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LIBRARY
Michigan State
University

 

 

 

This is to certify that the

dissertation entitled

Host and viral factors influencing
systemic infection and host
range of potyviruses in cucurbits

presented by

Zakir Ullah

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Plant Breeding and
Genetics/Horticulture

, ,,,/
Jd’f’t‘kfi -1/¢,«7..,-,_//

Major professor

Date 3:3/40

MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771

____.. ;-f — .

PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRC/DateDuepGS-p. 15

 

HOST AND VIRAL FACTORS INFLUENCING SYSTEMIC INFECTION
AND HOST RANGE OF POTYVIRUSES IN CUCURBITS
By

Zakir Ullah

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPY

Plant Breeding and Genetics Program
and Department of Horticulture

2000

ABSTRACT

HOST AND VIRAL FACTORS INFLUENCING SYSTEMIC INFECTION
AND HOST RANGE OF POTYVIRUSES IN CUCURBITS

By
Zakir Ullah

This study was directed toward investigation of host and viral factors that
influence successful systemic potyvirus infection. On the host side, I studied the
mechanism of resistance to zucchini yellow mosaic virus (ZYMV) in two cucumber
inbred lines, ‘Dina-l ’ and ‘TMG-l ’, both of which contain a single recessive gene but
show different phenotypes when young plants are inoculated on the cotyledons. ‘Dina-l ’
shows a distinct pattern of veinal chlorosis limited to one or two leaves while ‘TMG-l ’
remains virus free. I provide evidence that resistance to ZYMV in ‘Dina-l ’ plants is
developmentally regulated, occurs at the level of phloem loading or unloading, and that
the amino terminus (NT) of the ZYMV coat protein (CP) is involved in the veinal
chlorosis response.

The role of the highly variable NT of the CP also was investigated in systemic
infection and host range determination of potyviruses. Chimeric ZYMV infectious
constructs were made containing the NT of the CP of watermelon mosaic virus (having
overlapping host range with ZYMV), and tobacco etch virus (having non-overlapping
host range with ZYMV). Evidence indicated that despite substantial variability in the
length and sequence, the NTs of CPs of heterologous potyviruses could facilitate
systemic infection of ZYMV in susceptible cucurbit hosts. However, this substitution per

se was not sumcient to modify the host range of the virus. The CP-NT from a non-

cucurbit potyvirus triggered a host defense response in cucurbits resulting in recovery
from systemic virus infection.

Finally I characterized the interaction of a cucumber poly(A) binding protein
(PABP), with the RNA dependent RNA polymerase (RdRp) of ZYMV, and the RdRps
other viruses. My data suggest that the carboxy terminal end of the PABP is essential for
interaction with the ZYMV-RdRp, but the domains of the RdRp gene involved in this
interaction are more complex. The cucumber PABP also interacted with bean common
mosaic necrosis potyvirus and cowpea chlorotic mottle bromovirus but failed to interact

with certain other potyviruses.

I dedicate this work to the people who I left behind during this journey.

ACKNOWLEDGEMENTS

I would like to express my gratitude to my committee members Drs. James F.
Hancock, Rebecca Grumet, Richard Allison, and Wayne Loescher for helpful
suggestions and continued support throughout this program. I also thank Dr. John
Everard for his valuable help and suggestions during the initial stages of my research
program, and Drs. Amit Gal-On and Benjamen Raccah for giving me an opportunity
to work in their labs. I am particularly grateful to Dr. Rebecca Grumet, my major
professor, who helped me grow as a scientist and a person during my stay at
Michigan state university.

I also thank Dr. Mariam Sticklen for accepting me in her program and her
assistance during the first year of my graduate program. My special thanks to the
ministry of education, government of Pakistan, for providing me with financial
support to come to the United States in pursuit of higher education, and my family
members for being extremely supportive of my career goals.

All of my fiiends at MSU deserve special appreciation for making my life full of
fim. My colleagues in the lab, Sue Hammar, Eileen Kabelka, Katerina Papadoupolo,
Wang Xiaofeng, Holly Little, Benli Chai, and Mulmmmad Tawfik Saleh have all
shown me support and encouragement throughout these years and deserve my special

thanks. I also thank Dr. Mike F. Thomashow and his group for their help and

support.

TABLE OF CONTENTS

LIST OF TABLES ......................................................................... III
LIST OF FIGURES ........................................................................ IX
LIST OF ABBREVIATIONS ............................................................. XI
CHAPTER 1: REVIEW OF LITERATURE. ................................ 1
REVIEW OF LITERATURE .................................. 1
LITERATURE CITED ......................................... 21
CHAPTER II: RESISTANCE TO ZUCCHINI YELLOW MOSAIC

VIRUS AT THE zym LOCUS IN CUCUMBER:
LOCALIZATION OF VIRUS TO THE VEINAL
REGIONS AND THE ROLE OF COAT PROTEIN IN

VEINAL CHLOROSIS ....................................... 30
ABSTRACT .................................................... 30
INTRODUCTION .............................................. 3 1
MATERIALS AND METHODS ............................. 35
RESULTS ....................................................... 38
DISCUSSION ................................................... 51
LITERATURE CITED ........................................ 59

CHAPTER III: TESTING THE ROLE OF THE AMINO TERMINUS OF
THE COAT PROTEIN IN SYSTEMIC INFECTION
AND HOST RANGE DETERMINATION OF
POTYVIRUSES ................................................ 64

ABSTRACT .................................................... 64

CHAPTER IV:

INTRODUCTION .............................................. 65

MATERIALS AND METHODS ............................. 68
RESULTS ........................................................ 76
DISCUSSION .................................................... 88
LITERATURE CITED ......................................... 96

CHARACTERIZATION OF THE INTERACTION OF
THE CUCUMBER POLY(A) BINDING PROTEIN WITH

THE REPLICASE GENES OF VIRUSES ................. 102
ABSTRACT .................................................... 102
INTRODUCTION ............................................. 103
MATERIALS AND METHODS ............................ 105
RESULTS AND DISCUSSION ............................. 109
LITERATURE CITED ....................................... 114

VII

LIST OF TABLES

Page

Table 1. List of virus abbreviation and their fiill names used in this text. .................... 5
Table 2. Effect of prior inoculation of ‘Dina-l ’ plants with ZYMV-Ct on secondary

inoculation by ZYMV-NAA. ..................................................................... 49

Table 3. Effect of plant age (A) and prior inoculation (B) with ZYMV/TNT hybrid virus
on symptom development in squash plant ........................................................ 80
Table 4 (A and B) Summary of the inoculation experiments of various constructs using
infected leaves of squash (for ZYMV-derived constructs) or tobacco (for TEV) as a
source of inoculum onto squash, C. amaranticolor (C. amarant.), bean N. benthamiana
(N. benth) and tobacco. ............................................................................ 85

Table 5. Interaction of the poly(A) binding protein with viral RdRp proteins ............. 112

VIII

LIST OF FIGURES

Figure l. The potyvirus genome. ................................................................. 2

Figure 2. Response of ‘TMG-l’ (A), ‘Dina-l’ (B), and ‘Straight-8’ (C) to inoculation
with ZYMV. ....................................................................................... 40

Figure 3. Virus titer in the cotyledons (Cot) and true leaves (L) in ‘Straight-8’, ‘Dina-l ’
and ‘TMG-l ’ in response to inoculation with ZYMV-Ct. ................................... 41

Figure 4. Virus accumulation (as determined by ELISA) in different leaves of ‘Straight-
8’ as affected by plant age at the time of inoculation. ........................................ 42

Figure 5. (A and B) Time course experiments showing the accumulation of ZYMV in the
first systemic leaf of “Straigh-8” (top) and ‘Dina-l ’ (bottom) leaves at five, ten and thirty
days post inoculation. .............................................................................. 44

Figure 6. Virus titer in the cotyledons and true leaves of ‘Dina-l ’, at the time of removal
of cotyledons (A) and 14 days post inoculation (B). .......................................... 47

Figure 7. Infectious ZYMV-NAA constructs with the engineered PstI site (pZCPFL/P),
the NT of the CP of ZYMV-Ct (pCtNTFL/P), the core portion (pCtCoFL/P) of the CP of
ZYMV-Ct. .......................................................................................... 52

Figure 8. Alignment of the amino terminal portions of the CP OfZYMV(NAA), WMV
and TEV. ............................................................................................ 69

Figure 9. Chimeric constructs of ZYMV-NAA with the introduced changes in the NT of
the CP gene and their infectivity on susceptible squash plants. .............................. 70

Figure 10. Symptom expression on susceptible squash plants 6 weeks post inoculation.
Wild type ZYMV-NAA (left) and the chimeric construct containing the NT of the CP of
WMV (center) showed increasingly strong symptoms. ....................................... 78

Figure 11. Virus titer (as determined by ELISA) in susceptible squash and cucumber
plants at 30 days post inoculation (DPI). The top 7 or 8 leaves were sampled for ELISA
fiom 6 plants for each virus. ..................................................................... 79

Figure 12. Virus titer (as determined by ELISA) in resistant cucumber genotype ‘TMG-
1’, transgenic melon line 207 expressing the CP gene of ZYMV-Ct in ‘Hales Best

Jumbo’, and susceptible conunercial cultivar ‘Hales Best Jumbo’ (bottom) 26 days after
inoculation. .......................................................................................... 83

IX

Figure 13. Hybrid ZYMV-NAA constructs containing the core and carboxy terminus
(Co) of the coat protein of ZYMV-Ct (CtCoFL/P), WMV (WCoFL/P) or TEV
(TCoFL/P) and their infection on squash plants. .............................................. 87

Figure 14. Deletion analysis to determine regions in PABP and RdRP responsible for
the interaction. .................................................................................... 106

LIST OF ABBREVIATIONS

CC companion cell

CP coat protein

CT carboxy terminus

NT amino terminus

PD plasmodesmata

PTGS post-transcriptional gene silencing
SE sieve elements

XI

CHAPTER I

REVIEW OF LITERATURE

Potyviruses constitute the largest group of plant viruses and cause economic losses in
all major crops of the world (Shukla et al., 1994). In nature, potyviruses are
predominantly transmitted by aphids, but they can also be transmitted by mechanical
means, and in some cases through infected seeds (Shukla et al., 1994; Ding et a1 1992;
Maule and Wang, 1996). Potyviral genomes consist of a single positive sense RNA
molecule, ca.10kb in size, with a covalently attached viral protein (VPg) at the 5’ end and
a poly(A) tail encoded in the viral genome at the 3’ end (Figure l). The RNA is
encapsidated by ca. 2000 coat protein monomers to form a 750m long flexuous rod
shaped particle (Shukla et al., 1994). The whole genome is translated into a single
polyprotein which is subsequently cleaved into nine or more proteins by viral-encoded
proteinases (Dougherty and Semler, 1993; Riechmann et al., 1992), many of which are
multifunctional (Figure 1).

Functions that have been assigned to the different potyviral proteins are summarized
in Figure l. The P1 protein is a proteinase involved in processing the 5’ end of the
polyprotein: the second protein HC-Pro, was first named as helper component (HC) for
its role in facilitating aphid transmission (Pirone and Blanc, 1996). It was later found to
also act as a proteinase (Pro) that cleaves itself at the carboxy terminus. The HC-Pro also

has been shown to be involved in symptom expression, genome amplification, and virus

 

 

{‘P
L I Hm)

 

10kb positive sense single stranded RNA

 

 

Protein Function

P1 Proteinase; terminal step in polyprotein processing

HC-Pro Polyprotein processing; aphid transmission; cell to cell and systemic
movement; suppression of gene silencing

P3 Genome amplification

6K1 Unknown

CI Genome replication; RNA helicase; cell to cell movement

6K2 Membrane localization

VPg Genome replication; virus movement

NIa Proteinase; polyprotein processing

NIb RNA dependent RNA polymerase; genome replication

CP RNA encapsidation; aphid transmission; cell to cell and systemic movement

 

Sources: Shukla et al., 1994; Cronin et al., 1995; Rojas et al., 1997; Brigneti et al., 1998;

Kassclmu and Carrington, 1998; Fernandez et al., 1995; Carrington et a1, 1998; Schaad et

a1, 1997; Nicolas et al., 1997; Dolja et al., 1994, 1995; Rojas et al., 1997.

Figure l. The potyvirus genome. Individual components of the polyprotein are shown on

top of the lOkb genome and their fimctions indicated.

movement, and more recently it has been found to play a critical role in countering the
host defense mechanism of virus induced gene silencing by acting as a suppressor of gene
silencing (Shukla et al., 1994; Cronin et al., 1995; Rojas et al., 1997; Brigneti et al., 1998;
Kasschau and Carrington, 1998). P3 protein is involved in genome amplification
(Meritus et al., 1999). The 6K1 protein has yet to be assigned a function and the 6K2
protein is involved in membrane localization of the virus replicase (Schaad et al., 1997).
The cytoplasmic inclusion (CI) protein forms characteristic pinwheel shaped inclusion
bodies in the cytoplasm. It has been shown to have RNA helicase activity and also to be
involved in cell to cell movement and genome replication (Shukla et al.,1994; Fernandez
et al., 1996; Carrington et a1, 1998). The VPg is a 21KDa protein covalently linked to the
5’ end of the viral RNA and has roles in virus replication, translation and movement
(Shukla et al., 1994; Schaad et a1, 1997; Nicolas et al., 1997). The NIa protein is the
major viral proteinase and processes several proteins at the 3’ end of the polyprotein. The
NIb is an RNA-dependent RNA polymerase involved in viral replication. Both Ma and
NIb derive their names from the formation of inclusion bodies (NI) in the nucleus of
infected plant cells (Shukla et al., 1994). Finally the potyviral coat protein (CP) is
involved in encapsidation of the viral RNA, aphid transmission, and virus movement
(Jagadish et al., 1993; Pirone and Blanc, 1993; Dolja et al., 1994, 1995; Rojas et al.,
1997).

The viral proteins descrrbed above, allow for successful infection of a host plant via
translation and replication in the initially infected cells, cell-to- cell movement through

plasmodesmata (PD), and systemic movement of the virus through the phloem (Lucas

and Gilbertson, 1994; Carrington et al., 1996; Lucas et al., 1996). Host resistance
responses can interfere with the virus life cycle at one or more of these points. However,
the mechanisms of host resistance to viruses in general, and potyviruses in particular, are
not well characterized. Host range determinants of potyviruses also are not known.

In this project I sought to address questions concerning mechanisms of resistance to
potyviruses, and to examine viral factors influencing systemic infection and host range
determination. I characterized resistance to zucchini yellow mosaic virus (ZYMV) at the
zym locus in cucumbers and studied the role of potyviral coat protein (CP) in systemic
infection and host range determination. Therefore, this review will focus on recent
literature concerning the role of: 1. Potyviral proteins involved in viral movement, 2.
Host resistance to potyvirus infection including resistance to potyviruses in cucurbits, and
3. Host range determination of potyviruses.

Several reviews covering related aspects of plant virus interaction are available.
Readers are referred to Fraser (1990) and Provvidenti and Hampton (1992) for genetics
of resistance to potyviruses; to Leisner and Turgeon (1993), Lucas and Gilbertson,
(1994), Carrington et a1. (1996), Lucas et al. (1996), and Soren and Haenni (1996) for
plant virus movement; to Revers et a1. (1999) for plant potyvirus interaction, and
Dawson and Hilf ( l 992) for host range determination of plant viruses.

Throughout the literature review, references will be made to numerous viruses. A list

of abbreviations is shown in Table 1.

Table 1. List of virus abbreviation and their fiill names used in this text.

 

 

 

 

Abbreviation Full name

BCMV Bean common mosaic virus
BCMNV Bean common mosaic necrosis virus
BMV Brome mosaic virus

CMV Cucumber mosaic virus

CCMV Cowpea chlorotic mottle virus
LMV Lettuce mosaic virus

PepMoV Pepper mottle virus

PRSV Papaya ring spot virus

PSbMV Pea seedbome mosaic virus

PVA Potato virus A

PW Potato virus V

PVY Potato virus Y

SCMV Sugarcane mosaic virus

SPFMV Sweet potato feathery mottle virus
TEV Tobacco etch virus

TuMV Turnip mosaic virus

TVMV Tobacco vein mottling virus
WMV Watermelon mosaic virus

ZYMV Zucchini yellow mosaic virus

 

The role of potyviral proteins in cell to cell and systemic movement.

Most plant viruses move cell to cell through plamsodesmata (PD) and long
distance through phloem, and so can encounter a number of barriers during the course of
movement. PD maintain cytoplasmic continuity between adjacent cells and consist of a
central region occupied by an appressed form of endoplasmic reticulum (ER) which is
continuous with the ER of adjoining cells (Lucas, 1995). The space between the ER and
plasma membrane, called the cytoplasmic annulus, contains a number of proteins and is
the site for transport of metabolites and proteins. The number, structure and function of
PD may vary between different plant tissues and cell types. Most PD have size exclusion
limits of 800-1000da (Tucker, 1982; Robards and Lucas, 1990), which does not permit
passage of large macromolecules. Certain plant proteins that traffic through the PD
interact with the PD proteins to induce an increase in size exclusion limit (SEL) and
facilitate their movement into the sieve element (Balachandran et al., 1997; Beatriz et a1
1999).

Plant viral movement proteins (MP) also increase the PD SEL to facilitate their
own movement as well as movement of viral RNA and virions (Lucas et aL, 1996;
Carrington et al., 1996). Potyviruses do not encode a specific movement protein; instead
four multifunctional proteins i.e. HC-Pro, CI protein, VPg and CP, have been shown to
facilitate cell to cell and long distance viral movement (Revers et al., 1999). Each will be
discussed in turn.

HC-Pro has been implicated in both cell to cell and long distance movement, and
separate domains of the protein have been shown to be involved in facilitating each type

of movement (Rojas et al., 1997; Cronin et al., 1995). In microinjection studies, wild

type HC-Pro increased the PD SEL and facilitated its own movement as well as
movement of viral RNA (Rojas et al., 1997). Deletions in the C-terminal part of HC-Pro
affected cell to cell movement. A mutation in the central region of HC-Pro, however,
inactivated the long distance movement of TEV but had little effect on the genome
amplification and cell to cell movement (Cronin et al., 1995). In situ histochemical
analysis revealed that the mutant virus was capable of infecting mesophyll, bundle sheath
and phloem cells within the inoculated leaves, suggesting that the long distance
movement block was associated with entry into or exit fi'om sieve elements. Grafting
experiments have shown that HC-Pro is required for both entry into and exit fi'om the
phloem (Kasschau et al., 1997). TEV HC-Pro was required in both stock and scion
tissues for systemic virus infection to occur.

