Viral Resistance in Transgenic Plants | Genetics

In this article we will discuss about the viral resistance in transgenic plants.

Plant viruses are the potential candidate in affecting crop yield. Several conventional methods such as breeding for resistance, heat treatment are enforced to combat viral menace. Production of genetically modified plants with improved resistance to viral disease is one of the key goals in plant biotechnology.

Protection against viruses by employing improved and ad­vanced methods have been considered seriously. One of the earliest and controversial methods followed is cross protection. Plant biotechnology however, offered wide array of safest protec­tion systems.

Cross-Protection:

Cross-protection is a phenomenon in which plants are inoculated with mild strains of viruses or viroids to prevent more virulent strain viruses from infecting the plant. This practice of cross protection precludes yield losses in several crops such as potato and tomato from to­mato mosaic viruses and potato spindle tuber viroids, respectively.

The most positive implica­tion of cross-protection is the ability of mild strain virus to prevent or delay disease symptom caused by the more virulent strain otherwise known as challenge virus. Some cases replication of the challenge virus is prevented. In most cases, symptoms of challenge virus are appeared after certain period of protection.

Several mechanisms have been proposed to explain possible hypothesis of cross protec­tion. They include:

(i) Replication of inducer virus (mild strain) prevent availability of host cell component to challenge virus and prevent its replication,

(ii) Coat protein produced by inducer virus encapsulate the RNA of challenger, thereby preventing its replication

(iii) Uncoating or recoating of the challenge virus is blocked by free-coat protein of inducing virus.

(iv) Sense or antisense RNA annealing between inducer and challenger virus and thereby preventing repli­cation of the challenge virus.

Although the above said protection is manifested in reducing economic level. It however, requires substantial evidence as several mechanisms need to be assessed. The practice of crop protection raised several disadvantages. The apprehensions are mild strain, may become more virulent strain after period of time and risk of spreading in the field. Mild virus is one plant may be severe and virulent in another, thereby risk of spreading extensively in the field. Therefore, it is wise to adopt engineered plant by the expression of single viral gene rather than infecting plants with mild virus.

Coat Protein (CP) Mediated Protection:

Following certain risk factors associated with cross-protection, ultimately limits its use in control of viral infection. Plants can be genetically engineered and supress symptoms due to viral infection. A coat protein (CP) mediated protection follows certain simple rules. The patho­gen derived gene for coat protein is transferred into plants and expressed to counter affects replication of challenge virus. Coat protein mediated strategy resembles cross-protection.

Ex­pression of CP gene is thought to be primarily responsible for preventing particle disassembly or by re-encapsidating the incoming genome of the challenge virus. Collaboration between re­searchers at Monsanto and Washington University led to the first report of CP mediated protec­tion (CPMP) against tobacco mosaic virus (TMV) in tobacco in 1986.

Since then, CPMP have been reported over 20 viruses and at least 10 different taxonomic groups in wide variety of dicotyledonous species. Several technical difficulties have restricted efficacy of crop system in monocots. Presently, work on CPMP mediated are involved for single-stranded RNA and virtually no or little information on virus with double-stranded DNA.

Protection against tobacco mosaic virus (TMV) infection in transgenic plants that express chi­meric gene containing cDNA of the coat protein gene of tobacco mosaic virus has been described. The protection observed for the transgenic plants as genetically engineered cross-protection. It seems that there is considerable delay in the development of symptoms in transgenic tobacco plant that express a CP gene.

The symptom is reduced upto 95% on leaves of coat protein containing (CPt) plants when compared with coat protein devoid (CP-) plant. In addition to protection against TMV, CP expressing transgenic plants was protected against development of symptoms (chlorotic as nerotic) of infection after inoculation with severe TMV strain PV 230. Several groups of workers are attempting to produce viral resistance in cereals by CPMP especially in rice and maize.

The CP gene of rice stripe virus (RSV) was introduced into two Japonica varieties of rice by electroporation. The resultant transgenic rice plants expressed CP at high levels upto 0.5%. The resistance trait was observed to be stably transmitted the next generation of transgenic plants. These workers clearly indicated that CP mediated resistance to viral infection can be extended to monocot especially cereals.

