Functional Identification Of Three Effectors In The Rice False Smut Pathogen,Ustilaginoidea Virens

Rice false smut（RFS）is a fungal disease caused by Ustilaginoidea virens,which mainly damages the panicle of rice.In the rice mature stage,RFS balls are formed in the panicle.After the balls expand and rupture,the dark green powder will be scattered,thus contaminating rice grains.The RFS ball itself produces toxins,which can cause poisoning reactions after consumption by humans and animals.In recent decades,RFS has gradually increased from a secondary disease to a major disease in rice.Rice is one of the world’s three major food crops,which is vital to humans.In recent years,research institutions and scholars have paid more and more attention to RFS.However,most of these studies have focused on the study of the biological characteristics of the pathogen,host and infection cycle,and few studies on the molecular mechanism of the pathogenesis.Our previous study found that U.virens infects rice flowers and intercepts their pollination;however,rice grain filling related genes such as RISBZ1 can be activated,suggesting that U.virens may hijack rice nutrient reservoir for growth.Preliminary screening with Nicotiana benthamiana transient expression system indicated that three effectors from U.virens had the ability to activate the transcriptional activity of RISBZ1 promoter.In the present work,we carried out further study on these three effectors,so as to clarify their biological functions and confirm their relations with rice RISBZ1 gene.The major results are as following.（1）Bioinformatics analysis revealed that the amino acid regions 1 to 19 of the Uv₃746 protein domain were signal peptide regions,and the 51st to 148th amino acid regions were predicted microbial ribonucleases.The amino acid regions 1 to 23of the Uv₃667 protein domain are signal peptide regions,and the amino acid regions28 to 85 are predicted hydrophobins.The amino acid regions 1 to 19 of the Uv₇360protein domain are signal peptide regions,and the amino acid regions 37 to 383 are predicted various epidermal growth factor-like proteins.（2）By analyzing the expression patterns,it was found that the expression level of Uv₃667 reached two peaks at 4d and 10d after inoculation,and the maximum peak at 4d,and the expression of Uv₃746 reached two peaks at 5d and 11d after inoculation.The maximum peak value at 11d indicates that the effect factors Uv₃667and Uv₃746 may play a role in the early stage of RFS.The expression of Uv₇360reached the maximum at 8d and 12d after inoculation,and the expression of Uv₇360was higher than Uv₃746 and Uv₃667,indicating that the effector Uv₇360 may play a role after invading rice spikelets,and the expression of Uv₇360 is highly induced.（3）The subcellular localization of three effectors was studied by the N.benthamiana transient expression system.The results showed that Uv₃667 was located in the nucleus and cytoplasm.The fluorescence of the effector Uv₃746 was overlapped in the intracellular plastids,indicating that Uv₃746 may be localized on the plastid.Uv₇360 was distributed as dots in cytoplasm.（4）Three effector genes were heterologously expressed in rice cultivar ZH11,and the resultant transgenic plants were inoculated with U.virens PJ52.The data showed that only Uv₃746-expressing lines displayed enhanced susceptibility to RFS,indicating that Uv₃746 promoted infection of U.virens in rice.On the contrary,neither Uv₃667-nor Uv₇360-expressing rice displayed altered sensitivity to U.virens.RT-qPCR data demonstrated that the starch synthesis related gene AGPL2 was down-regulated in developing rice spikelets before flowering in effector-expressing transgenic lines,compared to the wild-type.However,the expression other grain filling replated genes were not consistently changed.In leaves of Uv₃746-expressing transgenic lines,RPBF,SSI,AGPL2,Glutlin1,Glutlin2 were up-regulated.（5）Soluble sugar and total protein were measured in seeds of effector-expressing lines and ZH11.The results showed that the amout of soluble sugars was significantly reduced,while total protein level was not changed.Investigation of agronomic traits indicated that the 1000-grain weight of effector-expressing lines was greater than that of ZH11,while other traits such as plant height,grain number,panicle number,and so on,were not consistently increased or decreased among multiple lines.Therefore,agronimic traits should be repeatly evaluated in the next seasons.（6）Over-expression or gene knock-out of three effector genes were carried out in U.virens PJ52.Multiple transformants over-expressing Uv₃667 or Uv₇360 were obtained.The colony growth rate of the over-expressors in PSA was not changed,compared to the wild-typle PJ52,while was faster in CA complemented with stachyose,sucrose or glucose.Under stresses such as congo red,SDS,H₂O₂,NaCl,the growth rates of PJ52 and the over-expressors were all inhibited.Moreover,the inhibition rate,the overexpression strain was inhibited by SDS stress and the inhibition rate was lower than that of the wild type strain PJ52.In addition,we obtained multiple knock-out transformants for Uv₃746,facilitating further work on functional identification of these genes.In summary,the expression pattern,subcellular localization,functional identification in rice and in U.virens of Uv₃746,Uv₃667,Uv₇360 have been extensively investigated.These data proved that Uv₃746 contributed to virulence of the pathogen,probably via disturbing the sugar distribution in rice spikelets and facilitating the nutrient acquisition of U.virens.Moreover,our work provides valuable materials for further understanding the molecular mechanisms of rice-U.virens interactions.