and the possibility of new genotypes of the pathogen with enhanced
virulence and greater resistance to chemical control. Prior to the 1980’s, P. infestans
populations in Europe and North America consisted solely of the U81 genotype and the

Al mating type. However, the U88 genotype (A2 mating type) was reported in EurOpe

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during the mid 1980’s and in North America in 1992 making sexual reproduction a threat
to late blight control.

Since the devastation of the Irish Potato Famine, research efforts have lead to
disease management programs that integrate cultural practices and chemical controls. It
is believed that asexual spores of P. infestans can only overwinter in living plant tissue.
Thus, an important control measure is to eliminate cull piles and volunteer potatoes.
Other cultural practices such as planting certified seed and regular scouting help to
minimize the development and spread of late blight. Chemical control of late blight
began during the 1880’s with the development of the Bordeaux mixture and other copper-
based compounds. Since then several classes of residual and systemic, fungicides have
been commercialized and are being used to prevent late blight infection in potato cr0ps.
Although chemical control is an important component of integrated management of late
blight, there is growing public concern over the use of chemicals. Special permission
(Section 18 registration) is required in order to use certain fungicides in the United States.
In addition, the US8 genotype, that is now present in North America, is resistant to the
systemic fungicide, metalaxyl, which had been the predominant chemical used for late
blight prevention and eradication. There is also concern that the heavy use of chemicals
will lead to the development of chemical-resistant late blight genotypes.

Another component of late blight management in potatoes has been to breed
resistant cultivars. The first efforts to develop late blight-resistant potato cultivars
emphasized vertical, or specific resistance, conferred by major resistance genes (R
genes). These R genes were initially found in wild Solanum species from Mexico where

the P. infestans populations are mixtures of many races and provide ample opportunity

for the host plant to evolve various resistances to the pathogen. S. demissum, a Mexican
hexaploid wild species, was initially the predominant source of late blight resistance used
for breeding (Ross, 1986). A series of crosses between cultivated potato and S. demissum
resulted in backcross hybrids that were completely resistant to late blight. Although S.
demissum-based resistance was highly successful it was short-lived as P. infestans
quickly evolved virulence to the deployed R genes. The rapid breakdown in this
resistance coinciding with the development of effective chemical controls for late blight
discouraged progress in late blight-resistance breeding for many decades.

Due to the presence of the USS genotype of P. infestans in Europe and North
America and the concern over the use of chemicals in agriculture, breeding for late blight
resistance is a priority once again. However, the emphasis now is on exploiting
horizontal resistance. Also referred to as general or field resistance, horizontal resistance
is polygenic in nature and is assumed to be more stable than a monogenic system such as
vertical resistance. The genetic basis for horizontal resistance to late blight has yet to be
resolved. However, studies have shown that both general (GCA) and specific combining
ability (SCA) contribute to the variability of horizontal resistance to late blight. SCA has
been reported for foliar resistance (Killick and Malcolmson, 1973) and for tuber
resistance (Stewart et al., 1992; Bradshaw et al., 1995) and several studies (Malcolrnson
and Killick, 1980; Wastie et al., 1993; Bradshaw et al., 1995) have reported GCA for
foliar and tuber resistance. In addition, Tai and Hodgson (1975) showed GCA to be the
largest source of variability for horizontal late blight resistance but suggested that there
was also an important SCA component as well. The importance of maternal effects and

the influence of R genes on horizontal late blight resistance is also unclear. Killick and

10

Malcolrnson (1973) noted no maternal effects in crosses involving a late blight resistant
parent. However, Lishak and Nazar (1977, cited in Umaerus et al., 1983) demonstrated
that the resistance of the progeny was better if the late blight resistant parent was female.
Some authors have suggested that horizontal and vertical resistance are not genetically
distinct mechanisms but that "minor" genes (quantitative trait loci or QTL) are alleles of
"major" (R) genes (Nelson, 1978; Robertson, 1985). Recent studies have provided
molecular evidence to support this theory. Leonards-Schippers et al., (1994) found a
QTL for late blight resistance that mapped to the same chromosome region as the
dominant allele, R1, conferring race specific resistance to P. infestans. In addition, the
clustered R genes R3, R6 and R7 on chromosome XI are also linked to a QTL for
quantitative late blight resistance (Gebhardt, 1999).

One of the difficulties in breeding for horizontal resistance to late blight is that it
is hard to maintain during crossing and backcrossing which is often necessary to recover
commercially acceptable tuber types (Black, 1970). The mean resistance of the progenies
is usually less than the parental mean. Another problem encountered in breeding for
horizontal resistance is the positive correlation between late maturity and late blight
resistance. Late maturity is not suitable for potato growing regions in North America but
has been closely associated with late blight resistance from many sources. It is also more
difficult to evaluate horizontal late blight resistance than it is to evaluate vertical
resistance. Horizontal resistance can be influenced by environmental factors such as day
length and light intensity (Schumann and Thurston, 1977) and can be masked by the

presence of R genes, which are found in most cultivars and wild species.

11

Although most potato breeding programs emphasize the use of horizontal late
blight resistance, it is important to note that single gene resistance sources are still being
explored. Helgeson et al. (1998) developed late blight resistant breeding lines via the
somatic fusion of Solanum bulbocastanum with S. tuberosum cultivars. Backcross
progeny of these somatic hybrids have been tested for several years in the Toluca Valley
of Mexico and have thus far demonstrated durable late blight resistance in the presence of
numerous P. infestans races (Helgeson et al., 1998; Helgeson, personal communication).
Subsequent evaluation of the resistance has implicated a single gene coming from S.
bulbocastanum that is currently being cloned to evaluate the sequence and identify any
known functional sequence motifs (Helgeson, personal communication). In addition
Douches et al (unpublished data) have developed a highly resistant breeding line derived
from the Mexican cultivar ‘Tollocan’. This cultivar has been identified as having durable
resistance in the Toluca Valley of Mexico (personal communication, Niederhauser).
However, the transmission of this resistance would indicate that it is monogenic or at
most oligogenic (Douches, unpublished data). Similarly, the horizontal late blight
resistance in the cultivar 'Sterling' has been mapped to a few major effect quantitative
trait loci (QTL) (Collins et al., 1999). Thus, it may be unwise to completely abandon the
use of what has been considered to be major -gene resistance to late blight.

Pyrarniding resistance genes has been proposed as a means of stabilizing
resistance against variable plant pathogens (Nelson, 1978). This breeding strategy
involves incorporating several different resistance genes into one cultivar in order to
expand the range of pathogen genotypes to which a cultivar is resistant and/or to increase

the durability of resistance. Gene pyrarniding has been highly successful for controlling

12

stem rust of wheat for 40 years in Canada and in the north-central United States (Line and
Chen, 1995). This strategy has also been developed and utilized to breed for resistance to
anthracnose (Young and Kelly, 1996; Young and Kelly, 1997), bean common mosaic
virus (Haley et al., 1994a; Haley et al., 1994b) and rust (Miklas et al., 1993; Haley et al.,
1993) in dry bean (Phaseolus vulgaris). For more complex resistance systems, an
analogous strategy would be to combine resistance components fi'om various sources.
There are several different components of horizontal resistance to fungal infection
including resistance to infection, slower growth of the pathogen through tissues, an
extended incubation period and a reduction in the number of spores produced (Hide and
Lapwood, 1992). The predominant mechanism of resistance may vary from species to
species. Ross (1986) noted that some Polish cultivars resisted infection by P. infestans,
whereas others resisted spread and sporulation. Additionally, in a study of eight wild
potato species, Colon et al., (1995) reported that the species differed in the importance of
various components of resistance. By crossing different sources of late blight resistance
it may be possible to combine several resistance mechanisms and thus improve upon the
level and durability of late blight resistance. The International Potato Center (CIP) in
Peru has developed an R gene free population (population B) in which it has been
possible to identify and combine sources of horizontal resistance (Landeo et al., 1997).
Many other sources of late blight resistance have been reported (Douches et al., 1997a;
Douches et al., 1997b; Colon and Budding, 1988; Helgeson et al., 1998; Bamberg et al.,
1994; Huaman, 1987) and several groups are utilizing this broad genetic base of materials

to deve10p late blight resistant potato cultivars.

13

Although late blight is the most significant disease of potato worldwide, the
potato is prone to more than one hundred diseases caused either by bacteria, fimgi,
viruses or mycoplasms (Hide and Lapwood, 1992). Fortunately, only a few of these
diseases become serious problems in any one growing region. Some of the most
important diseases of potato in North America other than late blight include: early blight
(Alternaria solani), early die (Verticillium dahliae), dry rot (Fusarium sp.), sofi rot
(Erwim'a carotovora ssp.), common scab (Streptomyces scabies), potato virus X and Y,
and potato leaf-roll virus. In addition to these pathogens, the potato also falls prey to
insect pests such as the Colorado potato beetle, which severely defoliates the plants,
aphids, which spread viruses, and potato leaf-hoppers which damage foliage. Many of
these pathogens and insect pests can currently be controlled with chemicals however the
development of chemical resistant genotypes is possible if not probable. Therefore,
potato breeders also focus on developing cultivars with resistance to multiple pathogens
and/or insect pests. As with late blight, the sources for resistance to these diseases and
pests are most ofien wild Solanum species.

In addition to the sources of disease resistance previously mentioned, it may also
be possible to utilize engineered resistance mechanisms. The potato is very amenable to
tissue culture propagation, to infection by Agrobacterium tumefaciens and to
regeneration. Numerous potato transformation and regeneration protocols have been
published utilizing both leaf and stem sections (Yadav and Sticklen, 1995; DeBlock,
1988; Wenzler et al., 1989; Hulme et al, 1992). Thus, transformation of potato with
foreign genes has been relatively easy. In recent years, potatoes have been genetically

modified to incorporate transgenes for resistance to Colorado potato beetle (Perlak et al.,

14

1993), potato virus Y (PVY) (Pehu et al., 1995; Hefferon et al., 1997) and potato leaf roll
virus (PLRV) (Brown et al., 1995; Kawchuck et al., 1991) as well as transgenes affecting
carbohydrate metabolism (Stark et al., 1992) and disease resistance (Allefs et al., 1996,
Liu et al., 1994). As our understanding of plant disease resistance mechanisms develops,
more and more defense-related genes are being cloned and thus becoming available for
insertion into plant genomes. Many of these transgenes show promising results in
tobacco or tomato, which are members of the same family (Solanaceae) as the potato.
By utilizing some of these promising genes alone or in combination with various sources
of resistance from wild species and existing cultivars it may be possible to develop potato
cultivars that are not only resistant to late blight but also resistant to pests and other

diseases as well.

15

LITERATURE CITED
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Allefs, S., E. Jong, D. Florack, C. Hoogendoom and W. Stiekema. 1996. Erwinia sofi
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Bamberg, J .B., M.W. Martin and J .J . Schartner. 1994. Elite selections of tuber-bearing
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Colon, L.T., D. J. Budding, L.C. Paul Keizer & M.M.J. Pieters, 1995 Components of
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DeBlock, M. 1988. Genotype-independent leaf disc transformation of potato (Solanum
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Douches, D., W. Kirk, K. Jastrzebski, C. Long and R. Hammerschmidt. 1997a.
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Douches, D., J. Bamberg J.B. 1997b. Fine screening of Solanum wild species for
resistance to late blight. PAA meeting abstract.

