Functionalanalysis Of UvTcd1 And SIX Proteins In Ustilaginoidea Virens

Rice false smut caused by Ustilaginoidea virens is a fungal disease in rice panicles,causing serious damage for rice yield and quality.U.virens secretes effectors to promote infection,but the corresponding functions are not clear.Among them,UvTcd1 and SIX effectors of rice smut fungus have related phenotypes in our laboratory’s previous research.This article further studied the two,and the main results are as follows:（1）UvTcd1 is an orphan protein of U.virens with the characteristics of effectors.It could trigger cell death,active oxygen burst and callose deposition in Nicotiana benthamiana.Subcellular localization results showed that it gradually transferred from cell membrane and nucleus to chloroplast,and the chloroplast localization was necessary for triggering host cell death.The mutation at the 26^thmyristoylation site of UvTcd1 lost the function of triggering cell death,indicating that the myristoylation was essential for UvTcd1 to regulate the host immunity in which HSP90 was involved.UvTcd1 was knocked out by homologous recombination combined with CRISPR/Cas9 technology and PEG-mediated protoplast transformation method.In addition,UvTcd1 was over-expressed and related biological characteristics were investigated in both knockout and over-expression transformants.Results showed that UvTcd1 affected the mycelial growth,sporulation,but did not affect the conidia germination.It was also found that UvTcd1negatively regulated the tolerance to stresses and positively regulated the pathogenicity in U.virens.UvTcd1 could interact with rice protein Os196 which was verified by various methods both in vitro and in vivo.Os196 was up-regulated in Ubp::UvTcd1 rice young panicles,and could inhibit cell death caused by UvTcd1 in N.benthamiana.After inoculation with U.virens,the expression of resistance genes and grain filling related genes in transgenic rice decreased.These results indicated that UvTcd1 played the important role in the pathogenicity through direct interaction with Os196 to regulate the rice immunity.（2）A total of 9 SIX proteins have been identified in U.virens,of which 2 have been preliminarily analyzed in previous study,and the remained 7 SIX proteins were studied in this project.Based on the sequence profiles and phylogenetic analysis,it was found that one was SIX2 protein and the other six were SIX1 proteins.Using the yeast secretion system,all seven SIX proteins were verified to be secretion proteins.Expression of the full length or sequences without signal peptide encoding regions of SIX genes in N.benthamiana could completely or partially inhibit the cell death caused by BAX,INF1and XEG1.Furthermore,all 7 SIX proteins could promote the infection of Phytophthora capsici on N.benthamiana.The homologous recombination combined with CRISPR/Cas9 technology and PEG-mediated protoplast transformation were used to knock out UvSIX1-2.Compared with the wild type parental isolate and complemented transformants,the mycelial growth,sporulation and virulence decreased in knockout transformants,indicating that UvSIX1-2 played important roles in the mycelial growth,sporulation and virulence in U.virens.The functional analysis of the orphan protein UvTcd1 and conserved SIX effectors provide new clues for understanding the mechanisms of the pathogenicity in U.virens,the corresponding proteins may be used as the molecular targets to design highly specific nucleic acid fungicides which is very important in practice of the prevention and control of rice smut disease.