Untersuchungen zur Interaktion des Pathogenitätsfaktors P25 des beet necrotic yellow vein virus mit Proteinen der Zuckerrübe (Beta vulgaris L.)

Abstract

Characterisation of physical interactions between pathogenicity factor P25 of beet necrotic yellow vein virus and the sugar beet proteome (Beta vulgaris L.) In this study protein-protein interactions were performed between P25 pathogenicity factor of the beet necrotic yellow vein virus (BNYVV) and sugar beet proteins in order to elucidate the sugar beet resistance mechanism against the virus. BNYVV is transmitted by the soil-borne plasmodiophorid Polymyxa betae and induces severe lateral root proliferation, necrosis and strong root and sugar yield reductions. Only the growth of partial resistant genotypes carrying monogenic dominant resistance genes stabilises yield but does not prevent virus infection and replication entirely. The viral RNA3 encodes the gene product P25 which is responsible for the induction of symptoms and virus translocation in the root system as well as for symptom development and yield reduction in susceptible sugar beet genotypes. The molecular basis of the virus-host interactions and P25 functions involved therein are unknown. To better understand P25 functions and the molecular basis of the virus-host interactions, the BNYVV encoded P25 was applied in a yeast two-hybrid screen of an Rz2 resistant sugar beet cDNA library. This screen identified several candidate proteins which were isolated and characterised for their involvement in BNYVV infection and plant defense response. Nucleotide sequencing of 36 identified sugar beet cDNAs and their database analysis resulted in predictions of putative functions and functional domains. Some interactions may be necessary for the virus life cycle or might serve to suppress the sugar beet anti BNYVV defense. Among the candidates are members of the plant ubiquitin/proteasome system and proteins involved in phytohormone signalling, cell cycle and structure as well as stress and pathogen response. The interaction of the candidates with P25 was confirmed by applying bimolecular fluorescence complementation (BiFC) assay in order to exclude false positives. Furthermore a detailed characterisation of one promising candidate was carried out. This candidate showed high significant homology to a kelch repe at-containing F-box (F-box) protein from A. thaliana. The involvement of F-box in the resistance machinery is plausible because F-box proteins are known for the recruitment of transcription factors and the induction of resistance reactions. Additionally, the interaction of P25 with F-box was re-tested and confirmed using a second YTH system and an in vitro pull-down assay based upon glutathion-s-transferase. Because of a cell death reaction induced by F-box during the BiFC application, the expression of pathogenesis related (PR-) proteins from tobacco was verified. The positive PR-protein induction confirmed the cell death reaction as hypersensitive reaction. Finally the function of the F-box protein was confirmed by an additional YTH interaction applying the F-box and known interaction partners of the SCF- (SKP1-Cullin1-F-box) complex which is known as E3 ligase.