Preliminary Study Of The Pathogenic Differences Between T And MT Type Ustilago Esculenta And The Genes Related To Teliospores Formation

Jiaobai,the swollen stem of Zizania latifolia,formed by infection with the biotrophic basidiomycete fungus Ustilago esculenta,has become the second largest aquatic vegetables in China.Traditional methods of Jiaobai in planting and breeding not only cost a lot of labor and financial resources but also cause variety degeneration,producing inedible grey Jiaobai and male Jiaobai in the fields,which seriously affect the economic benefits.An Artificial System for the formation of Jiaobai was established and optimized and gray Jiaobai which contain a large amount of black masses of teliospores were formed successfully.According to the virulence differences among T/MT type strains during the early infection,this study used the dual RNA-Seq to assess the transcriptome profiling of the two fungal pathogens T/MT type strains and the different stress response of the plant.Genes Itd1,Uepac2 and g5243 related to the formation of formation of the teliospores were screened by analyzed the transcriptome data about normal Jiaobai and gray Jiaobai and their functions were analyzed by gene clone,knock-out and artificial inoculation.As a typical dimorphic fungus,closely related to Ustilago maydis,compatible haploid strains of scale pathogenicity were first selected and identified from different strains and combinations of U.esculenta status.Next,leaves,leaf sheaths and tubular roots were inoculated,separately to determine the best inoculation site-leaf sheaths.Then,the inoculation method?stem base injection method,tissue infiltration method with stem base and tissue infiltration method?inoculation serious suspension culture concentration pathogen cell(OD₆₀₀ value of 2.0,1.5,1.0,0.5)and the cultivation environment of seedlings after inoculation were screened.Results showed that the appropriate inoculation method was stem base injection method,the appropriate cell concentration was OD₆₀₀ value of 2.0,and the better cultivation environment was keeping in greenhouse or transplanted to field in mid-March or mid-September.An optimized artificial inoculation system was established,which can induce the swollen of stems.Firstly,tubular roots of Z.latifolia were germinated in greenhouse under a 12/12 h light/dark cycle at 25/22°C for 10²0 d;Secondly,compatible strains UET1 and UET2 of U.esculenta with an OD₆₀₀ value of 2.0 were syringe-inoculated into the base of seedling stems,keeping in greenhouse or transplanted to field in mid-March or mid-September.Under this artificial inoculation system,plant survival rate was about 80%,and the rate of formation gray Jiaobai was higher than 80%.After infection with T/MT type strains,a plenty of defense-related pathways and genes in the diseased plant tissue were induced and showed distinct differences expression in the two strains and temporal dynamics.As the infection process progresses,fatty acid biosynthetic process,Phenylalanine,tyrosine and tryptophan biosynthesis,Plant hormone signal transduction,steroid dehydrogenase activity,cofactor binding,oxidoreductase activity were significant different expression in infected Z.latifolia with both strains.We also characterized the T/MT type strains transcriptomes.We found that in the early stage of plant pathogen infection,fatty acid metabolic process and cellulase activity were up-regulated in specific phase.Moreover,T/MT type strains responded differently to plant defense,that T type strains showed stronger adaptability to the plant defense response.In order to stop the telispores formation,MT strains from white Jiaobai?swollen stem without telispores?and T strains from grey Jiaobai?swollen stem with full of telispores?were collected and analysed by transcriptome.Differently expressed genes were screened and selected for functional verification.Fortunately,7 genes may be related to the formation of teliospores,5 genes may be related to the dimorphic transition were screened out.3 genes?g5243,g4070,g1449?which the most significant differences related to teliospores formation in gray Jiaobai and normal Jiaobai were identified.3 genes were cloned,and the open reading frames were 2876,2001,2670 bp,encode 925,666,852 amino acids,respectively.Phylogenetic analysis showed that g4070 and g1449 were belong to WOPR family,and closed relative to U.maydis,named Uepac2 and Itd1,respectively.Itd1 deletion mutants from the compatible U.esculenta strains UET1 and UET2 showed a defect in telisorores formation for inoculation.Combining artificial inoculation method and successful genetic modification,an artificial system for U.esculenta-induced swollen stem formation without black teliospores was established.While Uepac2and g5243 deletion mutants from the compatible U.esculenta strains UET1 and UET2 showed no difference to control and gray Jiaobai formed successfully.The above study explored a new efficient and stable breeding way for white Jiaobai and provided a reliable theoretical and technical basis for the artificial breeding of white Jiaobai.At the same time,it laid a foundation for further research on the dimorphism transition,filamentous growth and teliospores formation of U.esculenta.