Rodriguez-Cerezo et al. (1997), observed CI protein of potyviruses associated
with connections between plant cells at early stages of infection suggesting that CI might
also be involved in the virus movement. Alanine scanning mutagenesis of the CI gene in
TEV identified two mutants with substitutions in the N-terminal region of the gene,
which amplified to the level of the parental virus but were restricted to single cells in the
inoculated leaves (Carrington et al, 1998). Several other CI mutants showed either a slow
cell to cell movement or were unable to move long distance through the vasculature.
Cells across fiom an advancing infection front in pea cotyledons infected by PSbMV
contained CI and CP, providing further evidence for the role of the CI protein in virus
movement (Roberts et al., 1998). In microinjection studies, however, the BCMNV CI

protein did not exhibit viral movement protein properties (Rojas et al., 1996). Carrington

et al. (1998), have suggested that the CI protein may direct intracellular translocation of a
viral transport complex into plasmodesmata.

The genome linked viral protein (VPg) facilitates long distance movement of the
virus. Schaad et al. (1997), made chimeric constructs between TEV-HAT and TEV-
Oxnard, two strains of tobacco etch virus (TEV) that show differential response on
tobacco cultivar V20. Both strains can replicate and move cell to cell in V20, but only
TEV-Oxnard shows systemic infection. Full length cDNA constructs of TEV-HAT
containing the CP, HC-Pro or both CP and HC-Pro of TEV-Oxnard were not able to
cause systemic infection. However, the chimeric constructs containing the VPg of TEV-
Omard were able to cause systemic infection thereby suggesting a possible role for VPg
in systemic infection. VPg of TVMV-S is responsible for breaking this va gene-mediated
resistance in tobacco cultivar TN86 which also occurs at the level of movement (Gibb et
al., 1989; Nicolas et al., 1997). Virus titer and symptoms produced by TVMV-S in TN86
are identical to those produced on susceptible cultivars. When chimeric constructs were
made between the two strains, resistance breaking properties were localized to four
amino acids in the VPg gene indicating that this gene is required for virus movement. The
va gene in tobacco also confers resistance to PVY at the level of movement. A single aa
change in the VPg correlated with the resistance breaking in virgin A mutant (V AM)
tobacco containing the va gene (Masuta et al., 1999).

The potyviral CP is involved in both cell to cell and vascular movement (Dolja et
al., 1994, 1995; Rojas et al., 1997). The core portion of the CP is highly conserved
among potyviruses while the amino- and carboxy terminal (NT and CT) portions are

variable in length and sequence (Shukla et al., 1994). Both NT and CT are exposed on

the surface of virions and are not essential for virion assembly. The conserved core
portion is necessary for both encapsidation and cell to cell movement of the virus (Dolja
et al., 1995, Rojas et al., 1997). Amino acid substitution in the core region affected cell
to cell movement of TEV in tobacco plants (Dolja et al., 1995). In microinjection
experiments wild type CP of BCMNV and LMV increased the PD SEL and facilitated
transport of viral RNA, while amino acid substitutions in the core region either impaired
or abolished cell to cell movement (Rojas et al., 1997).

Deletion of the NT and CT of the CP abolished the systemic movement of TEV
(Dolja et al., 1994, 1995). Amino acid substitutions in the NT of the CP have variable
effects on virus movement depending upon the particular amino acid being substituted.
The conserved DAG motif has been studied most extensively due to its role in aphid
transmission (Lopez-Maya and Pirone, 1998; Gal-On et al., 1992; Atreya et al., 1991).
When Lys or Arg were substituted for Asp in the DAG motif of TVMV, the mutant
viruses failed to move systemically in tobacco plants but replicated and produced virions
in the protoplasts (Lopez-Moya and Pirone, 1998). TEV mutants with similar
substitutions for Asp in the two DAG motifs in the CP-NT also failed to infect tobacco
plants systemically, suggesting that besides its role in the aphid transmission, this motif is
also involved in the systemic virus movement (Lopez-Moya and Pirone, 1998).
However, substitution of Ala for Thr (DAG to DTG) in the NT of CP of ZYMV-NAA
and Gly for Glu (DAG to DAB) in CP-NT of TVMV (Gal-On et al., 1992; Atreya et al.,
1991) had no effect on accumulation of the virus in systemic leaves. Both these
mutations are associated with the loss of aphid transmissibility and occur when the virus

is maintained by rub inoculaton rather than aphid transmission. In each case aphid

transmissibility was regained after the conserved DAG motif was restored (Gal-On et al.,
1992; Atreya et al., 1990, 1991). A reduction in the accumulation of PVA was associated
with mutation of DAS to DAG in the conserved motif, but there was no efiect on the
systemic movement of the virus (Andrejeva et al., 1999). Andersen and Johansen,
(1998), observed that a single amino acid substitution (Ser 47 to Pro) in the NT of the CP
of PSbMV was sufficient to permit systemic spread in C. quinoa, suggesting that this
region of the CP might also be involved in systemic movement in strain and host specific

manner.

Host resistance mechanisms

Plants have evolved a number of mechanisms to protect themselves against virus
infection, ranging fiom complete immunity against virus infection, to restriction of virus
movement (White and Antoniw, 1991; Dawson and Hilf, 1992; Pennazio et aL, 1999).
Phenotypic responses of plants to virus infection vary from systemic symptoms in
susceptible plants, to recovery from initial infection, to a hypersensitive response
resulting in necrotic or chlorotic lesions at the site of infection, or a lack of symptoms due
to failure of the virus to, replicate in or move from the initially infected cells. Certain
plants tolerate virus infection without any showing visible symptoms even though the
virus is present at detectable levels in systemic tissues. A number of resistance genes
have been identified that act at various points in the virus life cycle preventing either
virus replication, cell to cell spread, or systemic movement (Fraser, 1990; Provvidenti

and Hampton, 1992; Dawson and Hilf, 1992)

10

Plant protoplasts provide a useful tool to distinguish between resistance genes
operating at the level of movement or replication. Inability of the virus to accumulate in
the protoplasts of resistant plants suggests that resistance is occurring at the level of
replication (or translation) of the viral genome. On the other hand, virus accumulation in
the protoplasts of resistant plants that do not show detectable symptoms and virus
accumulation in the inoculated leaves indicates a block at the level of cell to cell
movement. For example, viral coat protein or RNA could not be detected in the
protoplasts of homozygous recessive sbm-I peas transfected with the P-l isolate of
PSbMV, suggesting that resistance is occurring at the level of replication (Keller et al.,
1998). Similarly, the resistance conferred by the single recessive gene (ef’) in Capsicum
annum cv. Dempsey to TEV, is due to interference with virus RNA accumulation (Deom
et al., 1997). Infection foci were not detected in the inoculated leaves when ‘Dempsey’
plants were inoculated with TEV-GUS, and northern blot analysis of protoplasts
inoculated with TEV RNA did not show virus accumulation. Interference with the
accumulation of RNA of PepMoV and TEV-HAT in plants and protoplasts has also been
reported in two accessions of Capsicum chinense containing the pvrI gene.

In other cases virus replication was observed in protoplasts indicating that
resistance is occurring at the level of virus movement. For example in the homozymous
state, the y" (pr-2’) gene of pepper plants (cultivar Yolo Y) restricts cell to cell movement
of PVY (pathotype O: Arryo et al., 1996). When protoplasts fi'om healthy Yolo Y plants
were inoculated with PVY-O a high percentage of protoplasts (80-85%) showed virus
accumulation. PVY-0 was not detected (with ELISA) in the inoculated or systemic

leaves, and when protoplasts were prepared from inoculated leaves, only a small number

11

of protoplasts (0.06%) showed immunoflorescence. The va gene in tobacco (N. tabacum)
appears to restrict cell to cell movement of potato virus Y (PVY) and tobacco vein
mottling virus (TVMV) (Masuta et al., 1999; Gibb et al., 1989). Both the viruses
replicate in the protoplasts of resistant plants. In resistant tobacco cultivar TN86
(containing the va gene) TVMV is restricted to a few inoculated cells or small groups of
cells in the epidermal strips from inoculated leaves (Gibb et al., 1989). Virus
accmnulation was not detected in the mesophyll cells by irnmunostaining or ELISA,
suggesting that resistance in TN86 occurs primarily due to restriction of virus cell to cell
movement. PVY also was not detected in the inoculated leaves of tobacco cultivar VAM
(containing the va gene: Masuta et al., 1999). A reduction (ca. 30%) in virus replication
in the VAM protoplasts compared to protoplasts from susceptible plants was observed
indicating that impairment of replication also contributes to the resistance.

Resistance to long distance movement can occur at the level of phloem loading or
unloading resulting in reduced or no virus accumulation in systemic tissues. The
structure and number of plamsmodesnmta connecting sieve element-companion cells
(SE-CC) and those connecting mesophyll cells, differ suggesting that the viruses
encounter different levels of barriers in their cell to cell and long distance movement
(Leisner and Turgeon, 1993; Lucas et al., 1996; Carrington et al., 1996; Soren and
Haenni 1996; Mclean et al., 1997; Sjolund, 1997). Studies with phloem loading fi'om
inoculated leaves of several plant species showed that among the cells surrounding sieve
elements in the minor veins, vascular parenchyma cells were the predominant cells to
become visibly infected with poty- and tobamoviruses (Ding et al., 1998). It was

observed that a barrier exists for virus entry into mature smooth walled companion or

12

transfer cells. Viral proteins (or domains of the same protein) involved in the cell to cell
and long distance movement (e.g. CP and HC-Pro) also difi‘er, suggesting different levels
of interactions for the long distance vs. cell to cell movement (Carrington et al., 1996;
Revers et al., 1999).

Several cases of resistance to potyviruses occurring at the level of systemic
movement have been reported (Murphy and Kyle 1995; Schaad and Carrington 1996;
Hinrichs et al., 1998; Mahajan et al, 1998). For example use of an infectious TEV clone
engineered to express GUS gene showed tlmt the rate of replication and cell to cell
movement was nearly identical in tobacco line V20 showing strain-specific resistance to
TEV and the susceptible line Havana 425, however, systemic movement was markedly
restricted in the resistant line (Schaad and Carrington, 1996). Immunocytochemical
analysis of V20 tissues from infection foci indicated that the block in the long-distance
movement was associated with entry into or exit from the sieve elements. Similarly,
PVY and TEV-GUS replicated in, and moved cell to cell in a resistant tobacco cultivar
containing the Rl’m resistance gene, but systemic infection of both viruses was prevented
by a resistance reaction that also resulted in necrotic streaks on some cultivars (Hinrichs
et al., 1998). In another example, Murphy and Kyle (1995), examined the accumulation
and movement of pepper mottle potyvirus (PepMoV) in the resistant Capsicum annum L.
genotype ‘Avelar’, using immunotissue blot analysis. Although the viral antigen was
detectable in the stem below the inoculated leaves and in the first internode above the
inoculated leaves of the resistant genotype, virus was not detected in the first pair of
uninoculated leaves, even at 25 days post inoculation. Inoculated leaves also showed

reduced accumulation of virus compared to the susceptible control, thus although the

13

resistance appeared to be operating at the level of movement, intereference with
replication may also play a role. Finally, Mahajan et aL (1998) identified a dominant
locus conditioning the restricted systemic movement of TEV (RT M) in the Arabidopsis
ecotype Colombia. They proposed that RTMI mediates a restriction of long distance
movement through a mechanism that differs substantially from those conditioned by the
dominant genes associated with gene for gene interaction. Characterization of the gain of
susceptibility mutants with RT MI suppressed phenotypes revealed that at least two loci
RT MI and RT M2 cooperate to condition a restricted TEV movement phenotype (Witham
et al., 1999). Interestingly, the predicted amino acid sequence of the RTMI gene shows
homology to a family of proteins involved in defense against viruses, fungi and insects
(Chisholm et al., 2000).

Hypersensitive response (HR) is another type of plant defense observed in
response to a large number of viruses (Ponz and Bruening, 1986; White and Antoniw,
1991; Brunt et al., 1996). An HR restricts virus movement from the initial infection foci,
and results in the appearance of necrotic or chlorotic local lesions at the site of infection
(White and Antoniw, 1991). Local lesions are associated with an induced resistance
response and expression of pathogenesis related proteins. Most potyviruses have
experimental local lesion hosts (Brunt el al., 1996), but reports of Specific
hypersensitivity resistance genes for potyviruses are less common. Several wild potato
species, and cultivars have been reported to show HR response to PVY, PW and TEV
(V alkonen, 1997; Barker, 1997). For example, when potato cultivars containing the Nv
gene are graft inoculated with PVV, necrotic lesions and necrotic streaks in the veins

develop. The HR response eventually leads to death of the shoot tips and severe necrosis

leading to the collapse of the lower leaves (Barker, 1997). The RIG“) gene, which also
confers resistance to a number of potyviruses in potato plants restricts movement and
accumulation of TEV and PVY through a hypersensitive response-like mechanism
(Hinrichs et al., 1998). However, unlike the typical HR for viruses, where individual
local lesions are produced, necrotic lesions were observed along the veins when the lower
leaf surface was inoculated, but were either absent or their number significantly reduced
when the upper surface of the leaf was inoculated. In bean (Phaseolus vulgaris), the I
gene has been demonstrated to confer resistance to a wide range potyviruses through a
hypersensitive response (Kyle and Provvidenti, 1993; Scully et al., 1995).

Recovery from successful initial infection is found in response to infection by a
number of viruses, including some potyviruses (reviewed by Pennazio et al., 1999).
Strong initial symptoms are followed by a gradual reduction in symptom severity and
virus accumulation. The recovered plants are resistant to secondary infection by other
strains of the same virus or homologous viruses and show phenotypic similarities to post
transcriptional gene silencing (PTGS: Matzke and Matzke, 1995; Depicker and Montago,
1997). In transgenic plants showing PTGS, only low levels of transgene RNA could be
detected, even though transcription occurs at a relatively high rate. Recent evidence has
confirmed that PTGS-like host resistance is a generalized defense mechanism in plants
and neither recovery nor plant genome homology is essential for induction of this defense
mechanism (Rath et al., 1999). Small species of antisense RNA directed against the
transgene RNA in the case ofPTGS, and against viral genome in the case of virus
induced gene silencing WIGS) have been observed (Hamilton and Baulcombe, 1999).

As a counter defense strategy, a number of diverse plant viruses (e. g. cucumoviruses,

15

comoviruses, geminiviruses, potyviruses, tobamoviruses) encode proteins that suppress
PTGS-like resistance response of host plants against virus infection (Voinet et al., 1999).
In potyviruses the HC-Pro has been demonstrated to act as suppressor of PTGS (Brigneti

et al., 1998, Anandalakshmi et aL, 1998; Pruss et al., 1997; Voinet et al., 1999).

Cucurbit resistance to potyviruses.

In cucurbits, various sources of resistance to potyviruses have been identified
(Provvidenti 1985,1987; Gilbert-Albertini et al, 1993; Gibb et al., 1994; Kableka et al.,
1997), but in most cases the mechanisms of resistance have not been well characterized.
Interestingly, in several cases differences in response have been observed depending on
whether cotyledons or leaves were inoculated. The basis for this difference is not yet
understood. Resistance to the watermelon strain of PRSV in the muskmelon (Cucumis
melo) cultivars ‘Cinco’ and ‘Cinbo’ occured at the level of movement (Gibb et al., 1994);
only 3-7 cells were infected in the inoculated leaves. Cultivar ‘Cinco’ was highly
resistant and only occasionally showed mild symptoms when the cotyledons were
inoculated, however, when cotyledons of ‘Cinbo’ were inoculated mild systemic
symptoms were observed but plants remained symptomless when true leaves were
inoculated. Resistance to WMV in cucumber (C. sativus) line ‘TMG-l ’ involves two or
more resistance factors with tissue specific expression, one expressed in both cotyledons
and leaves and the other expressed only in the leaves (Wai and Grumet, 1995b). Limited
systemic movement of WMV and ZYMV to the first one or two leaves has also been
Observed in Cucurbita moschata line Menina 15 (Gilbert—Albertini et al, 1993) following

cotyledon inoculation.

l6

In cucumbers two inbred lines, ‘Dina-l ’ and ‘TMG-l ’, exhibit resistance to an
array of potyviruses including ZYMV, WMV, Moroccan watermelon mosaic virus (M-
WMV) and the water melon strain of papaya ringspot virus (PRSV-W: Provvidenti, 1987,
1985; Kableka et a1 1997; Wai and Grumet, 1995a). Both ‘Dina-l’ and ‘TMG—l ’ contain
a single recessive gene for resistance to ZYMV, but show different responses following
cotyledon inoculation. Limited spread of ZYMV was observed when the cotyledons of
young ‘Dina-l ’ (ca. 7 days old) plants were inoculated, resulting in a distinct pattern of
veinal chlorosis limited to the first or second systemic leaf while the rest of the plant
remained symptom free (Kabelka et al., 1997). Virus accumulation was detected in the
cotyledons and the chlorotic leaf but not in the upper non-symptomatic leaves. The
plants remained symptomless when true leaves were inoculated. TMG- 1 , however, did
not show veinal chlorosis following cotyledon inoculation and systemic spread of the
virus to the first leaf was only rarely observed when young plants are inoculated on the

cotyledons. Both the genotypes did not show symptoms following true leaf inoculation.

Non-hosts and host range determinants.

Many potviruses have very restricted host ranges, however, several have
intermediate host ranges and a few infect a wide range of host plants (Shukla et al.,
1994). In non-host species the virus is either unable to translate its genome, replicate in,
or move from the initially infected cells. The host range determinants ofpotyviruses are
poorly understood, and it is likely that several genes are involved depending on the nature
of block to virus infection (e. g. replication vs. movement). Virus replication has been

shown to be the target of non-host resistance against some potyviruses. For example,

17

Bak et al. (1998) observed that the non-host resistances to PSbMV in tobacco and to PVY
in pea operate at the single cell level. Both viruses were nimble to replicate in the
protoplasts of the non-host plants.

Inability to cause systemic infection also can determine host range, e.g. local
lesion hosts of viruses allow for initial virus replication, but prevent systemic spread.
Because of the extreme variability in the amino terminal portion of the potyviral CP and
its role in systemic movement, it has been suggested that the CP my play a role in host
range determination (Shukla et al., 1994; Ward et al., 1994). Xiao et al. (1993) observed
a correlation between duplication of amino acids (aa) residues in the amino terminus
(NT) of the CP and host range of several isolates of SCMV. Evidence for partial gene
duplication in the NT of CP was also found for most potyviruses that had CPs of 287 aa
residues or longer (Ward and Shukla 1993; Ward et al., 1994). Based on these
observations, Ward et al. (1994), suggested that variability in the CP-NT may have been
an important factor in the evolution of new species of potyviruses with new host and
vector specificities. However, the role of CP-NT in host range determination of

potyviruses has not been tested directly.

Research objectives for this project.

