Screening And Identification Of The Ustilaginoidea Virens Effectors That Can Induce Or Suppress Plant Immunity

Rice false smut （RFS）, caused by the ascomycetous fungus Ustilaginoidea virens（Cooke） Takah, has recently expanded rapidly and become one of major rice diseases in China. As a grain disease, RFS greatly affects rice yield. The pathogen can also produce large amount of mycotoxins, which are toxic to human and animals. At present, many studies have been performed on the epidemics of the disease, the infection cycle of U. virens, and purification and identification of mycotoxins. However, little is known about virulence and pathogenicity of U. virens. This study aims to screen the candidate effectors that can induce or inhibit plant immunity and to explore molecular mechanisms of U. virens pathogenicity in rice. First,30candidate effectors predicted in the genome of U. virens were isolated and transiently expressed in Nicotiana benthamiana using Agrobacterium-mediated transient expression system. The results showed that UV₅851, UV₆205and UV₇823can induce hypersensitive responses （HR） in N. benthamiana to different levels. Transient expression in rice protoplasts showed that UV₅851, UV₆205and UV₇823were also able to induce cell death in rice. Secretion experiments in yeast demonstrated that signal peptides of UV₅851and UV₆205were involved in protein secretion and that of UV₇823was not. It was also shown that signal peptides or C-terminal domains of UV₅851and UV₆205were required for their ability to induce HR in N. benthamiana. Second, the type III secretion system of rice bacterial pathogen Burkholderia glumae, together with pEDV vectors, was explored to deliver the U. virens effectors into plant cells. It was demonstrated that14out of30candidate effectors screened here were able to suppress HR induced by B. glumae in N. benthamiana to different degrees. Among these14effectors, UV₂964, UV₆039, UV₂508and UV₁261had the strongest suppressive effect. The signal peptides of UV₂964, UV₆039and UV₁261were shown to be associated with their secretion, but that of UV₂508was not. To further identify and characterize the functions of UV₂964, UV₆039and UV₁261, these genes were transformed into the rice cultivar Nipponbare by Agrobacterium-mediated transformation. At present, the transgenic resistant calli were obtained. Finally, based on RNA silencing system using N. benthamiana as materials,40candidate effectors were screened for RNA silencing suppressors. We found that the UV₅823has a weak effect to suppress gene silencing. However, the results are to be further verified. In summary, these results in this study lay the foundation for further study of the functions and targets of effectors and for molecular mechanisms underlying U. virens pathogenicity.