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Indian Journal of Biotechnology

Vol I , January 2002, pp 73-86

Virus Resistant Transgenic Plants for Environmentally Safe
Management of Viral Diseases
A Varma* , R K Jain and A I Bhat
Adv anced Center for Plant Virology, Division of Plant Pathology , Indian Agricultural Research Institute, New Delhi - 110012, India

Plant viruses are one of the major yield reducing factors for agricultural and horticultural crops. In India, most
destructive diseases are caused by gemini-, poty-, and tospoviruses. Virus resistant transgenic plants (VRTPs),
developed by the transfer of transgenes from virus, plant or other origins, have been found resistant to a wide range
of viruses. The most successful approach is the viral coat protein mediated resistance (CPMR). Other transgenes of
viral origin, which have shown promise are: replicase protein, movement protein, proteases, and antisense sequences.
'R'-genes from plants, plantibodies and yeast RNase genes are also useful for developing VRTPs. In a large number
of VRTPs developed using transgenes of viral origin, resistance is conferred by post-transcriptional gene silencing
(PTGS); in some cases PTGS has been overcome when plants are infected by a heterologous virus, indicating need
for cautious approach. Overall, the bio-safety concerns in the use of VRTPs get insignificant, but these must be
addressed scientifically. In India, initiatives have been taken for developing VRTPs to manage important plant viral
diseases. The present world area under VRTPs is about 0.4 mha. Judging from the success of various strategies, the
area under VRTPs is expected to grow at a fast rate in the coming years.
Keywords: virus resistant transgenic plants (VRTPs), CPMR, PTGS

Introduction
Plant viruses have emerged as a major yieldreducing factor for field and horticultural crops.
Losses caused by plant viruses are greater in the
tropics and semi-tropics, which provide ideal
conditions for the perpetuation of both the viruses and
their vectors. India, with its diverse climate ranging
from areas with world's highest rainfall to desert on
the one hand and snow-capped mountains to the
tropics on the other, is endowed with a large number
of viral diseases affecting various cropping systems.
The largest number of diseases, are caused by
potyviruses and geminiviruses, not only in India but
also all over the world. Other groups of viruses, which
are fast emerging as serious pathogens, are badna-,
ilar-, nano-, and tospoviruses. Losses caused by these
viruses are enormous. Potyviruses are a major
constraint in the production of a variety of crops like
sugarcane, potato, cucurbits, papaya, grain legumes
and vegetables (Varma, 1988). Geminiviruses cause
estimated economic loses of US $ 1300-2300 million
to cassava production in Africa (Thresh et ai, 1998),
US $ 5 billion to cotton production between 1992-99
in Pakistan (Briddon & Markham, 2000), US $ 300

*Author for correspondence
Tel : 5862134; Fax: +91-11-5813113
E-mail : anupamvarma@vsnl.net

million to grain legume production in India (Varma et
at, 1992) and US $ 140 million to tom ato production
In
Florida, USA (Moffat, 1999). In India,
badnaviruses cause serious diseases like rice tungro
(V arma et ai, 1999), ilarviruses are becoming
destructive in crops like sunflower and grain legumes
(Bhat et ai, 2001a), a nanovirus causes the most
damaging bunchy top disease of banana (Burns et aI,
1995) and tospoviruses are increasin gly becoming
most destructive virus disease problems in vegetables
and grain legumes (Bhat et aI, 2001b). Many other
viruses also commonly occur in the country (Varma
& Ramachandran, 1994).
Management of viral diseases is much more
difficult than that of diseases caused by other
pathogens as viral diseases have a complex disease
cycle, efficient vector transmission and no effective
viricide. Integration of various approaches like the
avoidance of sources of infection, control of vectors,
cultural practices and use of resistant host plants have
been used for the management of viral diseases of
plants. All these approaches are important, but most
practical approach is the use of varieties, which resist
vectors, seed transmission, symptom development,
cell-to-cell movement and virus multipli cation. The
mechanism of resistance varies from virus to virus
and host to host. Moreover, rapid development of
resistant breaking strains of viruses and lack of

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INDIAN J BIOTECHNOL, JANUARY 2002

