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Lecture 11&12 Virus resistant plants Plant viruses often cause considerable crop loss and significant crop

yield. Genetic engineering has been used to develop non conventional types of virus-resistant transgenic plants. Viral coat protein-mediated protection When transgenic plants express the gene for a coat protein (which usually is the most abundant protein of a virus particle) of a virus that normally infects those plants, the ability of virus to subsequently infect the plants and spread systemically is often greatly diminished. Although the precise mechanism by which the presence of coat protein genes inhibits viral proliferation is not understood, it is clear that the antiviral effect occurs early in the viral replication cycle and, as a result, prevents any significant amount of viral synthesis. This feature is an advantage because it decreases the probability of selecting for spontaneous viral mutants that can overcome this resistance and replicate in the presence of virus coat protein. The viral coat protein gene approach has been used to confer tolerance to a number of different plant viruses (Table 1). With this approach, researchers have developed virus-resistant transgenic plants for a number of different crops. Although complete protection is not usually achieved, high levels of virus resistance have been reported. Moreover, a coat protein gene from one virus sometimes provides tolerance to a broad spectrum of unrelated viruses. In both eukaryotes and prokaryotes, an RNA molecule that is complementary to a normal gene transcript (mRNA) is called antisense RNA. The mRNA, being translatable, is considered to be a sense RNA. The presence of antisense RNA can decrease the synthesis of the gene product by forming a duplex molecule with the normal sense mRNA, thereby preventing it from being translated. The antisense RNAmRNA duplex is also rapidly degraded, a response that diminishes the amount of that particular mRNA in the cell. Theoretically, it should be possible to prevent plant viruses from replicating and subsequently damaging plant tissues by creating transgenic plants that synthesize antisense RNA that is complementary to virus coat protein mRNA. In one of the studies, the efficacies of the virus coat protein gene and antisense RNA approaches were compared by cloning the cDNA for the coat protein of cucumber

mosaic virus (CuMV) into tobacco plants in two orientations (sense and antisense; one orientation per plant) and then testing transgenic plants for sensitivity to viral infection (Fig. 1).

Figure 1. Procedure for introducing Cucumber mosaic virus coat protein cDNA into plant cells. RNA4, which encodes the coat protein, is fractionated from a viral RNA preparation and used as the template for the synthesis of double-stranded cDNA. Linkers

are added to the cDNA preparations and the cDNAs are cloned into an E. coli plasmid vector. A full-length cDNA clone is identified, excised from the E.coli vector, and sub cloned into a Ti plasmid cloning vector between the 35S promoter from cauliflower mosaic virus (P35S) and the transcription terminator from the gene for the small subunit of ribulose bis phosphate carboxylase (tRBC). This cloning step creates two orientations for the RNA4 cDNA. In one case, the RNA that is transcribed is translated into coat protein (sense RNA), and in the other case, the transcribed RNA is complementary to the mRNA for the coat protein (antisense RNA). The Ti plasmid binary vector system was used to transfer both protein producing sense and antisense RNA producing cDNA sequences to separate tobacco cells, from which transgenic plants were regenerated(Fig 2).The transgenic tobacco plants that expressed CuMV coat protein were protected from viral particle accumulation and did not show any viral infection, regardless of whether the inoculum of the virus was high or low ,whereas the antisense orientation construct only protected transgenic plants against low viral doses.

Figure2.Ti plasmid binary vectors containing either the protein producing sense(A) or the antisense RNA producing (B) orientation of the CuMV coat protein cDNA. Ideally transgenic plants resistant to more than one virus were created. Ti plasmid binary vectors expressing one or more viral coat protein genes for CuMV, zucchini yellow mosaic virus, and water melon mosaic virus 2 were used to transform yellow

squash (Cucurbita pepo) (Fig 3) .Clearly, using more than one viral coat protein gene is an effective strategy should be useful in developing a range of transgenic plants that are resistant to all of the major viruses that normally inhibit their growth and development. The phenomenon of using a plant-encoded viral gene to disrupt the virus life cycle and thereby confer viral resistance is sometimes called homology-dependent gene silencing (cosuppression). In homology-dependent gene silencing, the addition of new copies of a gene to the genome inhibits expression of both the introduced gene and the previously present endogenous copies, or, in the case of viral genes, those genes that are synthesized after infection. In fact, in some cases the plants own defense mechanisms may include the possibility of homology-dependent gene silencing.

