Plant viruses and their vectors cause serious economic losses, limit crop production, and have negative effects on the quality and security of food supplies. Infection by viruses is a major cause of degeneration of potato seed stocks, particularly by aphid-borne viruses. Current approaches to the protection of plants from viruses are primarily based on poorly understood mechanisms and it is likely that more detailed knowledge will lead to improved virus management. Plant virus genomes are relatively small and therefore are physically unable to encode all the products needed for development of virus infection. When establishing infection, viruses recruit natural host factors to replicate and spread. These factors are candidate targets for novel virus resistance approaches.

Potyviruses

The aphid transmitted potyviruses (potato viruses Y, A and V) can spread readily during the growing season since application of insecticides to control vector aphids does not effectively control spread of these viruses (which are acquired and transmitted in seconds). The most effective potyvirus control can only be achieved by the deployment of resistant cultivars. Plants possess several different mechanisms to combat diseases. There has been much focus on genetic resistance that relies on pathogen recognition by single dominant resistance genes. Pathogen recognition results in a signalling cascade initiating rapid defence responses such as the hypersensitive response. Genes that confer extreme resistance to PVY or hypersensitive resistance to PVY, PVA and PVV have been identified. Of the two, extreme resistance is most effective but such genes have not been mapped for PVA or PVV and many commercial cultivars lack adequate levels of potyvirus resistance.

Another resistance mechanism relates to basic host incompatibility, where host factors, essential for virus multiplication and/or virus invasion, are not available. Research has shown that such host incompatibility can be controlled by recessive resistance genes. Approximately 40% of potyvirus resistances in a wide range of crops show a recessive character. A prime example of such recessive resistance is the eukaryotic translation factor 4E (eIF4E), which binds to the 5’-m7G cap of messenger RNAs and together with other components forms the translation initiation complex. In Arabidopsis thaliana, pepper, tomato and pea there are two paralogous eIF4E-related genes, eIF4E and eIF(iso)4E. A. thaliana deletion mutants in one or other gene grow normally indicating some functional redundancy. Potyvirus genomes contain a virus genome-bound protein (VPg) covalently attached to the 5’end of virus RNA. This protein has several functions one of which is likely to be as a cap-substitute in the initiation of translation. Hence, VPg has been shown to interact with eIF4E in yeast two-hybrid assays and in vitro, and provides a crucial step in the potyvirus life cycle. Resistances to a number of potyviruses in A. thaliana and crops such as lettuce, pepper, tomato, pea, and barley are linked to mutations in eIF4E (or iso4E), and there is a large body of evidence for the selective involvement of eIF4E alleles in resistance to potyvirus species and isolates.

Recessive resistance is not readily observable in tetraploid potato and there is no information about the occurrence of virus-resistant alleles of eIF4E. However, by analogy with other Solanaceae, it is almost certain that recessive resistance to potyviruses resides within the potato germplasm. Inspection of potato (Solanum tuberosum) EST databases has revealed natural variation in eIF4E and has identified five eIF4E family members. The existence of characterised monogenic recessive resistance in crop plants that carry multiple eIF4E genes means that not all of these genes have the potential to confer susceptibility to potyvirus infection; in many plants studied, eIF(iso)4E occurs as a single gene rather than a gene family. The challenge in potato therefore is to identify which eIF4E/(iso)4E supports pathogen infection and to identify allelic variants of this gene that confer resistance.

In this project we will investigate potyvirus resistance in the SCRI long-day-adapted population of diploid Solanum phureja (the Phureja core collection) from which we have bred the cultivar Mayan Gold. The collection will be screened for resistance to PVY in the first instance and resistance and susceptibility correlated with sequences of eIF4E and iso4E genes. Depending on the results we may use the identified resistant clones to produce homozygous pre-breeding material and design molecular markers to identify recessive resistance alleles for deployment in future selections of diploid potatoes.

Novel resistances

The laboratory headed by Peter Palukaitis is looking at identifying and characterising novel resistance approaches against viruses in potato, by examining the functions of proteins encoded by two genes shown to effect virus resistance in model plant species: one encoding an RNA-dependent RNA polymerase (RdR1) and the other encoding an inhibitor of virus replication (IVR). This project will examine the expression profiles and regulation of these genes during susceptible and resistant interactions after challenge by several important potato viruses. This project also will identify accessions in the Commonwealth Potato Collection that contain either constitutive or high level induced expression of these genes. The ultimate goal of this project is to provide markers for these genes that will be used in a breeding program to confer novel molecular resistance to a broad range of potato viruses.