Satellite RNA Mediated Resistance:

The ability of some satellite RNA to attenuate the symptoms of their helper virus has been established. Satellite RNAs are species of RNA associated with certain plant RNA virus. Its association with virus may not necessarily involved is required for replication. Satellites are replicated in cells infected with the particular virus.

The satellite RNA always depends on the virus for its replication and transmission and their nucleotide sequence seems to be unrelated to that of the viral genome. In nature, it has been observed that the presence of satellite RNA reduces intensity of defence symptoms caused by viral infection.

The utility of satellite RNA in plant protection against viral infection have been assem­bled by two groups, worked with different viruses. Harrison (1987) employed cucumber mosaic virus (CMV) with one of its satellites. The DNA version of the satellite was then ex­pressed in transgenic tobacco.

The replication and disease symptoms are well attenuated. A similar work has also been carried out by Gerlach (1987) in tobacco ring spot virus (TobRV) and its satellite (STobRV). They reported synthetic gene construct consist of short leader sequence of RNA inserted STobRV sequence and expressed in transgenic tobacco plants.

The transgenic plant which express full-length STobRV and confirmed excellent resistance when infected with TobRV. The increase in resistance is correlated with the application of satellite RNA to the greatest extent. In addition, cucumber mosaic virus satellite RNA also protected against the symptoms of tobacco aspermy virus without compromising virus replica­tion.

The resistance can be compromising in one of the abilities of attenuating sat RNA to prevent helper virus coat protein from entering chloroplast of infected cells. Satellite TRSV also interferes with replication and resistance to neovirus, cherryleaf rol virus. The protection against cherryleaf rol virus by sat TRSV was reported in transgenic walnut trees in California in 1988.

Animal virus contains D1 RNA, but it occurs naturally only in members of Tombus virus as satellite RNA. This can intensify simptoms of their helper virus and interfere with its repli­cation. The first demonstration of D1RNA in transformed plants involved a defective, sub genomic SS DNA of the B component of Cassava mosaic geminivirus. As a result, which interfere in replication of both cassava mosaic virus DNA component of A and B.

Plantibody Mediated Resistance:

After the first successful demonstration on the production of monoclonal antibodies in plants, there has been a wider interest in the production of transgenic plants expressing a appropriate IgG fab₂ fragment or single chain Fv antibody for possibility of providing protec­tion against viral and other diseases.

The overall strategy was further materialised after the expression of mouse monoclonal antibody single chain Fv in transgenic tobacco. It may be very effective to use monoclonal antibody targeted against catalytic (non-structural) viral proteins when the antigen concentrations are less and interfere virus replication.

Ribozyme Mediated Resistance:

Ribozymes, commonly known as catalytic RNA, are small RNA molecule derived from satellite tobacco ringspot virus (TRSV). These are also derived from viroids and viroids like sat RNA. Ribozymes exhibit specific catalytic cleavage of RNA. Ribozymes normally cleaves spe­cific target RNA (introns) intramolecularly.

However, the catalytic domain cleaves before GUC triplet codon. The efficiency of RNA cleavage depends on its kinetics. Expression of ribozyme in transgenic plants can targeted for the destruction of specific RNA have been demonstrated using protoplast culture. It was shown that neomycin phosphotransferase activity was com­pletely inhibited by transient expression of ribozyme.

Delay in challenge virus symptoms or replication in protoplast was noticed in tobacco expressing ribozymes targeted to TMV gene sequence. Hence, utility of ribozyme to enhance classical cross-protection is one of the most effective ways of controlling viral infection.

Movement Protein (MP) Mediated Resistance:

Successful viral infection will be established only when movement of plant virus is mobilizing from the initial site of infection into adjacent healthy cells. Recent studies have highlighted that plant virus move from one cell to another cell with the help of viral encoded protein known as movement protein (MP). Generally plant vims (or nucleic acid) move from cell to cell through plasmodesmata.

Which provides cytoplasmic continuity between adjacent cells? Establishment of viral infection and movement of viral progeny from one cell to another require modification of structure of plasmodesmata. Experiments with transgenic plants suggested that the 32 kD movement protein modifies the molecular exclusion limit of plasmodesmata.