sources of resistance make breeding of resistant
varieties difficult (Varma, 1993). Genes for resistance
to a large number of plant viruses have been identified
(Khetarpal et aI, 1999). Seventyeight per cent of the
res istant host-virus combinations are under the control
of monogenic resistance and a majority (65 %) of
these have dominant or incompletely dominant
resistance (Varma & Mitter, 2001), which are
overco me by resistant breaking strains .
Genetic engineering brings new hope for
overcoming various drawbacks associated with
conventional breeding for developing crop varieties
with durable resistance by 'pyramiding' genetically
engineered resistance over intrinsic plant resistance.
T his approach has been successfully applied to
generate viru s res istant tran sgenic plants (VRTPs).
VRTPs have been produced by transforming plants
with resi stance imparting transgenes from (a) the
AMV: Alfalfa mosaic alfamovirus ; ArMV: Arabis mosaic
nepov irus; AMCV: Artichoke mottle crinkle tombu svi ru s;
BGMV : Bean golden mosaic gemini virus; BNYVV: Beet necroti c
ye ll ow vein furoviru s; BMV: Brome mosaic bromovirus; BPMV:
Bean pod mottle co moviru s; BSMV : Barl ey stripe mosaic
hordeivirus; BYO V: Barley ye llow dw arf luteov irus; CaMV:
Cauliflower mosaic caulimovirus; CEVd: Citrus exocortis viroid;
CMMV : Chrysanthemum mil d mottle cucumovirus; CTV: C itrus
tristeza closterovirus: CuCLV: Cotto n leaf Geminivirus; CPMV:
Cowpea
mosaic co movirus;
CMV:
Cucumber mosaic
cuc um ovirus; CyRSV: Cymbidium rin g spot tombusvirus; GCMV
Grapev ine chrome mosa ic nepov irus; GFL V: Grapevine fan leaf
nepov irus; GRSV: Groundnut rin gspot tospovirus; INS V:
Impatiens necrotic spot tospov irus; LMV: Lettuce mosaic
potyvirus; MCMV : Mai ze ch lorotic mottl e machlomovirus ;
MOMV: Maize dwarf mosaic potyv irus; MYMV : Mun gbean
ye llow mosaic gemi ni virus; PRSV: Papaya ringspot potyvirus;
PEBV: Pea early browning tobrav irus; PEMV: Pea enation
mosaic enamovirus: PSbMV : Pea seed-borne mosaic potyvirus ;
PBNV: Peanut bud necros is tospovirus: PCSV: Pea nut chlorotic
st reak caulimovirus: PPV: Plum pox potyvirus; PLRV: Potato leaf
roll luteoviru s; PVM: Potato virus M ; PVS: Potato virus S ; PYX:
Po tato virus X; PVY : Potato virus Y; ROV: Rice d warf reo virus;
RSV: Rice stripe tenu ivirus; RTSV/RTBV: Rice tungro sp herical
and bac illiform vi ru s; RYMV: Rice yellow mottl e sobemovirus;
RgMV: Ryegrass mosaic rymovirus; SbMV: Soy bean mosaic
potyvirus; SMV: Sterility mosaic agent; SCMV: Sugarcane
mosaic potyvirus; SqMV Sq uash mosaic comovirus; TEV:
Tobacco etch potyv irus; TMGMV: Tobacco mi ld g reen mosaic
toba movirus; TMV: Tobacco mosaic tobamovirus; TRV : Tobacco
rattle tobrav irus; TRSV: Tobacco ringspot nepovirus; TSV:
Tobacco streak ilarvirus; TVMV : Tobacco veinal mottl e
potyvirus; TuMV: Turnip mosaic potyvirus; TYMV: Tobacco
yel low mosaic tymovirus; TCSV: Tomato ch lorotic spot
tospov irus: TGMV: Tomato golden mosaic gem ini virus; ToLCV:
To ma to leaf curl Geminivirus; ToMV : To mato mosaic
tobamov irus; ToRSV; Tomato ringspot nepovirus; TSWV:
To mato spotted wi lt lospovirus; TYLCV: Tomato yell ow leaf curl
geminivirus; WMV-2: Waterme lon mosa ic potyv irus-2; ZYMV :
Zucchini ye ll ow mo saic potyvirus.

virus for which resi stance is to be developed, (b)
plants, and (c) other sources (Table 1). Progress in the
development of VRTPs have been extensively
reviewed by Beachy et' al (1990); Hull & Davies
(1992); Fitchen & Beachy (1993 ); Grumet (1994);
Lomonossoff (1995) ; Pappu et af. (1995); Varma
(1997); Prins & Goldbach (1998) ; Reimann-Philipp
(1998); Jain & Varma (2000); Bendahmane &
Beachy, 1999 and Callaway et ai (2001). In thi s
review we analyse re lative significance of vari ous
approaches in the li ght of practical application of
VRTPs as an important component of integrated
management of serious plant di seases caused by
vIruses.

Virus-derived Resistance (VDR)
The concept that the viral genes, either as a wild
type or mutant, could confer resistance in host pl ants
(Hamilton, 1980; Sanford & Johnston, 1985),
stimulated research fo r generating VRTPs throu gh
genetic engineering. The first VRTP using VDR was
produced in the mid 1980s by expressing TM V coat
protein gene in transgen ic tobacco plants (Powe llAbel et aI, 1986). Since then , many different viral
genes and viral associated RNAs have been used as
transgenes to confer resistance in plants, and VDR
became a reality against a range of plant vi ru ses
having positi ve-sense ssRNA, ambisense RNA or
ssDNA (Grumet, 1994; Pappu et aI, 1995; Jain &
Varma, 2000). T his approach has also been used to
develop resistance to viroids (Atkins et aI, 1995 ).
Viruses depend on the host machinery for
rep lication. T he genome of plant viruses is ss DNA,
dsDNA , ssRNA or dsRNA. Most of the plant viruses
have positive sense ssRNA genome that replicate by
vi rus encoded RNA dependent RNA po lymerase and
form dsRNA repli cative intermed iate (Varma &
Ramachandran, 200 I). The vira l genome is
encapsidated in particles having icosahedral or sp iral
symmetry formed by compact arrangemen t of coat
protein subunits in specific pattern. The viral genome
could be either monopartite (undivided) or bi or
multipartite (divided into two or more molecules),
Irrespective of the number of genomic molecules,
each geno me has open reading frames (ORFs) to
produce structural and nonstructu ral proteins fo r
various funct ions like replication, cell-to-ce ll
move ment, vector transmission, encapsidati on, etc.
The events that fo llow infection include disassembly
of virus particles, synthesis or transcri ption of mRNA
(where required), tran slation of protei ns coded by
vira l genome for variolls function s, mat uration of

VARMA et al: VIRUS RESISTANT TRANSGENIC PLANTS

75

Table I-Possible transgenic interference with major events during plant-viru s-vector interactions
Transgene