16

Gebhardt, C. 1999. Genetic analysis of quantitative and qualitative resistance to late
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Hawkes, JG. 1990. The potato: evolution, biodiversity and genetic resources.
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Hawkes, JG. 1992. History of the potato. p. 1-12. In, P. Harris (ed.), The Potato Crop.
Chapman and Hall, New York.

Hawkes, JG. 1967. The history of the potato. J. Roy. Hort. So'c. 92:288-302.

Haley, S. P. Miklas, J. Stavely, J. Byrum and J. Kelly. 1993. Identification of RAPD
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Haley, S., L. Afanador and J. Kelly. 1994a. Selection for monogenic resistance traits
with coupling- and repulsion-phase RAPD markers. Crop Sci. 34:1061-1066.

Haley, S., L. Afanador, and J. Kelly. 1994b. Identification and application of RAPD
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Hefferon, K., H. Khalilian and M. Abouhaidar. 1997. Expression of the PVY(O) coat
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three potato cultivars. Theor. Appl. Genet. 94:287-292.

Helgeson, J.P., J.D. Phohnan, S. Austin, G.T. Haberlach, S.M. Wielgus, D. Ronis, L.
Zambolim, P. Tooley, J.M. McGrath, R.V. James and W.R. Stevenson. 1998.
Somatic hybrids between Solanum bulbocastanum and potato: a new source of
resistance to late blight. Theor. Appl. Genet. 96:738-742.

Hide, GA. and DH. Lapwood. 1992. Disease aspects of potato production. pp. 403-
437. In, P. Harris, (ed.). The potato crop. Chapman and Hall, New York.

Hosaka, K. 1986. Who is the mother of the potato?—restriction endonuclease analysis
of chloroplast DNA of cultivated potatoes. Theor. Appl. Genet 72:606-618.

Hosaka, K. and RE. Hanneman. 1988. The origin of the cultivated tetraploid potato
based on chloroplast DNA. Theor. Appl Genet. 76:172-176.

Huaman. Z. 1987. Inventory of Andean potato cultivars with resistance to some pests

and diseases and other desirable traits. International Potato Center (CIP), Lima,
Peru.

17

Hulme, J ., E. Higgins and R. Shields. 1992. An efficient genotype-independent method

for regeneration of potato plants fiom leaf tissue. Plant Cell, TissueOrgan Cult.
31:161-167.

Kawchuck, L., R. Martin, and J. McPherson. 1991. Sense and antisense RNA-mediated
resistance to potato leafioll virus in Russet Burbank potato plants. Mol. Plant-
Microbe Interact. 4:247-253.

Killick, R.J. and J .F . Malcolmson. 1973. Inheritance in potatoes of field resistance to
late blight (Phytophthora infestans. (Mont) de Bary). Physiol. Plant Pathol.
3:121-131.

Kinealy, C. 1996. How politics fed the famine. Natural Hist. 105233-35.

Kirk, W. 1996. Potato late blight in Michigan. In, Potato Late Blight a Guide to
Control. Michigan Potato Industry Commission.

Lacy, M. and R. Hammerschmidt. 1995. Diseases of potato: late blight. Extension
Bulletin E-1802. Michigan State University.

Landeo, J.A., M. Gastelo, G. Forbes, J.L. Zapata and F]. Flores. 1997. Developing
horizontal resistance to late blight in potato. pp. 122-126. In, International Potato
Center Program Report. International Potato Center, Lima, Peru.

Leonards-Schippers, C., W. Gieffers, R. Schafer-Pregl, E. Ritter, S. Knapp, F. Salamini
and C. Gebhardt. 1994. Quantitative resistance to Phytophthora infestans in

potato: a case study for QTL mapping in an allogarnous plant species. Genetics
137:67-77.

Line, R. and X. Chen. 1995. Successes in breeding for and managing durable resistance
to wheat rusts. Plant Dis. 79:1254-1255. 0

Liu, D., K. Raghotharna, P. Hasegawa and R. Bressan. 1994. Osmotin overexpression
in potato delays development of disease symptoms. Proc. Natl. Acad. Sci.
91:1888-1892.

Malcolmson, J.F. and R.J. Killick. 1980. The breeding values of potato parents for field
resistance to late blight measured by whole seedlings. Euphytica 29:489-495.

Miklas, P., J. Stavely, and J. Kelly. 1993. Identification and potential use of molecular
marker for rust resistance in common bean. Theor. Appl. Genet. 85:745-749.

Nelson, R. 1978. Genetics of horizontal resistance to plant diseases. Annu. Rev.
Phytopathol 16:359-378.

18

Pehu, T., T. Maki-Valkama, J. Valkonen, K. Koivu, K. Lehto and E. Pehu. 1995. Potato
plants transformed with a potato virus YPl gene sequence are resistant to PVY.
Amer. Potato J. 72:523-532.

Perlak, F, T. Stone, Y. Muskopf, L. Peterson, G. Parker, S. McPherson, J. Wyman, S.
Lowe, G. Reed, D. Biever and D. Fischhoff. 1993. Genetically improved
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22:313-321.

Robertson, BS. 1985. A possible technique for isolating genie DNA for quantitative
traits in plants. J. Theoret. Biol. 117:1-10.

Ross, H. 1986. Potato breeding-problems and perspectives. Verlag Paul Parey, Berlin.

Salaman, RN. 1946. The early European potato; its character and place of origin. J.
Linn. Soc. (Bot.) 53:1-27.

Salaman, RN. 1949. The history and social influence of the potato. University Press,
Cambridge, p. 685.

Schumann, G. and H. Thurston. 1977. Light intensity as a factor in field evaluations of
general resistance of potatoes to Phytophthora infestans. Phytopathology
67:1400-1402.

Simmonds, NW. 1966. Studies of the tetraploid potatoes. 111. Progress in the
experimental re-creation of the Tuberosum Group. J. Linn. Soc. (Bot.) 59:279-
288.

Stark, D., K. Timmerman, G. Barry, J. Preiss and G. Kishore. 1992. Regulation of the
amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science
258:287-292.

Stewart, H., J. Bradshaw, and R. Wastie. 1992. Inheritance of resistance to late blight in
foliage and tubers of progenies fiom parents differing inresistance. Potato Res.

35:313-319.

Tai, G.C and W. Hodgson. 1975. Estimating general combining ability of potato parents
for field resistance to late blight. Euphytica 24:285-189.

Umaerus, V, M. Umaerus, L. Efjefalt, and B. Nillson. 1983. Control of Phytophthora by
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Barnicki-Garcia and PH. Tsao, (eds.). Phytophthora Its Biology, Taxonomy,
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19

Umaerus, V. and M. Umaerus. 1994. Inheritance to resistance in late blight. pp. 36-401.
In, J. Bradshaw and F. MacKay, (eds.). Potato Genetics. AB International,
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Wastie, R., J. Bradshaw and H. Stewart. 1993. Assessing general combining ability for
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transformation method for the production of large numbers of transgenic potato
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Yadav, N. and M. Sticklen. 1995. Direct and efficient plant regeneration from leaf
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Young, R. and J. Kelly. 1996. RAPD markers flanking the Arc gene for antrhacnose
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20

Chapter II

PRODUCTION, MOLECULAR CHARACTERIZATION AND LATE BLIGHT
REACTION OF TRANSGENIC POTATO PLANTS (SOLAN UM TUBEROS UM L.)
EXPRESSING A F UNGAL GLUCOSE OXIDASE GENE

ABSTRACT

Late blight, (Phytophthora infestans (Mont.) De Bary), is one of the most
devastating diseases of potato worldwide and the development of resistant cultivars for
late blight management in commercial production is important. Current emphasis is on
the utilization of horizontal resistance rather than vertical resistance conferred by specific
R genes. Our research was undertaken to determine if enhanced resistance to late blight
could be obtained by combining natural resistance and engineered resistance conferred by
the fungal transgene glucose oxidase from Aspergillus niger. This gene was selected due
to its reported effect against multiple pathogens including P. infestans, Erwinia
carotovora, Alternaria solani, and Fusarium sambucinum. Late blight susceptible
cultivars 'Atlantic', 'Snowden' and 'Spunta' and partially resistant cultivars 'Zarevo' and
'Libertas' were transformed with the glucose oxidase gene under the control of the Gelvin
"super promoter". Putative transgenic lines were verified and copy number determined
via Southern blotting. Copy number ranged from 1 to 8. Northern and western analyses
were used to determine which transgenic lines transcribed and/or translated the glucose
oxidase transgene. Only those lines in which the glucose oxidase protein was detected
via western blotting were included in disease evaluations. Glucose oxidase enzyme

activity was estimated for each line that was positive in the western analyses and ranged

21

from O to 93.96 Units/mg plant tissue * 105. There was no apparent correlation between
level of transgene mRNA and enzyme activity or between copy number and level of
transgene mRNA.

In agronomic trials, 44% of the transgenic lines were phenotypically "off-type"
compared to non-transgenic controls based on plant and/or tuber characteristics. Because
several off-type lines did not express the glucose oxidase protein, this phenomenon
cannot be attributed solely to the glucose oxidase transgene.

Lines expressing the glucose oxidase transgene were evaluated for reaction to P.
infestans in growth chambers and inoculated field studies. Results from trials conducted
in grth chambers suggest a small effect in 'Spunta'-derived and 'Libertas' derived
transgenic lines but high coefficient of variation (CV) values prevented the identification
of any significant differences between the transgenic lines and their respective non-
transgenic controls. Field studies revealed no significant effect of the glucose oxidase
transgene in either late blight susceptible or partially late blight resistant cultivars.

These results indicated that the glucose oxidase gene alone or in combination with
natural host plant resistance will not produce completely late blight resistant cultivars.
Potato cultivars with moderate levels of late blight resistance may be useful for reducing
the amount of fungicide (active ingredient) used in late blight control programs.
Therefore, transgenic lines that exhibit lower levels of late blight infection in growth
chamber trials may be useful in combination with reduced rates of fungicide application.
This potential benefit would need to be evaluated against the time and cost required for
development of transgenic potato cultivars and the potential for off-type tubers and

plants.

22

INTRODUCTION

Late blight, caused by the oomycete, Phytophthora infestans (Mont.) deBary, is
the most serious disease of potato (Solanum tuberosum subsp. T uberosum L.) in many
areas of the world. Unchecked, this disease can rapidly devastate entire potato crops as
witnessed by the Irish potato famine of the 1840’s. Bourke (1964) noted that “In about
ten weeks, potato murrain (as the disease was sometimes called) spread unchecked
through Belgium and became widespread in the Netherlands, France, Britain and Ireland
where almost half of the staple food crop was lost. In the following year, the blight
reappeared first in Ireland and by August 1846 the entire Irish crop was destroyed.”

Economic losses due to this disease occur by reduced photosynthetic capacity
resulting in yield reduction and by tuber infection resulting in storage losses. Foliar
infection first appears as water-soaked lesions that enlarge rapidly under moist, cool
conditions and become brown and irregular in shape. White mycelial growth is often be
observed on the lower leaf surfaces. Under continuously wet conditions, the disease
develops rapidly resulting in the destruction of both leaves and stems. Tuber infection is
indicated by irregular, dark patches on the tuber surface and by reddish-brown
discoloration extending into the tuber tissue. During storage, infected tubers are often
invaded by secondary pathogens leading to tuber decay.

