Die Erysiphe necator Resistenzen Ren3 und Ren9 aus ’Regent’ – Eingrenzung, Analyse von Kandidatengenen und differentielle Genregulation

Abstract

The work presented here confirms the two recently described resistance loci Ren3 and Ren9, which confer resistance to grapevine powdery mildew in two independent crosspopulations. The interval of Ren3 could be delimited to a 200 kb region by parallel work. Further it could be shown that this locus enables the plant to react with a HR upon powdery mildew infection. It was possible to reproduce the two described resistance loci by QTL analysis with phenotypic field data in the two populations ’Regent’ x ’Cabernet Sauvignon’ and GF.GA-47-42 x ’Villard Blanc’. Also it was shown that a classification into resistant and susceptible genotypes according to observed SSR marker alleles of the resistance-linked genetic markers was possible. By genotypic and microscopic analysis of specifically selected F1 individuals with recombination on chromosome 15 from different crosses, the genomic interval of the resistance locus Ren9 was reduced to around 300 kb. The genetic markers GF15-08, GF15-10 and Indel-7 where identified to be closely linked to the locus conferring resistance to powdery mildew. Furthermore, HR associated with appressoria after infection with powdery mildew could be confirmed for Ren9. Parallel works on Ren3 enabled an analysis of genes encoded in this resistance locus. Hereby a cluster of four CC-NBS-LRR genes was identified. Transcript analysis of Ren3-1 revealed two different splice variants for this gene. Differential gene expression experiments after inoculation with powdery mildew showed that the two genes Ren3-1 and Ren3-4 are exclusively transcribed in the grapevine cultivar ‘Regent’ and that transcript levels of Ren3-4 are elevated upon infection with powdery mildew. The elevated transcript levels of Ren3-4 could give a hint that this gene might confer the resistance to
powdery mildew associated with the resistance locus Ren3. A comparison of Ren3 from ‘Regent’ and the reference genome PN40024 12Xv0 revealed major differences. Here an approximately 110 kb insertion in the coding region of Ren3-4 was identified comprising several retroelements. Also in the promoter region of Ren3-1 an additional retroelement was identified in PN40024 12Xv0. These two insertions probably inactivate the resistance genes in the susceptible PN40024. Their absence explains the exclusive transcription of these two genes in ‘Regent’. PN40024 has a pure V. vinifera background. This grapevine species evolved without the presence of powdery mildew. The insertion of the found retroelements could therefore be a way to silence not needed resistance genes. This silencing mechanism is thought to help prevent autoimmunity conferred by resistance genes. Analysis of differential gene expression upon inoculation with powdery mildew revealed major differences between the resistant cultivar ‘Regent’ and the susceptible cultivar ‘Chardonnay’. Especially by looking at genes of the pathogenesis-related group it could be shown that the resistance response in ‘Regent’ is faster and longer lasting. Additionally, it was possible to analyze possible orthologous genes involved in HR regulation, which were already described in A. thaliana. The differential experession of these genes in ‘Regent’ compared to ‘Chardonnay’ after inoculation with powdery mildew, could indicate the first steps in characterizing the observed HR on a molecular level. Furthermore, it was possible to prepare for the functional analysis of Ren3-1 by generating amiRNA constructs and transforming them into ‘Regent’ for a knockdown of the gene. In addition, it was possible to transform susceptible genotypes with the gene Ren3-1.