Transgenic Interference

Virus-derived
Coat protein
Replicase protein
Movement protein
Viral Protease
Helper protein
Seed transmission factor
Non-coding region
Antisense RNAIDNA
Ribozyme
Satellite
Defective interfering RNA s

Transmission, Uncoating, Assemb ly
Replication
In vasion
Protein processing
Delays symptom development
Seed transmission
Competition for viral replicase
Replication, tran slation , assemb ly
Replication, tran slation , assemb ly
Replication
Replication

Plant-derived

Antiviral proteins
Host 'R' genes

Multiplication
Multiplication

Plantibodies
Yeast RNase

Replication, protein processing, assembly
Cleaving of dsRNA

Others

particles, systemic infection and vector transmission.
Strategies directed to interference with any of these
function s result in the development of VRTPs
(Table 2).
Both the coding and non-coding regions of viral
genomes have been used for developing VRTPs. For
some groups of viruses, like potyviruses, resistance in
plants has been obtained using almost all the viral
genes as transgenes . CP gene is the most commonly
used transgene for developing VRTPs against viruses
belonging to different groups followed by the
replicase protein and movement 'protein genes
(Table 2). Transgenic plants have been developed for
a large number of crops (Table 3).

Coat Protein
Coat protein (CP) gene is the most widely used
transgene to generate VRTPs. The strategy is
commonly referred to as coat protein-mediated
resistance (CPMR) and occasionally as genetically
engineered cross-protection. CP genes from at least
37 RNA/DNA viruses belonging to 17 virus groups
(Table 2) have been used to confer resistance into
many different plant species (Grumet, 1994; Pappu
et ai, 1995; Jain & Varma, 2000; Callaway et ai,
2001). CPMR has been found dfective irrespective of
virus particle morphology (rigid rod, flexuou s,
isometric,
bacilliform, lipoprotein
enveloped),
genome organization (positive-sense, negative-sense,
ambisense; monopartite, multipartite) and mode of
transmission (mechanical, seed, pollen, and vector).

Unlike cross-protection (Gibbs & Skotnicki , 1986),
which is effective against a strain of the protecting
virus, CPMR may also provide protection against
other viruses of the same virus group. The degree of
heterologous protection depends on the amino acid
sequence homology between CP genes of the
challenging and protecting virus. Plants transformed
with CP gene of CMV are also resistant to CMMV
(Nakajima et ai, 1993). Similarly CP gene of SbMV is
effective against TEV and PVY, and of TMV against
ToMV and TMGMV (Nejidat & Beachy, 1990).
Tomato transformed with CP gene from CMV -WL is
resistant to all the strains of CMV except one (Fuchs
et ai, 1997) making generali zation difficult. However,
the efficacy of protection under field conditions is
greater for the homologous than for the heterologous
virus (Table 4). In order to broaden the resistance to
viruses belonging to different virus groups, CP genes
from more than one virus have been used very
effectively (Table 4). Like the mUltiple CP gene,
nucleocapsid protein (NP) gene of TSWV can also be
used with CP gene of other viruses. Nicotiana
benthamiana transformed with TSWV NP gene and
TuMV CP gene were resistant to both the viruses in
T1 plants but not in T2 plants (Jan et ai, 2000a)
showing the possibility of developing multiple virus
resistance including the tospoviruses, which have
emerged as devastating pathogens in various parts of
the world. VRTPs against TSWV have been
developed in chrysanthemum, peanut and tomato
using NP gene as transgene. A recent study has shown
that complete gene is not always necessary for

[NDIAN J BrOTECHNOL, JANUARY 2002

76

Table 2-Spectrum of transgenic techno logy against viruses
Virus group
Virus-derived
Rep
MP

CP

Transgene
Plant-derived
Sat
AS
Rz

Pro

Alfamo
+
+
Bromo
+
Carla
+
Clostero
Como
+
+
Cucumo
+
+
Enamo
+
Furo
+
Gemini
+
+
+
liar
+
Luteo
+
+
+
Nepo
+
Potex
+
+
+
Poty
+
+
+
+
Reo
Rymo
+
Tenui
+
Tobamo
+
+
+
Tobra
+
+
Tombus
+
+
Tospo
+
+
Viroids
CP, coat protein ; Rep,Replicase protein; MP, Movement protein ; Pro,
Avp, Antiviral protein; Pb, Plantibodies; Rase, Yeas t nuclease
+, Technology demonstrated; -, not tested

Avp

Pb

Others
Rase

+
+

+

+

+

+

+
+
+
+

+

+

+
+

+

+

+

+

+

+

+

+
+

+
+

+
+

+

Protease; Sat, Satellite RNAs; AS, Anti sense RNA; Rz, Ribozyme;

Table 3-Crops and the viruses affecting them for which virus resi stant transgenic plants ha ve been developed
Crops

Virus

Alfalfa
Apricot
Bean
Cantaloupe

Crops

Virus

AMV

Peanut

TSWV

PPV

Petunia

CMV

BGMV
ZYMV

Plum

PPV
PLRV, PVM, PVS, PYX, PVY

Potato

Cauliflower

CaMV

Rape seed

Chrysanthemum

Rice

Chinese cabbage

TSWV
TMV

Citrus

CTV

Soybean

BPMV
SCMV

Corn

MDMV, MCMV

Sugarcane
Sweet pepper

Grape
Lettuce

GCMV, GFLV, ToRSV

Tobacco

LMV, TSWV
PSbMV, PEMV

Tomato

Pea

CaMV
RDV, RSV, RYMV

Wheat

inducing resistance, and genome segment as small as
110 bp long could induce resistance (Jan et of,
2000b).
The mechanism of resi stance induced by CP gene
is either throu gh the protein encoded by the tran sgene
(pro tei n-medi ated) or by the transcript of the
transgene (RNA-mediated) (Lomonossoff, 1995;
Reimann-Ph ilipp, 1998) or both (Yusibov et 01,
1998). CP gene induced resistance is protein-mediated

