Functional Analysis Of Genes Associating With Ras Signaling Pathway Mediated By RasGTPase-activating Protein UvGap1 In Ustilaginoidea Virens

Rice false smut,caused by Ustilaginoidea virens,is a fungal panicle disease of rice.This disease is becoming one of the major diseases of rice,which seriously threaten the safe production of rice.In the previous study,a T-DNA insertion mutant library of U.virens was constructed by Agrobacterium tumefaciens-mediated transformation（ATMT）,and a mutant strain,named as B2510 with loss of sporulation and the weakened of pathogenicity was screened.Subsequently,one T-DNA insertion site in this mutant was mapped as the promoter region of the gene Uv8b₁386 whose product encodes a RasGTPase activating protein UvGap1.On the basis of our earlier study,the present work is aimed to investigate function of genes associating with the UvGapl-mediated Ras signaling pathway（U.virens contains two Ras proteins,UvRasl and UvRas2）and the downstream cAMP pathway-related protein kinase A（UvCpka）,the acid cyclase-related protein（UvCap1）,and the backbone protein UvSte50 of the MAPK（mitogen-activated protein kinase）signaling pathway.Major findings of the present wrok were described as follows:1.The function of UvGap1 in the rice false smut was preliminary analyzed.The CRISPR-Cas9 system was introduced to U.virens for gene knockout.The CRISPR-gRNA vectors and the homologous recombination vectors of UvGAPl、UvRAS1 and UvRAS2 were co-transformed into the wild type strain P1 using the PEG-mediated protoplast transformation method.After screening,the UvGAP1 knockout mutant was obtained,but the UvRAS1 and UvRAS2 knockout mutants were not obtained.By analyzing the expression levels of the genes UvGAP1,UvRAS1 and UvRAS2 in the infection stages of U.virens,it was found that UvGAPl and UvRAS1 had the highest expression at 3 days of inoculation,and UvRAS2 had the highest expression at 2 days of inoculation.The gene UvGAP1 was replenished into the gene deletion mutants by Agrobacterium-mediated transformation（ATMT）to obtain complementary strains,which recover the defect of pathogenicity.2.The function of Uvcpka and Uvcapl in the rice false smut were preliminary analyzed.Using the CRISPR-Cas9 technology to get gene knockout mutants of UvCPKA and UvCAP1 We analyzed the phenotypes changes of these mutants,the resulting data showed that ΔUvcpka had no significant difference in mycelial growth compared with the wild type strain.ΔUvcpka could form a few smut balls after inoculation,which revealed that UvCPKA is essential for the pathogenic process of U.virens.Compared with the wild type strain,the mycelial growth rate and conidia production were reduced in ΔUvcapl,but the formation of smut balls was consistented with the wild type strain,UvCAP1 is mainly involved in the regulation of mycelial growth and conidia production process in U.virens.3.The function of Uvste50 in the rice false smut was preliminary analyzed.Gene knockout mutant of UvSTE50 was also obtained using CRISPR-Cas9 system.ΔUvste50 has no significantly different with the wild-type strain in mycelial growth and conidia production.Inoculation experiments showed that ΔUvste50 completely lost the pathogenicity ability.The results of yeast two-hybrid experiments showed that there was an interaction between UvGap1,UvRas1 and UvRas2.UvSte50,UvRas2 and UvCap1 also have interaction with each other,moreover,UvRas1 interact with UvCap1.The UvGap1-mediated Ras signaling pathway is closely related to the pathogenic proteins of the downstream cAMP and MAPK pathways,and regulates the development and pathogenic process of U.virens together.This study has important value for revealing the signal transduction in the pathogenic process of rice false smut,and provides a theoretical basis for formulating the scientific prevention programs of rice false smut.