It is obvious fi'om this review of literature that potyvirus-host plant interaction is
very complex and requires different levels of interactions between viral and host factors
at different stages of infection. Only a few components of this interaction, mainly on the
virus side have been identified and numerous questions remain unanswered. For

example, how the different viral components interact with each other and with host

18

components to accomplish multiple tasks is not known Highly conserved and extremely
variable regions in the potyvirus genome have been identified, and functions of several
conserved regions have been demonstrated, but the significance of variability is still
speculative. Identification and characterization of host factors involved in virus infection
have only recently become the focus of plant virus research. In most cases, host
resistance mechanisms are not well characterized, and the determimmts of host specificity
also are poorly understood. The purpose of this dissertation was to address some of these

issues using zucchini yellow mosaic virus with the following specific objectives.

Objectives for chapter 1.

Before this project was initiated, the cucumber lines ‘Dina-l ’ and ‘TMG-l ’ were
reported as sources of resistance to a number of potyviruses including ZYMV. Different
phenotypic responses of the two genotypes to inoculation with ZYMV were observed in
our lab (Kableka et al., 1997). ‘Dina-l ’ remained free of symptoms while ‘TMG-l ’
showed veinal chlorosis limited to a Single leaf. These two responses were shown to be
due to different alleles at the same locus (Kableka et al., 1997). It remained to be
investigated why initial systemic infection occurs in ‘Dina-l ’ and why further systemic
spread of the virus is then restricted. Is the virus localized to the veinal regions in
chlorotic leaves? Why does inoculation of true leaves not result in a similar phenotypic
response? Why do certain strains of the virus fail to show veinal chlorosis on ‘Dina-l ’,
and what are the viral factors involved in the veinal chlorosis response? These questions

and several others formed the basis of the first chapter of my dissertation research.

19

Objectives for chapter 2.

The role of the amino terminus (NT) of the coat protein (CP) ofpotyviruses in
systemic movement was previously demonstrated, and based on extreme variability in the
NT, its role in role in host range determination was suggested (Dolja et al., 1994, 1995;
Shukla et al., 1994). However the significance of variability in the CP-NT has not been
established and its role in host range determination was not tested directly. In the second
part of my dissertation project I focused on further understanding the role of the NT of
the CP in potyvirus infection, particularly in facilitating systemic infection and host range
determination by making chimeric infectious constructs of ZYMV. The role of the core
portion of the CP and the whole CP in host range determination of potyvirses was also

investigated.

Objectives for chapter 3.

An understanding of the role of the host factors in facilitating virus infection is
one of the main focuses of current virus research. Our lab has initiated projects to
identify host proteins interacting with virus proteins in an effort to better understand
potyvirus infection in susceptible hosts. We have demonstrated that a poly(A) binding
protein (Cs-PABPl) fi'om susceptible cucumber host interacts with the RNA dependent
RNA polymerase (NIb) gene of ZYMV (Wang et al., submitted). I worked on an
additional project to further characterize this interaction as well as to study the interaction

of the Cs-PABPl with RdRp from an array of other viruses.

20

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25

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Phytopathology 85:561-566.

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26

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28

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29

CHAPTER II

RESISTANCE TO ZUCCHINI YELLOW MOSAIC VIRUS AT THE zym LOCUS
IN CUCUMBER: LOCALIZATION OF VIRUS TO THE VEINAL REGIONS

AND THE ROLE OF COAT PROTEIN IN VEINAL CHLOROSIS

ABSTRACT

The zym locus in cucumber (Cucumis sativus L.) is marked by multiple alleles conferring
resistance to zucchini yellow mosaic potyvirus (ZYMV) and is tightly associated (< 1
cM) with resistance to at least three other potyviruses. One allele, zymD'M, fiom the
inbred line ‘Dina- 1 ', confers an unusual phenotype that was studied to gain insight into
the mechanism of resistance. Following inoculation of cotyledons, ‘Dina-l' plants
showed a distinct pattern of veinal chlorosis limited to one or two leaves; the rest of the
plant remained symptom free. Inoculation of leaves generally did not result in veinal
chlorosis or virus accumulation in systemic leaves. Immunoblot analyses indicated that
the veinal chlorosis phenotype in ‘Dina-l' plants reflected virus distribution within the
leaf. Like in susceptible control plants, ZYMV moved fiom the inoculated coytledons to
the predominant sink at the time; however, unlike in susceptible plants, the virus in the
sink leaf remained localized to the veinal regions up to 30 days post inoculation

Cotyledon removal experiments indicated that the inability of the virus to spread within

30

the leaf exhibiting veinal chlorosis, or to establish infection in leaves, was not due to an
inability to replicate in the leaves. The pattern of virus accumulation along the veins
suggested a block in the ability of the virus to load into or unload fiom the phloem.
Production of chimeric viruses with switches in either the amino- or the central and
carboxy-terminal portions of the ZYMV coat protein (CP) showed that the amino
terminus of the CP appeared to play a role in the veinal chlorosis response. This is further
evidence suggesting that resistance in ‘Dina-l' operates at the level of systemic
movement as the CP-NT is involved in systemic virus movement. In contrast to the
greenhouse, where inoculation of leaves generally did not cause veinal chlorosis, veinal
chlorosis was observed with leaf inoculation in the growth chamber. Inoculation of the
first, but not the second, leaf resulted in a high percentage of plants showing veinal
chlorosis, indicating developmental control over expression of the resistance factor(s).
Together these observations suggest that resistance in ‘Dina-l' is developmentally

regulated and occurs at the level of phloem loading or unloading in the leaves.

INTRODUCTION

Potyviruses form the largest and economically most important group of plant
viruses; almost all the major crops of the world are infected by one if not several
members of the Potyviridae (Shukla et al., 1994). Naturally occurring sources of
resistance to potyviruses have been identified in a large number of plant species and can
interfere with virus replication, cell-to-cell spread, or systemic infection (Providentii and
Hampton, 1992; Carrington et al., 1996, Revers et al., 1999). Examples of interference

with replication include the recessive sbm-I allele of pea (Pisum sativum L.) which

31

prevents replication of pea seed borne mosaic potyvirus (PSbMV: Keller et al., 1998),
and the ef’ allele of pepper (Capsicum annum cv Demsey) which interferes with
accumulation of tobacco etch potyvirus (TEV) RNA (Deom et al., 1997). Interference
with the accumulation of RNA of pepper mottle virus (PepMoV) and TEV has also been
reported in plants and islolated protoplasts of two Capsicum chinense accessions
containing the recessive pvrI resistance gene (Murphy et al., 1998).

Cell-to-cell movement of potato virus Y (PVY) is restricted by the y" gene in
pepper plants (Arryo et al., 1996) and the va gene in tobacco (N. tabacum) plants, which
also restricts cell to cell movement of tobacco vein mottling virus (TVMV: Masuta et al.,
1999; Gibb et al., 1989). The Arabidopsis ecotype Bay-O is resistant to tin-nip mosaic
potyvirus via interference with cell to cell movement (Martin et al., 1998). In the
resistant tobacco line V20, long distance movement of TEV is restricted at the level of
entry into or exit from the sieve elements (Schaad and Carrington, 1996). Tobacco
cultivars containing the RIG“, gene also restrict systemic movement of TEV and PVY
(Hinrichs et al., 1998) and in Arabidopsis two loci, RT M1 and RT M2, cooperate to
condition a restricted long distance movement phenotype of TEV (Mahajan et al, 1998;
Witham et al., 1999). The RT M1 protein showed similarity to a group of plant proteins
that are implicated in defense against viruses, fimgi and insects (Chisholm et al., 2000).

Other types of resistances to potyviruses include hypersensitive reaction response
and recovery from initial infection (White and Antinow, 1991; Pennazio et al., 1999).
Examples of resistance genes conferring hypersensitive response to potyvirus infection
include the I gene of beans (Physiolus vulgaris) to multiple viruses (Kyle and

Provvidenti, 1993; Scully et al., 1995), the Nv gene of potato to PVV, (Barker, 1997) and

32

the RY,” gene of potato to TEV and PVY (Hinrichs et al., 1998). Recovery of plants
from initial virus infection is a host defense response resulting in a gradual reduction in
symptom severity, virus accumulation, and protection against secondary infection by
homologous viruses. The mechanism of recovery of host plants from virus infection
show homology to post transcriptional gene silencing (PT GS) and both responses are
associated with the appearance of small species of antisense RNA directed against the
transgene RNA in the case of PTGS, and against viral genome in the case of virus
induced gene silencing (Hamilton and Baulcombe, 1999; Ratclifl‘ et al., 1999; Pennazio et
al., 1999).

In a few cases, specific potyviral factors have been shown to play a role in
stimulating or overcoming host resistance, presumably through interaction with host
resistance factors. For example, the VPg gene of potyviruses is involved in overcoming
host resistances operating at the level Of replication (Keller et al., 1998) and movement
(Nicolas et al., 1997; Schaad et al., 1997; Masuta et al., 1999; Rajamaki and Valkonen,
1999). A single amino acid (a) substitution (Ser47 to Pro) in amino terminus (NT) of
the CP of the NY isolate of pea seed borne mosaic virus (PSbMV), allowed the virus to
move systemically in Chenopodium quinoa (Andersen and Johansen, 1998). Our studies
have shown that the CP-NT of a non-pathogenic potyvirus could induce a resistance
response in cucurbits resulting in recovery from systemic virus infection (chapter 3).
Solomon (1989) observed cleavage of the NT of the CP of sweet potato feathery mottle
virus (SPFMV) in the recovered leaves of Ipomea nil plants.

In cucumbers, two sources of naturally occuring resistance to zucchini yellow

mosaic potyvirus (ZYMV), have been identified (Provvidenti 1985, 1987; Abul-Hayja

33

and Al-Shahwan, 1991; Kabelka et aL, 1997). Although each is due to a single recessive
allele occurring at the same locus, they differ in phenotypic response. One source of
resistance comes fiom ‘TMG-l ’ (zymTMG), an inbred line derived from the Chinese
hybrid ‘Taichung Mou Gua’, that also is resistant to watermelon mosaic virus (WMV),
the watermelon strain of papaya ringspot virus (PRSV-W) and Moroccan watermelon
mosaic virus (MWMV: Providenti 1985, 1987; Kabelka and Grumet, 1997). The second
(zymD'M) has been described in ‘Dina-l ’, an inbred line derived fi'om a Dutch hybrid
which is also resistant to above listed viruses, allows for initial infection of cotyledons
followed by limited systemic spread (Abul-Hayja and Al-Shahwan, 1991; Kabelka et al.,
1997). The zym locus is of particular interest because in both ‘TMG-l ’ and ‘Dina-l ’, the
zym allele appears to be the same as, or tightly linked to genes conferring resistance to
MWMV, PRSV-W and WMV (Kabelka and Grumet, 1997; Kabelka et al., 1997; Wai
and Grumet, 1995a and b; Grumet et al., in press).

In response to cotyledon inoculation with ZYMV, ‘Dina-l ’ plants show a
pronounced pattern of veinal chlorosis that may provide insight into the mechanism of
resistance conferred by the zym allele. The veinal chlorosis is restricted to leaf-l or leaf-2
only, while subsequent leaves remain symptom flee. Segregation analyses indicate that
the veinal chlorosis is not due to a separate gene fiom the zym resistance gene (Kabelka et
aL, 1997). Although the the veinal chlorosis phenotype was observed with a number of
ZYMV isolates of diverse origin, it is unique to inoculation with ZYMV and was not
exhibited in response to inoculation with WMV, MWMV, or PRSV (Kabelka et al.,

1997).

34

In this work we sought to understand the mechanism of resistance to ZYMV
mediated by the zym resistance alleles in ‘TMG-l ’ and ‘Dina-l ’ and to investigate the
role of the coat protein of ZYMV in the resistance response. We provide evidence that
the veinal chlorosis phenotype of ‘Dina-l ’ is associated with the accumulation and
distribution of virus in the symptomatic leaves and that resistance operates at the level of
virus movement. We also show that the NT of the CP is involved in the veinal chlorosis

phenotype of ‘Dina-l ’ observed in response to inoculation with ZYMV.

MATERIALS AND METHODS

Plant material, virus stock and inoculation.

Plant material: The three cucumber genotypes used in this study are: the
susceptible cultivar ‘Straight-8’ (W. Atlee Burpee and Company, Warminster, Pa), and
the resistant inbred lines, ‘TMG-l’ and ‘Dina-l’. ‘TMG-l’ and ‘Dina-l’ were initially
provided by Dr. J. Staub (USDA, University of Wisconsin, Madison) and Dr. K. Owens
(Seminis Peto Seed Company, Woodland, Calfornia), respectively, and subsequently
maintained by self pollination in the greenhouse. Plants were grown in the greenhouse in
15cm clay pots using Baccto soil mix. During winter (October to March) the plants
received supplemental light (16h day). Seeds were pre-germinated for 24hr at 30C before
sowing. Growth chambers were set at 16hr day, 24C day, 20C night temperatures with
an average light intensity of 150 micro Einstein.

Experiments were performed using the Connecticut (Ct) isolate of ZYMV

(ZYMV-Ct; Provvidenti et al., 1987; Grumet and Fang, 1990) unless otherwise indicated.

35

Maintenance of virus stock and rub-inoculation procedures were as described in Wai et
al. (1995). Plasmid DNA of the infectious constructs (see below) were prepared using
the Wizard mini prep kit (Promega Madison, WI) and directly inoculated onto cotyledons
of 7-10 days old plants using the particle bombardment procedure of Gal-On et al.
(1995). Infected leaf material (passage 0, P0) was stored at —80F and used to inoculate
squash plants (P1) which were then used as a source of inoculum for all subsequent
experiments. ELISAs (enzyme linked irnmunossorbant assay) were performed with leaf
discs using the procedure of Wai et a1. (1995). Polyclonal antibodies prepared to ZYMV-
Ct coat protein were used for both ELISA and immunoblot analyses. Randomized

complete block designs were used for experiments when applicable.

Immunoblot analyses.

Leaves harvested for immunoblot analyses were frozen at -80C, thawed at room
temperature and used to form a sandwich of three layers of: filter paper; a nitrocellulose
membrane (Protran, PH79 pore size 0.1um) prewet with distilled water, the leaf, and a
single layer of paper towel. The whole sandwich was placed in a plastic sample bag
(Nasco WHIRL-PAK 180z) and rolled through a pasta machine (Atlas, Italy) using
pressure setting 4. Immediately after the run, the membrane was removed fi'om the
sandwich, placed on a piece of paper towel and allowed to dry at room temperature. The
blotted membrane was then developed using the western blot protocol of Blake et al.,

(1984).

36

Making chimeric ZYMV infectious constructs.

The full length infectious clone of ZYMV-NAA was kindly provided by Drs. A.
Gal-On and B. Raccah (Volcani Center, Bet Dagan, Israel). The amino terminus (NT) of
the CP of the infectious ZYMV-NAA construct was substituted with the CP-NT of
ZYMV-Ct (Grumet and Fang, 1990) by cloning the SacI-MIuI fragment from pCtCPSPX
(containing the CP of ZYMV-Ct, Chapter 3) into the infectious firll length ZYMV-NAA
construct digested with the same enzymes. The core and CT portions of the CP were
exchanged between the two constructs using the restriction sites of MIuI and AW”. MIuI
is a unique restriction site near the start of the core region of the CP of ZYMV (NAA and
Ct). AvrII is a unique site in the 3’ NTR and Sac] restriction is unique in the NIb gene of

ZYMV-NAA infectious construct.

Cotyledon removal experiments.

To test virus replication in the leaves, 7 day old plants were inoculated on their
cotyledons. On successive days after inoculation (1-5 dpi), the cotyledons and first leaf
(when present) of five plants were sampled for ELISA. The cotyledons were then
removed from the stem with a razor blade. Two weeks post inoculation, the first and
second true leaves of all the plants were sampled for ELISA. For immunoblot
experiments, seven day old ‘Dina-l ’ plants were inoculated on the cotyledons with
ZYMV-Ct. Both the cotyledons and one halfof the first true leaf were removed fi'om
five plants at 3, 4, and 5 dpi and stored at —80F. Ten days post inoculation (after the
appearance of the veinal chlorosis symptoms) the intact halves of the first leaves were

harvested for immunoblotting as described earlier. Both halves of each leaf were

37

immunoblotted and probed at the same time. Healthy ‘Dina-l ’ plants were used as
negative controls for each experiment while positive controls included plants fi'om which

cotyledons were not removed after inoculation.

Sequential inoculation experiments.

‘Dina-l ’ plants were inoculated on the cotyledons with ZYMV-Ct and ZYMV-
NAA when seven days old. Two to three weeks post inoculation (depending on growth
conditions during individual experiments) one halfof leaf-4 or leaf-5 from ZYMV-Ct
inoculated ‘Dina-l ’ plants was harvested and used to inoculate two susceptible squash
plants. The other half of the leaf was inoculated with ZYMV-NAA at the same time.
Healthy ‘Dina-l ’ plants were also inoculated on leaf-4l-5 with ZYMV-NAA. Two weeks
later one halfof leaf-7 and —8 were harvested and used to inoculate young squash plants.

Appearance of symptoms on squash plants was monitored up to 4 weeks post inoculation.

RESULTS

Symptom expression and virus accumulation in the resistant genotypes ‘Dina-l’ and
‘TMG-l’.

When cotyledons of 7-9 day old plants were inoculated with ZYMV, ‘TMG-l ’ plants
remained fi'ee of symptoms while ‘Dina-l ’ plants showed distinct veinal chlorosis on
leaf-1 or leaf-2 approximately 10 days post inoculation (dpi) (Figure 2B). Subsequent
leaves of ‘Dina-l ’ remained symptom free. This response was observed with a number

of isolates of diverse origin (Kabelka et al., 1997). The susceptible genotype, ‘Straight-

38

8’, responded with systemic mosaic symptoms. Two weeks post cotyledon inoculation,
‘TMG-l ’ plants did not show virus accumulation in cotyledons and leaves (Figure 2 and
Figure 3), while ‘Dina-l ’ plants showed high virus titer in the cotyledons and the
chlorotic leaf (leaf 1 or —-2) but not upper leaves, as detected by ELISA (Figure 3). On
rare occasions, TMG-l showed detectable levels of virus in the cotyledons at two weeks
post inoculation (data not shown).

Four to six weeks post inoculation (wpi), an additional one or two leaves often showed
detectable levels of virus accumulation and mild symptoms in ‘Dina-l ’. At that time
‘TMG-l ’ also showed virus accumulation in cotyledons and sometimes in the first
systemic leaf, indicating that resistance is not completely effective in preventing systemic
virus infection. ‘Straight-8’ showed systemic mosaic throughout the plants, however,
symptom severity varied on individual leaves.