Subcellular fractionation of homogenation of transgenic leaf tissue showed that the MP was abundantly present in the cell wall of older leaves. The ability of tobacco mosaic virus MP to modify the molecular structure of plasmodesmata in tobacco has been characterized. Thus, TMV MP can determine pathogenecity virulence and host range.

As movement protein is non-structural viral protein known to function in viral movement. It has been exploited to produce transgenic plants expressed defective or mutant movement protein. Accumulation of movement 32 kD movement protein in transgenic tobacco have been shown to reduces the amount of tobacco mosaic virus after infection. High level of accumulation of defective MP in transgenic plants can compete with native movement protein of virus and reduces its movement.

Replicase Mediated Protection:

This type of protection is otherwise known as non-structural mediated resistance. In the genomic organisation of TMV replicase enzyme is probably responsible for the synthesis of genomic and subs genomic RNA’s. The genomic RNA encodes 126 and 183 kDa protein, consid­ered as component of the replicase and role in replication of the TMV genome.

Golmboski (1990) discovered that TMV contains third sub genomic RNA in which at 5 terminuses has nucleotide residue 3405 containing open reading for 54 kDa protein. Interestingly, plants transformed with TMV non-structural gene sequence for 54 kDa protein exhibit considerable resist­ance to infection with TMV.

Presence of 54 kDa gene in transgenic tobacco precludes disease symptoms in the plant. According to one hypothesis 54 kDa proteins is a component of the mem­brane replicase. This 54 kDa protein non-structural proteins contains a Gly-Asp-Asp motif con­served sequence probably interfere with TMV replication.

RNA Mediated Resistance:

RNA mediated resistance is one of the remarkable strategy observed/adopted in plants in which it provides excellent protection against virus. A research investigation into RNA medi­ated virus resistance and co-suppression has provided broader insight into the interactions between plant viruses and their hosts.

Expression of transgene appears to have conferred re­sistance through its mRNA rather than by its translatable coat protein. The first evidence on RNA mediated virus resistance was shown in 1992, when virus-resistance plants expressing untranslatable coat-protein mRNA. RNA mediated resistance involved gene silencing process in plants.

Homology Dependent Gene Silencing:

Transgene silencing phenomena occurs in virus resistance plants. Which has furnished plant scientist with novel tools to understand epigenetic mechanism that regulates gene expres­sion (Table 20.1)? They have pioneered research into novel type of epigenetic regulation, termed as homology dependent gene silencing. Atleast two types’ transgene silencing phenomena have been distinguished.

The first type concerns with position effect, in which the flanking DNA or chromosomal location or both could negatively influence the expression of single copy transgene loci. This particular chromosomal insertion site (Heterochromatic domain) would repress transgene expression. The second type of gene silencing phenomenon concerns with new type of epigenetic silencing of the gene occurs when it is at the status of multiple copies of the same sequence in the genome.

This type of silencing occurs due to interaction between homology and complementary nucleic acid sequences. This type of silencing phenomenon is generally referred as homology dependent gene silencing (HDGS). Silencing of gene expression has shown that HDGS is due to the various control mechanism acting either at the transcriptional or at the post-transcriptional level.

Artificial MicroRNAs Mediated Resistance:

The micro RNAs (miRNAs) have been identified as vital component in several regulatory processes in plants (also in animals). The potential role of miRNAs, as antiviral agents in plant biotechnology have been realised recently. The researchers have engi­neered miRNA precursors of naturally available plant miRNAs to contain complementary se­quences to particular plant viruses.

After maturation of these artificial miRNAs, they target the genomic RNAs of plant viruses and destroy them within plant system (Fig. 20.6). The Arabidopsis thaliana transformed with the recombinant miRNAs precursors became specifi­cally immune to infection with viruses such as Turnip yellow mosaic virus (TYMV) and Turnip mosaic virus (TUMV).

Compared with homology dependent gene silencing, miRNAs has several advantages for generating viral immunity. Advantages are first this strategy is advantages with environmen­tal biosafety.

This is because of concern about transforming plants with viral sequences that might complement or recombine with non-target viruses do not apply to plants expressing artificial micro RNAs (amiRNAs). Another advantage is in the case of homology dependent gene silencing with siRNA, gene silencing and viral suppression are reduced at low tempera­ture. By contrast, amiRNAs is completely active at 15° C.