Mechanism Research On OsOPR7 Alternative Splicing Involved In The Interaction Between Rice And Xanthomonas Oryzae Pv.oryzicola

Plant diseases harm crop yield and food safety seriously in the world.Thus,analyzing the interaction mechanism between plants and pathogens is instructive for molecular breeding and disease control.Rice bacterial leaf streak(BLS)by Xanthomonas oryzae pv.oryzicola(Xoc),is a serious bacterial disease mainly distributes in central,south and east China.In recently years,BLS has spread to the northern rice region,and occurs in Africa and southeast Asia as well.During the occurrence of BLS,the pathogen spreads along the rice veins,causing the leaves to withered,resulting in a significant reduction in rice yield.In order to explore the resistance towards Xoc during rice growth and development,we analyzed the change of rice resistance in the growth process of 21-35 days.The results showed that the resistance of rice to Xoc increased gradually as time went by.Further study showed that JA content also decreased during growth process of 21-35 days,while its biosynthesis precursor OPDA content increased at the same time.We found that the rice was more susceptible to Xoc after treated with exogenous Me-JA.We further analyzed the disease resistance of the transgenic rice lines with over expression of JA biosynthesis genes AOS1 and AOS3,and aos1 mutant and JA receptor COI1-RNAi line to Xoc.The results showed that the transgenic lines of AOS1-OE and AOS3-OE were more susceptible to BSL,while mutant aos1 and COI1-RNAi lines were more resistant to Xoc,which indicated that JA negatively regulated the resistance of rice to Xoc.To further reveal the mechanism of how JA decreasing with rice growing period,we sequenced the transcriptome of rice leaves at 3 and 4 weeks(w).The results of data analysis and qPCR verification showed that the gene expression of JA downstream pathway in rice leaves at 3 w was significantly upregulated than 4 w,whereas no significant differences were found in the expressions of JA biosynthesis genes.As mentioned earlier,although the content of JA decreased,the JA biosynthesis precursor OPDA increased gradually.To explain this phenomenon,we further analyzed the related genes in the transformation process from OPDA to JA,and found that there were two transcripts of OsOPR7(a reductase catalyzes the conversion of OPDA to OPC:8): one was OsOPR7.1,which was composed of five exons,the other was OsOPR7.2 with unknown function,without exon 2.Although there was no significance in OsOPR7 transcription level between 3 w and 4 w rice leaves,we found that the peak value of exon 2 of OsOPR7 in 3 w was significantly higher than that in 4 w through genome visualization tool IGV software,but no significance in other exon peak values.Thus,we speculated that OPR7 undergoing alternative splicing from 21 d to 28 d.Using specific primers,we detected the expression level of OsOPR7 two transcripts and found that the expression level of OsOPR7.1 decreased gradually during 21 d-35 d,while the expression level of OsOPR7.2 increased gradually.This change trend during 21 d to 35 d was consistent with the content of JA and OPDA,respectively.Therefore,we hypothesized that OsOPR7.1 had stronger ability to reduce OPDA,resulting in higher JA content and lower OPDA content in 3 w rice leaves,but weaker catalyze ability of OPR7.2 resulted in the decrease of JA content and the accumulation of OPDA in 5 w rice leaves.For comparing the differences between the function of OsOPR7.1 and OsOPR7.2,we cloned their encoding regions,respectively,and transferred them into Arabidopsis.The results showed that only overexpression of OPR7.1 inhibited the elongation of Arabidopsis seedling primary root,and replenished the phenotype of opr3 mutant,proving that OsOPR7.1 had the similar function as AtOPR3 in Arabidopsis.After knocking out OsOPR7 through crisp technology in rice,we found that the JA content of rice decreased and the resistance to BLS increased.Meanwhile,overexpression of OsOPR7.1 in rice resulted in the increasing of JA content but more susceptible to BLS.However,overexpression of OsOPR7.2 did not increase JA content and made no significance on rice resistance to BLS.These indicated that OsOPR7 was important for JA biosynthesis in rice,and OsOPR7.1 played a key role in JA biosynthesis in the two transcripts of OsOPR7.To further analyze the catalytic ability of OsOPR7.1 and OsOPR7.2 to OPDA,we prokaryotic expressed OsOPR7.1 and OsOPR7.2,respectively,and detected their enzyme activity after purification.The results showed that OsOPR7.1 had strong catalytic ability to OPDA,however,OsOPR7.2 has weak catalytic ability to OPDA.Therefore,we proved that OsOPR7.1 has stronger catalytic ability to OPDA than OPR7.2.In addition to invading plant cell through wounds,pathogens also use stomata to invade plant cells in healthy leaves,and more pathogens will gather near the opening stomata.Pseudomonas syringae pv.tomato DC3000(Pst DC3000)secretes a JA chemical analogue coronatine(COR)when it invades Arabidopsis leaves.COR can mimic JA to initiate the signaling pathway downstream of JA through the JA receptor COI1,further inhibit stomatal defense response to promote pathogen infection.Therefore,we speculated that JA could regulate the resistance of rice to Xoc by regulating the stomatal aperture.In order to prove this hypothesis,we detected the effect of exogenous Me-JA on the stomatal aperture of rice,and compared the differences of the stomatal aperture of JA overexpression lines,mutant lines and wild-type lines.The results showed that JA positively regulate the stomatal aperture,which was more conducive to the infection of pathogenic bacteria.Further study showed that the stomatal aperture gradually decreased during the growth and development of rice from 21 d to 35 d,which was consistent with the change trend of JA content and the change trend of resistance of rice to BLS,preliminarily proving that JA modulates the resistance of rice to BLS via regulating the stomatal aperture during different growth period of rice.In the process of rice production,our research results provide technical guidance for the prevention of BLS.In the growth stage of rice seedling,exogenous spraying of JA biosynthesis inhibitor DIECA could inhibit the content of JA in rice leaves,so as to protect the rice seedling from Xoc.