Currently, the strategy for controlling potato late blight in the United States is a
combination of cultural and management practices such as frequent field scouting and
elimination of cull piles and the use of protectant fungicides. Until recently, the systemic
fungicide metalaxyl was used to prevent the spread of late blight once the disease was

identified in a field. However, in 1992 the metalaxyl resistant, US8 genotype of

23

P. infestans was identified in North America making this important chemical virtually
obsolete. At present there are no commercially acceptable late blight resistant potato
cultivars. However, many different sources of moderate to high levels of resistance have
been identified in wild species and various cultivars (Douches et al., 1997a; Douches et
al., 1997b; Helgeson et al., 1998; Bamberg et al., 1994). By combining these different
sources of resistance, it may be possible to pyramid different mechanisms of resistance
into one genotype and thereby develop stronger and more durable resistance to potato late
blight. Different sources of resistance might also be combined through the use of genetic
engineering. Combining a transgenic source of resistance with moderate levels of natural
late blight resistance may provide another means to develop a potato cultivar with
stronger more durable resistance.

One gene that has been used to increase disease resistance is the glucose oxidase
(B-D-glucosezoxygen l-oxidoreductase, EC 1.1.3.4) gene isolated from the fungus,
Aspergillus niger. Frederick et a1. (1990) cloned and sequenced this gene and it was
subsequently transformed into the potato cultivar ‘Russet Burbank' (Wu et al., 1995).
Transgenic plants were shown to have increased resistance to bacterial soft rot caused by
Erwinia carotovora ssp. carotovora, Verticillium wilt caused by V. dahliae and to late
blight caused by P. infestans. This increased resistance has been attributed to the
accumulation of hydrogen peroxide from the oxidation of B-D-glucose by glucose
oxidase (glucose ---- g1ucono-6-lactone + H202 ). Further support for the role of H202 in
host plant resistance was demonstrated using catalase deficient tobacco plants. When
induced by overexposure to light, these plants accumulated H202, acidic and basic

pathogenesis related proteins (PR proteins) and salicylic acid as well as demonstrating

24

increased resistance to the bacterial pathogen Pseudomonas syringae pv. syringae
(Chamnongpol et al., 1998).

The chain of activity that occurs within a plant upon contact with a pathogen has
been studied in depth. One of the first plant defense responses to both compatible and
incompatible pathogens is a rapid oxidative burst resulting in the production of active
oxygen species (AOS) including superoxide anion (02'), hydrogen peroxide (H202) and
hydroxyl radicals (OH') (Mehdy, 1994; Baker and Orlandi, 1995; Low and Merida,
1996). In a compatible reaction, a second, prolonged oxidative burst occurs following
hypersensitive cell death (Baker and Orlandi, 1995; Low and Merida, 1996). Two
mechanisms have been proposed for the production of H202 during the oxidative burst:
production via a plasma membrane bound NAD(P)H oxidase and production via cellular
peroxidases. Both mechanisms have been demonstrated in different plant species
(Bolwell, et al., 1998).

The A08, in particular H202, play several important roles in plant defense
systems. Hydrogen peroxide has been shown to have direct antifungal activity at low
concentrations (Peng and Kuc, 1992; Wu, et al., 1995) and to alter cell wall components.
Structural changes in the cell wall include lignification (Dean and Kuc, 1987;
Hammerschmidt and Kuc, 1982) and cross-linking of abundant, hydroxyproline-rich cell
wall proteins (Brisson et al., 1994; Bradley et al., 1992). Such alterations in cell wall
structure could delay pathogen invasion and/or trap pathogens in cells that will undergo
hypersensitive cell death. Although this would not provide complete resistance, it may
give the plant time to mobilize transcription-dependent defense mechanisms. Hydrogen

peroxide also contributes to hypersensitive cell death via lipid peroxidation and

25

generation of lipid free radicals (Keppler and Baker, 1989). Tenhaken et al. (1995)
reported that endogenous H2O2 produced by the oxidative burst is sufficient to trigger cell
death in an infected cell and serves as a diffusable signal for induction of cellular
protectant genes in neighboring cells. Expression of protectant genes encoding
glutathione S-transferase, glutathione peroxidase and ubiquitin limits the spread of cell
death by detoxifying and or metabolizing many products of oxidative stress (Tenhaken et
al., 1995). In contrast, Baker et al., (1997) demonstrated that it was possible to elicit the
oxidative burst without causing hypersensitive cell death. It has also been suggested that
in the cowpea-cowpea rust fungus pathosystem the oxidative burst does not trigger
hypersensitive cell death (Heath, 1998). In catalase deficient plants low levels of H202
were sufficient to activate defense mechanisms such as PR proteins without causing cell
death. However, the strength and speed of the induction was impaired compared to
plants in which high levels of H202 were present and cell death occurred (Chamnongpol
et al., 1998).

Activation of phytoalexin synthesis has also been reported as a role of H202 in the
plant disease response. Phytoalexins are low molecular weight antimicrobial compounds
formed during a hypersensitive reaction to a compatible pathogen. Activation of
phytoalexin synthesis by H202 has been reported in suspension cells (Apostol et al., 1989)
and in potato tubers (Chai and Doke, 1987). However, recent evidence suggests that
pathways independent of the oxidative burst also contribute to the regulation of
phytoalexin accumulation (Mehdy, 1994).

Hydrogen peroxide has not only been implicated in the hypersensitive response

but also in systemic acquired resistance (SAR) in plants. SAR is an induced defense

26

response triggered by infection with a necrosis-causing pathogen or by organic
compounds such as salicylic acid (SA). Establishment of SAR has been correlated with
the expression of nine gene families coding for proteins whose possible causal role in
resistance has attracted considerable attention (Ward et al., 1991). These genes, called
pathogenesis-related (PR) genes, are induced upon infection with a compatible pathogen,
but are also induced by SA via a secondary messenger. Chen et al., (1993) suggested that
H202 is the secondary messenger by which PR genes are activated. These authors found
that salicylic acid (SA) bound a protein (SABP) which was capable of catalyzing H202 to
form 02 and H20. When bound to SA, the catalytic activity of SABP would be disabled,
H202 would accumulate, and PR genes would be activated. Conrath et al. (1995)
provided support for this theory by demonstrating that a synthetic inducer of SAR also
inhibited catalase activity. However, Bi et al., (1995) found that H202 and the catalase
inhibitor 3-AT were weak activators of PRl-a in wild type tobacco and were not able to -
activate PRl-a in transgenic tobacco that lacked the ability to accumulate SA.
Neuenschwander et al. (1995) reported similar results suggesting that H202 does not
function downstream of SA in SAR signaling but that SA inhibition of catalase may be
important for the hypersensitive response. Alvarez et al., (1998) provided further
evidence for the role of H202 in SAR. These authors reported that the primary oxidative
burst leads to secondary oxidative bursts in small collections of cells distant from the
infection site resulting in low frequency systemic micro-hypersensitive responses (micro-
HR). Blocking the primary oxidative burst inhibited the formation of micro-HRS and the
induction of glutathione-S-transferase (GST) (an indicator of SAR) in uninfected leaves.

These authors speculated that micro-oxidative bursts mediate cell death, defense

27

induction and signal propagation in parallel with the primary oxidative burst and that
both the primary and secondary oxidative bursts are necessary for SAR.

Other molecules such as nitric oxide (NO) and ethylene have also been implicated
in oxidative cell death and may function in a complex cycle designed to perpetuate and
control H202 during the plant disease response. As with H202, NO may have several
important roles. One function of NO is to inhibit cytochrome c oxidase activity in
mitochondria which may increase electron flow towards oxygen and thus stimulate
superoxide and H202 formation (VanCamp et al., 1998). Nitric oxide was also found to
enhance H202-mediated cell death and the removal of NO by either NO scavengers or
biosynthetic inhibitors blocked the HR as well as the induction of phenylalanine
ammonia lyase (PAL) and chalcone synthase (CHS) in pathogen challenged plants
(Delledone et al., 1998). GST and PR1 genes were also induced by NO but the induction
was dependent on both H202 and SA (Levine et al, 1994). Thus, NO appears to function
in two pathways, one leading to phenylpropanoid (PAL) and flavanoid (CHS)
biosynthesis and one leading to antioxidant (GST) and PR gene expression (VanCamp et
al., 1998). In addition, Noritake et al, (1996) reported that NO was capable of inducing
synthesis of the phytoalexin rishitin in potato tissues. Ethylene has also been implicated
in oxidative cell death. Lund et al., (1998) reported that ethylene promotes lesion
formation around infection sites but doesn't seem to be present in leaves distant from the
infection site.

Although the exact function of H202 in the plant disease response process is not
fully elucidated, there appears to be complex interactions between H202, SA, NO and

ethylene “'which serve to carefully control cell death and pathogen defense pathways

28

involving hazardous molecules such as NO and H202. VanCamp et al., (1998) suggested
a model for oxidative cell death (OCD) in which H202, SA, and cell death are linked in a
self-amplifying process which is reiterated in distant leaves (Figure 2.1). In this model,
H202 alone or in combination with SA activates cell death, which leads to enhanced
production and accumulation of A08. This cycle would perpetuate H202 and lead to
expression of various defense related genes. SA and ethylene may enhance H202-
mediated cell death as indicated by dotted lines. The model depicts NO as functioning in
the OCD cycle (SA/ H202 dependent pathway) and separate from the OCD cycle leading
to PAL and CH8 expression (SA/ H202 independent pathway).

Regardless of the precise function of H202 in plant disease resistance,
manipulation of H202 levels in potatoes via the incorporation of the glucose oxidase gene
has been shown to provide some resistance to late blight and other diseases (Wu et al.,
1995; Wu et al., 1997). To date only one cultivar (Russet Burbank) has been transformed
with the glucose oxidase gene and these transfonnants had only a reduced rate of
infection when challenged with P. infestans and other pathogens (Wu et al., 1995; Wu et
al., 1997). Developing potato cultivars with less susceptibility to late blight may be
useful for reducing the amount of fungicide necessary for control. However, the ultimate
goal is to develop completely resistant cultivars. One way this may be accomplished is
by pyrarniding engineered resistance mechanisms with natural host plant resistance.
Therefore, we attempted to build stronger resistance to late blight by combining the
glucose oxidase gene with horizontal resistance to late blight found in existing potato

cultivars.

29

 

Emmionot
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1
. Expnssionot
/ odchRprotm
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,1

 

 

 

 

 

 

 

Infection

 

 

 

 

 

L ) Expression 0‘
No i PALnndCHS

Figure 2.1. Hypothetical model showing the role of H202 and NO in the induction of
gene expression and cell death during plant-pathogen interactions. H202, salicylic acid,
and cell death are linked in a self-arnplifying process, termed the oxidative cell death
(OCD) cycle. The OCD cycle is reiterated in distal leaves, leading to SAR. Cell death in
infected leaves is initiated through several routes, including, besides the OCD cycle,
ethylene- and NO-dependent pathways. Broken arrows marked by a plus indicate
putative agonistic effects of salicylic acid and ethylene on H202 mediated cell death

(VanCamp et al., 1998).

30

MATERIALS AND METHODS

Clo—mpg of the glucose oxidm

The glucose oxidase gene was cloned from Aspergillus niger using the
polymerase chain reaction (PCR) and primers designed according to Frederick et a1.
(1990) to which Smal and XbaI restriction sites had been added. The resulting DNA
fragment was sequenced for verification, compared to the published sequence (Frederick
et al., 1990) and incorporated into the pE1102 vector. The resulting plasmid, pSPUDll
(Fig. 2.2), had the glucose oxidase gene under the control of the Gelvin "super promoter"
(Ni et al., 1995). This promoter is a combination of a trimer octopine synthase upstream
activating sequence (ocs); joined to a mannopine synthase (mas) activator and promoter.
PSPUDll also contained the Nos terminator sequence, right and left borders for plant
transformation purposes and the NptII gene (kanamycin resistance) as a selectable
marker. The pSPUDll plasmid was transformed into Agrobacterium tumefaciens strain

LBA4404 (Ooms et al., 1982) via tri-parental mating.

