Virulence Mechanism Of Secreted Protein Uv5918 From Ustilaginoidea Virens

Plant floral diseases seriously threaten the safety of agricultural production,because these diseases not only reduce crop yield and quality,but also introduce food toxins.Rice is one of the main food crops in the world.In recent years,rice is suffering from a serious floral disease,rice false smut.By infecting the developing spikelet of rice,the pathogen Ustilaginoidea virens formed a false smut ball of which the size was several times of rice grains.The formation of rice false smut balls not only leads to the increase of blighted grain rate in panicle and affects yield,but also produces a variety of mycotoxins,which affects quality and are poisonous to humans and animals.However,the understanding of the pathogenic mechanism of U.virens and its interaction with rice is very limited.In our previous work,a candidate gene Uv5918 was found to be highly expressed during the infection of rice spikelet by U.virens.In this work,the biological function and virulence mechanism of Uv5918 were extensively studied.The role of Uv5918 in the pathogenic process of U.virens was elucidated.The mechanism of Uv5918 targeting host protein to inhibit plant immune response was analyzed.The important immune components and disease resistance signal pathways in rice flower organs were explored,which provided an important theoretical basis for revealing the pathogenesis and genetic mechanism of disease resistance of rice false smut.The main results are as follows1.Uv5918 is an important virulence factor of U.virensBiochemical experiments indicated that Uv5918 encoded a secreted protein with chitin binding ability and chitinase activity.Uv5918 was significantly up-regulated during U.virens infection of rice spikelet,and the expression peaked when the pathogen extended to inner floral organs.Genetic evidence showed that loss-of-function of Uv5918 resulted in impaired pathogenicity of U.virens to rice,that is,failure of developing rice false smut balls,although the hyphae of the mutant strain could still extend to all floral organs inside the spikelet and form a white colony.By contrast,there was no significant difference between the growth of the deletion mutant and wild type strain on solid medium.2.Uv5918 can bind to chitin and inhibit chitin-induced immune responses in riceHeterologous expression of the full-length of Uv5918 in rice could significantly inhibit chitin-induced expression of defense-related genes and promote the infection of U.virens.In vitro experiments showed that the recombinant protein Uv5918 could competitively bind to chitin with rice chitin receptor OsCEBiP,thus weakening chitin-induced burst of reactive oxygen species（ROS）and expression of defense-related genes.3.Uv5918 interacts with OsCEBiP and OsCERK1 and interferes with chitin-triggered immune responsesIn vivo and in vitro experiments showed that Uv5918 interacted directly with the chitin receptor protein OsCEBiP and its co-receptor OsCERK1,and interfered with the formation of homo-or hetero-oligomers of OsCERK1 and OsCEBiP,thereby inhibiting the chitin triggered immune responses,such as ROS burst and defense-related gene expression.It is worth noting thatthe interaction between Uv5918 and OsCEBiP or OsCERK1 and the interference of OsCEBiP-OsCERK1 association do not depend on the catalytic pocket conserved site of Uv5918,indicating that Uv5918 has dual functions in inhibiting host immunity.In addition,the amino acid sequences of OsCEBiP and OsCERK1 were highly conserved in the natural population of rice,and the amino acid sequences of Uv5918 were identical among more than 50 tested strains of U.virens from different regions,indicating that the interaction module of Uv5918-OsCEBiP-OsCERK1 was highly conserved in the rice-U.virens pathosystem.This may be an important reason for the universal sensitivity of rice to U.virens.4.Uv5918 can be translocated into rice cells and suppress host immune responsesImmunofluorescence and immunocolloidal gold localization showed that Uv5918could be secreted by U.virens and translocated to the rice stamen filament cells.Heterologous expression of the mature sequence of Uv5918（without the signal peptide-encoding sequence）in rice(Uv5918^△SP)significantly inhibited chitin-triggered callose deposition,ROS burst,and defense-related gene expression.Uv5918^△SPshowed increased susceptibility to U.virens,accompanied by decreased accumulation of hydrogen peroxide and expression of defense-related genes.These results indicate that Uv5918 can enter into the host cells to dampen plant immunity.5.Uv5918 targets OsRACK1A to weaken its interaction with OsRBOHB and reduce production of ROSYeast-two-hybrid assay identified that OsRACK1A interacted with Uv5918 directly,which was further confirmed by co-immunoprecipitation,GST pull-down and Bimolecular Fluorescence Complementation assays.Uv5918 had no significant effect on the subcellular localization and protein stability of OsRACK1A,but could competitively reduce the interaction between OsRACK1A and OsRBOHB,and ultimately affect chitin-induced ROS burst.6.OsRACK1A and OsRBOHB regulate rice resistance to false smutOverexpression of OsRACK1A significantly enhanced the resistance of rice to U.virens.On the contrary,OsRACK1A RNAi plants were more susceptible to U.virens than wild type plants.Loss-of function of OsRBOHB led to enhanced susceptibility to U.virens.OsRACK1A-overexpressing rice displayed changes of immune responses,including more hydrogen peroxide accumulation and higher expression levels of defense-related genes in spikelets than wild-type control.By contrast,OsRACK1A RNAi and osrbohb mutants had the opposite trends.These results suggest that OsRACK1A and OsRBOHB are involved in rice floral immunity against U.virens.In conclusion,the secretory chitinase Uv5918 is a conserved pathogenic factor of U.virens,which plays multiple virulence functions in the interaction between U.virens and rice.On one hand,Uv5918 inhibits the extracellular immunity of rice by binding to chitin and targeting chitin receptor.On the other hand,Uv5918 is translocated into rice floral cells and targets the scaffold protein OsRACK1A in an immune complex to inhibit the intracellular immunity.The results of this study will provide a new theoretical basis for revealing the susceptibility mechanisms of rice false smut,and a new clue for dissecting the resistance mechanisms of rice false smut.Future work will be conducted on exploring the new allelic variation of OsCEBiP,OsCERK1 and OsRACK1A,which are not targeted by Uv5918,so as to engineer resistant materials to rice false smut.