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

Advanced 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 yield­

reducing 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 tomato production In Florida, USA (Moffat, 1999). In India, badnaviruses cause serious diseases like rice tungro (Varma 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 increasingly 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 multiplication. 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

74 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 overcome 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. This approach has been successfully applied to generate virus res istant transgenic plants (VRTPs). VRTPs have been produced by transforming plants with resistance imparting transgenes from (a) the

AMV: Alfalfa mosaic alfamovirus ; ArMV: Arabis mosaic nepov irus; AMCV: Artichoke mottle crinkle tombusvi rus; BGMV: Bean golden mosaic gemini virus; BNYVV: Beet necrotic ye llow vein furovirus; BMV: Brome mosaic bromovirus; BPMV: Bean pod mottle comovirus; BSMV: Barley stripe mosaic hordeivirus; BYOV: Barley ye llow dwarf luteovirus; CaMV: Cauliflower mosaic caulimovirus; CEVd: Citrus exocortis viroid; CMMV : Chrysanthemum mild mottle cucumovirus; CTV: Citrus tristeza closterovirus: CuCLV: Cotton leaf Geminivirus; CPMV: Cowpea mosaic comovirus; CMV: Cucumber mosaic cuc umovirus; CyRSV: Cymbidium ring spot tombusvirus; GCMV Grapev ine chrome mosaic nepovirus; GFL V: Grapevine fan leaf nepov irus; GRSV: Groundnut ringspot tospovirus; INS V: Impat iens necrotic spot tospov irus; LMV: Lettuce mosaic potyvirus; MCMV: Maize ch lorot ic mottle machlomovirus ; MOMV: Maize dwarf mosaic potyvirus; MYMV: Mungbean yellow mosaic gemi ni virus; PRSV: Papaya ringspot potyvirus; PEBV: Pea early browning tobravirus; PEMV: Pea enation mosaic enamovirus: PSbMV: Pea seed-borne mosaic potyvirus ; PBNV: Peanut bud necros is tospovirus: PCSV: Peanut chlorotic st reak caulimovirus: PPV: Plum pox potyvirus; PLRV: Potato leaf roll luteovirus; PVM: Potato virus M; PVS: Potato virus S ; PYX: Potato virus X; PVY : Potato virus Y; ROV: Rice dwarf reovirus; RSV: Rice stripe tenuivirus; RTSV/RTBV: Rice tungro spherical and bac illiform vi rus; RYMV: Rice yellow mottle sobemovirus; RgMV: Ryegrass mosaic rymovirus; SbMV: Soybean mosaic potyvirus; SMV: Sterility mosaic agent; SCMV: Sugarcane mosaic potyvirus; SqMV Squash mosaic comovirus; TEV: Tobacco etch potyvirus; TMGMV: Tobacco mi ld green mosaic tobamovirus; TMV: Tobacco mosaic tobamovirus; TRV : Tobacco rattle tobrav irus; TRSV: Tobacco ringspot nepovirus; TSV: Tobacco streak ilarvirus; TVMV : Tobacco veinal mottle 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: Tomato leaf curl Geminivirus; ToMV: To mato mosaic tobamovirus; ToRSV; Tomato ringspot nepovirus; TSWV: Tomato spotted wi lt lospovirus; TYLCV: Tomato yell ow leaf curl geminivirus; WMV-2: Watermelon mosaic potyvirus-2; ZYMV : Zucchini ye ll ow mosaic potyvirus.

virus for which resistance 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 light 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 through genetic engineering. The first VRTP using VDR was produced in the mid 1980s by expressing TM V coat protein gene in transgenic 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 ruses having positive-sense ssRNA, ambisense RNA or ssDNA (Grumet, 1994; Pappu et aI, 1995; Jain & Varma, 2000). This approach has also been used to develop resistance to viroids (Atkins et aI, 1995 ).