Ignsformation of potato cultivars

The cultivars 'Spunta', 'Snowden', 'Atlantic', 'Zarevo' and ’Libertas' were
micropropagated in GA-7 Magenta vessels each containing 25 ml of modified MS basal
medium (Douches et al., 1998). Of these cultivars, 'Spunta', 'Snowden' and 'Atlantic' are
susceptible to P. infestans, whereas 'Zarevo' and 'Libertas' have shown moderate levels of
resistance in greenhouse and/or field experiments (Douches unpublished data).

Transformations of potato cultivars were done according to Li etal., (1999) using the

31

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32

A. tumefaciens culture harboring the pSPUDll vector. Regenerating shoots (> 5 mm)
were excised and transferred individually into 25 x 100 mm glass tubes each containing
20 ml of MS medium (Douches et al., 1998) supplemented with 200 mg'L'l Timentin
(SmithKline Beecham, Philadelphia, Pa.) and 50 mg'L'l kanamycin. Rooted plantlets
were designated as SGO-# (derived from 'Spunta'), AGO-# (derived from 'Atlantic'),
SNDGO-# (derived from 'Snowden'), ZGO-# (derived from 'Zarevo') and LGO-#
(derived from 'Libertas'). Named plantlets were subcultured and subsequently
transplanted into 10 cm pots containing Bacto potting medium (Michigan Peat Co.
Houston, Texas) and grown in the greenhouse under fluorescent lights (16 h photoperiod)
at 23 to 27°C. Leaf tissue from these plants was used for the molecular expression

assays.

Molecular characterization of transgenic potato lines

Southern Analysis

DNA was extracted from each putative, transgenic line (Saghai-Maroof et al.,
1984) and used for Southern blotting to verify incorporation of the glucose oxidase
transgene and to determine copy number. Genomic DNA (20 pg) was digested with
BamHI to excise the glucose oxidase gene for hybridization with a glucose oxidase DNA
probe. Digestion with XbaI was used to determine copy number. DNA fragments were
electrophoretically separated through a 1% agarose gel and transferred onto a nylon
membrane (Hybond N, Amersham, U.K.). Prehybridization was conducted for 2 hr. at
42°C followed by an overnight hybridization (Douches et al., 1998). The hybridization

solution contained a probe that was random primed labeled using DIG-1 l-dUTP

33

according to the manufacturer’s instructions (Roche Molecular Biochemicals, Germany).
Following hybridization, the membrane was washed twice in 2x SSC, 0.1% SDS for 15
min. at room temperature and then twice in 0.5x SSC, 0.1% SDS for 15 minutes at 65°C.
Chemiluminescent detection was according to Li et al., 1999. Each line was scored for

the presence or absence of the glucose oxidase gene and the number of inserts.

Northern analysis

Greenhouse-grown plants were also used as the source for RNA. Young, healthy
leaf tissue (1 mg) was collected from each transgenic potato line and frozen immediately
in liquid nitrogen. Frozen tissue was stored at —80°C. Total RNA was isolated using
Qiagen RNeasy Plant Total RNA Kit (Qiagen, Chatsworth, CA) and quantified by
spectrophotometer. Northern analysis was conducted according to Li et al. (1999) to
verify the presence of the glucose oxidase transcript. To prepare a DIG-1 l-dUTP labeled
RNA probe, the first half of the glucose oxidase (~1.5 kb) was transferred to the
Bluescript®SK+/' plasmid (Stratagene, LaJolla, CA). The resulting vector was
transformed into Eschericia coli strain DHSa and the glucose oxidase RNA probe was
synthesized by in vitro RNA transcription using an RNA labeling kit (Roche Molecular
Biochemicals, Germany). The entire gene sequence could not be used in this vector/host
combination because the glucose oxidase gene was expressed and toxic to the bacteria.
After detection of the glucose oxidase transcript via northern analysis, each blot was then
hybridized with a DIG-1 l-dUTP labeled 188 ribosomal probe to determine if the amount
of total RNA in each lane was approximately equal. For this probe, a

prehybridization/hybridization temperature of 42°C was used but all other conditions

34

were as described by Li et al. (1999). Relative levels of glucose oxidase transcript were

determined on each individual blot.

Western Analysis

Total protein was extracted from fresh leaf tissue (0.2 g) by grinding in 800 ml of
extraction buffer (50 mM Tris HCl pH 8.0, 1 mM EDTA, 10 mM diethyldithiocarbamic
acid, 0.05% Tween 20). A Bradford protein assay (BioRad, Hercules, CA) was
conducted on the soluble leaf extracts to quantify the amount of protein in each sample.
Based on the estimated amount of protein in each sample, equal amounts (200 mg) of
total protein were loaded on a 10% SDS-PAGE gel in addition to 50 ng purified glucose
oxidase enzyme (Sigma, St. Louis MO). Identical samples were loaded onto a second gel
and both gels were subjected to electrophoresis at 45 v and run overnight. One of the
gels was stained with Coomassie Brilliant Blue R-250 solution to visually compare
loading. The other gel was transferred, via western blotting, to a membrane and probed
with a 1:40,000 dilution of glucose oxidase rabbit polyclonal antibody (Polysciences,
Inc., Warrington, PA) and an alkaline phosphatase conjugated antirabbit IgG (Li et al.,
1999). Antibody binding was detected with CSPD (disodium 3-(4-methoxyspiro{1,2-

dioxetance-3,2 ’(5 ’-chloro)tricyclo[3 .3. 1 .13’7

]decan}-4-yl) phenylphosphate) according to
the manufacturer’s instructions (Roche Molecular Biochemicals, Germany) and the blot

was exposed to X-ray film (Hyperfilm, Amersharn).

35

Glucose oxidase en_zy_g;e activig assay

 

Transgenic plants that produced the glucose oxidase protein (based on western
analysis) were evaluated for the amount of glucose oxidase enzyme activity using a
method modified from Gallo (1981) by Wu et al. (1995). Enzyme activity in this assay is
indicated by the production of a rose colored pigment. Proteins were extracted from 200
mg fresh leaf tissue by grinding in a potassium phosphate buffer (25 mM, pH 7.0, 5 mM
EDTA) at 4°C. A dilution series was made from each sample by mixing 10, 20, 30 or 40
pl of sample with 990, 980, 970 or 960 pl reagent mixture respectively (70 mM KH2P04
buffer, pH 5.8, 0.57 mM 4-aminoantipyrine, 0.35 mL/L Triton X-100, 10 mM crystalline
phenol, 23 units/mL horseradish peroxidase and 175 mM glucose) for a total reaction
volume of 1.0 mL. Known concentrations of purified glucose oxidase were also mixed
with the reagent mixture in order to create a standard curve. Reaction mixtures were
incubated at 22°C for at least 10 minutes but not longer than 90 minutes as the color
begins to fade. Following the incubation period, the absorbance of each sample was
measured at 510 nm. Using the standard curve, the amount of enzyme (mg enzyme/mg
of fresh tissue) was determined for each dilution of a sample. One mg of the glucose
oxidase enzyme that was used to create the standard curve has 15 to 25 Units (U) of
activity/ mg enzyme without added oxygen (Sigma-Aldrich Co.). Therefore, we used the
average value, 20 U/mg enzyme, to convert mg enzyme/mg fresh tissue into units of
enzyme activity. Because the resulting numbers were small, all values were multiplied
by 105 in order to simplify analysis. The value of the non-transgenic control was then

subtracted from each of the transgenic lines. Using dilutions as replications, ANOVA

36

was done using SAS proc glm (SAS Institute) and lines were compared to non-transgenic

controls with both Dunnett's T test and Tukey's Studentized Range Test (SAS Institute).

Late blight screening of transgenic lines

F oliar resistance

Foliar resistance to late blight was tested both in greenhouse mist chambers and in
isolation plots at the MSU Muck Soils Research Farm (Bath, MI). Transgenic potato
plants were subcultured in 20 mL of MS medium (Douches et al, 1998) and then planted
in the greenhouse 10 cm pots containing Bacto potting medium (Michigan Peat Co.,
Houston, TX). Prior to flowering, plants were placed on trays in the mist chamber in a
randomized complete block design (RCBD) with four replications and were inoculated
with zoospore suspension cultures of P. infestans (US8) as outlined by Douches et al.
(1997). Plants were rated over time for percent area infected. Percentage data were
normalized by transformation using the arcsine function. Analysis of variance (ANOVA)
was conducted using SAS proc glm and transgenic lines were compared to the non-
transgenic control cultivar from which they were derived using both Dunnett's T Test and
Tukey's Studentized Range Test (SAS Institute). Some plantlets did not survive the
transfer from tissue culture to the greenhouse and therefore not all lines tested were
included in all four replications. Therefore, least-squares means (LSmeans) were used
for comparisons.

Transplants from tissue culture were also used for field studies with the exception
of SGO plants in the 1999 field-season which were grown from tubers produced at the

Montcalm Research Farm the previous year (1998). Plants were grown in a RCBD with

37

four replications and five plants per plot with two feet between plots to facilitate
evaluation. Forty-two days (1998) and 55 days (1999) after planting, plants were
inoculated with zoospore suspension cultures of P. infestans via a sprinkler irrigation
system. Moisture was maintained on the foliage via frequent misting irrigation. Plots
were evaluated over time for percent area infected. These data were used to calculate
area under the disease progress curve (AUDPC) (Shaner and Finney, 1977). The
AUDPC was converted to relative area under the disease progress curve (RAUDPC) by
dividing the calculated AUDPC by the maximum area under the disease progress curve.

Statistical analyses and comparisons were conducted as noted above for greenhouse data.

Tuber resistance

Tuber late blight response was evaluated in each of the transgenic lines. Ten
field-grown tubers of each line were removed from cold storage (3 months at 56°C) and
brought to room temperature for 24 hrs. The tubers were surface sterilized by immersion
in a 10% Clorox solution for 10 minutes and then rinsed 3X with distilled water. In
1998, tubers were injected with homogenized mycelia] cultures after surface sterilization
and then incubated at 12°C and 95% relative humidity for 48 days. Tubers were visually
rated using a 1-9 scale (Niemira et al., 1999) and then tuber slices (apical, middle and
terminal slices) were scanned and digitally analyzed for reflective intensity (Niemira et
al., 1999). In 1999, tubers were prepared for inoculation as in 1998. However, instead
of injecting the inoculum, ten tubers of each line were scuffed with a wire brush to
simulate harvest damage and were submerged in a zoospore suspension culture (1 x 105

sporangia/ml) for 3 minutes (Platt and Tai, 1998). All tubers were incubated as in 1998

38

and evaluated using the 1-9 scale. Numerical data were analyzed using SAS proc glm
and transgenic lines were compared to their respective non-transgenic controls using both
Dunnett's T Test and Tukey's Studentized Range Tests (SAS Institute). Ordinal data
from visual ratings were analyzed by ordinal analysis (Karcher and Schabenberger, 2000;

Karcher, 2000). Photographic images in this dissertation are presented in color.

