PLANT VIRUSES II

• Some plant viruses have a very limited host range and others attack numerous species.

• Plant viruses can multiply only within living cells.• Plant viruses usually multiply only within living plant

cells, but some may be able to multiply within the bodies of aphids and nematodes.

• A given plant virus may be able to multiply only within the living cells of one species or genera of plants, but some can multiply within the cells of a wide group of plant families.

• Plant viruses do not attack animals and vice versa.

• Temperate viruses embed themselves within the hosts nucleic acid and are transmitted generation to generation just like genes of the host.

• Often viruses reside in their host without causing any disease or symptom. Such latent viruses are undiscovered.

• Plant viruses vary in their mode of transmission. Usually only a single mode of transmission is important, but some viruses are transmitted by more than one mode.

• Aphids or other sap-suckers are the most common mode of transmission

• Nematodes living in the soil transmit some viruses.• Sap transmission is important for only a few viruses and occurs on

cultivators, pruning, hands of workers, and clothing of workers. Important for some potato viruses.

• Pollen transmission from male flower to female occurs for a few viruses. Such viruses are seed borne.

• Most viruses are do not get into the gametes and therefore are not seed-borne.

Detection

• Until a virus is detected, its presence is not known. Nearly everyone who received an early polio vaccine is now a carrier of Simian Virus 40.(SV40). The monkey cells used to grow that vaccine were infected by SV40 but no one knew SV40 existed.

• Many plants are carriers of plant viruses but show no disease. The yield might be reduced but that would not be visible either.

• The methods for detecting plant viruses include:

• Use of antibodies to the virus in the ELISA assay.

• Graft a leaf from the suspected plant to an indicator plant. The virus moves into the host plant and causes symptoms in the indicator plant.

• Sap transmission. Place a drop of sap from the suspected plant on an intact leaf of an indicator plant. Add some grit to the sap and rub so the leaf is scratched and the virus, if any, can get into the leaf. If the indicator plant show symptoms then we know the virus came from the donor plant. The indicator plant for sweetpotato viruses is Brazilian Morning glory, tabacco, tomato, lambsquarter, and other plants are used as indicators in the sap and grafting assays.

• It is not possible to cure a plant of viruses, but one of the following methods may give a virus free clone. Since the "virus-free" plant might contain a virus you did not know about, it is proper to call them virus-tested or virus indexed plants.

• Often the terminal bud of a plant is free of virus. Remove the 0.03 to 0.05 mm bud tip and grow it in sterile agar contain sucrose and everything the bud needs. 70 to 99.9% of the buds will rot or fail. If some grow to plants, test that plant for virus as above and you might find one which is free of virus. Use it to start more plants. Be sure to test the plant for every known virus.

• Some viruses infect the tip also and the above method will not work. In such cases try growing the plant in an incubator that is so hot the plant barely grows. Often you must supply extra carbon dioxide at a very precise level. Then try to start a new plant as above and test it for all the known viruses.

• Most viruses are not seed-borne. Therefore, a new seedling may be free of virus. Test the seedling for all known viruses.

• Once you have a virus-indexed plant keep it in a screened cage where it can't be infected via a vector. Alternatively, keep it on sugar agar in a sterile glass jar under fluorescent light at a low temperature where it grows slowly. Use scions from this microplant to grow plants for the field.

• Expression strategies in plant RNA viruses• Genome of plus strand viruses can be translated

directly upon 80 S ribosomes to produce virus specific polypeptides.

• The basic anatomy of these are similar to host mRNA with which they must compete.

• Variations are seen in the modifications to RNA termini, in translation and protein processing strategy.

• 3 different structures have been identified at the 5’end.

• m 7 G 5 ppp 5’ seen in at least nine groups of plant viruses, similar to those seen in eukaryotic mRNAs.

• Existence of a small protein of mol.wt 3000-7000 , covalently linked to the 5’ end, called VPg.( polio virus also has this.)

• Unmodified 5’ termini with di or triphosphate groups.