Viruses depend on the host machinery for rep lication. The genome of plant viruses is ssDNA, dsDNA, ssRNA or dsRNA. Most of the plant viruses have positive sense ssRNA genome that replicate by vi rus encoded RNA dependent RNA polymerase and form dsRNA replicative intermed iate (Varma & Ramachandran, 200 I). The vira l genome is encapsidated in particles having icosahedral or spiral symmetry formed by compact arrangement 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 genome has open reading frames (ORFs) to produce structural and nonstructural proteins fo r various funct ions like replication, cell-to-ce ll movement, vector transmission, encapsidation, etc. The events that fo llow infection include disassembly of virus particles, synthesis or transcription of mRNA (where required), translation of proteins coded by vira l genome for variolls function s, maturation of

VARMA et al: VIRUS RESISTANT TRANSGENIC PLANTS 75

Table I-Possible transgenic interference with major events during plant-virus-vector interactions

Transgene

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

Plant-derived • Antiviral proteins • Host 'R' genes

Others • Plantibodies • Yeast RNase

particles, systemic infection and vector transmission. Strategies directed to interference with any of these functions 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, flexuous, isometric, bacilliform, lipoprotein enveloped), genome organization (positive-sense, negative-sense, ambisense; monopartite, multipartite) and mode of transmission (mechanical, seed, pollen, and vector).

Transgenic Interference

Transmission, Uncoating, Assembly Replication Invasion Protein processing Delays symptom development Seed transmission Competition for viral replicase Replication, translation , assembly Replication, translation , assembly Replication Replication

Multiplication Multiplication

Replication, protein processing, assembly Cleaving of dsRNA

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 generalization 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

76

Virus group

Alfamo Bromo Carla Clostero Como Cucumo Enamo Furo Gemini liar Luteo Nepo Potex Poty Reo Rymo Tenui Tobamo Tobra Tombus

CP +

+

+ + + + + + + + +

+ + + + +

[NDIAN J BrOTECHNOL, JANUARY 2002

Table 2-Spectrum of transgenic technology against viruses

Virus-derived Rep MP

+ +

+ +

+

+

+ +

+ + +

+

+

+

+ +

+

Pro

+

Transgene Plant-derived

Sat AS Rz

+

+

+

+

+

+ + + +

+

+ +

+

Avp

+

+ +

+

Others Pb Rase

+

+

+

+

+

+ +

Tospo + + + + Viroids + +

CP, coat protein ; Rep,Replicase protein; MP, Movement protein ; Pro, Protease; Sat, Satellite RNAs; AS, Anti sense RNA; Rz, Ribozyme; Avp, Antiviral protein; Pb, Plantibodies; Rase, Yeas t nuclease +, Technology demonstrated; -, not tested

Table 3-Crops and the viruses affecting them for which virus resistant transgenic plants have been developed

Crops Virus Crops Virus

Alfalfa AMV Peanut TSWV

Apricot PPV Petunia CMV

Bean BGMV Plum PPV

Cantaloupe ZYMV Potato PLRV, PVM, PVS, PYX, PVY

Cauliflower CaMV Rape seed CaMV

Chrysanthemum TSWV Rice RDV, RSV, RYMV

Chinese cabbage TMV Soybean BPMV

Citrus CTV Sugarcane SCMV

Corn MDMV, MCMV Sweet pepper CMV

Grape GCMV, GFLV, ToRSV Tobacco CMV, PVY, TRSV

Lettuce LMV, TSWV Tomato CMV,TMV, ToMV, TSWV, TYLCV

Pea PSbMV, PEMV Wheat BYDV, WSMV

inducing resistance, and genome segment as small as 110 bp long could induce resistance (Jan et of, 2000b).

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 transgenic plants. Transgene undergoes transcription and transl ation, resulting in high 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.

