Virus Resistance Plants
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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 RNA—mRNA 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 plant’s 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 plantswith 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 ribosome-
inactivating 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
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