Functional Analysis Of A Small GTPase VdRAC1 And Its Interacting Proteins In Verticillium Dahliae

Verticillium wilt of cotton caused by Verticillium dahliae is a major fungal disease in cotton production, resulting in reduction on both yield and fibre quality. Root penetration is the first step during the infection processes. After gain entry into the root surface, the hyphae proceed toward the root cortex and the vascular tissues, in which the hyphae grow rapidly and produce large quantity of spores, resulting in the block of water and nutrition transport and leading to the whole plant death. At present, chemical method hardly control the disease. Broad host ranges and the long survival in the soil of microsclerotia produced by the fungus make crop rotation ineffective. When condition is suitable, such as sense of exists of plant roots, microsclerotia germinate to produce hyphae and initiate a few infection cycle.Cell polar growth is a specific character in fungal development, and is required for plant penetration and therefore the pathogenicity. This research focuses on the role of small GTPase Racl and its potential partner Cla4 on cell polarized growth and the pathogenicity. △ Vdracl and △ Vdcla4 mutants have similar phenotypes. Racl and Cla4 disruption results in severe defects on colony morphology, hyphae production and produces abnormal spores that lack cell polar growth. The virulence to both Arabidopsis and cotton was drastically reduced. Further experiments suggest that the reduction in virulence may be due to the defects in root adhesion, penetration and plant colonization of the mutants. Gal4-based yeast two-hybrid tests show that Racl interacts with Cla4. qPCR results revealed that the expression of Racl and Cla4 was down-regulated in △Vdcla4 and △Vdraclmutants respectively, suggesting the coordination action of the two proteins in controlling cell polarized growth. The effects of nutrition on spore production were also investigated. It was found that trace elements have major contribution to spore production.The relation of ROS production by the fungus to the pathogenicity was also investigated. We focus on two ROS production enzymes, VdNoxl and VdNox2 (NADPH oxidases). Though there are no phenotypic effects, the virulence of both △Vdnoxl and △Vdnox2 mutants to cotton and Arabidopsis is greatly compromised, suggesting a role of ROS on fungal pathogenicity. Bioinformatic analysis show VdNoxl and VdNox2 are transmembrane proteins. qPCR results revealed that the expression of Racl,Noxl and Nox2 is mutually affected in respective mutants. GAL4-based yeast two-hybrid tests showed that Racl have weak interactions with both VdNoxl and VdNox2. These primary results revealed that ROS production by the fungus play an important role on virulence pathway in which VdRacl may be a partner protein.