CMV
CMV, PVY , TRSV
CMV,TMV, ToMV, TSWV, TYLCV
BYDV, WSMV

when a si ngle copy of transgene is inserted.
Resistance so expressed is of moderate level against a
broad range of related viruses and influenced by the
level of CP expressed in transgeni c plants. Transgene
undergoes transcription and transl ation, resulting in
hi gh levels of protein. The protein inhibits
disassembly of the infecting virus and forces the
assembly/disassembly equilibrium towards assembly.
This seems likely for the viruses belonging to potex-,
tenui- and tobamoviruses.

Y ARMA el ai: VIRUS RESISTANT TRANSGENIC PLANTS

77

Table 4-Reaction to coat protein mediated resistance in transgenic plants under field conditions
Degree of
protection*

Reference

Crop

Resistance to virus (es)

Transgene (source)

Tomato

TMY (U 1 and PY230)

CP (TMY Ul)
CP (ToMY)
CP (TMY UI)
CP (ToMY)
CP (CMY WL)

+++
+++
+
+++
+++

Nelson et ai, 1988
Fuchs et ai, 1997
Fuchs et ai, 1997

CP (ZYMY)
CP(ZYMY)
CP(ZYMY)+ WMY2
CP (ZYMY+WMY2+CMY)

+++
+
+++
+++

Fuchs & Gonslaves, 1995
Fuchs & Gonslaves, 1995
Fuchs et ai, 1997
Fuchs et ai, 1997

CP (CMY+
ZYMYand
WMY2)
CP(PYX)
CP (PYYN)
CP(PYY(N)
CP(PYX+PYY)
CP (PRSY-Hawaii)
CP (PRSY- Hawaii)

+++
+++
+++
+++
+++
++
+++
+++
++

Fuchs el ai, 1997

ToMY

Squash

Cantaloupe
(Cucumis meia)

CMY (many strains
except one)
ZYMY
WMY2
ZYMY+WMY2
ZYMY+WMY2+CMY

CMY;
CMY+ZYMY
CMY+ZYMY+WMY2
Potato
PYX
PYY (N)
PYY (0)
PYX+PYY
PRSY (Hawaii)
Papaya
PRSY (Asia)
* high: +++; medium: ++; low: +

CP gene induced resistance is RNA-mediated when
of
transgene
are
inserted
multiple
copies
(Lomonossoff, 1995). Resistance so expressed is of
high level and virus-specific and is attributed to lower
levels of transcripts. Transgene expression is upto
mRNA level with little or no transgenic protein.
When mRNA accumulation exceeds threshold level ,
co-suppression (silencing) is initiated, affecting
transgene expression and virus replication. Thus,
limited expression of CP gene is a pre-requisite for
virus-specific resistance. CPMR in many virus-host
combinations (Table 4) is caused by posttranscriptional gene silencing (PTGS). This plant
defense system results in degradation of mRNA
produced both by the transgene and the viru s
(Waterhouse et ai, 2001). CPMR has also been shown
to work for gemll1lvlruses. Tobacco plants
transformed with ToMo V CP gene modified by the
deletion of 30 nu cleotides in the 5' end showed
res istance varying from susceptibili ty to immunity .
Resistance in these plants seems to be mediated by
transgene tran script as no transgene product was
detected (SinisteIT8 et al, 1999). PTGS based
resistance, however, may be suppressed foll owing
infection by another virus . For example, virus specific
resistance induced by PV A CP gene in transgenic
plants was suppressed by PVY infection (Savenkov &
Val konen, 2001). Similarly resistance to PVY is
suppressed by CMV infection (Mitter et al, 2001) .
Breakdow n in resistance by infection with a

Gonsalves et ai, 1992; Fuchs el ai, 1997

Fuchs et ai, 1997
Fuchs et ai, 1997
Fuchs el ai, 1997
Kaniewski et ai, 1990
Gonsalves, 1998
Gonsalves, 1998

heterologous virus limits the use of PTGS as a
strategy for virus disease management.
Since the first field testing of CPMR against TMV
in tomato plants in 1987 (Beachy et al, 1990), there
have been increasing number of field tests in different
host-virus systems (Table 3). All the VRTPs
commercialized so far are based on CPMR. Squash
val'. Freedom II is resi stant to WMV-2 and ZYMV
(Tricoli et aI, 1995), squash val'. CZW-3 is resistant to
CMV , WMV -2 and ZYMV (Fuchs et aI, 1997) and
papaya vars SunUP and UH Rainbow are resistant to
PRSV (Gonsalves, 1998).
Resistance to PRSV in papaya: a success sto ry.
Gonsalves (1 998) and Swain & Powell (2001) have
produced informative reviews on the success of
transgenic papaya resistant to PRSV in revivin g
Hawa iian papaya industry. Hawaii is a major
commercial papaya producer in the world. Initiall y
papaya production started on island of Oahu in 1940
but was abandoned by 1950 due to the high incidence
of PRSV and relocated in PRSV free Puna region of
Hawaii islands. In 1992, PRSV appeared in thi s
region too res ulting in a decreased papaya production
by 38% hetween 1993 and 1997. Anticipatory
resea rch was initiated in 1987 at the Uni versity of
Hawaii to develop tran sgen ic papaya res istant to
PRSV using the CP gene. Tn field trials from 1991 to
1993 transgenic line 55 - 1 of papaya var. Sunset
remained free for 25 months whereas 95% of nontransgenic plants were infected within 2.5 month s.