When cotyledons of slightly older plants were inoculated (leaf-l unfolded),
‘TMG-l ’ remained symptom free, while ‘Dina-l ’ plants showed veinal chlorosis on leaf-
2 or leaf-3. These results indicated that the specific leaf that developed veinal chlorosis
varied with the age of the plant at the time of cotyledon inoculation. The mean leaf
position showing veinal chlorosis was 2.43 (n=16) for plants inoculated when 10-12 day
old vs. mean leaf position of 1.15 (n=20) for plants inoculated when 7-9 day old.
Regardless of whether 7-9 day or 10-12 day old plants were inoculated on the cotyledons,
in almost all the cases symptoms remained restricted to a single leaf. In analogous
experiments with ‘Straight-8’, when cotyledons of different ages were inoculated (Figure
4), symptoms and virus accumulation were first detected at progressively higher leaf

positions (i.e. in leaves that were newly expanding at the time of inoculation). When 7

39

 

Figure 2. Response of ‘TMG-l ’ (A), ‘Dina-l ’ (B), and ‘Straight-8’ (C) to inoculation
with ZYMV. Plants were inoculated on the cotyledons when 7 days Old and pictures
were taken 12 days after inoculation. ‘TMG-l ’ remained symptom fiee, ‘Dina-l’ showed
a distinct pattern of veinal chlorosis on the first systemic leaf, while ‘Straight-8’ showed
systemic mosaic throughout the plant. D., E., and F. Irnmunoblots of the first systemic
leaves of ‘TMG-l ’ (D) ‘Dina-l ’ (E) and ‘Straight-8’ (F) at 10 days post inoculation,
using ZYMV coat protein antiserum. ‘TMG-l ’ did not show detectable levels of virus
accumulation, ‘Dina-l ’ showed distribution of virus along the veins while ‘Straight-8’
showed uniform distribution throughout the leaf. Punch holes in the leaf (F) indicate that

smearing of leaf sap did not occur during blotting.

40

Absorbance Asorbance

Absorbance

times with similar results.

'2 * Straight-8

A —O—(-) Ctrl

  
 
  

   

03 l +Cotlnoc
" +TL1 lnoc
? —X—TL2 lnoc
04+
1
o t —+—-+-* +-~4—— -
Cot L1 L2 L3 L4 L5 L6
1.2 .- Dina-l
08 A
|
|
04 .
i
o L. -- . _- .
Cot L1 L2 L3 L4 L5 L6
12
lTMG-l
0.8
04 l

 

Cot L1 L2 L3 1.4 L5 L6
LeafNumber

Figure 3. Virus titer in the cotyledons (Cot) and true leaves (L) in ‘Straight-8’, ‘Dina-l ’
and ‘TMG-l ’ in response to inoculation with ZYMV-Ct. Young seedlings were
inoculated on the cotyledons (Cot lnoc), first (TLl) or second (TL2) true leaf. Virus titer
was measured by ELISA 14 days post inoculation. The X-axis shows the number of
leaves from the base to the top of the plant and the Y-axis shows absorbance at 405nm.

Each point represents the mean of 4 replications. The experiment was repeated three

41

7 Da 8
1°80 " y 10 Days

1.50 A / 16 Days
I

 

Absorbance
o
e
O

 

 

 

 

0.60 A
0.30 i
- M
0.00 ()Ctrl + i +-—- - l i #-
L1 L2 L3 L4 L5 L6
Leaf Number

Figure 4. Virus accumulation (as determined by ELISA) in different leaves of ‘Straight-
8’ as affected by plant age at the time of inoculation. Each point represents the mean of 5

replications. The experiment was repeated twice and similar results were observed.

42

day old plants were inoculated, the virus peaked in leaf-l (L1) first, when 10 day old
plants were inoculated, this peak moved to leaf-2 (L2) and to leaf-3 (L3) when 16 day old
plants were inoculated. Together these observations suggest that in both ‘Dina-l ’ and
‘Straight-8’ the virus moved from inoculated cotyledons to the predominant sink leaf at
the time.

The distribution of virus within the first leaf of ‘TMG-l ’, ‘Dina-l ’ and ‘Straight-
8’ was examined by immunoblotting with antiserum to the ZYMV coat protein at 10 dpi
(Figure 2B). No virus accumulation could be detected in ‘TMG-l ’ while in ‘Dina-l ’ the
virus appeared to be present along the veins. Non-symptomatic leaves of ‘Dina-l ’ did
not show virus accumulation (data not shown). ‘Straight-8’ showed uniform distribution
of virus throughout the leaf. These results suggest that the veinal chlorosis pattern in
‘Dina-l ’ plants corresponds to virus distribution within the leaf, and that the symptomless
phenotype of ‘TMG-l ’ is associated with the absence of virus accumulation.

If sampled at an earlier time (e.g. 5 days post inoculation) (Figure 5), ‘Straight-8’
also showed a predominantly veinal pattern of virus distribution, but at 10 dpi there was
more uniform distribution, throughout the leaf, indicating virus exit fi'om the phloem and
subsequent cell-to-cell spread. ‘Dina-l ’ plants, on the other hand, showed very low
levels of virus in the first leaf at 5 dpi and a veinal distribution at 10 dpi that continued up
to 30 dpi (Figure 5).

Unlike the response to cotyledon inoculation, when the first or second leaves were
inoculated, ‘Dina-l ’ plants remained symptom fi'ee and did not show virus accumulation

(Figure 3). Occasionally very mild veinal chlorosis symptoms were observed (on leaf-3)

43

Mannie leaf

 

 

mm.
Sdpi 10dpi 14de
S .1 S
(I , . ‘ '
'\ A :- I-J'flf'x: f ,1 1:31 ‘V _ 1‘
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Figure 5. A. Time course experiments showing the accumulation of ZYMV in the first
systemic leaf of “Straigh-8” (top) and ‘Dina-l ’ (bottom) leaves at five, ten and thirty days
post inoculation. Plants were inoculated on the cotyledons when seven days old.

B. Inoculated first leaf immunoblotted at two weeks post inoculation.

when leaf-1 was inoculated (in the greenhouse: 12%; 8/64), while no symptoms were
observed when leaf-2 was inoculated (0/30). ‘TMG-l ’ plants also remained symptom
and virus free, while ‘Straight-8’ plants showed typical systemic mosaic and virus
accumulation. Immunoblots showed accumulation of virus along the veins in the
inoculated leaves of ‘Dina-l ’ and a more uniform distribution in the inoculated leaves of
‘Straight-8’ (Figure 5). Virus accumulation in the inoculated leaves Of TMG-l could not
be detected with immunoblotting (data not shown). The observed differences in the
appearance of veinal chlorosis phenotype following true leaf vs. cotyledon inoculation
suggested tlmt the resistance response in ‘Dina-l ’ plants is tissue specific or
developmentally regulated. We sought to investigate the nature of the block observed in

the true leaves, and the timing of the expression of resistance.

Nature of the block to successful virus infection in the resistant genotypes.
Limitation of veinal chlorosis to a single leaf following cotyledon inoculation in
‘Dina-l ’ plants, localization of virus along the veins, and failure to see symptoms or virus
accumulation (Figure 3) with leaf inoculation, raised the possibility that the virus my not
be able to replicate in the leaves. The veinal chlorosis phenotype would thus result fiom
the limited supply of virus transported from the cotyledons to the predominant sink leaf.
Other leaves either do not receive virus, or import only a limited quantity, which is
insufficient to cause the veinal chlorosis phenotype.
To investigate virus replication in the true leaves, the inoculated cotyledons were

removed at various time intervals afier inoculation, and the plants were scored for veinal

45

chlorosis and virus accumulation with ELISA and immunoblotting. Although virus titer
was not detectable in the cotyledons or leaves at 1, 2, or 3 dpi (Figure 6A), plants fi'om
which the cotyledons were removed at 2 or 3 dpi showed veinal chlorosis and high virus
titer in leaf-1 (or some cases on leaf-2) at 14dpi (Figure 6B). This indicated that virus
replication had occurred in the absence of cotyledons.

Virus replication in ‘Dina-l ’ plants in the absence of cotyledons was further
confirmed by immunoblot experiments (Figure 6C). One half of leaf-l and the
cotyledons were removed at 3dpi, the other half of leaf-1 was removed 7 days later when
symptoms appeared. Both the halves of leaf-1 were immunoblotted at the same time
using coat protein antiserum. No virus accumulation could be detected in the halfleaf
removed at the time of cotyledon removal while the other half of the same leaf (sampled
seven days later) showed detectable levels of the virus (Figure 6C). Together these
results suggested that virus replication in ‘Dina-l ’ plants can occur in the absence of
cotyledons, and that the block to systemic spread of virus in ‘Dina-l ’ is occurring at the

level of movement.

Nature of leaf specific expression of the resistance response in Dina-1.

The observed response, replication in and movement from the cotyledons,
followed by restricted movement of the virus after reaching the true leaf might be due to
inherent structural differences between leaves and cotyledons, developmental changes in
the host, or a delay due to time required for induction of a resistance response. If the

delay in observed resistance following cotyledon inoculation was due to an induced

46

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Figure 6. Virus titer in the cotyledons and true leaves of ‘Dina-l ’, at the time of removal
of cotyledons (A) and 14 days post inoculation (B). X-axis shows days post inoculation
(DPI) at which cotyledons were removed. Y- axis shows absorbance at 405nm. The
Data were normalized by subtraction of background ELISA readings. COT = cotyledon,
L1 and L2 = leaf-1 and leaf-2 from the base of the plant. Each column represents the
mean of five replications. C. and D. Inununoblots showing virus replication in the true
leaves of ‘Dina-l ’ plants. Plants were inoculated on the cotyledons with ZYMV-Ct when
7 days old and the cotyledons and one halfof the first leaf were removed 3 days post
inoculation (C). The other halfof the leaf was removed 10 dpi when veinal chlorosis
symptoms appeared (D). Both halves of the leaf were immunoblotted at the same time
with ZYMV coat protein antiserum. Both the experiments were repeated three times with
similar results.

47

response, a similar pattern of symptom expression would be expected following true leaf
inoculation, as was Observed with cotyledon inoculation. However, as indicated earlier,
when ‘Dina-l ’ plants were inoculated on the leaves, veinal chlorosis generally was not
observed suggesting that in the true leaves the resistance mechanism is active prior to
inoculation.

Another characteristic of induced resistance is protection against secondary
inoculation. This possrbility was tested using the NAA isolate of ZYMV (Gal-On et al.,
1991, 1992) that did not cause veinal chlorosis or systemic symptoms, but occasionally
showed limited systemic movement as detected by ELISA (data not shown), or more
frequently by the more sensitive assay of back inoculation on to susceptible squash plants
(Table 2). When cotyledons of 7 day old ‘Dina-l ’ plants were inoculated with ZYMV-Ct
typical veinal chlorosis symptoms were observed on leaf-1 or leaf-2 at 10 dpi. However,
less than 3% (1/ 36 plants) of ‘Dina-l ’ plants, contained ZYMV-Ct in leaf-4 or —5 when
tested by back inoculation onto susceptible squash plants (Table 2A). As observed
previously, healthy ‘Dina-l ’ plants inoculated with ZYMV-Ct on leaf-4 or —5 did not
show veinal chlorosis, and only 2/20 plants (5%) tested had virus in leaf-7 or —8 based on
back inoculation. In contrast, following inoculation with ZYMV-NAA on leaf-4 or —5,
72% of the inoculated plants (32/40) showed virus accumulation as detected by back
inoculation of squash plants (Table 2B).

To test whether the ZYMV-Ct inoculated plants exhibited induced protection
against secondary inoculation, ‘Dina-l ’ plants showing veinal chlorosis on leaf-1 in
response to ZYMV-Ct, were inoculated with ZYMV-NAA on leaf-4 or -5 (Table 2C).

Half of leaf-4 or —5 was removed for back inoculation to squash (Table 2A) the other

48

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49

half was inoculated with ZYMV-NAA. Two weeks post secondary inoculation with
ZYMV-NAA, susceptible squash plants were back inoculated fi‘om leaf-7 or leaf-8 of
‘Dina-l ’. Similar to the results with ZYMV-NAA alone (72%: 32/40), a large percentage
of the plants showed systemic infection with ZYMV-NAA afier secondary inoculation
(52%; 22/40). Thus, prior inoculation with ZYMV-Ct did not prevent subsequent
infection by ZYMV-NAA. This provides further evidence against an induced resistance
response.

Finally, environmental conditions appeared to influence the appearance of the
veinal chlorosis on ‘Dina-l ’ plants following true leaf inoculation. When experiments
were performd in the growth chambers instead of the greenhouse, 81% of the plants
( 13/ 16) showed veinal chlorosis following the first true leaf inoculation. Inoculation of
leaf-2, however, resulted in a greatly reduced number of plants showing veinal chlorosis
(19%; 3/16 plants). These observations argue against inherent structural differences in
the leaves preventing virus spread, and instead suggest developmentally controlled

expression of resistance.

Role of the coat protein of ZYMV in the veinal chlorosis response of Dina-l.

Data presented so far indicated that ‘Dina-l ’ plants interfere with the movement
of ZYMV. Since the multifunctional CP of potyviruses is also involved in virus
movement (Dolja et al., 1994. 1995; Rojas et al., 1997) and host defense responses
(Andersen and Johansen, 1998; chapter 3), we decided to investigate the role of the

ZYMV CP in the resistance response of ‘Dina-l ’ plants. The conserved core region of

50

the CP is involved in cell to cell movement, while the highly variable, surface exposed
amino terminus of the CP is essential for systemic movement and is suggested to play a
role in host adaptation and host range determination (Dolja et al., 1994, 1995; Shukla et
al., 1994). The carboxy terminal (CT) portion of the CP also is essential for long distance
movement. This known separation of the cell to cell and systemic movement fimctions
of the CP allowed us to further dissect the potential role of CP in the resistance response
of ‘Dina-l ’.

Chimeric ZYMV-NAA infectious constructs were made containing either the NT
of the CP (CtNTFL/P) or the core and carboxy terminal portions of the CP (CtCoreFL/P)
of ZYMV-Ct (Figure 7). Both constructs produced systemic symptoms on susceptible
squash and cucumber plants. When tested on the resistant genotype ‘Dina-l ’, the
chimeric construct containing the NT of CP of ZYMV-Ct (pCtNTFL/P) showed veinal
chlorosis on either leaf one or two of approximately 58% (25/43) of the inoculated plants
while CtCoreFL/P and ZYMV-NAA inoculated plants remained largely symtomless
(Figure 7) and only a few plants showed veinal chlorosis. These results suggest that the

NT of the CP of ZYMV plays a role in the veinal chlorosis response of ‘Dina-l ’ plants.

DISCUSSION

Resistance to ZYMV infection in ‘Dina-l ’ plants is controlled by a single

recessive allele (zymDm) that confers an unusual phenotype. When cotyledons of young
plants were inoculated with ZYMV, a distinct veinal chlorosis pattern developed on the
first or second leaf. Subsequent leaves remained symptom fiee, and inoculation of leaves

generally, did not result in veinal chlorosis. Immunoblot analyses revealed that the

51

a ”infib‘»

 

 

 

(1 P1 HC-Pro P3 CI 6K VPg le Nlb e,» /
l
I I l I L I l l r I ll—A(n)
YMV-NAA iIdT
pl (V Veilnl Cholmsis

 

l_ I l ] ll“

  

 

25/43

 

4/42

 

 

Isolate Veini Cholroels
Z-Ct scror’rv 5 man - macs G EKTVAAVTKDKD +
Z-NAA A S -

Figure 7. Infectious ZYMV-NAA constructs with the engineered Psi] site (pZCPFL/P),
the NT of the CP of ZYMV-Ct (pCtNTFL/P), the core portion (pCtCoFL/P) of the CP of
ZYMV-Ct. All the constructs were infectious on susceptible squash and cucumber

plants. The veinal chlorosis response of ‘Dina-l ’ to these constructs is indicated at the

right. Comparison of the amino acids sequence of the CP-NTs is shown at the bottom.

52

pattern of veinal chlorosis reflected the pattern of virus distribution within the leaf. In
leaves showing veinal chlorosis, virus accumulation was localized along the veins; non-
symptomatic leaves did not show detectable virus accumulation. In susceptible ‘Straight
8’ plants, virus was initially associated with the veins (at 5 dpi), but by 10 dpi the virus
was more uniformly distributed throughout the leaf. In ‘Dina- 1' plants, the virus
remained largely restricted to the veinal regions even at 30 dpi.

Age of the plant at the time of inoculation influenced which leaf showed veinal
chlorosis. When older plants were inoculated, veinal chlorosis appeared at progressively
higher leaf positions. Analogous patterns showing a relationship between plant age at the
time of inoculation and first leaf to accumulate virus, were observed with the ‘Straight 8'
plants. This suggested that in both ‘Straight 8' and ‘Dina-l', the virus replicated in the
inoculated cotyledons, and, as is typical for virus infection (Leisner and Turgeon, 1993;
Andrianifahanana et al., 1997), was then transported to the predominant photosynthetic
sink at the time. Consistent with movement from the cotyledons to the predominant sink,
the observed pattern of veinal chlorosis and virus accumulation closely resembled the
pattern observed for phloem unloading of carboxyflorecein molecules in sink leaves
(Roberts et al., 1997). Chlorosis was predominantly observed along class II and HI veins,
which are the main sites of phloem unloading (Roberts et al., 1997; Oparka and Turgeon,
1999).

Thus, the observed ‘Dina-l' phenotype includes initial infection of the cotyledons
followed by restriction to veinal regions of the systemic sink leaf, and in most cases

inability to establish infection when directly inoculated onto leaves. Localization of virus

53

to the veinal regions indicated that the virus was either unable to unload from the phloem,
or to replicate or move cell to cell following initial unloading in the leaf. Failure to
observe the resistance response in the cotyledons could be due to structural difl'erences
between cotyledons and leaves, induction of resistance in the leaves, or developmental
regulation of the resistance response.

Although an inability to replicate in the leaves could explain the observed veiml
chlorosis phenotype (a limited amount of virus is exported fi'om the cotyledons to the
leaves), cotyledon removal experiments indicated that ZYMV could replicate in ‘Dina- 1'
leaves. When the cotyledons were removed at 2-3 dpi, detectable levels of virus were not
present in the leaves or cotyledons, yet measurable virus accumulation was observed in
the leaves one week later. Similarly, in growth chamber experiments, veinal chlorosis
symptoms following inoculation of the first true leaf were observed even when
cotyledons were removed before inoculation, confirming that the block to systemic
infection was not resulting fi'om inability of ZYMV to replicate in ‘Dina-l' leaves, and
instead indicates a restriction in virus movement.

Several lines of evidence favor a block in long distance movement rather than cell
to cell movement. The pattern of veinal chlorosis and localization of virus to the veinal
regions suggests a block in phloem unloading, and immunoblots of inoculated leaves that
did not lead to subsequent veinal chlorosis, showed accumulation of virus adjacent to the
veins. This indicates movement of the virus to the veinal regions, followed by inability
to load into the veins. Consistent with this Observation, Schaad and (hrrington (1996)

observed that in the resistant tobacco line V20, which interferes with long distance

54

movement of TEV, virus accumulation in the inoculated leaves appeared as an apparent
tracking along the primary veins.