The mechanism of resistance induced by CP gene is either through the protein encoded by the transgene (pro tei n-mediated) 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

Y ARMA el ai: VIRUS RESISTANT TRANSGENIC PLANTS 77

Table 4-Reaction to coat protein mediated resistance in transgenic plants under field conditions

Crop Resistance to virus (es) Transgene (source) Degree of Reference protection*

Tomato TMY (U 1 and PY230) CP (TMY Ul) +++ Nelson et ai, 1988 CP (ToMY) +++ Fuchs et ai, 1997

ToMY CP (TMY UI) + Fuchs et ai, 1997 CP (ToMY) +++

CMY (many strains CP (CMY WL) +++ Gonsalves et ai, 1992; Fuchs el ai, 1997 except one)

Squash ZYMY CP (ZYMY) +++ Fuchs & Gonslaves, 1995 WMY2 CP(ZYMY) + Fuchs & Gonslaves, 1995 ZYMY+WMY2 CP(ZYMY)+ WMY2 +++ Fuchs et ai, 1997 ZYMY+WMY2+CMY CP (ZYMY+WMY2+CMY) +++ Fuchs et ai, 1997

Cantaloupe (Cucumis meia)

Potato

Papaya

CMY; CMY+ZYMY CMY+ZYMY+WMY2 PYX PYY (N) PYY (0) PYX+PYY PRSY (Hawaii) PRSY (Asia)

* high: +++; medium: ++; low: +

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

CP gene induced resistance is RNA-mediated when multiple copies of transgene are inserted (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 virus (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 nucleotides in the 5' end showed res istance varying from susceptibili ty to immunity . Resistance in these plants seems to be mediated by transgene transcript 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) . Breakdown in resistance by infection with a

+++ 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 resistant 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 story. Gonsalves (1 998) and Swain & Powell (2001) have produced informative reviews on the success of transgenic papaya resistant to PRSV in revivin g Hawaiian papaya industry. Hawaii is a major commercial papaya producer in the world. Initially 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 resulting in a decreased papaya production by 38% hetween 1993 and 1997. Anticipatory research was initiated in 1987 at the Uni versity of Hawaii to develop transgen 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 months.

78 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. putting several farmers out of business. In Puna region mainly papaya var. Kapoho was grown. It was also transformed but the transgenic plants were susceptible. Therefore. F-l hybrid (UH Rainbow) between var. Kapoho and transgenic line of var. Sunset. designated as 'UH SunUP' was developed. Hybrid UH Rainbow plants. a lthough susceptible at early growth stages became fully res istant to PRSV at about 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 remarkable 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 developing solutions to the ex isting and expected problems through the application 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 transgene 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 track approval 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 (Golemboski 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 transformed N. benthamiana plan ts either remain sy mptoml ess or produce delayed and attenuated symptoms (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. Interfering wi th cell-to-cell or long distance transport would thus be an ideal strategy for developing virus resistance as has been demonstrated for at least six different groups of viruses (Tabl e 2) . In order to achieve thi s goal, defective movement proteins have been expressed in transgenic plants. 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 defective TMV MP expressed in transgenic tobacco plants was shown to confer varying levels of resistance to a number of viruses th at are not members of the tobamovirus group, includ ing AMV, CMV, PCSV, TRV and TRSV (Cooper et ai, 1995) . Similarly, transgenic potato lines expressing mutant PLRV pr 17 movement protein exhibited resi stance against unrelated viruses PVY and PYX (Tacke et ai, 1996). On the other hand, transgenic 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 potyviruses

use polyprotein strategy for gene expression (Varma & Ramachandran, 2001) . A single large protein is first produced which is subsequently processed by a viral protease to yield functional proteins. The processing of a polyprotein is thus an important step during the course of viral infection. It, therefore,

VARMA el ai: VIRUS RESISTANT TRANSGENIC PLANTS 79

Table 5- Promising viral resistant transgenic plants developed using transgene from diverse sources

Crop Resistant to virus Transgene Mechanism of resistance Reference

Melon WMV2 Ribozyme Cleaving of viral RNA Huttner el al. 200 I Plum PPV CP PTGS Ravclonandro el al. 2000

CI (RNA hel icase) Wittner et al. 1998 Potato PLRV Replicase PTGS Vazquez et al. 200 1

PLRV CP Virus replication Barker et al. 1992 PV Y(o) PI Mak i-Val kama el £Ii. 2000

