Identification of apple scab avirulence gene AvrVg candidates

by Giovanni Antonio Lodovico Broggini

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ABSTRACT

A major goal of plant pathology research is to understand the functioning of disease resistances and how pathogens overcome these resistances. The ascomycete Venturia inaequalis, causal pathogen of apple scab, underlies a gene-for-gene relationship with its host apple plant (Malus spp). Probably almost every apple cultivar carries resistance genes V. inaequalis avirulence genes underlying such relationship. To day fourteen apple scab resistance genes have been identified in different apple cultivars. Nevertheless most of these resistance genes have been overcome by the pathogen. The efficacy of the R-genes that were overcome depends mostly on the frequency of the matching avirulence gene in the pathogen population. In other plant-pathogen interactions, resistance circumvention was shown to occur mainly by the mutation of the avirulence alleles, so that the avirulence gene encoded proteins escape host recognition. In all nineteen avirulence genes have been postulated in the past for V. inaequalis. However little is known at molecular level about V. inaequalis avirulence genes. The genes involved in apple scab avirulence on hosts carrying the Vf gene from M. floribunda 821 and Vg from ‘Golden Delicious’ have been previously mapped in the genome of V. inaequalis at INRA Angers and called AvrVf and AvrVg, respectively. We decided therefore to try the positional cloning of one of these avirulence genes. Since the number of markers in the region surrounding both avirulence genes was low, we started an enrichment of these regions by mean of bulk segregant analysis coupled with amplified fragment length polymorphism technology. This allowed to develop three new markers linked to AvrVg and one to AvrVf. Simultaneously, two SSR markers were mapped tightly linked to AvrVg and one to AvrVf, respectively, at INRA Angers. Upon construction of a genetic library consisting of BAC clones of a double avirulent isolate of V. inaequalis, a chromosome walking in the region of AvrVg was performed isolating eleven overlapping BAC clones spanning the estimated 330 kb region of AvrVg. The four clones which allowed spanning the whole region with minimal overlap were sequenced by shotgun sequencing technique. The sequenced region was 330 kb long and revealed a total of 61 genes predicted by two distinct gene prediction software. Since most of fungal avirulence genes encode for proteins secreted extracellularly, we analyzed the whole set of predicted genes in the AvrVg region and recognized a total of 40 regions which encode for genes secreted extracellularly and were therefore considered as candidate avirulence genes. Two candidate avirulence genes, which were encoded in the same sequence but differed in one of their two splicing sites, were found to share high homology (98% on 105bp) to two cDNA clones from a library derived from apple leaves infected with Venturia inaequalis. Moreover identity was found in the 5’-untranslated region. Since the cDNA encoded proteins are expressed during apple scab infection, we speculate that one of the two predicted variants of candidate avirulence gene might also be expressed under similar conditions. Moreover the predicted candidate genes show other typical features of fungal avirulence genes: they are small and cysteine rich proteins. The expression of these candidates avirulence genes during infection, as well as the other encoded in the remaining 39 regions that were found to encode extracellular secreted proteins, has to be proven and will allow identifying their complete open reading frames. Complementation experiments with expressed genes will allow identifying the one conferring avirulence on apple cultivar ‘Golden Delicious’.