Functional Research Of OsF3H03g And OsF3H04g In Rice And Xanthomonas Oryzae Pv.oryzicola Interaction

Rice is the important food crop worldwide,while the disoperation of pathogens has been the main limition during rice cultivation.Rice bacterial leaf streak(BLS)caused by Xanthomonas oryzae pv.oryzicola(Xoc)has gradually been the primary bacterial diseases in West Africa and the south and central of China.However,there are little rice cultivars which carry the BLS major resistance genes.Thus,the investigation of interaction between Xoc virulence factors and rice that to reveal the pathogenic mechanism is instructive and meaningful for the exploration of putative resistance genes and cultivation of disease-resistant varieties.This research had identified a Xoc strain from rice planting region in Huanggang city,Hubei province,China,and then named as HGA4.The pathogenicity test of HGA4 had revealed that in the contrast to the Chinese model Xoc strain RS105,the HGA4 showed stronger pathogenicity in 8 japonica rice varieties and 6 indica rice varieties.To reveal the stronger pathogenicity of HGA4,the third-generation sequencing technology was used to sequence the genome of HGA4 to discovery the pathogenic factors.The transcription activator-like effectors(TALEs)which have transcriptional activation are mian pathogenic factors in Xoc,thus this research had analysised all TALEs in HGA4 genome.It showed that HGA4 contained 28 TALEs,and 24 of these TALEs had the homologues in RS105 which gomone has been sequencing and contains only 24 TALEs.And there were another four TALEs which were Tal2 b,Tal2c,Tal2 d and Tal2 e in HGA4 gonome.Homology analysis revealed that Tal2 b of HGA4 was homologous to the Tal2 c of abroad model Xoc strain BLS256,and previous research has indicated that Tal2 c of BLS256 may target the rice gene OsF3H03 g which encodes a 2-oxoglutarate and Fe(II)-dependent dioxygenase(2OGD).The gene expression revealed that OsF3H03 g was significant induced by HGA4 but not RS105.To verify that wheather Tal2 b directly targets OsF3H03 g gene,the whole Tal2 b gene was cloned from HGA4 genome,and then it was proved that Tal2 b directly bound to OsF3H03 g gene promoter through tobacco transient expression and EMSA.Simultaneously,Tal2 b was introduced into RS105 to generate RS105/Tal2 b.In contrast to wild type RS105,RS105/Tal2 b had ability to induced OsF3H03 g gene expression,and it caused longer lesion length than RS105 in rice leaves notably.Above results indicate that Tal2 b directly regulates OsF3H03 g gene expression and works as a pathogenicity factor of Xoc to promote invasion.To understand the role of OsF3H03 g in interaction of rice and Xoc,the genetic transformation was performed in Zhonghua 11(ZH11)rice to obtain the OsF3H03 g overexpression and suppression rice and the gene mutant mediated by CRISPR geneediting system.The further evaluation of resistance had revealed that OsF3H03 g overexpression rice was more susceptible to BLS,while OsF3H03 g suppression rice and gene-editing rice were more resistant to BLS.This indicated that OsF3H03 g is a negative regulator in rice resistance to BLS.To expound the action of OsF3H03 g on rice resistance,the yeast two-hybridization screen was performed and identified the OsF3H03g-interacting protein OsUGT which encode an UDP-glycosyltransferase.And then the facticity of interaction between OsF3H03 g and OsUGT was verified by Pull-down and bimolecular fluorescence complementation assays.Meanwhile,the overexpression,suppression and CRISPR mediated gene-editing mutant transgenic operation of OsUGT were performed in ZH11 rice,and it showed that the OsUGT overexpression rice was more susceptible to BLS than ZH11 and the OsUGT suppression rice and gene-editing rice were more resistant to BLS.Thus,OsUGT also negatively regulates rice resistance to BLS.In addition,SA related genes were upregulated in OsF3H03 g and OsUGT suppression rice lines,indicating that the function of OsF3H03 g and OsUGT may be related to SA.Additionally,this study found that Tal2 c of HGA4 was homologous to the Tal2 d of BLS256,and Tal2 d of BLS256 is predicated to target another 2OGD gene OsF3H04 g from previous report.The expression pattern in response to Xoc had showed that OsF3H04 g was not induced by RS105 but significantly induced by HGA4.To verify that weather OsF3H04 g takes part in regulating the interaction of rice and Xoc,the genetic transformation was performed in ZH11 rice and obtained OsF3H04 g overexpression rice,and the insertion mutant rice osf3h04 g was obtained from Korea rice mutant library.And evaluation of Xoc resistance showed that overexpression of OsF3H04 g resulted in more susceptibility to BLS,while the insertion mutant rice osf3h04 g were also more susceptible to BLS.These results indicate that the function of OsF3H04 g in regulating interaction of rice and Xoc is specific and intricate.To reveal the susceptible function of OsF3H04 g,the transcriptomic analysis of osf3h04 g was performed and it showed that SA and JA related genes and pathogenrelated genes were down-regulated in osf3h04 g.Futhermore,the hormone determination showed that JA content in osf3h04 g was decreased which was consistent with the depressed expression of JA synthetic genes OsLOX9 and OsAOS2.Surprisingly,SA content was dramatically decreased while the SA synthetic genes OsPAL and OsICS showed no significant change in osf3h04 g.Subsequently,a 2OGD gene OsS3 H was found drastically enhanced expression in osf3h04 g.Then,the OsS3 H overexpression rice and CRISPR mediated gene-editing rice showed decreased and increased resistance to BLS respectively.Above findings indicates that the deficiency of OsF3H04 g which mediated rice susceptibility to Xoc may be related to the elevated expression of OsS3 H.In conclusion,this research reveals the putative susceptible mechasim of the 2OGD genes OsF3H03 g and OsF3H04 g in response to BLS,which would provide guidance for disease-resistance cultivation in rice.