The RDR1 gene has been cloned out of the potato genome and its sequence and organization has been determined. Silencing of this gene in tobacco was shown to reduce resistance to tobacco mosaic virus (TMV) as was silencing of the IVR gene in tobacco, while constitutive expression of the IVR gene was shown to provide resistance to TMV (Figure 1).

Figure 1
Left: Non-transgenic plant showing no virus spread from lower leaves. Middle: transgenic plant in which RDR1 was silenced, showing spread of the virus to upper leaves. Right: transgenic plant in which the IVR gene was silenced showing spread of the virus to upper leaves.

Resistance to infection by viruses also can be achieved by expression of sequences of those viruses in transgenic plants. We have demonstrated resistance to PVY by expression of the gene (NIb) encoding the replicating enzyme of this virus (Figure 2). However, the resistance relied on RNA silencing of the expressed NIb gene and was found to be overcome by co-infection with an unrelated virus. Therefore, a different strategy is being explored, in which resistance is conferred to multiple viruses rather than against single viruses. This strategy, called pyramiding of resistance genes, involves making fusions from three or more viruses and incorporating these into the genome of transgenic plants. Potato is being used as the target crop as a demonstration of the feasibility of obtaining resistance to multiple viruses.

Figure 2
Symptom expression in non-inoculated upper leaves of NIb transgenic tobacco plants resistant (left) and susceptible (right) to infection by PVY.

Transgenic plants in the field often use the cauliflower mosaic virus (CaMV) 35S RNA promoter to facilitate gene expression. However, there are some issues related to what might happen to genes regulated by this promoter if the plants became infected by CaMV. Therefore, we have undertaken studies to examine these potential adverse effects of using the CaMV 35S RNA promoter to facilitate gene expression. We have screened transgenic plants expressing a green fluorescent protein under the control of the CaMV 35S RNA promoter and infected these plants with CaMV.

In four species (tobacco, oilseed rape, Nicotiana benthamiana and Arabidopsis thaliana) we did not see transgene expression being affected by infection, nor did we see any evidence of integration of the virus into the viral 35S RNA promoter sequences present in the plant genomes. We have also transformed A. thaliana plants with the complete CaMV genome and examined the effects of this on plant gene expression in comparison with non-transgenic plants and plants infected by CaMV. The number and types of genes affected are being analyzed. These transgenic plants also were assessed for the ability of the virus to excise itself from the genome and to replicate autonomously. This did happen, and seemed to be caused by aging or development rather than environmental stress.

The cucumber mosaic virus (CMV) 2b protein is involved in the suppression of a defence response against infection by CMV. This involves the ability of the 2b protein to interfere with the processing of microRNAs (miRNAs), which themselves regulate gene expression. Recent work in collaboration with J.P. Carr at Cambridge has shown that different strains of the CMV, which differ in their virulence on host species also differ in the extent to which their corresponding 2b protein interferes with the processing of plant miRNAs, affecting plant development and root growth.

The roles of the capsid proteins of PLRV in assembly of virus particles in planta, in movement of the virus through plants and in the transmission of the virus by the aphid vectors of PLRV are being assessed in collaboration with S.M. Gray at Cornell. Mutants were made in several regions of the major capsid protein and these were found to affect some or all of the above processes. These mutants have been useful for developing a structural model for PLRV particles and predicting likely points of interaction of capsid protein amino acids with each other and with their environment.

Virus resistance in raspberry

Raspberry production is a major part of scottish agriculture and SCRI is heavily involved in research for raspberry breeding and pest management. we are investigating the mechanism of natural resistance to Raspberry bushy dwarf virus, as well as collaborating with SCRI geneticists who are mapping the reaction of selected raspberry varieties to different pests and pathogens.

References

Lewsey, M., Robertson, F.C., Canto, T, Palukaitis, P. and Carr, J.P. 2007. Selective targeting of miRNA-regulated plant development by a viral counter-silencing protein. The Plant Journal 50, 240-252.

Lee, L., Kaplan, I.B., Ripoll, D.R., Liang, D., Palukaitis, P. and Gray, S.M. 2005. A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission. Journal of Virology 79, 1207-1214.

Kaplan, I.B., Lee, L., Ripoll, D.R., Palukaitis, P., Gildow, F. and Gray, S.M. 2007. Point mutations in the potato leafroll major capsid protein alter virion stability and aphid transmission. Journal of General Virology 88, 1821-1830.