A Preliminary Study On The Mechanism Of OsHIN1 Negatively Regulates Rice Resistance Against Bacterial Blight

Bacterial blight,which causes serious harm to rice production and food security,is a worldwide bacterial disease of rice.Therefore,it is very important to study the mechanism of disease resistance.The previous results showed that OsmiR1858a over-expression transgenic rice significantly increased resistance to bacterial blight and OsHIN1 is OsmiR1858a's target gene.On the contrary,OsHIN1 over-expression lines showed reduced resistance to bacterial blight while RNAi and knock-out lines showed contrary phenotype.These results demonstrated that OsHIN1 decreases resistant to bacterial blight,but the mechanism remains elusive.Therefore,it is necessary to study how OsHIN1 negatively regulates resistance against bacterial blight.To address this question,we examined several characteristics of OsHIN1,including the regulation of gene expression,subcellular localization and possible interacting proteins.The results were described as follows:?1?The 2kb OsHIN1 promoter was cloned and the pOsHIN1::GUS expression vector was constructed and genetically transformed into rice.Twelve positive transgenic rice were confirmed by PCR detection.Then,GUS staining was performed,and results showed that expression of OsHIN1 was higher in the tender tissue of rice,including middle part of leaf nodes,the joint of rhizome,and the active part of root hair,but lower in the mature tissue.After inoculation with P6 and simulated inoculation with ddH₂O in the same lines,the expression of GUS in the same tissue inoculated with P6 was stronger than that in the control.?2?When rice protoplasts were co-transformed with 35S::eGFP::OsHIN1 vector and Marker?mCherry?,which locates in plasma membrane,the yellow is observed after 3 d,indicating that GFP overlapped with Marker,so OsHIN1 was located in the cell membrane.?3?Genetic transformation was carried out by agrobacterium-mediated rice embryo,and both 10 35S::OsHIN1::eGFP transgenic lines and a 35S::eGFP transgenic line were obtained by PCR detection.These positive materials provided the important basis for subsequent identification of interaction proteins by immunoprecipitation.?4?Yeast membrane protein library was constructed from rice samples at 3 day post inoculation with P6.When OsHIN1::BD was used for prey to screen yeast library,38 positive clones were obtained.12 candidate clones were choosed for testing one-to-one after sequence of these positive clones were analyzed.7 of 12 candidate genes showed positive result in yeast two hybrids.On this basis,we cloned the ORF of the 7 genes,and yeast two hybrids and BiFC were performed respectively.The results showed that CPT interacted with OsHIN1 in both yeast two hybrids and BiFC,however,four genes only interacted with OsHIN1 in Nicotiana benthamiana,but not in yeast.