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INDIAN J BIOTECHNOL. JANUARY 2002

This remarkable demonstration coincided with the
emergence of PRSV. which affec ted more than 50%
of the area in Puna region by late 1994. puttin g
several farmers out of business. In Puna region mainly
papaya var. Kapoho was grown. It was also
transformed but the transgenic plants were
susceptible. Th erefore. F-l hybrid (UH Rainbow)
between var. Kapoho and transgenic line of var.
Sunset. designated as 'UH SunUP' was developed.
Hybrid UH Rainbow plants. although susceptible at
early growth stages became fully res istant to PRSV at
abo ut three month s age. In 1995 , UH Rainbow
yielded 1,12,000 kg/ha marketable fruits compared to
5600 kg/ha frrom non -transgenic plants
a
re markable twenty times increase in yield. The
environmental and toxicological concerns were also
favourably addressed resulting in deregulation of
PRSV CP transgenic papaya in the US in 1998.
There are lessons to be learnt fro m the transgenic
papaya success story . (1) Anticipatory research for
develop ing solutions to the ex isting and expected
problems through the applicati on of modern science is
important. (2) The efficacy of a particular strategy
may not work for all the strains or varieties of
transformed host. For example, PRSV CP transgen e
was effective in trangenic papaya var. Sunset but not
in var. Kapoho, and even in transgenic Sunset it is not
effective for PRSV isolates from outside Hawaii. (3)
Resistant transgenic plants are valuable genetic
resource. (4) Fast trac k approv al sys tem is an essential
component of achieving success in such efforts.

Replicase Protein
Replicase protein gene is the second widely used
transgene to confer resistance against plant viruses
and the strategy is referred to as replicase protein mediated resi stance (RPMR). Since its first
demonstration in TMV (Gol emboski et ai, 1990), it
has been successfully used from 16 RNA/DNA
viruses representing 11 plant virus groups (Table 2;
Palukaitis & Zaitlin, 1997; Jones et ai, 1998). RPMR
gives nearly immune type and highly specific
resistance for the virus from which the transgene is
isolated . It is more effective than CPMR and is not
influenced by inoculum levels. Molecular mechanism
of RPMR is not fully understood. However, it seems
to be either protein- or RNA-mediated. RPMR to
PLRV in potato appears to be caused by PTGS
(Table 5). Since the concept is relatively new , its
commercial application is just being initiated. Potato
lines combining RPMR to PLRV and Bt-resistance to

Colorado potato beetle have also been developed
(Thomas et ai. 1995; 1998). RPMR also shows
promise for developing VRTPs resistant to
gemini viruses, which are emerging at a fast rate and
are a major growth limiting factors in a wide range of
crops in various parts of the world (Varma & Malathi,
2001). Transient express ion of ACI (required for viral
DNA replication) of ACMV gave significant
reduction in viral DNA replication. The tran sformed
N. benthamiana pl an ts either re main sy mptoml ess or
produce delayed and attenuated sym ptoms (Hong &
Stanley, 1996).

Movement Protein
Cell-to-cell or long di stance transport of viruses in
plants is brought about by movement proteins (MPs)
(Mushegi an & Koonin, 1993) or coat protein (Dolja et
ai, 1995) or helper component-proteinase (HC-Pro)
protein (Cronin et ai. 1995 ) encoded by viruses.
Interferin g wi th cell-to-cell or long distance transport
would thu s be an ideal strategy for developing virus
resistance as has been demonstrated for at least six
different gro ups of viruses (Tabl e 2) . In order to
achieve thi s goal, defective movement proteins have
been expressed in transgenic pl ants. In contrast to
other strategies, th is approach offers attractive
possibility to confer broad- spectru m resistance to
related and unrelated viruses. For example, a
defectiv e TMV MP expressed in transgenic tobacco
plants was shown to confer varying lev els of
resistance to a number of viru ses th at are not members
of the tobamoviru s group, includ ing AMV, CMV,
PCSV , TRV and TRSV (Cooper et ai, 1995) .
Similarly, transgenic potato lines expressin g mutant
PLRV pr 17 movement protein exhibited resi stance
against unrelated viruses PVY and PYX (Tacke et ai,
1996). On the other hand , transgeni c plants expressing
the putative movement protein gene (NSm) from
TSWV were resistant to infection by TSWV strains
only (Prins & Goldbach, 1998). T his demonstrates
variability in the cell-to-cell movement amongst the
viruses of different groups .

Proteases
Certain viruses like como-, nepo- and poty viruses
use polyprotein strategy for gene expression (Varma
& Ramachandran, 2001) . A single large protein is
first produced which is subseque ntly processed by a
viral protease to yield functi ona l proteins. The
processing of a polyprotein is thu s an important step
during the course of viral infection. It, therefore,

VARMA el ai: VIRUS RESISTANT TRANSGENIC PLANTS

79

Table 5- Promi sing viral resistant transgenic plants developed using transgene from diverse sources
Crop

Resistant to virus

Transgene

Mechanism of resistance

Reference

Melon
Plum

WMV2
PPV

Cleaving of viral RNA
PTGS

Potato

Rice
Rice
Ryegrass
Sq uas h
Tobacco

PLRV
PLRV
PV Y(o)
RDV
RYMV
RgMV
SqMV
CMV

Ribozyme
CP
CI (RNA hel icase)
Replicase
CP
PI
Ribozyme
Polymerase
Untran slatable CP
CP
RNAI

