Molecular and functional characterization of potential pathogenicity related genes from <i>Verticillium longisporum</i>

Abstract

The genus Verticillium includes many plant pathogenic species that cause serious economic losses through the infection of host plants, and it belong to one of the most widespread groups of plant pathogens in the world. Molecular studies on pathogenicity-related genes provides the opportunity for a better understanding of the disease caused by the fungus and thereby help in finding new strategies for the prevention or the control of infection. This thesis mainly focuses on the results of analysis of two genes detected in the genome of Verticillium longisporum putatively involved in the pathogenic life-cycle of the fungus while infecting natural host plant Brassica napus. During the research for this thesis, many candidate genes of V. longisporum were efficiently silenced by RNA interference (RNAi), triggered by the intracellular expression of hairpin (HP) cassettes. We tested different approaches published in recent years for the construction of HP fragments by PCR based methods. All these methods utilize Overlap extension-PCR (OE-PCR) to assemble sense and antisense strands of a candidate gene with an intervening spacer, and do not depend on time-consuming restriction enzyme-based cloning steps or require costly consumables. Problems encountered during establishment gave new insights into the applicability of OE-PCR for construction of HP fragments. Nevertheless, for all generated silencing-mutants of V. longisporum candidate genes, we could show a reduction in candidate gene expression of at least 90 % so that we obtained a reliable tool for the characterization of putative pathogenicity related genes to evaluate their role in the life cycle of the fungus. During our research, we demonstrated that downregulation of a gene with a high homology to the necrosis and ethylene inducing peptide (NEP)- family, which we therefore designated as Vl-NEP-1, was connected to symptom severity caused by V. longisporum on the natural host plant B. napus, and thereby indicated a reduced ability of the fungus to spread into the plant. In summary, we detected five NEP genes, designated as Vl-NEP-1 to 5, in the genome of V. longisporum from which we could measure strong expression levels of three NEP genes (Vl-NEP-1, -2, -5) in planta by qRT-PCR. This makes a contribution of these genes to the pathogenic life cycle of V. longisporum while infecting B. napus acceptable. Our results for Vl-NEP-1 suggest that the related gene product is secreted in the xylem by the fungus and permeabilizes the surrounding cell membranes of the xylem cylinder, causing leakage of monomers from adjacent tissue into the xylem sap.Our findings strongly suggest that Vl-NEP-1 is a true virulence factor of V. longisporum during infection of B. napus. The second candidate gene which we analysed during the research for this thesis show high homology to fungal type I polyketide synthase (PKS) genes of the wA type, and we therefore named this gene Vl-PKS-1. PKS catalyze the series of small carboxylic acids into polyketides (PK) which belong to a large group of fungal secondary metabolites that are described as compounds not primarily needed for essential processes concerning the survival and the reproduction of the fungus. Polyketides from phytopathogenic fungi are known to often play a role during the hostpathogen interaction as toxins, or as pathogenicity or virulence factors. Gene silencing mutants of Vl-PKS-1 show major differences in their growth characteristics as compared to those of V. longisporum wild type. As compared to the wild type, a significant increased growth rate of Vl-PKS-1 mutants was shown, their partial loss of competitive capacity was demonstrated by an fungal-interaction assay and a delayed pigmentation could be observed. Despite the highly upregulated gene expression of Vl-PKS-1 during all stages of the pathogenic life cycle of V. longisporum, we did not observe that silencing of the gene expression led to any alteration on the phenotype of infected B. napus plants. Therefore it is most likely that Vl-PKS-1 is not a virulence or pathogenicity factor of the fungus in the interaction with B. napus.