1.12.2
THE RESISTANCE RESPONSE OF POTATO BEARING THE RYSTO GENE IS ELICITED BY THE POTATO VIRUS Y COAT PROTEIN

J HINRICHS-BERGER1, S BERGER1, H BUCHENAUER1 and JG SHAW2

1Universität Hohenheim, Institut für Phytomedizin (360), 70593 Stuttgart, Germany; 2University of Kentucky, College of Agriculture, Plant Pathology, S-305 Agricultural Science Building North, Lexington, Kentucky 40546-0091, USA

Background and objectives
Potato virus Y (PVY) is the type member of the potyvirus group and causes severe losses in potato worldwide. The dominant gene Rysto from Solanum stoloniferum was introduced in potato cultivars and confers a stable, extreme resistance to PVY and tobacco etch virus (TEV). Both PVY and TEV replicate in initially infected cells of extremely resistant potato cultivars and move into neighbouring cells before a hypersensitive reaction (HR)-like mechanism eventually stops the infection [1]. In order to determine which PVY gene can trigger the resistance response, we have utilized a transient expression assay [2].

Materials and methods
The PVY coat protein (CP) gene and the uidA gene (encoding GUS), respectively, were cloned into the plant expression vector pKYLX80 under the control of a modified 35S promoter. The vector combinations pKYLX80-GUS/pKYLX80 or pKYLX80-GUS/pKYLX80-CP were co-precipitated in equimolar amounts on gold particles that were delivered by a biolistic device into leaflets of extremely resistant (cv. Pirola) or susceptible (cv. Quarta) potato plants. After the bombardment, the leaflets were incubated in a controlled environment chamber at 21°C for 3 days and then subjected to a histochemical GUS staining protocol using X-Gluc as substrate at 37°C overnight. The blue-stained cells were counted under a microscope after clearing the leaflets with 70% ethanol.

Results and conclusions
We have used the uidA gene as an indicator of transiently transformed cells. The efficiency of the transient transformation was determined by the number of cells expressing a level of GUS activity that allowed the visible formation of a blue precipitate in the cell. While no cells were stained when the leaflets were bombarded only with pKYLX80, more than 100 cells in Pirola or Quarta leaflets became blue after co-bombardment with the vectors pKYLX80 and pKYLX80-GUS. Essentially the same number of cells were stained after co-transformation of Quarta leaflets with the vector combination pKYLX80-GUS/pKYLX80-CP. However, using the identical vector combination for Pirola leaflets, we detected a GUS activity by in situ staining in only about 10 cells.

These results suggest that the CP of PVY inhibited the expression of GUS activity in extremely resistant potato plants. In these plants, the CP possibly triggered an HR leading to rapid cell death and low levels of GUS enzyme accumulation, so that GUS activity was not detectable by in situ staining. Other PVY genes will be checked in the same transient expression assay regarding their resistance-eliciting ability.

References
1. Hinrichs J, Berger S, Shaw JG, 1998. Journal of General Virology 79, 167-176
2. Mindrinos M, Katagiri F, Yu G-L, Ausubel FM, 1994. Cell 78, 1089-1099