Huttner el al. 200 I
Ravclonandro el al. 2000
Wittner et al. 1998
Vazquez et al. 200 1
Barker et al. 1992
Mak i-Val kama el £Ii. 2000
Han et ai, 2000
Pinto el ai, 1999
Xu et al. 200 1
Jan et ai. 2000c
Canto & Palukaiti s, 200 I

Tobacco
Tobacco
Wheat

TEV, PVY
PVY
BYDV

Oryza cystatin
PVYCP
Bacterial ribonuclease fII
(RNase III)

PTGS
Virus replication
Transgene si lencing
PTGS
PTGS
PTGS
Suppresses Vara (RNA-I )
acc umul ati on
Inhibition of polyprotein processing
PTGS
Degradation of viral RNA

seems likely th at one could si mulate resistance to
viruses using a polyprotein strategy by expressing
defective proteases th at could interfere with the action
of the incoming functional protease and inhibit the
cleavage of the polyprotein. Such a strategy has been
tested only fo r potyviruses (Table 2). Transgenic
plants that expressed the nuclear inclusion (NIa)
protease of TVMV or PVY were resistant to
subsequent infection by respective viruses (Maiti et
aI, 1993; Vardi et at, 1993). The resistance seems to
be virus-specific as heterologous protection against
TEV was not observed.
Satellite and Defective lntelfering RNAs

Some viruses have specific satellite RNA
molecules (sat-RNA), which are considered viral
parasites being dependent on helper virus for
multiplication (Matthews, 1991). Some sat-RNA s
exacerbate the disease caused by the helper virus,
whereas some other sat-RN As ameliorate the disease.
The sat-RNAs of the latter type have been used for
developing VRTPs for resi stance to cuc umo- and
nepoviruses (Table 2) . Transgenic tobacco plants
expressing sat RNAs of CMV or TRSV on ch allenge
inoculation exhibited attenuation of disease symptom s
(Harrison et aI, 1987; Gerlac h et aI, 1987). This
strategy has not gained much acceptance as (i)
resistance is incomplete; and (ii) minor mutations in
sat-RNA migh t turn a benign sat-RNA into a disease
exacerbating sat-RNA as has bcen shown for CMV
sat-RNA. There is, however, a pussibility of
combining sat-RNA-mediated resistance with CPMR
for developing stable resistance (Yie et aI, 1992).
Defective interfering (D!) RNAs (or DNAs) also
potentially can be used to engineer virus resi stance.

Gutterrez-Campos et al. 1999
Han et al. 1999
Zhang el al. 200 I

The first demonstration of thi s approach in plants was
fo r ACMV, a member of the geminivi rus group
(Stanley et at, 1990). DI RNA has been shown to
reduce disease severity due to CyRSV in transformed
N. benthamiana plants (R ubino et al, 1992). Further,
synthetic DI RNAs have been developed in BMV
(Mars h et ai, 1991). Whether synthetic DI molecul es
wi ll be efffecti ve in transgenic plants remains to be
tested. It may become a widely appl icable strategy.
Antisense RNA

Antisense RNAs are RNAs that are complementary
to the coding or m-RNA strand. Antisense technology
provides protection either by inhibiting gene
expression or viral replic~tion . Although th is
approach has been used wi th varying success aga inst
RNA and DNA viruses and viroids (Table 2), it has
considerable potential in the case of viruses confined
to ph loem tissues (luteoviruses) and the viruses,
which replicate in th e nucleus (geminiv irw;es) (Tabler
et aI, 1998). Transgenic bean (Phaseolus vulguris)
plants engineered with genes Rep-TrAP-Ren and BC 1
of BGMV , a gcminivirus, in ~lI1tisense orientati on
show attenuated symptoms to the virus (Fuchs er al.
1997).
Ribozyme

The term ribozyme refcrs to the ability of a RNA
mol ecule to cleave targe t RNA molec ules. Althou gh
ribozyme specific for any unwanted gene can be
designed and integrated into plant genome (Haseloff
& Gerlach, 1988), its use in vivo has been limi ted.
Ribozy me-derivecl resi stance has recentl y bee n
demonstrated in transgen ic tomato against CEVd
(Atkins et at, 1995), tobacc o against TMV (De Feyter

80

INDIAN J BIOTECHNOL. JANUARY 2002

et ai, 1996), rice against ROV (Han et ai, 2000), and
melon against WMV 2 and ZYMV (Huttner et ai,
2001).

(LRR) genes (Lawrence et ai, 1995). It is possible that
such genes might confer resistance in transgenic
plants to taxonomically distant pathogens.

Vector Transmission Protein
Transmission of potyviruses by aphid vectors is
mediated either by a helper component protein
encoded by HC-Pro region of the genome or OAG
sequence near the amino-terminus of capsid protein
(Atreya et ai, 1995; Blanc et ai, 1998). Thus,
resistance could be engineered by transforming plants
either with a capsid protein construct mutated in the
OAG region or a helper component construct mutated
in the KITC region.

Antiviral Proteins
Natural resistance to virus infectio n in non-hosts
has been attributed to the presence of anti viral
proteins (Verma et ai, 1998). These novel biomolecules have been identified from several plant
species and genes coding them have been tran sferred
to other plants . For example, tobacco and potato
plants transformed with PAP/PAPII (Pokeweed
antiviral protein) genes exhibited resistance not only
against the viruses like TMV, PYX, PVY and PLRV
but, also against fungus (Rhizoctonia solani) infection
(Lodge et ai, 1993 ; Wang et ai, 1998). The genes
coding for antiviral proteins in other plants like
Boerhaavia, carnation, Clerodendron, Mirabilis ,
etc.may also be useful. A major difficulty in using
such genes is the adverse effect of their products in
transformed plants.