In cucurbits the phloem is bicollateral in nature (Schmitz et al, 1987). The
adaxial phloem consists of sieve elements (SE), and companion cells (CC) separated by a
single tracheid and a single vascular parenchyma (VP) cell. Unlike CC of the adaxial
phloem, the CC3 of the abaxial phloem are modified into intermediary cells,
characterized by extensive plasmodesrnatal connections with the adjacent bundle sheath
(BS) cells. Larger veins contain many more xylem and phloem elements and are
associated with ordinary CC (Schafl‘er et al., 1996). Thompson et al. (1998) observed
functional differences between PD connecting mesophyll cells and those connecting
mesophyll cells with BS cells in cucumber plants. Using a pseudorecombinant cucumber
mosaic virus strain (FFT), they observed that FFT infection was arrested at the BS cells
and was not detected in intermediary or other phloem cells suggesting that the BS-
phloem interface is the boundary for systemic movement. It is also possible that ‘Dina-l ’
plants restrict movement of ZYMV in the leaves at the BS-phloem interface in an
analogous fashion by preventing exit fi'om the phloem when the cotyledons are
inoculated or entry into the phloem when the leaves are inoculated.

Further evidence for a block in long distance movement in ‘Dina-l' leaves comes
from the use of chimeric ZYMV viruses with switches in the coat protein (CP) amino
terminus (NT). The conserved core portion of the potyviral CP is involved in the
encapsidation of viral RNA and cell to cell movement of the virus while the variable
surface exposed N- and C-temiinal portions are required for systemic movement (Dolja et

al. 1994, 1995; Shukla et al. 1994; Rojas et al. 1997). When the NT of the CP of the

55

infectious construct of ZYMV-NAA, which did not produce veinal chlorosis on ‘Dina-l'
plants, was substituted for the respective region of the CP of ZYMV-Ct, the chimeric
construct induced veinal chlorosis on ‘Dina-l' plants. Substitution of the core and CT
portion of the CP did not cause a veinal chlorosis response. This suggests that the CP-NT
is involved in the differential response of ‘Dina-l' to these two isolates of ZYMV, and
firrther suggests that the block to successfiil infection in ‘Dina-l' is occurring at the level
of systemic movement. It appears that the ZYMV-NAA isolate is able to partially escape
the block to systemic movement. Like ZYMV-Ct on ‘Dina-l ’ the virus distribution of
ZYMV-NAA within the inoculated and systemic leaves shows a veinal pattern (data not
shown); however, ZYMV-NAA can be detected in systemic leaves by back inoculation
onto susceptible squash plants. Comparison of the amino acid sequence of the CPs of
ZYMV-Ct and ZYMV-NAA shows two amino acid differences (Ser7 to Ala and Gly32
to Ser) located in the N-terminal region. Thus it is possible that the CP-NT is involved in
interaction with the host resistance factor, and that the amino acid differences between
the CP-NTs of these two isolates influences this interaction.

The observation of resistance in leaves but not cotyledons of ‘Dina-l' might result
from inherent structural differences between the two tissues. On some occasions,
however, inoculation of leaf lresulted in mild chlorosis on leaf 3 in the greenhouse, and
in the growth chamber, a majority of the plants showed veinal chlorosis following
inoculation of the first leaf. These observations argue against structural differences in the
leaves vs. cotyledons. On the other hand, chlorosis was not observed following

inoculation of second or third leaves in the greenhouse, and a much lower percentage of

56

plants in the growth chamber (19%) showed symptoms when the second leaf was
inoculated, indicating developmentally regulated expression of the resistance factor(s).

‘Dina-l ’ plants do not appear to exhibit either a classical recovery phenotype or
induced resistance. Recovery of susceptible plants fiom virus infection has been
observed for several viruses including potyviruses and is associated with a gradual
reduction in symptom severity and protection from secondary infection by strains of the
same virus or homologous viruses (Pennazio et al., 1999). We have observed a similar
recovery phenotype and protection against secondary virus infection in susceptible
cucumber and squash plants in response to inoculation with a chimeric ZYMV containing
the amino terminus of the coat protein of a non-cucurbit potyvirus, TEV (chapter 3). The
veinal chlorosis phenotype in ‘Dina-l ’ plants, however, differed fi'om virus induced
recovery response in susceptible plants in that the recovery was not gradual, symptoms
were almost always restricted to a single leaf, non-symptomatic leaves did not show
detectable virus accumulation, and were not protected against secondary virus infection.
Similarly the failure of ZYMV-Ct inoculated plants showing veinal chlorosis to be
protected against subsequent infection by ZYMV-NAA, and the generally constitutive
resistance of ‘Dina-l ’ leaves to ZYMV-Ct infection argues against an induced resistance
response.

Collectively these results suggest developmental control of a resistance factor that
restricts systemic ZYMV infection at the level of loading or unloading from the phloem.
This may be contrasted with resistance conferred by the allele in ‘TMG-l' plants, zymTMG,
which appears to be expressed earlier in development. Although not generally apparent

by veinal chlorosis, developmental differences in expression of resistance have been

57

observed in other cucurbit potyvirus-interactions (Gilbert-Albertini, 1993; Gibb et al.,
1994; Wai et al., 1995b). For example when cotyledons of muskmelon (Cucumis melo)
cultivar ‘Cinbo’ were inoculated with PRSV-W mild systemic symptoms were observed,
but plants remained symptomless when true leaves were inoculated (Gibb et al., 1994).
Limited systemic movement of WMV and ZYMV to the first one or two leaves also has
been observed in Cucurbita moschata line Menina 15 (Gilbert-Albertini et al, 1993). In
‘TMG-l ’ two independently assorting resistance factors to WMV were identified, one
expressed in both cotyledons and leaves and the other expressed only in the leaves (Wai
and Grumet, 1995b). It will be of interest to determine whether similar mechanisms or
resistance gene products are involved in each of these examples.

The differences in developmental expression of the zymD'm locus, and interaction
with the ZYMV CP-NT may provide approaches to allow for cloning and further
characterization of the zym locus. Tight linkage of this locus to resistance against other
potyviruses could lead to cloning and characterization of other resistance factors, and
thereby provide insights into plant potyvirus interaction and systemic movement in

cucurbits.

Future Directions

We have shown in this project that resistance in Dina-l plants is occurring at the
level of phloem loading or unloading, and that the amino terminus of the coat protein
plays a role in the veinal chlorosis response. The specific cellular location of ZYMV in
the chlorotic veins remains to be determined. Although the viral CP determinant

involved in the veinal chlorosis response was identified, other regions of the viral genome

58

were not tested. Cloning of the zym gene firom ‘Dim-l ’ or ‘TMG-l ’ plants will be the
ultimate target for any future project and will certainly reveal valuable insights into our

understanding of the potyvirus resistance in cucumbers.

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Leisner, S. M., Turgeon, R, and Howel, S. H. 1992. Long distance movement of
cauliflower mosaic virus in infected turnip plants. M01. Plant-Microbe Interact.
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Leisner, S. M., and Turgeon, R 1993. Movement of virus and photoasimilate in the
phloem: A comparative analysis. BioEssays 1:741-748.

Mahajan, S. K., Chisholm, S. T., Witham, S. A., and Carrington, J. C. 1998.
Identification and characterization of a locus (RTMl) that restricts long-distance
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Martin, A. M, Poh, H. L. C., Herrera, D. M., and Ponz, F. 1998. Resistance to turnip

mosaic potyvirus in Arabidopsis thaliana. Mol. Plant-Microbe Interact. 12:1016-
1021 .

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Masuta, C., Nishimura, M., Morishita, H., Hataya, T. 1999. A single amino acid change
in viral genome-associated protein of potato virus Y correlates with resistance
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Murphy, J. F., Blauth, J. R, Livingstone, K. D., Lackney, V. K., and Jahn, M. K. 1998.
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Nicolas, O., Dunnington, S. W., Gotow, L. F., Pirone, T. P., and Hellmann, G. M. 1997.
Variation in the VPg protein allow a potyvirus to overcome va gene resistance in
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Oparka, K. J. and Turgeon, R 1999. Sieve elements and companion cells-traffic control
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Pennazio, S., Roggero, P., Conti, M. 1999. Recovery of plants from viral diseases:
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Provvidenti, R 1987. Inheritance of resistance to a strain of zucchini yellow mosaic
virus in cucumber. HortScience 22:102-103.

Provvidenti R, and Hampton, R O. 1992. Sources of resistance to viruses in Potyviridae.
Arch. Virol. 5:189-211.

Rajamaki, M. I., and Valkonen, J. P. T. 1999. The 6K2 protein and the VPg of potato
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Capsid protein and helper component-proteinases fimction as potyvirus cell to cell
movement proteins. Virology 237:283-295.

62

Schaad, M. C., and Carrington, J. C. 1996. Suppression of long-distance movement of
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Critical Reviews Plant Sci. 9:443-455.

63

CHAPTER III

TESTING THE ROLE OF THE AMINO TERMINUS OF THE COAT PROTEIN
IN SYSTEMIC INFECTION AND HOST RANGE DETERMINATION OF

POTYVIRUSES

ABSTRACT

The role of the amino terminal (NT) region of the potyviral coat protein (CP) in systemic
infection and host range determination was investigated. Chimeric zucchini yellow
mosaic virus (ZYMV) constructs were made by substitution of the native CP-NT with the
CP-NTS of watermelon mosaic virus (WMV; overlapping host range with ZYMV) and
tobacco etch virus (TEV; non-overlapping host range with ZYMV). Both the constructs
produced strong initial symptoms on ZYMV-susceptible cucurbit plants and were aphid
transmissible. A gradual reduction in symptom severity was observed in squash and
cucumber plants infected with TEV CP-NT chimeric virus. Four to six weeks post
inoculation the newly developing leaves were either symptomless or showed markedly
reduced symptoms. The recovery phenotype was specific to the TEV CP-NT chimeric
virus: 1) did not result from a mutation in the virus, 2) provided protection from
secondary virus infection in a sequence specific manner, and 3) showed characteristics
associated with virus induced post transcriptional gene silencing (PTGS). The chimeric
viruses did not overcome naturally occurring resistance to ZYMV in the resistant
cucumber line ‘TMG-l ’ or ZYMV coat protein-mediated resistance in transgenic melon

plants. The host range OfZYMV also was not affected by these substitutions, and the

chimeric viruses did not systemically infect bean, N. benthamiana, or tobacco plants.
Together these results show that despite substantial variability in the length and sequence,
the NTs of CPS of heterologous potyviruses can facilitate systemic infection of ZYMV in
susceptible cucurbit hosts. However, this substitution per se is not sufficient to modify
the host range of the virus. The CP-NT fi'om a non-pathogenic potyvirus could trigger a

host defense response in cucurbits resulting in recovery fi'om systemic virus infection.

INTRODUCTION

Potyviral coat proteins (CPS) are multifunctional proteins involved in several
stages of the viral life cycle. Approximately 2000 CP monomers assemble to encapsidate
the 10 kb single stranded RNA genome into long flexuous rod shaped particles (Shukla et
al., 1994; Jagadish et al., 1991,1993). Potyviral CPs have been shown to be involved in
cell to cell movement through the plasmodesmata (Dolja et al., 1995; Rojas et al., 1997),
long distance movement through the phloem (Dolja et al., 1994, 1995), aphid mediated
transmission (Pirone and Blanc, 1996), and possibly host mediated defense responses and
host range determination (Shukla et al., 1994; Solomon, 1989; chapter 2). The CP is
composed of variable amino and carboxy-terminal (NT and CT) regions and a highly
conserved core portion (Shukla et al., 1994). Both N- and C-terminal regions are surface
exposed and can be cleaved by protease treatment without affecting virion morphology
(Shukla et al., 1988; Shukla and Ward, 1989).

The domains of the CP involved in various functions have been identified. The
conserved core region is involved in encapsidation of RNA and cell-to-cell movement of

the virus (Dolja et al., 1994, 1995; Jagadish et al., 1993). A tobacco etch virus (TEV)

65

mutant with a substitution of the highly conserved Ser122 residue (S122W) within the
core domain was confined to single cells in tobacco plants (Dolja et al., 1995). The cell-
to-eell movement defect was restored efficiently in transgenic plants expressing the wild
type CP gene. Mutations in the core region of the CP of bean common mosaic necrosis
(BCMNV) and lettuce mosaic (LMV) potyviruses afl‘eeted cell to cell movement in
microinjection studies (Rojas et al., 1997). Both the NT and CT of the CP are not
essential for the assembly of virions, but are required for systemic movement (Shukla et
al., 1988; Jagadish et al., 1991; Dolja et al, 1994, 1995). Deletions of the N- and C-
terminal regions affect long distance movement of the virus (Dolja et al., 1994, 1995),
and certain amino acid substitutions in the conserved DAG motif in the NT of CP of
tobacco vein mottling potyvirus (TVMV) abolished the ability of the mutant viruses to
cause systemic infection, but did not affect replication and virion assembly in protoplasts
(Lopez-Moya and Pirone, 1998).

The CP in concert with the viral encoded helper component-proteinase (HC-Pro)
is also involved in the aphid transmission of potyviruses. The conserved DAG motif in
the NT of the CP interacts with conserved motifs in the HC-Pro gene to facilitate aphid
transmission (Blane et al., 1997; Peng et al., 1998; Thombury et al., 1990). In binding
assays, the DAG motifin the CP and the PTK motifin the HC-Pro were shown to be
involved in CP-HC-Pro binding (Blane et al., 1997; Peng et al., 1998). If the virus is
maintained by rub inoculation rather than aphid transmission, there is a fi'equently
observed loss of aphid transmissibility that is associated with amino acid substitutions in
the CP-DAG motif (Rajamati et al., 1998; Gal-On et al., 1992; Atreya et al., 1990, 1991)

or motifs within the HC-Pro (Pirone and Blane, 1996; Llave et al., 1999; Legavre et al.,

66

1996, Grumet et al., 1992). Both CP and HC-Pro are also involved in virus movement
(Revers et al., 1999), suggesting that the CP-HC-Pro interactions might have implications
beyond aphid transmission.

Viral CPS, and in the case of potyviruses, the NTs of CPS may also be involved in
interaction with the host leading to resistance responses or host range determination. In
Ipomea nil plants showing recovery fi'om virus infection Solomon et al. (1989) observed
a proteolytic activity that cleaved the NT of the CP of sweet potato feathery mottle
potyvirus (SFMV) and we have shown that the NT ofthe ZYMV CP is involved in the
veinal chlorosis phenotype of the resistant cucumber genotype ‘Dina-l ’ (chapter 2). The
CPS of brome mosaic (BMV) and alfafa mosaic (AlMV) bromoviruses and cucumber
mosaic cucumovirus (CMV), which are also involved in virus movement, have been
shown to play a crucial role in host range determination (Misc et al., 1993; Spitsin et al.,
1999; Ryu et al., 1998). Potyviruses can vary a great deal in the breadth of their host
ranges; some (e.g., tobacco etch virus, TEV) infect only a limited number of plant species
while others (e.g., watermelon mosaic virus, WMV) infect a wide variety of species
(Brunt et al., 1996; Shukla et al., 1994). It has been suggested that the extreme variability
in the NT of the CP might be involved in the host range determination of the virus
(Shukla et al., 1994). A correlation between the length of the NT of the CP and the host
range of different isolates of sugarcane mosaic virus (SCMV) was observed by Xiao et al.
(1993).

In this project we sought to further understand the role of the NT of potyviral CPS
in facilitating systemic infection and host range determination. Chimeric ZYMV viruses

were created by exchanging the NT of the CP of ZYMV with the respective regions of

67

the CP of either TEV (non overlapping host range with ZYMV) or WMV (overlapping
but broader host range) and tested for infectivity on a range of plant species. Comparison
of the NT of the CP of these three potyviruses shows only 13 amino acid (a) identity
(Figure 8). Theamino terminusoftheCPofTEV isonly29aalong comparedto44aaof
ZYMV and 46 a of WMV. For WMV and ZYMV, which have overlapping host

ranges, there is only 50% aa identity between the CP-NTs. Our results indicate that
despite limited homology, NTs of the CPS of heterologous viruses facilitated systemic
infection, but failed to modify the host range of the chimeric viruses. Interestingly, the
NT of the CP fi'om the non-overlapping host range virus TEV appeared to play a role in

induction of host defense responses.

MATERIALS AND METHODS

Engineering cloning sites into the infectious ZYMV construct.

Two restriction sites (Pstl and Kpnl) were introduced in the infectious ZYMV-
NAA construct (Gal-On et al., 1991, 1992) to facilitate substitutions in the coat protein
region (Figure 9). The PstI site was introduced at the NIb-CP junction by PCR
amplification (using Vent DNA polymerase with proof reading activity, NE Biolabs Inc.
Beverly, MA) of the CP gene and the l93bp of the 3’NTR using NIb-CP junction primer
RG81(5’-ATGCTGCAGTCAGGCACCAGCCA-3’;(restriction site is underlined) and 3’
NTR primer RG40 (5’-AGTGAATTCTCGAGCTTATTCGTGA-3’) including an
engineered EcoRI and a natural XhoI site with a single base overlap. The amplified

fragment was digested with PstI and XhoI and cloned into PstI-Xhol digested pBlueScript

68

ZYMV SG TQP TV A ------ DAG AT ----- KD KB DDK GKNKDVTGS ------ GSSE
WMV SG KE - TV ENL--- DAG KES-K KD AS DK G NKPQNSQVGQGSKE
TEV SG ------ TV DAGA DAG K----K KD QKD DK VAEQ

 

ZYMV —KT VAAVT KD—-KD
WMV PTKT-GT---VS KD
TEV AS KD --RD

Figure 8. Alignment of the amino terminal portions of the CP of ZYMV(NAA), WMV
and TEV. Amino acids showing sequence identity among the three viruses are shown in
bold letters. Amino acids showing sequence identity between ZYMV and WMV only are
italicized.

 

69

4a,,”

a s a
of 4" $831-me

r)” [11ng minim (I,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pZYMV-NAA (Wild Type) 5"“:
a is:
f I l l J Strong
pZNAAFL/P ‘_ ‘_
so 40
f: I
a a.
n3 41!; ..
pZNTFl/P [ I I J 1 Strong
4— <—
49 40
plCtNTPFI/P I r‘ I I I Strong
8;,
pWNTPFL/P I r... ‘11.: J 1 Strong
‘—
46
89+
p'l'NTFl/P l I I l I Mild/Recovery
‘—

Figure 9. Chimeric constructs of ZYMV-NAA with the introduced changes in the NT of
the CP gene and their infectivity on susceptible squash plants. The introduced restriction
sites are shown at the top of the control constructs. Shaded areas indicate the NT of the
CP swapped with the respective regions of ZYMV-CT (pCTNTFL/P), WMV

(pWNTFL/P), or TEV (pTNTFL/P) using the introduced restriction sites.