Rice RDV Ribozyme Transgene si lencing Han et ai, 2000 Rice RYMV Polymerase PTGS Pinto el ai, 1999 Ryegrass RgMV Untranslatable CP PTGS Xu et al. 200 1 Squash SqMV CP PTGS Jan et ai. 2000c Tobacco CMV RNAI Suppresses Vara (RNA-I ) Canto & Palukaiti s, 200 I

accumulati on Tobacco TEV, PVY Oryza cystatin Inhibition of polyprotein processing Gutterrez-Campos et al. 1999 Tobacco PVY PVYCP PTGS Han et al. 1999 Wheat BYDV Bacterial ribonuclease fII Degradat ion of viral RNA Zhang el al. 200 I

(RNase III)

seems likely that one could simulate resistance to viruses using a polyprotein strategy by expressing defective proteases that 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-RNAs exacerbate the disease caused by the helper virus, whereas some other sat-RNAs ameliorate the disease. The sat-RNAs of the latter type have been used for developing VRTPs for resi stance to cucumo- and nepoviruses (Table 2) . Transgenic tobacco plants expressing sat RNAs of CMV or TRSV on challenge inoculation exhibited attenuation of disease symptoms (Harrison et aI, 1987; Gerlach et aI, 1987). This strategy has not gained much acceptance as (i) resistance is incomplete; and (ii) minor mutations in sat-RNA might 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 resis tance (Yie et aI, 1992).

Defective interfering (D!) RNAs (or DNAs) also potentially can be used to engineer virus resi stance.

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 (Marsh et ai, 1991). Whether synthetic DI molecules 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 against 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 the 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

molecule to cleave targe t RNA molec ules. Although 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. Ribozyme-derivecl resi stance has recentl y been demonstrated in transgen ic tomato against CEVd (Atkins et at, 1995), tobacco 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).

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.

Seed Transmission Factors Viral determinants associated with seed

transmission have been determined in BSMV (Yb gene) (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

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

Antiviral Proteins Natural resistance to virus infection 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 transferred 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.

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 plantibodies (antibodies, antibody fragments or antibody fusion proteins). Transgenic plants expressing antibody specific to AMCV CP were found resistant to virus infection (Tavladoraki et ai, 1993). Similar approach has been attempted to generate transgenic 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 81

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 than 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 transencapsidation. 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

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 the transgenic plants (Hammond et ai, 2000). A recombinant arising in a transgenic 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

Table 6- Potential ri sks in the use of VRTPs for integrated virus disease management

SI No Potential risk Environmental impact

Transfer of transgenes to related species Favourable

2 Development of resistance breaking strains(s) Not greater than the use of conventional resistant hosts of the virus

3 Transgene of viral origin may result in Not greater than the normal evolution of viruses emergence of new recombinant viruses which

4

5

6

7

8

may be more virulent and have enhanced host range

Transencapsidation may occur

Adverse effect of marker genes like the antibiotic resistance genes Toxicity of transgene products

Insertion of transgene into structural gene

Loss of biodiversity due to replacement of the traditional varieties

Not greater than the normal circumstances

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

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

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

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

82 INDIAN J BIOTECHNOL, JANUARY 2002

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

Crop Transgene 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 Tomato leaf curl , tomato mosaic IAR) , New Delhi (CMV), CP (ToMV)

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 source 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 subcontinent (Varma & Ramachandran , 1994). Besides these , tospo- and ilarviruses 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, but for a large number of host-virus combinations either sui table sources of resistance are not available 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, mungbean , 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 generation although the 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 level 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 1 s 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 tospoviruses could be engineered by simultaneously expressing NP and NSm genes in plants. Similarly, CP and satellite RNA sequences could be used simultaneously to engineer durable resi stance against cucumoviruses. In general, CPMR is more effective than the other approaches. Now that VRTPs have also shown promise in monocoty ledonous plants li ke rice, ryegrass and wheat, and perennial plants like plum (Table 3) , more extensive effort s would be required in various parts of the world to minimize the losses caused by plant viruses through wider use of VRTPs. At present, of the 44.7 m ha area under transgenic crops about 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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