Fig 3. T-DNA constructs with a neomycin phosphotransferase gene (NPTII) as a selectable marker ,GUS as reporter gene, with coat protein genes of CuMV(CMV), zucchini yellow mosaic virus(ZYMV), and water melon mosaic virus 2(WMV2). Protection by expression of other genes Engineered resistance to plant viruses generally as a result of expressing a viral coat protein or other viral gene in the transgenic plant is usually an effective strategy only against closely related viruses. Since there are a large number of different viruses that could potentially infect a crop, it would be advantageous if plants could be engineered to be resistant to a broad spectrum of viruses. To do this, a strain of wheat was engineered to express the E. coli gene for ribonuclease (RNase) III (i.e., rnc), an enzyme that cleaves

only double-stranded RNA; most plant viruses have double-stranded RNA as their genetic material. When tested, transgenic plants that expressed the rnc gene were resistant to several different RNA plant viruses. Unfortunately, plants that expressed this gene were often stunted and did not develop normally. This is probably a result of the interaction between the plant RNA and the enzyme. To overcome this problem, a mutant of RNase III was used. The mutant enzyme was still able to bind to double-stranded RNA, but it no longer cleaved this substrate. The mutant gene (rnc70) was introduced into wheat, under the control of a corn ubiquitin gene (Fig. 4), by micro- projectile bombardment. Transgenic plants that expressed mutant RNase III developed normally and exhibited a high level of resistance to infection by barley stripe mosaic virus. In this instance, binding of the mutant RNase III to replicating barley stripe mosaic virus prevented viral replication. In addition to being useful with RNA viruses, this approach is an effective strategy for eliminating viroid infection of plants. Viroids are circular, single-stranded RNA disease-causing agents and form a highly base-paired double_stranded-like structure by intrastrand base pairing. Plant viroids are difficult to control because they do not encode any proteins; therefore, the viroid nucleic acid must be targeted.

Figure 4. A portion of the genetic construct used to transform wheat plants with the gene for the mutant form of E. coli RNase III. The construct includes the maize ubiquitin promoter, first exon and intron in front of a mutant form of E. coli RNase III and a transcription terminator region from a nopaline synthase gene. Anti viral protiens In addition to immunizing plants against damage from viruses by expressing viral proteins in the plant cells, protection can be conferred by antiviral plant proteins. For example, pokeweed (Phytolacca americana) has three antiviral proteins in its cell wall: pokeweed antiviral protein PAPI, which is found in spring leaves; PAPII, which is

found in summer leaves; and PAP-S, which appears in seeds. Although they are only 40% identical at the protein level and antibodies directed against PAP do not react with PAPT II, they employ a similar mode of action. Both PAP and PAPII are ribosomeinactivating proteins that remove a specific adenine residue from the large ribosomal RNA of the 60S subunit of eukaryotic ribosomes. When pokeweed plants are infected with viruses, either PAP or PAPII is synthesized, depending on the season, and the ribosomes in the infected cells are inactivated. Based on their mode of action, PAP and PAPI are good candidates for developing transgenic plants that are resistant to a broad spectrum of plant viruses. After a cDNA encoding PAP was isolated, it was introduced, under the transcriptional control of the 35S promoter, into tobacco and potato plants with binary Ti plasmid vectors. Transformants that expressed PAP produced less number of lesions than non transformed plants. TABLE 1. Transgenic plants with viral coat protein mediated protection