70

KS (Stratagene, La Jolla, CA) to produce pZPX. To clone the lkb 3’ fragment of the NIb
gene, the forward primer (RG69: 5’-GGTCATACG TATGATGTG-3’) priming at
position 7280 in the ZYMV genome and the reverse primer (RG80 5’-TGA_C_3_TGC_A_G_CA
TTACAGTGCTCC-3’) introducing a PstI Site at the NIb-CP junction were used. The
PCR product was cloned into pZPX digested with Sac] (a unique restriction site in the
NIb) and PstI resulting in the construct pZCPSPX. pZCPSPX contained the 2kb, 3’
fiagment of the infectious ZYMV-NAA construct with an engineered PstI site at the NIb-
CP junction. The engineered PstI site resulted in substitution of two bases (CTCCAA to
CTGCAG), but did not change the amino acid sequence.

The KpnI site was introduced near the beginning of the core portion of the CP
(position 8691: amino acid motif NAGSH) resulting in a substitution from Ser at position
50 in the CP to Thr. The amino acid sequence at this position in the CP of potyviruses is
not highly conserved (Shukla et al., 1994). The core and CT portion of the CP (including
193 bp of the 3’NTR) were amplified using RG41 (5’-GCTGGTACCCATGGGAAA
ATTGTG-3’ including a Kpnl Site) as forward primer (priming at position 8691) and
RG40 (3’ NTR with EcoRI site), and cloned into pUC119 (Stratagene) digested with
Kan] and EcoRI resulting in pZNTKE. The NT of the CP of ZYMV-NAA was amplified
using RG81 as forward primer and a reverse primer, RG49 (5’-ATGGGTACCAGCAT
TACATCCTTGT-3’), designed to prime 15bp in the core region of the CP (position
8691) and introduce a Kpnl site. The PCR product was digested with PstI and K0121 and
cloned into pZNTKE resulting in the construct pZNTPKE. The resulting construct

pZNTPKE contained the entire CP and an engineered Kan] site at the beginning of the

71

CP core region. pZNTEKP was digested with Pstl and Aer (a naturally occurring
unique restriction 15bp from the CP stop codon), and the CP cloned into pZCPSPX to

form pZNTSPX. Both the constructs (pZCPSPX and pZNTSPX) were confirmed by

sequencing and restriction enzyme analyses.

Substitutions in the CP region.

The WMV construct used as a template was kindly provided by Dr. Hector
Quemada (Quemada et al., 1990). The NT of the WMV CP was amplified by using
primers RG 82 (5’-TCTCTGCAGTCAG GAAAAGAAACAGTT-3’ with PstI site) and
RG46 (5’-TTTG MAGCATTTACATCCTT GCT-3’ with KpnI site) and cloned
into PstI- KpnI digested pZNTSPX to make in pWNTSPX. The NT of the CP of TEV
was amplified fiom the construct (Allison et al., 1986) using primers RG83 (5’-TATfl
_C_A_(_iAGTGGCACTGTGGAT-3 ’ with PstI Site) and RG51 (5’-TGA@TACCAGCATT
AAC ATCCTT ATCCTT-3’ with KpnI site) and cloned as PstI-Kvn! fi'agment into
pZNTSPX to form pTNTSPX. No amino acid changes were introduced into the NT of
the CP of WMV or TEV by PCR The entire CP of WMV was amplified by using RG82
as forward primer and RG58 (5’-AACCCTAGGCAGTTT ACCTAGTCTTTA-3’) as
reverse primer designed to prime 15bp downstream fi'om the CP stop codon and
introduce an AvrII site. The PCR product was cloned into pZCPSPX resultng in
pWCPSPX. The core and CT portion of the CP of WMV was PCR amplified by using
RG84 (5’-GTTGGTA CCAAAGGAAAAGAAGTCCCA-3’) as a forward primer which
introduced a KpnI site 15bp in the core and RG58 as a reverse primer and cloned into

pZNTSPX to make pWCoSPX.

72

The CP and the core and CT portions of the CP of TEV were cloned analogously
using primer pairs RG 83 (as a forward primer) and RG57 (5’-GAATCTAGAGACACG
CAGAAACTATCA-3’), and RG85 (5’-GCTGGTACCTCAGG AACAT TCTC-3’
introducing Kpnl site) and RG57 respectively and resulting in the constructs pTCPSPX
and pTCoSPX. The primer and restriction enzyme sites for the full CP and core
substitutions are shown in Figure 13. Primer RG57 introduced XbaI site 15bp in the
3’NTR, which is compatible with AvrII, occru'ring at the same position in ZYMV (TEV
has an interml Avril Site in the CP). The full length CP gene of the Connecticut isolate
of ZYMV (ZYMV-Ct) was cloned by using primer pair RG81 and RG56 (S’-TAAEI‘_A
_GQTAGGCGACC-3’ priming 15bp from the CP stop codon in the 3’ NTR at the
naturally occurring AvrII site) and resulted in the construct pCtCPSPX. Using the same
PCR based approach a second set of WCP, WCore, TCP and TCore SPX constructs were
made which contained only the coding sequence of the CP or the core portion of the CP
ofWMV and TEV. The same 5’ primers used for making these constructs while two
new 3’ primers RG92 (5-AACCCTAGGTAGGCGACCTACC CTTTACTGCGG TGG
ACCCAT-3’ for WMV) and RG91 (5-GAATCTAGATAGGCG ACCTACCCT'I‘TA CT
GGCGGACCCCTAA-3 for TEV) were designed which were complementary to ZYMV
in the 3’NTR region and WMV or TEV in the CP coding regions. The templates used for
PCR were pWCPSPX and pTCPSPX. All the constructs were confirmed by sequencing
and restriction enzyme analyses. Chimeric fiill length constructs were made by digesting
the subclones with SacI and XbaI (a unique restriction in the 3’NTR) and cloning into the

infectious ZYMV-NAA construct.

73

A chimeric full length construct containing the NT of the CP of ZYMV-Ct was
made by digesting pCtCPSPX with Sue] and [Wu]. MIuI is a unique restriction site close
to the beginning of the core portion of the CP (position 8746). Thus, unlike other NT
chimeric constructs, CtNT construct lacked the engineered KpnI site. The core portion of

the CP of ZYMV-Ct was cloned by using the unique restriction sites of Mia] and XbaI.

Virus Stocks, plant material and growth conditions.

ZYMV and chimeric virus stocks were maintained in the growth chamber in sthe
usceptible squash (Cucurbita pepo) cultivar Midas (Willhite Seeds Incorporated,
Poolville, TX). TEV was maintained on the tobacco (Nicotiana tabacum) cultivar
Burley. Growth chambers were set at 16/8 hrs 24/20C day/night conditions and an
average light intensity of 150 uEinstein. Experiments were conducted in the greenhouse
using a randomized complete block design and the plants received supplemental light
during winter months (October-April). Numbers of replications in each block varied
fi'om experiment to experiment and are indicated in the results section. Plant genotypes
used in this study were: resistant cucumber (Cucumus sativus) line ‘TMG-l ’
(Provvidenti, 1987, Seeds provided by J. Staub, Univ. of Wisconsin), susceptible
cucumber cultivar ‘Straight-8’ (Burpee Seed Co, Warminster, PA), CP transgenic melon
(Cucumus melo) line 207 (Fang and Grumet, 1993), susceptible melon cultivar ‘Hales
Best Jumbo’ (Hollar Seeds, Rocky Ford, CO), Black turtle-H beans (Phaseolus vulgaris:
Provvidenti and Gonsalves, 1984), Nicotiana benthamiana, and Chenopodium

amaranticolor. All the experiments were repeated two to three times except the

74

experiment with the CP transgenic melon plants, which was done once with 5 replications
due to limited supply of seeds.
Plant inoculations.

Plasmid DNA of the full length constructs was purified using the Wizard plasmid
miniprep system (Promega, Madison, WI) and directly inoculated onto the plants using
the particle bombardment method of Gal-On et al, (1995). After the plants showed
systemic symptoms, young symptomatic leaves were harvested and stored at —80°F as
passage 0 (PO) stock. All the tissue used for subsequent inoculation was either fi'om the
original P0 plants or fiom stock (Pl) plants inoculated with P0 inoculum. Plants were
rub inoculated on leaves or cotyledons dusted with carborundum using inoculum
prepared from young symptomatic leaves ground in 0.02M Phosphate buffer (PH 7.0).
For back inoculations fi'om beans, N. benthamiana, and tobacco plants, inoculated or
systemic leaves were harvested from each inoculated plant, ground separately in

phosphate buffer, and used to inoculate two susceptible squash plants.

PCR, RT-PCR and ELISA tests of the inoculated plants

PCR amplifications were run for 25 cycles using Vent DNA polymerase (NE
Biolabs). For RT-PCR, total RNA was extracted from inoculated and healthy plants
using Trizol RNA extraction reagent (Grbco BRL Grand Island, NY) according to the
manufacturer’s instructions. Two pg of total RNA was used for first strand cDNA
synthesis using AMV or MMuLV reverse transcriptase in a 201.11 reaction mixture. Five
pl of the first strand cDNA reaction mix was used as a template in $01.11 PCR reaction

using Vent DNA polymerase and virus specific primers for 35-40 cycles. The resulting

75

PCR products were run on a 0.8% agarose gel. ELISAS were performed according to the
leaf disc procedure as described by Wai and Grumet (1995). Tissue printing was
described in chapter 2. Plants were scored for symptom severity on the five youngest
leaves on a scale of zero to five: 0=symptom free, 5=highly symptomatic, including

severe mosaic and laminar distortion.

Aphid transmission.

Aphid (Myzus persicae) cultures were maintained on tobacco plants in the grth
chamber. Using a soft camel hair brush, the aphids were collected in a petri dish and
allowed to feed on virus infected squash leaves for 2-3 minutes. Alter acquisition
feeding, 8-10 aphids were transferred to individual healthy squash plants (2 weeks old)
and given a minimum of two hours of inoculation feeding time. All the aphids were then
hand removed; the plants were transferred to growth chambers and sprayed to kill any

escaped aphids.

RESULTS

Tests of the CP-NT chimeric constructs on susceptible squash and cucumber plants.
The chimeric ZYMV-constructs containing the NT of the CP of WMV
(pWNTFL/P) and TEV (pTNTFL/P) were infectious on susceptible squash and cucumber
plants (Figure 9). Symptoms appeared 5-7 days afier inoculation on plants inoculated
with the control or pWNTFL/P constructs, while a delay of 1-3 days was observed when
susceptible plants were inoculated with pTNTFL/P. The inoculated plants showed strong

systemic symptoms at 2-3 weeks post inoculation with all the constructs. Thus despite

76

limited sequence homology, the heterologous NTS were able to facilitate systemic
infection.

Interestingly, 4-6 weeks after inoculation there was a gradual reduction in
symptom severity on TNTFL/P infected susceptible squash and cucumber plants. Newly
emerging leaves were either completely fiee of symptoms or showed very mild
symptoms, while older leaves showed strong symptoms, comparable in severity to the
control constructs (Figure 10). The recovery phenotype was associated with reduced virus
titer in the younger leaves (Figure 11). Plants inoculated with the control constructs or
pWNTFL/P developed increasingly severe symptoms throughout the course of the
experiments: the newly formed leaves were severely distorted, had significantly reduced
leaf lamina (Figure 10), and showed high virus accumulation (Figure 11).

The observed recovery in TNTFL/P inoculated plants does not appear to be
caused by the developmental changes in the host associated with the age of the plant.
When squash plants were inoculated with TNTFL/P at different developmental stages
strong symptoms were observed 2-3 WPI irrespective of the age of the plant at the time
of inoculation (Table 3A). The recovery also was not due to a loss of infectivity of the
virus. Although virus titers were reduced in the recovered leaves, when these recovered
leaves were used as an inoculum source to infect squash plants, typical symptoms were
again observed (data not shown).

When the recovered leaves fiom TNTFL/P inoculated squash plants were
inoculated with ZYMV (strain NAA or Ct), the challenge virus was not able to produce
strong symptoms up to six weeks post secondary inoculation (Table 3B). Control plants

of the same age inoculated with ZYMV Showed typical symptoms 2-3 WPI (Table 3A).

77

 

Figure 10. Symptom expression on susceptible squash plants 6 weeks post inoculation.

Wild type ZYMV-NAA (left) and the chimeric construct containing the NT of the CP of
WMV (center) showed increasingly strong symptoms. However, the chimeric ZYMV
construct containing the NT of the CP of TEV (right) showed a marked reduction in

symptom severity.

78

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1_2 .. Squash (cv. Midas) “ TNT
—-)(—ZCPFL/P
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g . \r A
f 0.4, \
o
3 I
re 0.2+
A A A x
. e e e v v a
0 . - —+ . I
L1 L2 L3 L4 L5 L6 L7 L8
1.2 . .
. Cucumber (cv. Straight-8)
1 +-
E i
a 1
V3
o 0.8 s
Q
v
3 0.6 I
r:
a
"3 0.4 ..
o
(I)
.o
a 0.2 -
A A A A A A A
v v v v V v fi
0A -— ~+ — 4-4—+—————+- -a+——-»—r 1
LI L2 L3 L4 L5 L6 L7 L8

leaf number

Figure 11. Virus titer (as determined by ELISA) in susceptible squash and cucumber
plants at 30 days post inoculation (DPI). The top 7 or 8 leaves were sampled for ELISA
fi'om 6 plants for each virus. The X-axis shows leaf number from base to the top of the
plant. The Y-axis Shows absorbance at 405nm. Each point represents a mean of 6
replications. Experiments were repeated two times, each with 6 replications and similar
results were observed.

79

Table 3. Effect of plant age (A) and prior inoculation (B) with ZYMV/TNT hybrid virus
on symptom development in squash plants.

 

A Effect of plant age on symptom development.

 

Symptoms i3 weeks mst inoculation“

 

 

 

Number
Age at lnoc Plants Inoc. TNT NAA/CT
7 20 5.0 5.0
17 16 4.2 4.7
50 9 3.7 4.1

 

B. Effect of prior inoculation of squash plants with TNT hybrid virus on infection by

ZYMV or PRSV. Cotyledons were inoculated with TNT when 7 days old.

 

 

 

 

 

 

 

 

Detection by RT-PCR
Number Symptoms at Symptoms 2-3wks
Age at 20 lnoc. Plants Inoc tmf 2@. post 20 inoc. TNT ZYMV
17-ZYMV 10 5.0 1.5
SO-ZYMV 12 1.5 1.7 12/12 3/12
50-PRSV 13 1.5 4.7

 

Data are pooled from two experiments.
“ Symptoms were given a ranking from 0-5 with 5 being the most severely symptomatic.

80

RT-PCR on the recovered plants inoculated with the two viruses showed the presence of
the TNT hybrid CP in most of the plants (12/12 plants tested) but fewer plants (3/12)
showed the presence of ZYMV RNA, as determined by presence of the TEV-CP NT or
ZYMV-CP NT sequences, respectively. These results suggest that ZYMV could not
establish a successful infection in the recovered plants after secondary inoculation. The
observed protection was virus specific, and secondary inoculation with the watermelon
strain of papaya ring spot virus (PRSV) on the recovered leaves produced strong systemic
symptoms (1 1/13 plants tested). The above recovery phenotype and protection against
secondary inoculation was also observed when the non-recovered pTNTFL/P inoculated
plants were inoculated with ZYMV-NAA as early as ten days after initial inoculation
(Table 3B).

The chimeric viruses containing the NT of the CP of WMV or TEV also were
aphid transmissible: 10/ 12, 12/ 12 and 7/ 12 plants showed systemic infections 10-14 days
post-aphid inoculation with ZNAAFL/P, WNTFL/P and TNTFL/P viruses, respectively.
The NT of the CP of WMV and TEV in the hybrid viruses contained the DAG motif
known to be involved in the aphid transmission of potyviruses (Pirone and Blanc, 1996).
Aphid transmission of potyviruses requires an interaction between CP and HC-Pro (Peng
et al., 1998; Blane et al., 1997). These results Show that the NTS of the CPS of WMV and

TEV were capable of interaction with the ZYMV HC-Pro to facilitate aphid transmission

81

Tests on cucumber and melon plants with natural or genetically engineered
resistance to ZYMV.

The chimeric viruses were tested on the cucumber genotype, ‘TMG-l ’, possessing the
recessive zym resistance allele (Provvidenti, 1987; Kabelka et al., 1997) and on
transgenic melons engineered for resistance with the ZYMV CP gene (Fang et al., 1993).
Neither the control nor the chimeric viruses were able to produce symptoms on the
resistant cucumber genotype ‘TMG-l ’ or CP transgenic melons. Virus accumulation
could only be detected in the first one or two leaves of, TMG-l, at 4 weeks post
inoculation (Figure 12). All the viruses showed severe symptoms on the susceptible
melon cultivar, ‘HaleS-Best Jtunbo’. These results indicate that the NT of the CP of
WMV or TEV does not affect the resistance response of ‘TMG-l ’ or ZYMV CP-

transgenic melon plants to ZYM-NAA.

Tests on local lesion-hosts or non-hosts of ZYMV.

The chimeric viruses also were tested for infectivity on several additional
local lesion or non-hosts including: Black Turtle-II beans (Phaseolus vulgaris),
Chenopodium amaranticolor, Nicotiana benthamiana and tobacco (N. tabacum). ZYMV
produces local lesions on Black Turtle-II bean and Chenopodium amaranticolor plants
but does not infect N. benthamiana and tobacco (Brunt et al., 1996; Provvidenti and
Gonsalves, 1984). WMV is reported to systemically infect black Black Turtle-II beans
(Provvidenti and Gonsalves, 1984) and N. benthamiana (Brunt et al., 1996), while TEV
produces local lesions on C. amaranticolor and systemically infects N. benthamiana and

tobacco plants (Brunt et al., 1996).

82

Figure 12. Virus titer (as determined by ELISA) in resistant cucumber genotype ‘TMG-
1’, transgenic melon line 207 expressing the CP gene of ZYMV-Ct in ‘Hales Best
Jumbo’, and susceptible commercial cultivar ‘Hales Best Jumbo’ (bottom) 26 days after
inoculation Each point represents the mean of 5 replications. Cot = cotyledon, Ll-to L5
represents leaf number 1 to -5 from the base of the plant. The ‘TMG-l ’ and CP
transgenic melon plants did not Show symptoms while ‘Hales Best Jumbo', plants Showed

severe foliar symptoms and only three to four leaves could be sampled for ELISA.