Seed Transmission Factors
Viral
determinants
associated
with
seed
transmission have been determined in BSMV (Yb
ge ne) (Edward, 1995), PEBV (12 K gene-RNA1)
(Wang et ai, 1997) and PSbMV (5' UTR-PI-Pro-HCPro) (Johansen et ai, 1996). Potential of these factors
to provide resistance to seed transmission in
transgenic plants could be explored.
Plant-derived resistance

R-Genes
IMany plant genes imparting resistance to viruses
have been identified (Hulbert et ai, 2001; Gebhardt &
Volkonen, 2001; Varma & Mitter, 2001). Use of these
genes for conferring resistance would be more easily
acceptable. The 'N' gene from tobacco introgressed
into tomato gives good protection against TMV and
ToMV, which are serious constraint in tomato
production (Whitham et ai, 1994; Oinesh-Kumar et
ai, 1995; Oinesh-Kumar & Baker, 2000). Similarly
Rxl, and Rx2 genes from Solanum tuberosum have
been introgressed into potato breeding line to confer
resistance against PYX (Bendahmane et ai, 1999;
2000). Plant cystatins are strong inhibitors of virus
infection requiring processing of viral polyprotein, as
has been demonstrated for rice cysteine proteinase
inhibitor (rice cystatin), which resists infection by
PVY and TEV in transformed tobacco (GuttierrezCampos et ai, 1999). It has been observed that genes
(like L6 from flax for resistance to a fungal disease,
RPS2 from Arabidopsis for resistance to bacterial
infection and 'N' from tobacco for resistance to virus
infection), which control resistance to three widely
different pathogen types, share common structural
organization and are refened as leucine-rich region

Plantibodies
Antibody-based resistance is a novel strategy for
generating MRTPs (Martin, 1998; Schillberg et ai,
2001). Resistance can be engineered by the
expression of virus specific plantibod ies (antibodies,
antibody fragments or antibody fusion proteins).
Transgenic plants express ing antibody specific to
AMCV CP were found resistant to virus infection
(Tavladoraki et ai, 1993). Similar approach has been
attempted to generate tran sgenic tobacco lines
expressing antibody to TMV CP and TSWV
nucleocapsid and glycoprotein (G1) (Sherwood, 1994;
Francone et ai, 1999). The protection afforded by
plantibodies in transgenic plants has some advantages.
Firstly, it can be engineered against almost any target
molecule and no viral sequence is required to be
expressed in the transgenic plants. Secondly , this
approach could be applied at any stage of the virus
life cycle, although it would be more effective if
targeted towards early stages of the virus infection
cycle (Martin, 1998). A disadvantage in using the
approach is that a gene of animal origin would be
expressed in plants, which may not find favour with
the environmentalists.

Yeast RNase
A majority of plant viruses have positive sense ss
RNA as genome. They form ds RNA during their
replication, which are characteristic of virus infections

VARMA

el

al: VIRUS RESISTANT TRANSGENIC PLANTS

in plants. Yeast RNase has been shown to cleave
dsRNA in transgenic plants expressing yeast RNase
specific for dsRNA. Potato plants expressing yeast
RNase resist infection by CMV, PVY, PYX, TMV
and TRV (Lamontagne et ai, 2001). This approach
provides an excellent opportunity for developing
broad spectrum VRTPs.

Concerns about Field Release of Transgenic Plants
Potential risks in the use of VRTPs are essentially
similar to those posed by conventional biotechnology
and plant breeding (Varma, 1997). However, for
achieving acceptance of VRTPs various concerns
must be judiciously 'addressed on strong scientific
basis. Environmental risks related to the use of
VRTPs are not greater th an those caused by normal
infection of plants by viruses (Table 6). Main
concerns about the field release of VRTPs are about
the possibility of generation of new viruses/strains as
a result of recombination and/or tran sencapsidation.
Recombinants between TMV vector and TMV in
TMV transformed N. benthamiana plants have been
observed (Adair & Kearney, 2000). PPV with mutated
CP gene is able to cause systemic infection in
N.benthamiana, transformed with wild type PPV CP
but not in non-transgenic plants, suggesting
complementation of CP gene in transgenic plants
(Varrelmann & Maiso, 2000). Such events do occur in
nature during co-infection of two viruses of same or
different taxon, which occasionally lead to virulent

81

forms as has been found for gemini viruses (Varma &
Malathi, 2001). The recombinants must not only be
viable but also have some selective advantage in th e
transgenic plants (Hammond et ai, 2000). A
recombinant arising in a tran sgenic plant will be
inhibited or eliminated by the resistance mechanisms,
like PTGS, of the transgenic plant (Rubio et ai, 1999).
Further, Thomas et ai (1998) extensively examined
the possibility of recombination and its adverse
effects in the field release of transgenic potato plants
expressing either the CP or replicase gene of PLRV.
No evidence of new viruses resulting from large-scale
deployment of transgenic plants was observed .
Although the risks posed by deployment of transgenic
technology seem to be small, the potential risks must
be minimized by suitably tailoring the transgene
itself. This could be achieved by (i) use of defecti ve
forms of viral genes, (ii) use of untranslatable RNA
sequences ; (iii) use of genes from mild endemic
isolates, (iv) avoidance of replicase recognition
sequences, and (v) combining/pyramiding transgenes
with other types of resistance such as plant-expressed
antibodies, antiviral proteins or dsRNA specific
nucleases (Timmerman-Vaughan, 1998; Hammond et
ai, 2000).
Many a times, transfer of transgene to related
species is considered a serious risk in the field use of
transgenic plants as some weeds may become more
weedy and the transgene may move to non-targeted
crop species. Firstly, such flow of genes under natural