• Tobacco mosaic virus, • Monopartite genome, 6395 nt,rod shaped

particle.• Has a cap at the 5’ end of RNA , the 3’ end has

a tRNA like structure , which can be aminoacylated with histidine or valine.

• Encloses 4 polypeptides.

• 68 nt leader seq precedes 2 long ORFs . • First ORF is translated into a polypeptide of

mol wt ,126000, P126.• Suppression of amber termination codon at

the end of the first ORF permits translation readthrough into the second , producing P183.

• 2 smaller ORFs towards the 3’ end , P30, P18.

• P126, P183 appear early in the viral life cycle and thought to constitute the viral replicase.

• The internal ORFs coding for P30 , P 18, are not expressed as translation readthro.

• Instead subgenomic mRNAs are generated from each gene by RNA transcription from specific promoters.

• P30 is supposed to be the involved in cell to cell movement of the virus.

Cowpea Mosaic virus

• An RNA-containing virus with isometric particles about 28 nm in diameter.

• Has a limited host range, is transmitted mainly by beetles and readily by sap inoculation.

• Infected plants contain two kinds of nucleoprotein particle similar in size but differing in RNA content.

• Particles containing no RNA are also produced by most isolates. The RNA species in different particle types represent separate parts of the viral genome.

• Has a bipartite genome with 2 plus sense strand RNA molecules.

• Ultracentifuged purified CPMV shows 3 distinct spherical particles.

• Fast sedimenting Bottom (B ) component with single RNA species 5889 nt long.

• Intermediate sedimenting M component 3481 nt long

• Slow sedimenting top component has empty viral capsids.

• 5’ end of each RNA has a VPg attached to it and a poly A tail at the 3’end.

• Both the RNA s are translated into large polypeptides which are subsequently cleaved.

• Both the RNA s are required for infectivity

• B RNA can support its own replication independantly of MRNA( viral replicase)

• BRNA cannot move to adjacent plant cells in the absence of MRNA.

• The structure of CPMV is similar and has resemblance to that of animal picarnoviruses

• Both genome RNAs contain a small protein (VPg) at their 5'-end and a poly A tail at their 3'-end.

• Each RNA species contains one large open reading frame, and they are translated in vitro as well as in vivo into one (RNA1) or two (RNA2) polyproteins that are cleaved by a viral proteinase (encoded by RNA1) to give 15 intermediate and final processing products.

• RNA1 carries all the information for RNA replication, including the polymerase, VPg and a protein containing a RNA replication, including the polymerase, VPg and a protein containing a nucleotide binding site, whereas RNA2 codes for the two capsid proteins and the movement protein, which are involved in cell-to-cell transport of the virus.

Viral movement in plant cell

• Upon infection, viral RNA (vRNA) initiates synthesis of replicase, plus and minus vRNA stands, subgenomic RNAs and movement protein (MP), and coat protein. In response to infection, callose accumulates in the wall region surrounding the plasmodesmata (Pd) restricting the cytoplasmic sleeve.

• Stage II: MP, an integral endoplasmic reticulum (ER) membrane protein, functions as a protein raft binding vRNA on its cytoplasmic domains

forming a replication complex (VRC) that may also contain replicase.

Intracellular trafficking of the VRC to the cortical ER is either by diffusion in the ER lipids or by

vesicular trafficking.

• Stress-induced class I beta-1,3,glucanase traffics in the lumen of VRC vesicles to plasma

membrane (PM) with requisite docking protein (filled arrowheads). Stage III:• Cycling vesicles containing beta-1,3-glucanase

cargo fuse to PM and deliver beta-1,3-glucanase to the cell wall.

• Callose is hydrolyzed, allowing Pd to dilate.• Vesicles with attached VRC recycle back to the

cortical ER, in which vesicles fuse to cortical ER. VRC diffuses through the Pd to adjacent cells by diffusion in ER desmotubule continuum motivated by the concentration

gradient between a viral-infect cell and adjacent noninfected cells.