83

1.5
g 1
.D
‘5
B 0.5
<
0
1.5
8
s
.D
'5
B 0.5
<
O
1.5 -
8
g 1
.D
'6
B
<

-O- Contrl (-)

 

 

 

 

' +WNTFL/P
r Cucumber ’TMG-l +TNTFL/P
+ZCPFL/P
-.l_ 2
L1 L2 L3 L4 L5 L6 L7
Leaf number
7. CP transgenic melons
Cot. L1 L2 L3 L4 L5 L6 L7
Leaf number
Susceptible Melons

J. W
W

 

0 T—ma— l a i l l -—--———-++

 

Cot. L1 L2 L3 L4 L5 L6 L7
Leaf number

Table 4A. Summary of the inoculation experiments of various constructs using
infected leaves of squash (for ZYMV-derived constructs) or tobacco (for TEV) as a
source of inoculum onto squash, C. amaranticolor (C. amarant.), bean N. benthamiana
(N. benth) and tobacco.

No. of plants showing symptoms/ No. plants inoculated

Squash“ C.amarant. Beans N. benth. Tobacco

 

Sys.b Local Sys. Local Sys. Local Sys. Local Sys.

 

Construct

ZYCPFL/P 12/12° 3/7 0/7 18/26 0/52 0 0/30 0 0/9

TEV nt. 11. t. nt. n. t. nt. 0 13/13 0 18/18
WNTFL/P 12/12 5/7 0/7 15/26 0/54 0 0/32 nt. nt.
TNTFL/P 10/ 12 7/7 0/7 17/26 0/38 0 0/40 0 0/39

 

Table 4B. Back inoculation of susceptible squash plants fi'om inoculated and systemic
leaves of bean, N. benthamiana or tobacco plants

 

Source of inoculum

 

 

 

 

 

 

Bean N. benthamiana Tobacco
lnoc. L Sys. L Inoc. L Sys. L Inoc. L Sys. L
Virus
ZYMV 5/7 0/34 2/22 0/27 0/19 0/13
WNTFL/P 6/7 0/36 1/14 0/26 nt. nt.
TNTFL/P 5/7 0/20 3/22 0/36 0/15 0/26

 

3’ Squash plants did not Show local lesions;

b‘ Sys. = systemic, Inoc. = inoculated, and L=leaf, nt. = not tested

°' Ntunber of plants showing symptoms on squash plants/ number used as inoculum
source.

85

As expected, all ZYMV derived constructs produced systemic symptoms on squash
(Table 4A). When C. amaranticolor and Black Turtle-II bean plants were inoculated,
local lesions were observed on the inoculated leaves with both the control and chimeric
viruses; however, none of the viruses produced systemic symptoms (Table 4A).
Susceptible squash plants back inoculated fi'om the inoculated leaves of Black Turtle-II
beans, showed symptoms for all the constructs, but no symptoms were observed on plants
back inoculated from systemic leaves (Table 4B). No local or systemic symptoms were
observed for any of the ZYMV-derived constructs on N. benthamiana or tobacco plants.
Back inoculated squash plants from the systemic leaves of N. benthamiana or tobacco did
not show symptoms (Table 4B). A limited number of squash plants (ca. 10%) back
inoculated fiom the inoculated leaves of N. benthamiana showed symptom. These
results suggest tlmt the NT of CP of WMV or TEV is not sufficient to allow ZYMV to
cause systemic infection in C. amaranticolor, Black Turtle-II beans, N. benthamiana, or

N. tabacum.

Core and whole CP substitutions

We also investigated the effect of substitution of the core and carboxy terminus
(for simplicity will be referred to as core) and the fill] length CP of WMV and TEV, in
facilitating movement of the chimeric viruses (Figure 13). Substitutions also were made
with the Connecticut isolate of ZYMV (ZYMV-Ct). Bombardment of squash plants with
the original ZYMV-NAA construct gives 50-70% infection, on average. A comparable
percentage of the control constructs carrying the core regions (CtCoFL/P) or the entire

CP (CtCPFL/P) ZYMV-Ct showed symptoms 5-7 days after particle bombardment with

86

 

 

'3
n _ . or;
i? [W 1 m . M...

pZYMV-NAA (Wild Type)

 

 

 

 

 

 

 

 

 

 

 

 

 

Infection on
~
Tobacco Squash .3 E
I;
“no I F l I l pCtCoFL/P
"' 7
i i a
0/12 0/22 I I I ‘ '. I I pWCoFL/P
fi‘
. AZ
cm M cm I I I I I
5 if pTCol'FL/P
z E
ans I 1i l 1 pCtCPFL/P
. . . £31 pWCPFL/P
olii om L I? . .
£1
om om one I I]? I I pTCPFL/l’

Figure 13. Hybrid ZYMV-NAA constructs containing the core and. carboxy
terminus (Co) of the coat protein of ZYMV-Ct (CtCoFL/P), WMV (WCoFL/P) or TEV
(TCoFL/P) and their infection on squash plants. Hybrid viruses containing the entire CP
of ZYMV-CT, WMV and TEV are pCTCPFL/P, pWCPFL/P and pCTCPFL/P
respectively. Introduced restriction Sites are indicated at the top of the control constructs.
Numbers on the left indicate the number of plants showing symptoms/number of plants

inoculated by particle bombardment with hill-length cDNA constructs.

87

the full length cDNA constructs (Figure 13). The chimeric viruses containing the core or
the entire CP of WMV (WCoFL/P, WCPFL/P) and TEV (TCoFL/P TCPFL/P), however,
were not infectious on any of the plants tested including squash, N. benthamiana, and
tobacco (Figure 13). We were also unable to detect symptoms (up to two months after
inoculations), or virus titer with ELISA, tissue prints, RT-PCR or back inoculation (data
not shown) in these plants. To account for possible effects of a foreign 3’ NTR on
replication, a second set of constructs were made that retained the entire 3’ NTR of
ZYMV and only the coding sequences of the CP or core region were substituted with the
respective regions WMV or TEV. These constructs were also not infectious on squash,

N. benthamiana or tobacco plants (data not shown).

DISCUSSION

The NT of the potyvirus CP, which is essential for systemic movement and aphid
transmission of the virus, is highly variable in length and sequence (Shukla et al., 1994;
Dolja et al., 94, 1995; Atreya et al., 1990; Blane et al., 1997). Our results Show that
despite substantial variation, the CP NTS fiom heterologous potyviruses can facilitate
systemic infection of chimeric ZYMV viruses in ZYMV-susceptible cucurbits. This was
observed for chimeric viruses with CP-NTs fi'om viruses having both overlapping
(WMV) and non-overlapping (TEV) host ranges with ZYMV. The NT of the CP of
ZYMV is 44 arrrino acids (aa) long, compared to 46 a of WMV and only 29 aa of TEV.
There are only 13 a that are identical among the CP NTS of the three viruses, six of

which occur in KD pairs and three of them form the DAG triplet shown to be involved in

88

aphid transmission (Blane et al., 1997; Atreya et al., 1990). This suggests that only a few
key amino acids in the NT of the CP are required to facilitate systemic movement of
potyviruses, even in Species that are not typically hosts. Previous studies have shown
Asp in the conserved DAG motif to be critical for systemic movement. Substitution of
Lys or Arg for Asp in the DAG motif of TVMV and TEV affected systemic movement of
the virus (Lopez-Moya and Pirone, 1998). However, mutations in the second and third
positions of the DAG motif are fiequently observed in association with aphid

transmission and do not aflect systemic virus movement (Atreya et al.,1990, 1991; Gal-
On et al., 1992; Andrejeva et al., 1999).

The ZYMV CP-NT chimeric viruses were also aphid transmissible. Since
successfirl aphid transmission requires interaction between the CP-NT and HC-Pro
(Blane et al., 1997; Flasinski and Cassidy, 1998; Peng et al., 1998), this indicates that the
features necessary for CP - HC-Pro interaction to facilitate aphid transmission were
present in the heterologous CP-NTS. In binding assays, the conserved DAG motif in the
NT of CP and the PTK motif in the HC-Pro gene have been shown to be involved in this
interaction (Blane et al., 1997; Peng et al., 1998). A sequence of 7 aa (DTVDAGK) in
the NT of the CP was suflicient for binding HC-Pro (Blane et al., 1997). The chimeric
viruses used in this study contained the amino acids TV and DAG in different contexts in
the NT of the CP, but were all aphid transmissible suggesting flexibility in this motif for
interaction with the HC-Pro gene of ZYMV. These observations are also consistent with
previous studies showing facilitation of aphid transmission by heterologous helper
components (Atreya and Pirone, 1993; Hobbs and McLaoghlin, 1990; Lecoq and Pitrat,

1985; Sako and Ogata, 1981). Mixing experiments using purified virions from one virus

89

and HC-Pro from a different virus resulted in aphid transmission, although some
combinations were less efl°ective than others (Sako and Ogata, 1981; Lecoq and Pitrat,
1985; Hobbs and McLaoghlin, 1990). For example, HC-Pro from ZYMV facilitated
aphid transmission of WMV and vice versa, but the ZYMV and WMV HC-Pro were less
effective in facilitating transmission of the less closely related PRSV-W (Lecoq and
Pitrat, 1985). Similarly, aphid transmission of the NAT isolate of TEV was facilitated by
the aphid transmissible (AT) isolate of PVY, but not by WMV-AT (Simons, 1976). A
chimeric TVMV construct containing the HC-Pro gene of ZYMV also was aphid
transmissible (Atreya and Pirone, 1993).

Although the TNT chimeric virus produced strong symptoms at the initial stages
of infection, 4-6 weeks post inoculation the plants showed a marked recovery
characterized by a progressive reduction in symptom development and virus titer, and a
virus-specific resistance to secondary inoculation The reduction in symptom severity
was not a result of plant age or mutation of the infecting virus, and was unique to the
TNT chimeric virus; the parent ZYMV and the chimeric WNT viruses caused
increasingly severe symptoms over time. This indicates that the NT of the CP could be
the target of host defense responses and that the variability in the NT has a role in host
adaptation The role of the NT of potyviral CP in host adaptation and resistance
responses has previously been suggested (Shukla et al., 1994, Xiao et al., 1993, Solomon,
1989, chapter 2). A single aa substitution (Ser47 to Pro) in the NT of the CP of the NY
isolate allowed PSbMV to produce systemic symptoms in C. quinoa (Andersen and
Johansen, 1998) suggesting that the NT of the CP is involved in the localization of the

virus to the inoculated leaves in C. quinoa in a host specific manner. Evidence for the

90

role of the CP-NT in inducing host defense responses also comes from the response of
the resistant cucumber genotype ‘Dina-l ’ to inoculation with chimeric ZYMV infectious
constructs (chapter 2). Unlike ZYMV-Ct, ZYMV-NAA does not produce veinal chlorosis
symptoms on ‘Dina-l ’ plants. When the NT of CP of the infectious ZYMV-NAA
construct was substituted with CP-NT ZYMV-Ct, the resulting chimeric construct
produced veinal chlorosis on ‘Dina-l ’ plants. Substitution of the core region of the CP
between the two virus strains, on the other hand, did not result in veinal chlorosis. In
another example of recovery fiom potyvirus infection, a protease activity associated with
the cleavage of the NT of the CP of SPFMV was observed in Ipomia nil plants (Solomon,
1989). Another, not mutually exclusive, possibility is that the eventual recovery
observed with the chimeric TNT virus reflects sub-optimal interaction between the
heterologous CP-NT and other viral or host proteins thereby allowing host defenses to be
more effective. The TNT virus was slower in establishing initial infection, which could
have resulted fi'om this sub-optirnal interaction

Interestingly, the above described recovery phenotype also shows striking
resemblance to recovery of infected plants from infection by a number of viruses
including potyviruses (reviewed by Pennazio et al., 1999) and to recovery responses
observed in transgenic plants expressing RNA mediated protection Recent studies with
several types of viruses (e. g. Tobra-, Caulimo- and Nepoviruses) have Shown that the
natural recovery phenotype is associated with induction of a post transcriptional gene
silencing (PTGS) like resistance response which leads to specific degradation of the viral
RNA (Al-Kafl‘et al., 1998; Covey et al., 1997; Ratclifl‘et al., 1997, 1999; Hamilton and

Baulcombe, 1999). As a counter-strategy, many viruses encode specific proteins, which

91

suppress PTGS-like resistance of the host (Brigneti et al., 1998; Anandalaskshmi et
al.1998; Kasschau and Carrington, 1998; Voinnet et al, 1999). In potyviruses Pl-HC-
Pro acst as a suppressor of PT GS (Brigneti et al., 1998; Kasschau and Carrington, 1998;
Voinnet et al., 1999). Klein et al., (1994) observed a recovery phenotype in tobacco
plants associated with certain mutations in the P1 or HC-Pro genes of TVMV. The
mutated viruses caused strong initial symptoms on tobacco plants, however, by 25dpi the
newly emerging leaves were almost symptomless suggesting that these mutations might
interfere with the ability of the virus to suppress PTGS.

Unlike HC-Pro, CP has not been directly implicated in suppression of PTGS. It
will be of interest to determine whether the observed recovery associated with the TNT
chimeric virus infection is a result of a PTGS like resistance response. Interaction
between CP and HC-Pro has been well documented for aphid transmission (Blane et al.,
1997; Peng et al., 1998) and due to the overlapping functions of these proteins, e.g., both
proteins appear to be involved in cell to cell and long distance viral movement, it is
conceivable that the two proteins may interact at other stages in the viral life cycle.
Andrejeva et al., (1999), have suggested that potyvirus HC-Pro and CP have coordinated
functions in virus host interactions. They observed that the effects on virus accumulation
and movement caused by simultaneous mutations made in the HC-Pro and CP genes of
PVA were different from the expected ‘sum’ of phenotypic changes observed following
mutation of only one gene at a time.

The NT hybrid viruses did not overcome naturally occurring resistance to ZYMV
in the cucumber line ‘TMG-l ’. This is not entirely surprising as ‘TMG-l’ is also

resistant to WMV (Provvidenti, 1985, 1987; Kabelka and Grumet, 1995), and TEV is a

92

non-pathogen of cucumbers. Interestingly, however, the chimeric viruses also did not
overcome the ZYMV CP mediated resistance. Although the hill length CP confers a high
level of protection against ZYMV infection (Fang and Grumet 1993; this study), the core
portion of the CP is not suflicient to provide resistance (Fang and Grumet, 1993). This
suggests that although the NT is required for the high level of transgenic CP mediated
resistance, which is virus Specific and not RNA mediated (Grumet et al., 1995, 1998), the
resistance does not depend on the NT of the CP of the incoming virus. Interestingly, the
recovery phenotype associated with the CP-NT of TEV also did not appear to be targeted
against CP-NT in a sequence specific manner, and was effective to protect against
secondary infection by ZYMV-NAA and ZYMV-Ct. Together these results suggest that
the CP-NT could be involved in triggering host resistance responses targeted against
other parts of the virus genome.

Substitution of the CP NT did not appear to alter the host range of ZYMV. We
were unable to detect local or systemic infection in N. benthamiana and tobacco plants or
systemic infection in C. amaranticolor or P. vulgaris plants after inoculation with ZYMV
or the chimeric viruses. The failure to observe systemic infection by the chimeric viruses
in the local lesion hosts suggests that the host defense responses in these plants leading to
virus localization were not dependent on the specific CP-NT. Only a limited number
(ca.10%) of N. benthamiana plants Showed the presence of virus in the inoculated leaves.
The inability of ZYMV and chimeric viruses to cause local infection in N. benthamiana
and tobacco suggest impaired replication or cell-to-cell movement of the virus in these

non-hosts.

93

Although the chimeric constructs containing the CP or core region of the
Connecticut isolate of ZYMV were infectious on squash plants, when squash, N.
benthamiana, or tobacco were inoculated with the chimeric ZYMV constructs containing
the entire CP, or the core portion of the CP of TEV or WMV, no symptoms were
observed. This might be due to a number of reasons related to the functions of the core
of the CP functions, such as encapsidation or eell-to-cell movement, or more general
factors influencing virus viability such as the ability to replicate or processing of the
polyprotein. Although we can not rule out encapsidation or cell-to-cell movement,
transencapsidation among potyviruses has been observed in several systems (Sirnons
1976; Bourdin and Lecoq, 1991; Lecoq et al., 1993) and the cell-to-cell movement
mediated by the potyvirus CP did not appear to be responsible for host specificity in
micro injection studies (Rojas et al, 1997). The bean common mosaic necrotic virus
(BCMNV) moved cell-to-cell and increased plasmodesmata SEL in a non-host (lettuce)
and facilitated the movement of LMV CP RNA in N. benthamiana (Rojas et al., 1997).
Successful proteolytic processing also does not appear to be a problem. The infectious
CP-NT chimeric viruses used the same hybrid CP protease cut Sites as did the CP or core
substituted constructs, indicating that they were processed properly. However, the CP
coding sequence and the 3’ NTR have been shown to be co-adapted for genome
amplification through a requirement for base pair interactions leading to complex
secondary structures (Haldermn-Cahill et al., 1998; Mahajan et al., 1996). Chimeric
constructs in which only the coding sequence of the CP or core region was substituted
(leaving an intact ZYMV 3’ NTR) also were not infectious (data not Shown).

Comparison of the nucleotide sequence of the three viruses used in this study shows

94

considerable variability in the 3’NTR and the carboxy terrrrinus of the CP raising the
possibility that the chimeric constructs might be replication defective due to defective
secondary structure in this region

Our results Showed that the CP-NTS fi'om heterologous potyviruses could
facilitate systemic infection of ZYMV in susceptible cucurbit hosts, but this substitution
was not suficient to modify host range of the chimeric viruses. Substitution of the CP-
NT of ZYMV with a non-cucurbit potyviruses resulted in induction of a host defense
response leading to recovery from the chimeric virus infection, suggesting that the

variability in the CP-NT has a potential a role in host adaptation.

Future Directions.

Our data revealed that heterologous NT from the CP of a non-cucurbit potyvirus
could induce (or fail to suppress) a resistance response to the chimeric ZYMV in
cucumber and squash plants. The recovery phenotype was similar to the phenotype
associated with virus induced gene silencing. Further characterization of this recovery
response is important. It will also be useful to know if CP-NTS from other non-cucurbit
potyviruses could induce a similar resistance response and this will help us better
understand the nature of this resistance response. Identification of the domains within the
CP-NT involved in host recovery from chimeric virus infection will also be very usefirl in
firrther understanding the recovery response, and this could have practical implications in

engineering transgenic plants expressing viral coat proteins.