39

RESULTS

Transformgation and Molecular Analysis

Transformation of ‘Spunta’ and ‘Atlantic’ was successfiil resulting in 12
transgenic lines from ‘Spunta’ and 14 transgenic lines from ‘Atlantic’ (Table 2.1 and
Table 2.2). However, transformation of the cultivars ‘Libertas’ (7 transgenic lines)
(Table 2.3), ‘Zarevo’ (1 transgenic line) and ‘Snowden’ (2 transgenic lines) was more
difficult resulting in fewer transformed shoots. Regeneration of transgenic shoots from
‘Spunta’ and ‘Atlantic’ occurred within 3 to 5 weeks following co-cultivation and there
were multiple shoots per explant. Very few shoots were recovered from the other
transformed cultivars and regeneration occurred 8 to 12 weeks following co-cultivation
(data not shown). Both ‘Snowden’ and ‘Zarevo’ explants quickly developed a dry,
brown border along the cut edge of the leaf and were very sensitive to any mechanical
damage during the transformation process. In addition, ‘Zarevo’ explants would begin to
brown soon after transfer to selection media and would often die before regeneration
occurred

The copy number of the transgenic lines recovered in this study ranged from 1 to
as many as 7 copies in AGO-8 (Table 2.1; Table 2.2; Table 2.3; Figure 2.3). Gene
silencing often results from having multiple copies of the same gene (Napoli et al., 1990;
Van der Krol et al., 1990) and therefore transgenic lines with single gene insertions
are preferred. However, in this study there is no correlation, positive or negative,
between c0py number and gene silencing. For example, AGO-2 has only 1 copy of the
glucose oxidase transgene but northern analysis (Figure 2.4) revealed a lack of transgene

transcription whereas, AGO-8 contains 7 transgene copies and produces both the

40

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Figure 2.3. Southern blot of Barn
glucose oxidase gene (A) and XbaI digested DNA to determine transgene copy

number (B).

A

 

 

 

 

 

888
<<<

Atlantic

Figure 2.4. Northern blot of transgenic potato lines containing the
glucose oxidase gene hybridized with the glucose oxidase probe (A) and

a potato 18S ribosomal probe (B).

transgene transcript and the glucose oxidase protein (Table 2.2; Figure 2.5). There are
other lines (SGO-1, LGO-l, AGO-5) with only one copy which produce relatively high
levels of the transgene transcript as well as the glucose oxidase protein. In addition,
relative level of transgene mRNA did not always reflect the amount of enzyme activity
detected. Lines with relatively high transgene mRNA had both high (LGO-1, LGO-4)
and low (LGO-10, SGO-2) glucose oxidase enzyme activity (Table 2.1; Table 2.3).

In this study there is evidence suggesting both transcriptional and post-
transcriptional gene silencing. Lines in which the transgene is present but the transgene
transcript is absent suggest transcriptional gene silencing whereas lines in which both the
transgene and its transcript are present but the glucose oxidase protein is lacking suggest
post transcriptional gene silencing (Table 2.1; Table 2.2; Table 2.3).

Of all the transgenic lines recovered, there were 9 AGO lines, 5 LGO lines, 6
SGO lines and one SNDGO line that produced the glucose oxidase protein (Table 2.1;
Table 2.2; Table 2.3). The only ZGO line recovered did not express the glucose oxidase
protein. Further analyses were limited to AGO, LGO and SGO lines in which western
analysis revealed the presence of glucose oxidase protein (approximately 80 kDa).

Because elevated levels of H202 can have detrimental effects on plant cells, plant

growth habit as well as plant and tuber morphology were closely monitored for each of the
transgenic lines (Table 2.1; Table 2.2; Table 2.3; Figure 2.6). Lines SGO-3 and SGO-9
exhibited delayed shoot emergence in the trial at the Montcalm Research Farm.
Although tubers of SGO-3 were noted to be small with numerous knobs, tubers of SGO-9
were similar in shape and size to untransformed ‘Spunta’. Line SGO-4 did not express

the glucose oxidase protein, even so, it was stunted in growth and produced small,

45

 

 

 

 

 

97.4 kDa —> —

68kDa —> —

at YOG<
3 2-03
3 3-03
cm

3 2-03
5 2-03
3 «-03
E 703
8885

2355 Emma; 330202

Figure 2.5. Western blot showing leaf protein hybridized to glucose oxidase polyclonal

anubody.

46

 

Figure 2.6. Photograph showing the off-type tuber morphology of
several transgenic lines containing the glucose oxidase gene.

47

knobby tubers. None of the AGO lines differed from the untransformed ‘Atlantic’ in
growth habit or plant morphology but many had slightly elongated tubers. The exception
was AGO-2 which was off-type with very knobby and elongated tubers despite the lack
of glucose oxidase protein. Among the LGO lines, LGO-l, 3, 4 and 16 were stunted in
growth compared to the untransformed ‘Libertas’ whereas plants of LGO-10 were the
same as the control. Because ‘Libertas’ is a late maturing variety and tissue culture
transplants were used, the tubers from the LGO lines and the ‘Libertas’ control were
small. However, tubers of LGO-1, 4 and 16 were also knobby and elongated. Glucose
oxidase enzyme activity in lines LGO-l and 4 was high compared to other transgenic

plants but LGO-16 and LGO-3 had much lower enzyme activity (Table 2.3).

Foliar Late Blight Screening

The ‘Spunta’ derived SGO lines were tested in the greenhouse in three
consecutive trials (Table 2.1). In the first trial lines were a significant source of variation
only at 14 days post inoculation (P < 0.0075) and lines SGO-l, 2, 9 and 12 were less
infected than the 'Spunta' control based on LSmeans comparisons of percent infection.
Lines were not a significant source of variation at any evaluation point in the second or
third trials. Using data combined over trials one and three, lines were a significant
source of variation at 14 DPI (p<0.0215) and lines SGO-l, 2, 9, 12 and 14 were
significantly less infected than the 'Spunta‘ control based on Dunnett's T tests (Table 2.1).
Trial two could not be combined with either trial one or trial three due to non-

homogenous variation.

48

The ‘Atlantic’ derived AGO lines were tested twice in the greenhouse for
response to P. infestans (Table 2.2). In the first trial, lines were not significantly different
at 7 DPI but were significant different at 10 and 14 DPI (P < 0.0183 and P < 0.046
respectively). Line AGO-6 had a higher infection level than the ‘Atlantic’ control (P <
.0022) at 10 DPI and lines AGO-5 and 6 more infected than the control (P < 0.0224 and
P < 0.0166 respectively) at 14 DPI based on LS means comparisons. In the second trial,
lines were not significant at any point following inoculation. In the first greenhouse trial
of LGO lines, lines were a significant source of variation at 7 (P < 0.0112), 10 (P <
0.0001) and 14 (P < 0.0389) DPI. Transgenic line LGO-16 had more infection than the
‘Libertas’ control at 7 DPI (P < 0.006), LGO-3, 10 and 16 were more infected than the
control at 10 DPI and LGO-16 was the only line more infected than the control at 14 DPI.
In the second trial, lines were not a significant source of variation at any point after
inoculation (Table 2.3).

Field evaluations of response to P. infestans were done two years for SGO lines
and one year for all other transgenic lines. In 1998, lines were a significant source of
variation (P < 0.0117). However, based on both Tukey’s Studentized Range Test and
Dunnett’s T test none of the SGO lines were significantly different (P < 0.05) from the
‘Spunta’ control. Transgenic lines SGO-1, 12 and 14, which were significantly less
infected than the control in one of the greenhouse trials, did have a lower RAUDPC
value than the control, but the difference was not significant (Table 2.1). In 1999, lines
were not significantly different for the SGO ANOVA, however, when data were

combined over years, lines were significant. In the combined analysis, lines SGO-3 and

49

SGO-9 were significantly more infected than the 'Spunta' control (Table 2.4). This is in
contrast to the greenhouse trial in which SGO-9 ranked lower than 'Spunta' for percent
area infected. Lines were not significantly different in the AGO field trial (P < 0.2694)

and the LGO field trial (P < 0.0568) (Table 2.2 and Table 2.3).

Tuber Late Blight Evaluation

Tuber late blight evaluations were done in both 1998 and 1999 for SGO
transgenic lines and in 1999 for AGO and LGO transgenic lines (Table 2.4). In 1998
lines were a significant source of variation (P < 0.007) although none of the SGO
transgenic lines were different from the 'Spunta‘ control based on ordinal analysis of
visual ratings. Lines were significant in the digital analysis of tuber flesh for the apical
(P < 0.0096), middle (P < 0.0089) and terminal slices (P < 0.0147) as well as tuber
average (P < 0.0086). However, none of the transgenic lines were significantly different
from the 'Spunta' control. In the 1999 ordinal analysis, lines were not a significant source
of variation for either the SGO lines or the LGO lines but were significant for AGO lines
(P < 0.05). Lines AGO-6 and 8 were significantly less infected than the 'Atlantic'
control. AGO-6 was among the most susceptible lines in both greenhouse and field
evaluations of foliar response to late blight whereas AGO-8 was among the best lines in
all evaluations. However, based on limited infection of control tubers ('Spunta', 'Atlantic'
and 'Libertas') in 1999, the dipping method of inoculation did not provide adequate

infection for determination of tuber late blight resistance.

50

Table 2.4. Tuber late blight evaluations for 1998 and 1999.

 

1998 tuber late blight evaluations

 

 

 

 

 

Mean
visual
Line rating Apical Middle Terminal

SGO-14 1.7 163.1 154.8 162.8
SGO-9 3.5 146.4 142.3 149.2
SGO-2 2.8 139.0 131.6 135.0
SGO-3 2.7 127.1 123.9 137.8
Spunta 3.5 118.6 124.7 137.0
SGO-1 5.1 114.6 111.3 110.3
SGO-12 5.0 111.3 115.9 131.3
CV ----- 38.0 36.6 34.1

 

 

 

1999 tuber late

blight evaluations

Mean

visual

Line rating
SGO-l l .0
SGO-12 1 .4
SGO-3 1 .6
SGO-14 1 .7
SGO-2 2.2
Spunta 2.2
SGO-1 2.7
LGO-4 l .0
LGO-10 1 .2
Libertas l .4
LGO-l 1 .4
LGO-15 1.4
LGO-3 1 .5
LGO-16 1.5
AGO-7 l .3
Atlantic 1 .3
AGO-12 l .4
AGO-1 1 1 .5
AGO-5 l .8

AGO-6 22*

AGO-8 2.2*

 

*Significantly different from control based on Dunnett's T test (p<0.05)

51

Enzyme activity assay

Lines differed in the ANOVA for enzyme activity among AGO, LGO and SGO
transgenics (P < 0 0001). Based on Dunnet's T tests, all of the AGO lines except AGO-8
had higher glucose oxidase enzyme activity than the 'Atlantic' control (Table 2.2).
Among LGO and SGO lines, only LGO-l, 3 and 4 and SGO-1, 3 and 12 had higher

enzyme activity than their respective non-transgenic controls (Table 2.1 and Table 2.3).

52

DISCUSSION

The development of late blight resistant cultivars is an important objective in
potato breeding programs. However, since the late blight pathogen readily overcomes R-
gene based resistance, whether as single genes or as a pyramid of R genes (Ross, 1986), it
is important to identify more complex mechanisms of resistance. Therefore, this study
was undertaken to evaluate the effect of a fungal glucose oxidase gene in a late blight
susceptible potato cultivar and in a cultivar possessing quantitative genetic resistance to
blight.

