Epsilon Open Archive

Abstract

In this thesis molecular interactions between Potato virus A (PVA) and its hosts Nicotiana benthamiana and potato (Solanum tuberosum) have been studied using PVA recombinants, gene silencing and non-hypersensitive resistance (nnr). The full-length cDNA clone pPVA-B11 infects N. benthamiana systemically but is restricted to inoculated leaves in potato. Therefore, a new infectious clone based on the isolate PVA-U, which infects potato systemically, was constructed by replacing parts of pPVA-B11 with parts of PVA-U. Chimeric viruses produced during this process were used to study how recombination of two closely related viral strains might alter their virus-host interactions. Our results suggest that recombination between homologous viral genomes can result in new potyviral strains with novel phenotypic traits. The full-length infectious clone based on PVA-U, pUFL, was able to infect potato systemically and could be further used for studying nnr resistance in potato. To be able to study PVA movement in nnr plants gfp was inserted into the P1 region of pUFL. PVA-GFP could not infect any potato genotypes, but was still highly infectious in N. benthamiana. Infection of gfp transgenic N. benthamiana (line 16c) resulted in transgene silencing despite the strong silencing suppressor HC-Pro. In this model system systemic progression of gene silencing and antiviral defense was analyzed in a novel manner. Use of GFP as a visual marker revealed a mosaic-like recovery phenotype in the top leaves. Leaf areas appearing red or purple under UV-light (no GFP expression) corresponded to dark green islands under visible light and they contained little PVA and gfp mRNA. The surrounding green fluorescent tissue contained replicating viral deletion mutants that suppressed gfp silencing. In plants with nnr resistance PVA moves cell-to-cell and accumulates to high titres in inoculated leaves, but cannot move systemically and no necrotic lesions develop on inoculated leaves. Suppression subtractive hybridization was used to extract transcripts of genes that had higher expression levels in the inoculated leaves of nnr genotypes compared to susceptible (S) genotypes 24 hpi. Hybridization of the extracted transcripts to a cDNA array containing 10 000 potato cDNAs and sequencing of randomly picked clones identified 645 genes expressed at a higher level in nnr than S. The sequenced cDNAs were spotted to a microarray and used together with quantitative PCR to study differentially expressed genes in repeated experiments. According to microarrays and quantitative PCR a family of proteinase inhibitor 2 (pin2) were the only genes expressed to significantly higher levels in nnr than S plants. Since SSH is a more sensitive method than microarray it cannot be excluded that the genes detected by SSH can be involved in a basal defense activated by putative virus-induced molecular patterns resulting in a low resistance response that is high enough to inhibit spread of PVA in the nnr genotypes. PR-1 and SAR were not induced in systemic leaves in nnr plants.