OsmiR1858a Targets OsHIN1 And Positively Regulates Rice Immunity Against Bacterial Blight

Rice(Oryza sativa)is one of the most important food crops and bacterial blight is a serious bacterial disease of rice.Studying the interaction between rice and Xanthomonas oryzae pv.oryzae(XOO)not only clarify the molecular mechanism of plant resistance to bacteria but also provide an effective strategy for prevention and treatment of bacterial blight.As an important way of regulating gene expression,miRNAs are involved in the regulation of plant growth,development and disease resistance.The previous experiments results showed that OsmiR1858 a was associated with the resistance to bacterial blight and proved its target gene is OsHIN1(Harpin-induced 1 domain containing protein).The transformations,including the OsmiR1858a-overexpressin、OsHIN1-overexpressing in the resistant rice background(SH5)and OsmiR1858a-overexpressing in the susceptible background(8411)have been successfully obtained in our laboratory.In this study,we use available transgenic materials to further explore the function of OsmiR1858 a and its target gene OsHIN1 in resistance to bacterial blight.The results are as follows:(1)OsmiR1858a-overexpressing transgenic lines in each background were inoculated with the Xoo strains P6 and P3.The disease lesion length and area were measured,and bacterial growth curve was also established.The results showed that OsmiR1858a-overexpressing transgenic lines were more resistant to bacterial blight than corresponding wild type,proving that OsmiR1858 a positively regulates resistance to bacterial blight.(2)Transient expression system of Nicotiana benthamiana was used to prove OsmiR1858 a splicing OsHIN1.Following successfully constructing fusion vectors with 35S::YFP:OsHIN1 and 35S::YFP:mOsHIN1,these constructions were co-expressed with 35S::OsmiR1858a in Nicotiana.benthamiana.The results showed that,the YFP fluorescence intensity was decreased with increasing miR1858 a concentration when OsHIN1 co-expressed with OsmiR1858 a,whereas no significant differences were observed when mOsHIN1 co-expressed with miR1858 a.So,we give the conclusion that OsmiR1858 a can splice OsHIN1.(3)To confirm the function of OsHIN1 in resistance to bacterial blight,OsHIN1 knock-out transgenic rices have been obtained by gene-edit method.Target lines from transgenic rices was acquired by sequencing site-edited of OsHIN1.And OsHIN1-overexpressing lines、 knock-out lines were analyzed by inoculation with Xoo strains P6 and P3 at same time.The result showed that the over-expression lines were more susceptible than SH5 while knock-out lines showed contrary phenomenon,demonstrating OsHIN1 decrease resistant to bacterial blight.(4)35S::GFP:OsHIN1 construction was injected into N.benthamiana by Agrobacterium tumefaciens to study OsHIN1 protein subcellular localization.The results showed that the OsHIN1 proteins were localized on the cytoplasmic membrane.