The production of transgenic potato lines containing the glucose oxidase gene was
readily achieved for the susceptible cultivars 'Atlantic' and 'Spunta'. However, lines with
moderate resistance to late blight, 'Libertas' and 'Zarevo, were recalcitrant to
transformation resulting in few transgenic lines. In addition, the transformed lines were
recovered only after a lengthy time in in vitro culture. The difference between the
cultivars in recovery of transgenic lines may be due to genetic variation for regeneration
and transformation efficiency. Several authors have noted such a difference and have
developed protocols for efficient regeneration of many cultivars (DeBlock, 1988; Hulme
et al., 1992; ; Wenzler et al., 1989; Yadav and Sticklen, 1995). Another possible
explanation may be an increased sensitivity to kanamycin. Breeding line MSG274-3
from the Michigan State University Potato Breeding Program, responded similarly to
‘Zarevo’ upon transformation and no regenerates had been recovered from this line using
kanamycin selection (50 mg/L). However, transgenic regenerates from MSG274-3 were

obtained if the explants were cultured on a reduced concentration of kanamycin (25

53

mg/L) (Douches unpublished data). A more formal study is needed to support this
observation.

Although several of the plants recovered fi‘om the transformation process had the
glucose oxidase transgene, many of them either did not transcribe the inserted gene, as
indicated by Northern analysis, or did not translate the gene, as indicated by Westem
analysis. This phenomenon, known as gene silencing, results in the lack of expression of
a transgene and/or any genes with similar homology (endogenous or exogenous). Gene
silencing occurs either by prevention of transcription, transcriptional gene silencing
(TGS) or by alteration and/or destruction of the gene transcript, post-transcriptional gene
silencing (PTGS). TGS has been attributed to interactions between homologous
promoters present at different genomic locations (trans silencing), interactions between
different parts of a complex transgene locus, chromosome position into which a transgene
inserts and the sequences surrounding the insertion site (Taylor, 1997). PTGS often
occurs when transgene transcripts are abundant and it is generally thought to be triggered
at the level of mRNA processing, localization, and/or degradation (Taylor, 1997).
Several models have been proposed to explain this phenomenon. The “threshold model”
suggests that when a specific transcript reaches a threshold level it and any homologous
transcripts are targeted and degraded by the cell (Dougherty and Parks, 1995). Another
model suggests that methylated transgenes produce aberrant RNA molecules that are
selectively degraded along with related transcripts (Baulcombe and English, 1996). A
third model suggests that base pairing of transcripts triggers the targeted degradation of

homologous transcripts (Cameron and Jennings, 1991). Although the method of gene

54

silencing in our study has not been determined, it appears that both TGS and PTGS have
occurred.

Although many of the glucose oxidase—containing lines exhibited altered plant
growth habit and/or tuber phenotype, these changes cannot be attributed solely to the
effects of H202 because some of these lines did not produce the glucose oxidase protein
and would therefore not have elevated levels of H202. For example, tubers of the AGO-2
lines were highly off-type, but neither the glucose oxidase transcript nor the protein was
detected in this line (Table 2.1). Because morphological changes in either the plant or
the tuber are not clearly associated with transgene expression, it is possible that these
physical changes are due to the position of the transgene insertion and/or somaclonal
variation that occurred during the transformation process. Somaclonal variation is
defined as an increase of genetic variability in higher plants which takes place during in
vitro culture. Several factors can influence the probability that somaclonal variation will
occur including the method of vegetative propagation, type of tissue used, media
components, and genotype of the plant (Pierik, 1987). In potato, certain genotypes have a
greater tendency for somaclonal variation. Shepard et al. (1980) demonstrated that the
cultivar ‘Russet Burbank’ was very unstable when plants originated from protoplasts.
Similar results were found for the cultivar ‘Bintje’ (Ramula et al., 1984). Regardless of
the basis for the altered plant and/or tuber morphology, it is evident that an unusually
large percentage of the transgenic plants recovered in this study (44%) were considered
off-type. Transformation of the same cultivars used herein with other genes resulted in
very few off-type plants despite using identical transformation protocols and

transformation vectors (Douches, unpublished data). Therefore, if the glucose oxidase

55

gene is to be used in the development of potato cultivars, it may be necessary to produce
and screen a larger population of plants than is required with other transgenes.

In previous research, the glucose oxidase gene was shown to reduce the growth of
late blight lesions by 49% to 56% compared to controls in detached leaf assays (Wu et
al., 1995). In our study, greenhouse trials may indicate a reduction in late blight infection
among SGO and LGO lines but large experimental error (CV's from 20.1 to 71.5%)
limited our ablity to identify of differences between the control and transgenic lines.
However, field trials indicate little if any effect of the glucose oxidase gene in any of the
transgenic lines upon infection by the late blight pathogen P. infestans. It is not unusual
for a potato line to have resistance in a greenhouse evaluation and exhibit less resistance
in the field. A good example is the cultivar 'Zarevo' which is highly resistant in
greenhouse studies, but is only moderately resistant in the field (Douches et al., 2000). In
the case of 'Zarevo' a possible explanation for the difference between field and
greenhouse late blight evaluations is the behavior of P. infestans zoospores. Niemira et
al., (1998) demonstrated that on 'Zarevo', zoospores target stomata for penetration,
whereas, on susceptible cultivars the zoospores penetrate directly through the leaf
surface. Therefore, in 'Zarevo' the onset of infection may be delayed resulting in delayed
growth and sporulation of the pathogen. In a short (one to two-week) greenhouse
evaluation this may be detected as a high level of resistance. However, in a field trial,
which is longer in duration, the amount of disease in 'Zarevo' will eventually reach levels
at which the cultivar will be categorized as only moderately resistant.

There are several possible explanations for the disparity between the current

research and that done by Wu et al., (1995). Wu et al, (1995) measured the spread of

56

individual lesions in detached leaf assays and the area infected on four center leaves in
whole plant analyses allong them to limit error due to subjective measurements and
identify small differences. In our study whole plants were evaluated for area infected in
both the field and in the greenhouse. The subjectivity in this type of evaluation can lead
to high experimental error and limit the ability to identify small differences. In an attempt
to limit the subjectivity of these measurements all analyses were conducted by two or
three member teams. Another difference between the two studies is the severity of
environmental conditions in which whole plant analyses were conducted. Wu et al.,
(1995) maintained high humidity in the experimental chamber for only the first 48 hours
whereas we maintained high humidity for the entire experiment. Because high humidity
favors late blight development our experimental conditions must be considered more
severe and may have prohibited the measurement of small differences in disease severity.
Another source of difference may be in the biotype of P. infestans used. In this study,
several highly pathogenic isolates of the US-8 biotype of P. infestans were used. These
isolates had been recovered fi'om infected potato tissue discovered in various Michigan
farm locations and would therefore be similar to what potatoes in the field would
encounter. Wu et al., (1995) do not report the source or biotype of P. infestans used in
their research. Lastly, both studies used different glucose oxidase gene constructs for
transformation. Our construct used the Gelvin super promoter and utilized the
kanamycin resistance gene, NptII, as the selectable marker. The construct used by Wu et
al., (1995) used the constitutive figwort mosaic virus 35S promoter and a glyphosate
resistance gene as the selectable marker. The DNA sequence of the glucose oxidase gene

in both constructs should be the same as both were isolated from Aspergillus niger using

57

primers designed according to the published gene sequence (Frederick et al., 1990).
However, direct comparisons are not possible as the sequence of the construct used by
Wu et a1 (1995) was not published.

Based on this study, the glucose oxidase gene, even in combination with moderate
host plant resistance, will not provide complete protection against P. infestans but may
slightly reduce susceptibility. Although the ultimate goal is to produce potato cultivars
with complete resistance to late blight, cultivars with reduced susceptibility can lessen the
fiequency and quantity (active ingredient) of fungicide applied (Kirk et al., 1999).
Therefore, the glucose oxidase gene may be useful for slightly increasing the late blight

resistance of otherwise desirable potato cultivars.

58

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Chapter III
RESPONSE OF TRANSGENIC “POTATOES (SOLAN UM TUBEROSUM L.)
CONTAINING THE FUNGAL GLUCOSE OXIDASE GENE TO F USARI UM
SAMBUCINUM AND ER WINIA CAROTOVORA SSP. CAROTOVORA
ABSTRACT

Production of high quality, disease free potatoes is challenging due to the large
number of pathogens that can attack the growing plant and/or the stored tubers. Two
pathogens that are particularly troublesome during storage are F usarium sambucinum, a
fungus that causes dry rot of potato tubers, and Erwim'a carotovora ssp. carotovora, a
bacterium that causes sofi rot of potato tubers. Currently, there are no commercial potato
cultivars that are highly resistant to either disease and control measures are
predominantly cultural. Transgenic potatoes containing a fungal glucose oxidase gene
have been shown to have increased resistance to E. carotovora and several other
pathogens Therefore, three potato cultivars ('Atlantic', 'Libertas' and 'Spunta') were
transformed with the glucose oxidase gene and evaluated for their response to both E.
carotovora ssp. carotovora and F. sambucinum.

Greenhouse and field grown tubers of transgenic lines were used to assess
response to inoculation with both pathogens. Whole tubers were wounded and then
inoculated with agar plugs of growing F. sambucinum cultures or tuber slices were
inoculated with a suspension solution of E. carotovora ssp. carotovora (108 CFU'ml'l).
Disease progression was determined by measuring the diameter and depth of the lesion
for Fusarium dry rot (14 to 28 days post inoculation based on tuber size) and by

measuring lesion diameter 48 hours post inoculation for Erwinia sofi rot.

65

The glucose oxidase gene had a no effect on the resistance of the cultivars to
F usarium dry rot but had a small effect against Erwinia soft rot. However, differences
between lines were difficult to detect due to high error variations in many of the
evaluations. Lines SGO-1 and 2 were the only lines significantly different from the
non-transgenic control for response to Fusarium dry rot and lines SGO-9, 12, 1 and
LGO-3 were the only lines significantly different from the non-transgenic controls for
response to Erwinia soft rot. There does not appear to be any correlation between
resistance to F usarium and resistance to Erwinia among the transgenic lines. In addition,
many of the lines had abnormal tuber phenotypes and/or abnormal plant morphology.

The glucose oxidase gene may be usefirl to develop potato cultivars with
increased resistance to Erwinia soft rot. Future research will include evaluation of l) the
resistance of the transgenic lines to Erwinia soft rot under commercial storage conditions,
2) the resistance of the transgenic lines to Erwinia infection of the plant during the
growing season, and 3) the possibility of producing lines with increased resistance and

normal morphology.

66

INTRODUCTION

The potato is one of the most important crops worldwide ranking fourth in yield
with worldwide production of at 194,833,731 metric tons of which the United States
produces 21,725,182 metric tons (National Potato Council, 1999) many of which are held
in storage until marketed. Producing high quality, disease-flee potatoes is expensive and
time consuming due to the large number of pathogens and insect pests to which the
potato is susceptible. Hide and Lapwood (1992) estimated that there are over 100
pathogens that may infect the potato. Although many of these pathogens attack the
growing crop, there are several important diseases that destroy stored potato tubers. Two
of the most important of these are dry rot caused by the fungus F usarium sp. and soft rot
caused by the bacteria Erwinia carotovora.

Potato dry rot is often difficult to detect as tubers rot from the inside producing
discolored cavities (Figure 3.1). However, infected tubers may exhibit wrinkled, sunken
patches on the surface and fungal mycelia may be visible on the tuber surface as the
disease progresses (Rich, 1983). The causal agent of this disease may be one of several
F usarium species including, but not limited to, F. sambucinum and F. solam', (Rich,
1983). This pathogen relies on wounding during harvest or seed cutting to infect.
Therefore, careful handling of tubers during harvest and storage can reduce the
occurrence of dry rot in storage. Other cultural practices that can limit the incidence of
dry rot are harvesting at warmer temperatures (above 45°F) to avoid bruising,
bruise/wound healing prior to storage, and long-tenn storage at low temperatures as
Fusarium can develop at 40°F and above (Lacy and Hammerschmidt, 1993). The

fungicide thiabendazole has been widely used by growers since the 1970's to control

67

 

Figure 3.1. Photographs of tubers inoculated with F usarium sambucinum
showing internal (A) and external (B) symptoms of infection.

