Rhizoctonia solani and sugar beet responses : genomic and molecular analysis

Abstract: The soil-borne basidiomycete Rhizoctonia solani (strain AG2-2) incite root rot disease in sugar beet (Beta vulgaris). The overall objective of this thesis work was to enhance the genomic knowledge on this pathogen and induced responses in the host to promote breeding of better performing cultivars. The AG2-2IIIB R. solani isolate sequenced in this project had a predicted genome size of 56.02 Mb and encoded 11,897 genes. In comparisons with four other R. solani genomes, the AG2-2IIIB genome contained more carbohydrate active enzymes, especially the polysaccharide lyase group represented by the pectate lyase family 1 (PL-1). When predicting for small, cysteine rich and secreted-proteins (effectors) 11 potential candidates were found to be AG2-2IIIB strain specific. In parallel, transcript data was generated from sugar beet breeding lines known to express differential responses to R. solani infection. After extensive data mining of the achieved information a handful of genes with potential roles in sugar beet defence were identified. Particularly three Bet v I/Major latex protein (MLP) homologous genes caught the interest and were further investigated together with three R. solani (Rs) effector candidates selected based on their transcript profiles during infection of sugar beet seedlings. They are: a rare lipoprotein-A like protein (RsRlpA), the chitin-binding lysin motif effector (RsLysM) and a cysteine-rich protein (RsCRP1). The three fungal effectors were induced upon early infection and were heterologously expressed in Cercospora beticola, a sugar beet leaf spot fungus, facilitating functional analysis. RsLysM showed perturbation of chitin-triggered plant immunity as expected but did not protect fungal hyphae from degradation. RsRlpA is localized to the plant plasma membrane and has capacity to suppress the hypersensitive response. When monitoring cellular localization of RsCRP1 it was found to target both plant mitochondria and chloroplasts. RsCRP1 was also used in pull-down experiments followed by amino acid sequencing from which a potential interacting protein, a plasma membrane intrinsic protein, BvPIP1;1 was proposed to be a candidate. The studies on the fungal effectors and the potential plant defence candidates involving BvMLPs and BvPIP1;1 are on-going including assays of gene homologs in Arabidopsis to promote mechanistic understanding of the sugar beet – R. solani interactions together with protein-protein interactions and associated assays. Results to be implemented in resistance breeding.