95

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100

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101

CHAPTER IV

CHARACTERIZATION OF THE INTERACTION OF THE CUCUMBER

POLY(A) BINDING PROTEIN WITH THE REPLICASE GENES OF VIRUSES

ABSTRACT

Deletion Studies were performed to identify the domains involved in the interaction
between a cucumber poly(A) binding protein (PABP) and the RNA dependent RNA
polymerase (RdRp) gene of zucchini yellow mosaic virus (ZYMV) in the yeast two
hybrid system. Deletions of 47 amino acids (aa) fiom the carboxy terminus of the
cucumber PABP resulted in weak interaction with ZYMV RdRP, while deletions of 143
aa completely abolished the interaction, suggesting that the C-terminus of the PABP is
involved in interaction with the RdRp. Deletions from both the amino and carboxy
termini of the RdRp gene abolished the interaction in yeast, suggesting that a large
portion of the RdRp protein is required for the interaction Interaction of the cucumber
PABP was also studied with the RdRp proteins fi'om other viruses. Bean common
mosaic potyvirus and cowpea chlorotic mottle bromovirus RdRp genes interacted
strongly with the cucumber PABP protein, and the RdRp gene from poliovirus Showed
weak interaction However, RdRps of watermelon mosaic and tobacco vein mottling

potyviruses did not interact with the cucumber PABP.

102

INTRODUCTION

Successful systemic infection by a pathogen depends on compatible interactions
between the pathogen and its host; this is particularly true in the case of viruses, which
have extremely small genomes and limited protein coding capacity. Several host proteins
have been shown to be involved in replication of RNA viruses, either as components of
the viral replication complex, or by binding directly to the viral genome (reviewed in:
Lai, 1998; Strauss and Strauss, 1999). For RNA viruses, a majority of the factors found
in association with the viral replicase, the RNA dependent RNA polymerase (RdRp), are
subverted from the host RN A-processing and translation machinery. For example,
elongation factor EF-lor and different subunits of eIF3 have been found in association of
replicase complexes of an array of bacterial, plant, and mammalian viruses such as QB
phage, brome mosaic virus tobacco mosaic virus, vesicular stomatitis virus, measles
virus and poliovirus (Lai, 1998; Strauss and Strauss, 1999).

Potyviruses also have a small genome, and presumably interact with a number of
plant proteins for successful infection The 10kb genome of potyviruses, has a genome
linked viral protein (VPg) covalently attached to the 5’ end and a poly(A) tail at the 3’
end. The entire genome is translated into a Single polyprotein which is subsequently
cleaved into at least nine proteins by viral encoded proteases (Shukla et al., 1994). To
better understand host-potyvirus interaction, a project was initiated in the lab to identify
host factors interacting with the RNA dependent RNA polymerase (RdRp) gene of

zucchini yellow mosaic virus (ZYMV).

103

 

A leaf cDNA library was constructed fi'om the susceptible cucumber (Cucumis
sativus) host ‘Straight-8’, and screened for interaction with the ZYMV RdRp gene using
the yeast two hybrid system (Wang et al., submitted). Interestingly, a poly(A) binding
protein (PABP) was repeatedly isolated in these screens. This interaction was also
confirmed in subsequent in vitro binding experiments.

Potyvirus replication occurs in the cytoplasm and proceeds by copying of the
plus strand to minus strand followed by copying back to the plus strand (Martin and
Garcia, 1991; Schaad et al., 1997). Presence of a poly (A) tail is essential for replication
of picorrmviruses such as poliovirus, encephalomyocarditis virus and rhinovirus (Cui et
al., 1993, Todd and Semler, 1996; Todd et al., 1997; Agol et al., 1999) and recent
evidence suggests it is also necessary for potyvirus replication (Tacahashi and Uyeda,
1999). A study of the interaction of the viral RdRp gene with the host PABP could
therefore be of fundamental importance in understanding replication of plant viruses.

In this project I attempted to characterize the interaction the ZYMV RdRp protein
with the cucumber PABP by identifying the domains of the two proteins involved in this
interaction The interaction of the PABP with polymerase genes from other viruses was
also studied. Deletion of the carboxy terminal 150aa of the PABP abolished interaction
with the ZYMV-RdRp protein; however, the components of the RdRp protein involved in
the interaction were more complex. The cucumber PABP also interacted with the
replicase genes from bean common mosaic necrosis potyvirus (BCMNV) and cowpea
chlorotic mosaic bromovirus (CCMV), but failed to interact with certain other viruses

including some members of the potyvirus group.

104

MATERIALS AND METHODS

ZYMV-RdRp and Cucumber PABP deletions.

All the PABP deletions were node from N18 (Figure 14 B), the longest cDNA
obtained from the two hybrid screen of Wang et al (submitted). NI8A3 00 was amplified
by PCR using primer pair RG110 (5'-AAAGAA TTCGGCTTTGTAAATTTTGAG-3':
forward: position: 5’ end of N18) and RG157 (5’-TCTCTCGAGCAAATGTAGAAC
CTCAGT-3’: reverse, position 1801) introducing EcoRI and 2010] sites (underlined),
respectively. Position numbering is based on the full length Cs-PABPI gene (Wang et
al., submitted). The PCR product was cloned into the yeast two hybrid vector,
pADGAL4 (Stratagene) as an EcoRI-XhoI fragment, resulting in the construct
pADN18A300. NIAMIu was amplified with RG110 and RG115 (5’-GTACTC GAGCAT
GCGA GCTCAAAG GTACAGGCTGC TGG-3’: Position 1476 near MIuI site and
introducing XhoI, SphI, and Sac] sites at the 3 ’end). The PCR product was cloned as an
EcoRI-Xhol fi'agrnent into pADGAL4 to form pADNI8AMIu, and into pBlueScript to
form pBSNI8AMZu. pBSNISAMIu was then digested with SphI, and religated to form
pBSNI8ASph. The EcoRI-Xhol NI8ASph fi'agment was then subcloned to pADGAL4 to
form pADNISASph. pADNI8ASac was made by digesting pADNI8AMlu with Sad to
drop the 418 bp and religatirrg the plasmid band.

All the RdRp deletions were made from the hill-length RdRp clone (Figure 14).
RdRpAl, A4, A5, A6, A7, A8 were amplified by PCR using primers as indicated in Figure
14; all the PCR products were inserted into the yeast two hybrid vector pBDGAL4 as
EcoRI-Sall fiagments The sequences of primers RG100, 101, 121, 125, 126, 127 were as

follows, restriction sites are underlined: 5’-AGAGTCGA CCCTGA C I I ICTCAAGC-

105

A Interaction
with RdRp

N18‘I

NI8A300

 

NI 8AMlu
NI 8ASph

 

 

 

NI 8 ASac Interaction

with N18

 

2
1551

RdRpAl
RdRpA2
RdRpA3
RdRpA4
RdRpAS
RdRpA6
RdRpA7

 

RdRpA8

 

Figure 14 Deletion analysis to determine regions in PABP and RdRP responsible for
the interaction. A. Different sizes of cDNA NI 8 were amplified by PCR or generated
by restriction enzymes digestion All the deletions were tested against full-length RdRp
in the yeast two-hybrid system. B. Different sizes of RdRp was amplified by PCR or
generated by restriction enzymes digestion All the deletions were tested against NI 8 in
the yeast two-hybrid system. Positive interactions are Shown by + sign. ‘- Positions of
N18 were marked according to the full-length sequence.

106

3’, 5’-CCGGAA TI‘CTGCGCTGCGAT GATT-3’; 5’-ACTGAA TTCCT CGAGAAAGAG
AGAAT-3 ’; 5’-GTGGAA TTCCCAA TI‘CTI‘GCTCCTGA-3’; 5’-TTA GAA TTCGAG
CTCAGGCCGCTT-3’, 5’-T'l‘Cfl‘_C_‘G_ATCTCGAGTTTTGGAGTG-3’. RdRpA2 was
generated as follows: pBDRdRp was digested with NcoI, filled in with Klenow fiagrnent,
and digested with EcoRI. This RdRp fragment was ligated to pBD, which was digested
with Sal], blunted, then cut by EcoRI to form pBDRdRpAZ. pBDRdRpA3 was produced
by cutting pBDRdRp with EcoRI and XhoI, and then subcloning to EcoRI-XhoI digested

pBDGAL4.

Cloning of the RdRp genes from other viruses.

The infectious construct of tobacco vein mottling virus (TVMV) was provided by
Dr. Pirone (Univ. of Kentucky). The RdRp gene was amplified from this construct using
the primer RG133 (S-AAGAA 7TCCAAGGGGAGAAGCGAAAA-3) that primed at the
5’end of the RdRp and introduced an EcoRI site, and primer RG134 (5’-AACTCGAG
TGTATCACTI‘TGA AATCTCAC-3’) which introduced a 20101 Site at the 3’ end. The
PCR product was digested with XhoI followed by partial digestion with EcoRI (TVMV
RdRp gene has an internal EcoRI site) and cloned into pBDGAL4 cut with EcoRI and
SaII (compatible with XhoI). The CCMV (provided by Richard Allison, Michigan State
University) 2a gene was cloned analogously using the primer pairs RG122 (5’-GC_Q;AA
ZEATGTCTAAGTT CAT‘T CCAG-3’: forward) and RG124 (5’-GCCT CGA GIT A
TTTAGAAAGGGTC TTAC-3': reverse). Watermelon mosaic virus RdRp gene

(Quemada et al., 1990) was amplified using the primer pair RG145 (5-GCAGA ATTCAG

107

 

CAGAAAGGAAAGATG-3’: forward) and RG146 (5’-T'ITGTCG AC'I‘TGTA
AAGACACTGATTC-3’: reverse). The PCR product was cut with Sal] followed by
partial digestion with EcoRI and cloned into the bonding domain vector cut with the same
restriction enzymes. BCMNV RdRp was cloned analogously by using the primer pair
RG151 (5 ’-GAAGAA TI‘CGGTACCAGCAAGAAGGATAGATGG-3 ’: forward) and
RG152 (5’-T‘TCGTCGA CCCTAGGT'I‘GTGTTGACACGGATTC-3’: reverse). RG15]
and RG152 also introduced two useful cloning sites; a Kpnl (RG151) site adjacent to the
EcoRI site and an AvrII site (RG152) next to the SaII site. All the clones were verified by
restriction enzyme analyses and sequencing.

The BMV 23 gene (provided by Richard Allison, Michigan State University) was
amplified using primer RG142 (5-ATCGAA TI‘CATG TCTI‘CGAAAACCTGG-3’:
forward) and primer RG143 (5-AACCTCGA GTTTCAGATCA GAGGG CT-3’: reverse).
The PCR product was cut with EcoRI and XhoI and cloned into pBDGAL4 cut with
EcoRI and SalI. The 3D gene of poliovirus was cloned by digesting the construct pJG3D
prey (provided by Dr. Karla Kiukegaard, Stanford university) with EcoRI and XhoI to

drop the 3D gene, and cloned into pBDGAL4 cut with EcoRI and SaII.

Yeast transformation and study of the interaction between NIb and Cs-PABPl.
Yeast transformation, growth media, and X-gal (5-bromo-4-ehloro-3-indolyl B-D-
galactopyranoside) filter assays were performed following the supplier’s procedures
(Stratagene). To test the effect of deletions in the cucumber PABP on interaction with
ZYMV-RdRp, yeast cells were transformed with pBDRdRp (binding dormin vector

containing the wild type ZYMV RdRp gene), and each of the PABP deletions in

108

pADGAL4. The transformants were plated on SD (synthetic dropout) medium without
leucine (L), tryptophan (T), and histidine (H: SD-L-T-H). As a control for transformation
efficiency, transformants were also plated on SD-L-T. Colonies that grew on selection
medium were restreaked onto SD-L-T-H, transferred onto nitrocellulose membrane
(Schleicher & Schuell), and assayed for expression of B-galactosidase activity (Lac Z) by
X-Gal filter assay. Colonies that survived medium without histidine and turned blue in
the X-Gal assay were considered as positives. Deletions in the RdRp gene were tested
similarly by transforming competent yeast cells with Cs-PABPI in the pADGAL4 vector
and the RdRp deletions in the pBDGAL4 vector.

To test the interaction of the RdRp genes of different viruses with the cucumber
PABP, competent yeast cells were transformed with pBDGAL4 containing the RdRp
gene of the respective virus and pADGAL4 containing the Cs-PABPI gene. As a control
cells were also transformed with pBDRdRp constructs alone. The transformants were

plated on appropriate plates and tested for interaction as described above.

RESULTS AND DISCUSSION

Identification of the interacting domains of the cucumber PABP and ZYMV-RdRp.
To identify the domains of the CSPABP and the ZYMV NIb genes responsible for
interaction in the yeast two hybrid system, a series of deletions were made in both
proteins. The deletions made in the PABP are shown in Figure 14. When 47aa were
deleted from the carboxy (C-) terminal end (pADNI8A300), a very weak interaction was
observed as measured by the lower intensity of the blue color after X-gal test compared

to the wild type construct and the longer time required for the color to appear (four hours

109

 

vs. overnight). Deletions of l43aa fi'om this end of the protein (pADNI8AMZu)
completely abolished the interaction, suggesting that the C-terminal end of the PABP is
critical for interaction with the ZYMV RdRp protein in the yeast two hybrid system. The
smallest interacting PABP cDNA obtained fiom the two hybrid screen (N1 351) encodes
the last 130 amino acids, further suggesting that the carboxy terminal conserved (CTC)
domain might be involved in the interaction with RdRp (Wang et al., submitted).

The C-terminal third of the PABP protein is not as highly conserved among
PABPS as the amino terminus, and functions of the C-terminus are less well defined. At
the C-terminus, there is a 71 amino acid region (amino acids 553-624) that is conserved
among Arabidopsis, wheat, and cucumber. This CTC (C-terminal conserved) domain was
also found within other characterized PABPS (e. g. yeast, vertebrate) and has been
implicated in homodimeiization and eflicient poly (A) binding (Kuhn and Pieler, 1996).
The N terminal two-thirds of the predicted PABP contains 4 RRMS (RNA recognition
motifs), which are found in PABPS fi'om all sources, including yeast and animals (Le et
al., 1997) and directly interact RNA (Kuhn and Pieler, 1996). The two smaller
cucumber cDNAs (NI439 and NI359) lacked the RRMS, suggesting that the RRMS are
not involved in binding with ZYMV RdRp (Wang et al., submitted).

To identify the domains of the ZYMV RdRp involved in the interaction with the
Cs-PABPl , a total of eight deletions were made in the ZYMV RdRp protein (Figure
14B). Deletions fiom both the amino and carboxy termini of the RdRp abolished the
interaction in yeast. Only one protein with 156 aa deletions fiom the 5’ end showed mild
interaction. Although we cannot rule out the possibility that certain deletion products

were unstable, it may be that a large portion of the RdRp is necessary for the interaction

110

with PABP. RdRps, like other types of DNA and RNA polymerases, have been shown to
consist of finger-pahn-thumb domains resulting in intramoleeular interactions between
the arnino- and carboxy-terminal portions of the molecule (O’Reilly and Cao, 1998;
Lesburg et al., 1999) and such interactions also may be important for association with the
PABP. Similar problems in assigning functions to specific RdRp domains were observed
with the tobacco etch potyvirus (TEV) RdRp. Loss of interaction between the TEV
RdRp and the Ma protein were observed with both amino and carboxy terminal deletions
of the TEV RdRp, and nuclear localization capacity was eliminated by deletions from
either terminus and by small insertions at several positions in the protein (Li and

Carrington, 1993; Li et al., 1997).

Interaction of the cucumber PABP with RdRp genes from other viruses.
Replication of the potyvirus positive sense SSRNA genome is initiated at the 3’
end by the RdRp protein to make a minus sense RNA. Since the potyvirus genome
contains a poly(A) tail at the 3’ end, interaction of PABP with ZYMV RdRp raised the
possibility that this interaction might have implications for virus replication. To
investigate if the PABP has a more general role in virus replication, the interaction of the
cucumber PABP with the RdRp of other viruses containing a poly(A) tail (the
potyviruses: WMV, TVMV, and BCMNV, and the Picomavirus, poliovirus), or viruses
without poly(A) tail (Bromoviruses; BMV and CCMV) were studied (Table 5). Among
the potyviruses tested, the cucumber PABP interacted with the RdRp gene of BCMNV,
but not with the WMV and TVMV RdRps. This is intriguing because WMV is more

closely related to ZYMV and infects cucurbits, while BCMNV is more distantly related

11]

to ZYMV and does not infect cucurbits. Interestingly, the cucumber PABP also showed
mild interaction with the 3D gene of poliovirus. Poliovirus, like potyviruses, belongs to
the Picomavirus super group, and it also contains a poly(A) tail at the 3’ end.

Cowpea chlorotic mottle virus 2a protein also showed strong interaction with the
cucumber PABP protein; however the interaction with the closely related BMV 2a
protein could not be confirmed as the 2a gene from this virus also turned on the two
reporter genes, in the absence of the PABP. CCMV lacks a poly(A) tail suggesting that
the functional Significance of PABP-RdRp interaction may not depend on the presence of
a viral poly(A) tail.

The Significance of interaction between the PABP and viral RdRp and its effect
on the viral life cycle remain to be determined. It is possible that this interaction plays
more than one role in virus infection or has different roles in the infection of different
viruses. A number of possible roles for the cucumber PABP interaction with ZYMV
RdRp can be proposed. For example, it is possible that the association of the RdRp with

PABP interferes with the PABP polymerization (Kuhn and Pieler, 1996) and so

Table 5. Interaction of the poly(A) binding protein with viral RdRp goteins.

 

 

Source of RdRp gene Interaction with poly(A) binding protein
Growth on (-) His X-gal test

ZYMV 4+ 4+
BCNMV ++ ++
TVMV - -
WMV - -
BMV ? ?
CCMV ++ ++
Polio virus - -

 

 

112

might facilitate removal of PABP fi'om the poly(A) tail. Another possibility is that the
RdRp sequesters PABP from binding to hnRNA in the nucleus, inhibiting RNA
processing. Recent studies have Shown that viral-induced Shut down of host protein
synthesis, which is thought to facilitate viral infection by increasing accessibility of host
factors for viral purposes, can be mediated at least in part, by sequestration or cleavage of
PABPS (Piron et al., 1998; Chen et al., 1999; Joachirns et al., 1999). In the case of
potyvirus, inhibition of host gene expression and virus-mediated mRNA degradation has
been observed to occur in a reversible manner during pea seed borne mosaic virus
(PSbMV) infection (Wang and Maule, 1995; Aranda et al., 1996; Aranda and Maule,
1998). Such an observation would not be inconsistent with sequestration of PABP.
During potyvirus infection, RdRp is expressed in large quantities and can accumulate in
the nucleus as an inclusion body (NIb). Interestingly, the interaction between influenza
A N81 and human PABPII, which takes place in the nucleus and results in mRNAS with
poly(A) tails that are too short to allow for export, occurs via the carboxy terminus of
PAPBH (Chen et al., 1999).

Further experiments will need to be performed to determine the biological

significance of the RdRp-PABP interaction

113

 

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