Tab le 6- Potential ri sks in the use of VRTPs for integrated virus disease management
SI No

Potential risk

Environmental impact

Transfer of tran sge nes to related species

Favourable

2

Development of resistance breaking strains(s)
of the virus

Not greater than the use of conventional resistant hosts

3

Transgene of viral origin may result in
emergence of new recombinant viruses which
may be more virulent and have enhanced host
range

Not greater than the normal evolution of viruses

4

Transencapsidation may occur

Not greater than the normal circumstances

5

Marker genes could have adverse effect, but their use is avoidable

6

Adverse effect of marker genes like the
antibiotic resistance genes
Toxicity of transge ne products

7

Insertion of transgene into structural gene

It would cause phenotypic changes and such VRTP would not be used

8

Loss of biodiversity due to replacement of the
traditional varieties

No more than caused by the improved crop varieties in use.

Transgenes of virus origin are safe as their products are a part of normal
diet. Similarly ' R' genes are safe. Other transgene would need appropriate
biosafety tests

82

INDIAN J BIOTECHNOL, JANUARY 2002

Table 7-Research efforts for developing vi ral resistant transgenic plants in India
Crop

Transge ne

Resistant agai nst

Research centre

Cotton

Replicase (CuCL V)

Cotton leaf curl disease

lISe, Bangalore

Mungbean

CP/replicase (MYMV)

Mungbean yellow mosaic

Madurai University

Papaya

CP (PRSV)

Papaya ringspot

IARI, New Delhi

Potato

CP (PVY)

Potato virus Y

JAR!, New Delhi; CPRI, Shimla: BARC, Mumbai

Rice

CP (RTSV IRTBV)

Rice tungro

South campus, Delhi University

Soybean

Replicase (MYMV)

Soybean yellow mosaic

IARI, New De lhi

Tobacco

CP (PVY)

Potato virus Y

IARI , New Delhi

Tomato

Replicase (ToLCV) , CP
(CMV), CP (ToMV)

Tomato leaf curl , tomato mosaic

IAR) , New Delhi

ecosystem is remote and secondly, if it does occur it
would be environmentally favourable as build up of
virus inoculum in weed plants, which are a major
so urce of virus infection, would be reduced leading to
\
reduction in epidemics. As far as the non-target crop
species is concerned, this too would be of advantage
as virus infection is not desirable in any cropping
system. III effect of transgene on human, animal and
plant health is another area of concern. This should be
appropriately addressed when transgenes are used
from non-edible plants and other sources. Overall, the
bio-safety concerns in the use of VRTPs are
insignificant.

Indian Scenario
Certain groups of plant viruses like gemini, poty- ,
cucumo-, badna-, and tobamoviruses are major
constraints of crop production in the Indi an
su bcontinent (Varma & Ram achandran , 1994).
Besides these , tospo- and il arviruses are emerging as
threatening pathogens (Bhat el ai, 2001a, b).
Development of host plants resistance for the
management of vi ral diseases is the most practical
approach used in the country, bu t for a large number
of host-virus combinations either sui table sources of
resistance are not av ailable or the resistance genes are
linked to undesirable agronomic traits. It is therefore
essential to launch an aggressive programme for
developing VRTPs. Efforts are in progress at various
centres in the country to develop VRTPs of cotton,
mun gbean , papaya, potato, tomato and soybean
res istant to the important viru ses affecting these crops
(Table 7). High degree of resistance to PVY has been
developed in tobacco transformed with CP gene,
which has remained stable up to T4 ge neration
although th e transgene product has not been detected
(Maheshkumar el al, 2001 ), suggesting that the
resistance may be due to PTGS.

NBRI. Lucknow

Concluding Remarks
Transgenic technology holds promise in restraining
plant viruses by enhancing the leve l of intrinsic plant
resistance and increasing yield potential. Increase in
yields of tomato transformed with CP gene of CMV
has been shown to be 17 fold and in cantaloupe 7
folds (Fuchs el ai, 1997). The potential benefits of
VRTPs also include reduction in the use of pesticides
for vector control, improved crop quality, possibility
of developing varieties with multiple virus resistance,
and decreased seed certfication costs. In addition ,
VRTPs are important genetic source for plant virus
resistance (Varma et ai, 2001). The 1s f generation
transgenics are mostly under monogenic control. In
order to have durable transgenic resistance, next
generation of transgenics will have to be produced by
transforming with multiple genes. For example,
durable resistance to tospoviru ses could be engineered
by simultaneously expressing NP and NSm genes in
plants. Similarly, CP and satellite RNA sequences
could be used simultaneously to engin eer durable
resi stance against cucumoviruses. In general, CPMR
is more effective th an the other approaches. Now that
VRTPs
have
also
shown
promise
in
monocoty ledonous plants li ke rice, ryegrass and
wheat, and perenni al plants like plum (Tab le 3) , more
extensive effort s would be required in various parts of
the world to minimize the losses caused by plant
viru ses through wider use of VRTPs. At present, of
the 44.7 m ha area under transgenic crops abo ut 1% is
under VRTPs, 74% under transgenic crops resistant to
herbicides, 19% resi stant to insect pes ts and 7% with
combined resistance to herbicide and insect pests. In
the years to come the area under VRTPs in expected
to grow at a fas ter rate .
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