1.1.28
MOLECULAR CHARACTERIZATION OF THE PVX ELICITOR OF NB-MEDIATED HYPERSENSITIVE RESISTANCE IN SOLANUM TUBEROSUM

I MALCUIT1, M MARANO1, TA KAVANAGH2 and DC BAULCOMBE1

1Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR7 4UH, UK; 2Department of Genetics, Trinity College, Dublin, Eire

Background and objectives
In plants, the most common manifestation of resistance to pathogen attack is the hypersensitive response (HR). Activation of HR resistance is dependent upon specific recognition of the avirulent pathogen by the plant host and results in a localized activation of a programmed cell death. Two types of resistance to potato virus X (PVX) have been identified in potato: HR resistance is controlled by the genes Nx and Nb, and extreme resistance (ER) is provided by Rx1 and Rx2. Avirulence determinants eliciting both the Nx- and Rx-mediated resistance were mapped in the coat protein gene [1, 2]. The PVX coat protein was subsequently shown to be the viral elicitor of Rx-mediated resistance [3]. We are currently interested in the characterization of molecular and cellular events associated with the Nb-mediated resistance that is activated in response to PVX infection. Here we describe (i) the localization of the region in the PVX genome involved in the activation of HR in potato plants carrying the Nb gene, and (ii) further characterization of the viral elicitor at the molecular level by site-directed mutagenesis and deletion analyses.

Results and conclusions
Chimaeric virus genomes were constructed by exchanging the genes encoding the replicase or the 25-kDa movement protein between PVX-Rothl (avirulent strain) and PVX-UK3 (virulent strain). Analysis of the avirulence/virulence phenotype of the PVX hybrids was carried out using particle bombardment technology to directly target the plant cells with PVX CDNA transcription clones. HR was induced in resistant plants bombarded with the viral hybrid containing the 25K gene from PVX-Rothl in a virulent background, indicating that activation of the Nb-mediated resistance is dependent upon the 25K gene. PCR-directed mutagenesis experiments also showed that presence of an isoleucine residue at position 6 in the 25-kDa protein was required for activation of HR.The 25K genes of PVXRothl and PVX-UK3 were then placed under the control of the CAMV 35S promoter and terminator in a vector which also carries a 35S:GUS expression cassette, the presence of a reporter gene being required to monitor cell death. Biolistic transient expression of the avirulent 25K gene resulted in a reduction of GUS activity to an almost undetectable level in resistant potato leaves compared to the level of GUS expression in susceptible plants. GUS expression was not affected in resistant potato leaves bombarded with a construct containing an untranslatable version of the 25K gene. These results indicated that the PVX 25-kDa protein is the elicitor of Nb-mediated hypersensitive cell-death in potato. In order to define the minimal region in the 25-kDa protein required for elicitation of HR, full-length and truncated versions of the 25K sequence were fused to the gfp reporter gene. Biolistic transient expression of GFP fusions in potato cells was monitored by laser scanning confocal microscopy.

References
1. Santa Cruz S, Baulcombe DC, 1993. Molecular Plant-Microbe Interactions 6, 707-714.
2. Kavanagh TA, Goulden M, Santa-Cruz S et al., 1992. Virology 189, 609-617.
3. Bendahmane A, Kohm B, Dedi C, Baulcombe DC, 1995. Plant Journal 8, 933-941.