68

potato dry rot however, several F usarium isolates have demonstrated resistance to this
chemical (Hanson et al, 1996). In their study, Desjardin et al., (1993) found that all
Fusarium isolates collected between 1963 and 1986 were sensitive to thiabendazole,
whereas the majority of isolates collected in 1990 and 1991 were resistant. Hanson et al.,
(1996) found that 42% of the isolates collected in the northeastern United States were
resistant to thiabendazole. Currently, there are no commercial cultivars that are resistant
to F usarium dry rot thus, it is necessary to find alternative control measures for Fusarium
dry rot and to develop resistant potato cultivars.

Testing for response to F usarium dry rot is done either by wounding whole tubers
and inoculating them with the pathogen (Vaughn and Spencer, 1994), or by directly
inoculating freshly cut tuber surfaces with the fungus (Hanson et al., 1996). Disease
response is evaluated based on the presence and size of the lesion at the site of
inoculation.

Unlike dry rot, potato soft rot is readily detectable due to the offensive odor
produced through secondary infection by other bacteria such as Pseudomonas sp. E.
carotovora, the causal agent of this disease penetrates the tuber via natural openings and
wounds and destroys the tuber tissue through the activity of pectolytic enzymes (Bartz
and Eckert, 1987). Bacterial sofi rot is favored by warm temperatures (25° to 37°C), high
moisture levels in the soil or during storage, wounding (Cromerty and Easton, 1973) and
anaerobic conditions (DeBoer and Kelman, 1978). A number of chemicals, including
chlorine-based chemicals, antibiotics and organic and synthetic bacteriocides have been
tested for their ability to control soft rot in potato and other vegetables (Bartz and Eckert,

1987). However, public concern about the usage of toxic chemical on food has

69

encouraged the use of less controversial control measures to manage storage diseases
(Wilson and Wisniewski, 1992). Therefore, control of soft rot in potatoes is
predominantly cultural including precautions such as disinfecting storage bins and
harvest equipment, avoiding excessive irrigation, careful handling during harvest and
storage to avoid bruising and wounding, storage in cool dry conditions and air circulation
to prevent depletion of oxygen (Rich, 1983). Currently, there are no commercial
cultivars that are resistant to Erwinia soft rot.

Variations of two inoculation procedures have been used to evaluate potato tuber
resistance to soft rot: point titration and shoe inoculation. Point titration involves the
injection of the pathogen directly into intact tubers at a specified depth (Lojkowska and
Kelman, 1994) whereas, in the slice method bacterial suspensions on filter paper disks
are used to inoculate tuber slices (Lapwood, et al., 1984). The relative resistance of the
potato tuber is measured as the diameter of the macerated tissue for both inoculation
procedures. Several authors have compared the results of both inoculation methods.
Lojkowska and Kelrnan (1994) questioned the reliability of the tuber slice method due to
the high level of variability that is often observed. However, Koppel (1993) suggested
that the two methods measured different aspects of resistance. Point titration measured
the resistance to progressive rot under anaerobic conditions, whereas, the slice method
measured the reaction of the tuber to bacterial challenge at a wound site and the speed
that wound healing occurred. Haynes et al. (1997) concluded that the slice test was valid
provided a sufficient sample size was used and the appropriate parameters were

measured.

70

In addition to inoculation method the Erwinia pathogen used for resistance
screening is important. Erwim'a carotovora ssp. carotovora (Ecc) and Erwinia
carotovora ssp. atroseptica (Eca) are the predominant soft rot species in cooler potato
growing areas (DeBoer and Kelman, 1978) Whereas, Erwinia chrysanthenmi (Ech)
prevails in the warmer growing areas (Hidalgo and Echandi, 1983). Several authors
(Lapwood et al., 1984; Austin et al, 1988; Wolters and Collins, 1994) have reported
similar rankings of potato cultivars when inoculating with either Ecc, Eca or Ech.
Therefore, the choice of Erwim'a species used can be decided based upon the growing
area for which variety development is targeted.

Several alternative treatments have been suggested to control both F usarium dry
rot and Erwim’a soft rot in potatoes. Shashirekha and Narashimham, (1990) found that
potatoes dipped in solutions containing trace elements such as iron, copper and zinc prior
to storage significantly reduced the incidence of both dry and soft rots. Vaughn and
Spencer (1994) tested three sprout inhibitors (salicylaldehyde, cinnamaldehyde and
thymol) and found that they inhibited dry rot development if applied prior to penetration
of the fungus into the tuber tissue. Biological control of both diseases was effective
under experimental conditions (Wilson and Wisniewski, 1992; Bartz and Eckert, 1987),
and ultraviolet irradiation eliminated dry rot and delayed soft rot in potatoes stored at
37°C without adversely affecting tuber quality (Ranganna et al, 1997). However, to date
none of these control measures have found application at the commercial level.

As mentioned previously, no commercial potato cultivars have resistance to
F usarium dry rot and/or Erwinia soft rot. Various wild potato species have been reported

as having some level of resistance to these diseases (Carputo et al, 1996; Bamberg, et al.,

71

1994) and some somatic hybrids have been reported to possess resistance to Erwim'a soft
rot (Helgeson et al., 1993). In addition, Wu et al., (1995) reported nearly complete
resistance to E. carotovora ssp. carotovora in transgenic ‘Russet Burbank’ tubers
containing a fungal glucose oxidase gene. As the name suggests, this gene encodes a
protein that oxidizes glucose and produces hydrogen peroxide (H202) as a byproduct of
the reaction. Hydrogen peroxide has been reported to function at several points in the
plant disease response including: direct antimicrobial effect (Wu et al., 1995), structural
changes in the plant cell wall (Dean and Kuc, 1987; Brisson et al., 1994), production of
toxic, lipid free radicals (Keppler and Baker, 1989), activation of phytoalexin synthesis
(Apostol et a1, 1989; Chai and Doke, 1987) and induction of systemic acquired resistance
(Chen et al., 1993; Conrath et al., 1995). The glucose oxidase transgene has been
reported to be effective against a broad range of pathogens (Phytophthora infestans,
Alternaria solani, Verticillium dahliae, and E. carotovora) (Wu et al., 1995; Wu et
al.,1997). Therefore, transgenic glucose oxidase-containing potato plants, which were
developed to enhance foliar late blight (P. infestans) resistance (Felcher et al., 2000),

were also tested for their response to F usarium dry rot and Erwim'a soft rot.

72

MATERIALS AND METHODS

Cloning of the glucose oxidase gene

The glucose oxidase gene was cloned from Aspergillus niger using the
polymerase chain reaction (PCR) and primers designed according to Frederick et al.
(1990) to which SmaI and XbaI restriction sites had been added. The resulting DNA
fragment was sequenced, compared to the published glucose oxidase sequence (Frederick
et al., 1990) and was incorporated into the pE1102 vector. The resulting plasmid, named
pSPUDll (Figure 3.2), had the glucose oxidase gene under the control of the Gelvin
"super promoter" (Ni et al., 1995). This promoter is a combination of a trimer octopine
synthase upstream activating sequence (ocs)3 joined to a mannopine synthase (mas)
activator and promoter. The pSPUDll vector also contained the Nos terminator
sequence (Nos T), right and left borders for plant transformation purposes and the NptII
gene (kanamycin resistance) to be used as a selectable marker. The pSPUDll plasmid
was transformed into Agrobacterium tumefaciens strain LBA4404 (Ooms et al., 1982)

via tri-parental mating.

Transformation of potato cultivars

The cultivars 'Spunta', 'Atlantic', and 'Libertas' were micropropagated in GA-7
Magenta vessels each containing 25 ml of modified MS basal medium (Douches et al.,
1998). Transformations of potato cultivars were done according to Li et al., (1999) using
the A. tumefaciens culture harboring the pSPUDll vector. Regenerating shoots (> 5 mm)
were excised and transferred individually into 25 x 100 mm glass tubes each containing

20 mL of MS medium (Douches et al., 1998) supplemented with 200 mg'L'l Timentin

73

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(SmithKiine Beecham, Philadelphia, Pa.) and 50 mg‘L’l kanamycin. Rooted plantlets
were designated as SGO-# (derived from 'Spunta'), AGO-# (derived from 'Atlantic'), and
LGO-# (derived from 'Libertas'). Named plantlets were subcultured and subsequently
transplanted into 10 cm pots containing Bacto potting medium (Michigan Peat Co.
Houston, Texas) and grown in the greenhouse under fluorescent lights (16 h photoperiod)
at 23 to 27°C. Leaf tissue fiom these plants was used for the molecular expression
assays. All lines were characterized for transgene copy number, transgene transcription

and translation, glucose oxidase enzyme activity and plant morphology (Felcher et al.,

2000).

F usarium m rot screening of transgenic lines

Transgenic lines were tested for response to F usarium sambucinum (F usarium
dry rot) using both greenhouse tubers (1998) and field grown tubers (1999). F.
sambucinum cultures (isolate RNl), kindly provided by Dr. Hammerschmidt, were
started by subculturing agar plugs of older cultures onto freshly prepared potato dextrose
agar (PDA) medium. New cultures were incubated at 23°C in the dark under ambient
conditions for four days prior to use. Small sections of the tuber surface were peeled
back with a razor blade and the exposed tuber tissue was inoculated with an agar plug
fiom the growing edge of the F. sambucinum mycelia] culture. Tubers were labeled,
placed in trays and incubated in the dark at 25-26°C for 14 days in 1998 due to small
tuber size, and 28 days in 1999 with the exception of 'Libertas' and LGO lines (14 days)
which were small in size. Following incubation, tubers were sliced perpendicularly

through the inoculation site and lesion depth and diameter were measured (mm). Data

75

were analyzed using SAS proc glm and transgenic lines were compared to the respective
non-transgenic controls using least squares means (LSmeans) comparisons for
unbalanced data (1998) and Dunnett’s T Test and Tukey’s Studentized Range Test for

balanced data in 1999 (SAS Institute, Inc).

Erwinia sofi rot screening of transgenic lines

Cultures of Erwinia carotovora ssp. carotovora (Ecc) were provided by Dr.
Hammerschmidt and were maintained as glycerol stocks in a -80°C freezer. Prior to use,
a sample was taken from the stock, streaked onto solid PDA medium and incubated
overnight at 25-26°C. A single colony was selected from the overnight culture and used
to inoculate liquid LB media. The liquid culture was incubated overnight on a shaker
(100 rpm) and then diluted to (108 CFU‘ml") based on spectrophotometer readings.

Ten field-grown tubers of each transgenic line were tested for Erwim'a sofl rot
resistance. Tubers were removed from cold storage, washed and warmed to room
temperature for 24 h prior to inoculation. A slice was taken from the middle of each
tuber, placed in a Petri dish, and inoculated with a filter paper disk soaked in the Eco
suspension culture. In 1998, Petri dishes (15 x 100 mm) containing inoculated tuber
slices were closed and placed in sealed plastic sleeves to maintain a near-anaerobic
environment and maintain moisture levels. Lesion diameter (mm) was measured at 24
and 48 hrs. and lesion depth (m) was measured at 48 hrs by cutting the tuber slice
perpendicular to the previously cut surface. Due to variable data in 1998, the evaluation
was modified in 1999. Tuber slices were placed in Petri dishes (25 x 100 mm) in which a

thin layer of solid water agar had been poured. The dishes were stored and the tuber

76

slices evaluated as in the previous year. Data were analyzed in the same way as the

F usan'um dry rot data.

