1.12.17
USE OF VIRUS-INDUCED GENE SILENCING (VIGS) IN THE STUDY OF GENES INVOLVED IN DISEASE RESISTANCE

J PEART1, MT RUIZ1 and DC BAULCOMBE1

1Sainsbury Laboratory, John lnnes Centre, Norwich NR4 7UH, UK

Background and objectives
In recent years, the study of plant resistance to different pathogens has shown that such responses involve a variety of genes other than those initially identified as resistance genes. Pathways involving many genes are initiated by the recognition of incompatible pathogens and operate to trigger resistance responses. Our objective is to use VIGS as a rapid method of silencing genes in order to determine their involvement in these pathways. VIGS is achieved by infecting a plant with a viral vector carrying a sequence homologous to a target gene.

Results and conclusions
Nicotiana benthamiana plants transformed with the tomato resistance gene Pto under control of the 35S promoter became resistant to Pseudomonas syringae pv. tabaci carrying the avirulence determinant avrPto [1]. In tomato, the presence of an active Prf locus is required for Pto function. N. benthamiana plants transformed with Pto were infected with PVX viral vectors carrying a fragment of the N. benthamiana homologue of Prf. After 4 weeks they were tested for resistance to P. syringae and for the ability to display the hypersensitive response (HR) upon transient expression, via Agrobacterium tumefaciens, of the avrPto avirulence determinant. Transgenic plants infected with PVX-Prf supported growth of the previously avirulent Pseudomonas strain to similar levels as those of the virulent strain. Additionally, these plants were no longer capable of mounting a corresponding HR. These results demonstrate that the silencing of genes downstream of pathogen recognition in the signalling pathway can block the resistance response.

These results prompted us to extend our studies to other plant-pathogen interactions. N. benthamiana plants were transformed with the tobacco N gene, which confers resistance to tobacco mosaic virus (TMV). Transformants inoculated with GFP-tagged TMV did not show persistent fluorescent foci, demonstrating that components required for the function of N are present in N. benthamiana. Preliminary results show that silencing of N, an alternatively spliced gene [2], in these transgenic plants can be achieved by use of a PVX vector carrying a fragment of N common to both its full-length and truncated transcripts. Thus these transgenic plants are likely to be amenable to the study, by VIGS, of genes required for TMV resistance.

Other genes previously shown to play roles in resistance responses will also be targeted. These include NPR1, NDR1 and hinl. Subsequently we will target genes with speculative roles in resistance such as MAP kinases and oxidative burst enzymes. Eventually, it is hoped that an N. benthamiana EDNA library will be constructed in the PVX vector to enable large-scale screening for genes with resistance functions.

References
1. Rommens CM, Salmeron JM, Oldroyd GED, Staskawicz B, 1995. Plant Cell 7, 1537-1544.
2. Witham S, Dinesh-Kumar SP, Choi D et al., 1994. Cell 78, 1101-1115.