77

RESULTS

Egsarigm dry rot

In the ANOVA of the 1998 Fusarium dry rot evaluation of transgenic SGO lines,
lines were significant (P < 0.01) accounting for approximately 58% of the variation (R2 =
.581) in lesion diameter . LSmeans ranged from 12.78 mm (SGO-2) to 18.17 mm (SGO-
9) with only SGO-1 and 2 different from the ‘Spunta’ non-transgenic control (Table 3.1).
For lesion depth, lines were also a significant source of variation in 1998 (P < 0.01)
accounting for approximately 39% of the variation (R2 = 0.385). Transgenic line SGO-3
was the only line different from the non-transgenic ‘Spunta’ control (Table 3.1). In the
1999 trial, lines were not significantly different for lesion diameter (P < 0.56) or lesion
depth (P < 0.22). Lesion diameter ranged from 31.4 mm (SGO-9) to 24.9 mm (SGO-2)
and lesion depth ranged from 3.45 mm (‘Spunta) to 10.1 mm (SGO-9) (Table 3.1).

Ranking of lines varied between years and between lesion measurements within
years although the most resistant and most susceptible lines tended to be the same across
years and measurements (Table 3.1). For example, SGO-2 was the most resistant line,
SGO-14 was the third most resistant line and SGO-9 was the most susceptible line in
both 1998 and 1999 based on lesion diameter.

All other transgenic lines were only evaluated for response to F. sambucinum in
1999 due to the availability of tubers. Based on the ANOVA, lines were not significantly
different for either the AGO lines or the LGO lines when lesion diameter or lesion depth
was analyzed (Table 3.2 and Table 3.3). Lesion diameter ranged from 18.25 mm (AGO-
12) to 23.9 mm (AGO-8) for ‘Atlantic’-derived lines and from 15.8 mm (LGO-4) to 20.1

mm (Libertas) for ‘Libertas’-derived lines. Lesion depth ranged from 3.7 mm

78

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81

(AGO-6) to 6.0 mm (AGO-7) for ‘Atlantic’-derived lines and from 6.8 mm (LGO-4) to
10.1 mm (LGO-l4) for ‘Libertas’-derived lines. Ranking of lines based on lesion
diameter differed from that based on lesion depth for both AGO and LGO lines but the
rankings for LGO lines were more similar with LGO-4 ranked as the most resistant line

for both measurements.

Erwim'a soft rot

In 1998 two Erwinia sofi rot inoculation experiments were conducted
using tubers of SGO lines. The first experiment indicated that all transgenic SGO lines
had smaller lesion diameters than the 'Spunta' control whereas, in the second experiment,
lines did not differ . In 1999, a third experiment was conducted with results more similar
to the first experiment in 1998. The level of infection, based on lesion diameter, was
lower in the second experiment (16.7 mm) than in the first (23.5 mm) or third (29.10
mm) experiments. Based on this .inforrnation only the first (1998) and third (1999)
experiments were considered in the analysis. In both experiments, several SGO
transgenic lines had significantly smaller lesion diameters than the 'Spunta' control (Table
3.4). Analysis of data combined across experiments (Fm = 1.57) indicated that SGO-9
and 12 had smaller lesion diameters than the 'Spunta' control (Table 3.4).

Due to low tuber availability, LGO lines and AGO lines were tested only
following the 1999 field season. Transgenic lines LGO-l, 3, and 16 had significantly
smaller lesion diameters than the 'Libertas' control and lines AGO-4 and 5 had
significantly smaller lesion diameters than the 'Atlantic' control (Table 3.5; Table 3.6;

Figure 3.3 and 3.4).

82

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Libertas

LGO-3

LGO-4
LGO-16

 

Figure 3.3. Photograph of ‘Libertas’ and LGO lines inoculated with
Erwinia carotovora ssp. carotovora. All LGO lines shown had lesion
diameters significantly less than the ‘Libertas’ control (p<0.05).

86

 

Atlantic AGO-4

 

Figure 3.4. Photograph of ‘Atlantic’ and AGO-4 tubers inoculated with
Erwim'a carotovora ssp. carotovora. Lesion diameter of AGO-4 was
significantly less than that of the non-transgenic ‘Atlantic’ control
(P< 0.05).

87

DISCUSSION

For all of the 1999 Fusarium dry rot evaluations the coefficients of variation (CV)
for the ANOVA’s were quite high, ranging from 26.5 % for LGO lesion diameter to
93.7 % for SGO lesion depth. Therefore, identifying small differences between lines
was difficult. In contrast, CV’s for the 1998 trials were lower (ranging from 12.2 for
SGO lesion diameter to 16.1 for SGO lesion depth) and differences were detected
between lines for both measurements. However, only three AGO transgenic lines had
smaller mean lesion diameters and only two had mean lesion depths smaller than the non-
transgenic ‘Atlantic’ control. Therefore, even if the CV’s were lower it is not likely that
any of the lines would be significantly less infected than the control. In addition, glucose
oxidase mRNA was not detected in northern analysis of RNA samples from tuber tissue
of any AGO line. Thus, we would expect neither an increase in H202 levels in tubers nor
an increased resistance to pathogens in tubers detached from the plant. In addition,
previous research (Douches and Hammerschmidt, unpublished data) indicated that
'Atlantic' is highly susceptible to Fusarium dry rot and Erwinia sofi rot due to slow
suberization. Therefore, in 'Atlantic', even if endogenous H202 levels were increased it
might not be sufficient to deter the pathogen and result in detectable differences in lesion
size. Cultivars that are more tolerant of Fusarium might benefit more from the glucose
oxidase gene. All of the LGO lines had smaller mean lesion diameters than the ‘Libertas’
control and four of the LGO lines had smaller mean lesion depths than the control. Thus
for the LGO lines, lower CV’s might have permitted us to identify small differences

between lines. Northern analysis of LGO tuber tissues indicated the presence of glucose

88

oxidase mRNA in both LGO-1 and 3, two lines which had both small lesion diameter and
lesion depth.

Similar to the dry rot evaluations, the CV's for the analyses of the soft rot trials
were quite high ranging from 21.0%, for the first SGO trial in 1998, to 32.3% for the
LGO trial in 1999. However, despite the relatively high CV's in these trials, differences
existed among the SGO lines and the LGO lines. Two of the LGO lines with the smallest
lesion diameters in the sofi rot trials (LGO-1 and 3) also had some of the smallest dry rot
lesion diameters. These two LGO lines were the only ones that had the glucose oxidase
mRNA in the tubers. Taken together, these data suggest that the reduction in lesion size
in LGO-1 and 3 is not a false positive result and is due to the presence of the glucose
oxidase transgene. In contrast, glucose oxidase mRNA was not detected in any SGO line
although differences were observed between lines for soft rot lesion diameter. It is
possible that the glucose oxidase mRNA represents a small portion of total tuber RNA in
most of the transgenic lines that it is undetectable in the amount of total RNA used for
Northern analysis. Altemately, glucose oxidase mRNA may be transient, whereas the
protein persists and remains active. Among AGO and SGO lines there did not appear to
be any relationship between resistance to F usarium dry rot and resistance to Erwinia soft
rot. Wu et al. (1995), also found that glucose oxidase could inhibit E. carotovora,
however, their results indicated nearly complete inhibition in aerobic conditions. The
difference in these results could be due to differences in the amount of H202 produced in
the tubers and this difference could be due to the different gene promoters used in each
experiment, the lines used, the E. carotovora isolate used or any combination of these

factors. Wu et al. (1995) utilized the figwort mosaic virus 358 promoter and transformed

89

'Russet Burbank‘ whereas, we transformed three different potato cultivars with the
glucose oxidase gene under the control of the Gelvin "super promoter" (Ni er al., 1995).
Based on the methods used to clone the glucose oxidase, our gene sequences should be
identical to that of Wu et al., (1995) but differences are possible. Such differences would
only be detected through direct comparisons which were not possible as Wu et al., (1995)
did not publish their gene sequence.

Based on these data, expression of the glucose oxidase gene seems to have very
little effect on the response of potato lines to infection by F. sambucinum but may have a
small effect on response to infection by E. carotovora. Wu et al., (1995) found that the
concentration of H202 required to inhibit bacterial growth was less than the concentration
required to inhibit fungal growth. At 50 uM H202 bacterial growth was inhibited by
about 80%, whereas fungal growth was only inhibited by about 60%. Inhibition of both
bacteria and fungi was nearly 100% at 100 uM H202. Therefore, any increase in H202
levels in the transgenic glucose oxidase plants developed for this study may not be
sufficient to be directly fungicidal, but may still have detrimental effects on bacteria such
as E. carotovora.

Many of the transgenic lines containing the glucose oxidase gene exhibited tuber
characteristics that were off-type compared to non-transgenic controls (Figure 3.5). Of
the 33 transgenic plants evaluated, 16 (48%) were classified as off-type based on tuber
appearance and some of these were also off-type for foliar characteristics (data not
shown). When transgenic lines were compared to their non-transgenic controls based on
shape and size, lines that had smaller dry rot and/or soft rot lesions than the controls

(SGO-2, LGO-1 and -4) were often misshapen and severely off-type. All sectors of the

90

 

Figure 3.5. Photograph showing the off-type tuber morphology of
several transgenic lines containing the glucose oxidase gene.

91

potato market have specific requirements for tuber shape and any new cultivar must
conform if it is to be successful. Therefore, it would be necessary to develop and test
many more transgenic lines in order to identify ones with an effect against soft and/or dry
rot and an acceptable tuber appearance.

Based on these results some concerns arise about the use of the glucose oxidase
gene to increase the resistance of stored potato tubers to F. sambucinum or E. carotovora
ssp. carotovora. The effect of the glucose oxidase gene against both diseases appeared to
be minimal under the experimental conditions that we employed. Therefore, the time,
expense and effort required to develop transgenic lines with this gene may not prove
worthwhile. However, small improvements gained under laboratory conditions may
translate into more significant differences under commercial storage conditions. For
example, the cultivar 'Snowden' is slightly less susceptible to both dry and soft rot in
laboratory experiments but when combined with proper cultural practices, the use of
'Snowden' can significantly reduce the occurrence of these diseases in storage
(Hammerschmidt, personal communication). Thus, it would be useful to test our
transgenic lines in a commercial storage to determine the future role of the glucose
oxidase gene in a breeding program. Secondly, because glucose oxidase produces H202,
which is involved in several plant physiological processes, there is a stronger likelihood
that it will adversely affect the physiology and/or morphology of transgenic plants than a
gene that has a very limited and specific function.

If both of these concerns can be addressed then using the glucose oxidase gene in
a potato breeding program could be beneficial. In addition, Erwinia can cause wilting of

potato plants in the field and transgenic plants expressing the glucose oxidase gene in the

92

leaf tissue may be less susceptible to infection by these pathogens. Therefore, future
research should include evaluation of 1) the resistance of the transgenic lines to Erwz'nia
soft rot under commercial storage conditions, 2) the resistance of the transgenic lines to
Erwinia infection of the plant during the growing season, and 3) the possibility of

producing lines with increased resistance and normal morphology.

93

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