Study On Mechanism Of Osa-miR167d And RPW8.1 In Regulating Plant Immune Response

Rice is one of the important crops in the world,and rice blast is one of the devastating diseases of rice.In the past decades,increasing evidences have shown that mi RNAs and traditional R genes play important roles in the regulation of rice and rice blast interactions.Therefore,it is necessary to further study the roles of mi RNA and R genes in the resistance against rice blast.In previous studies,we collected the samples at the indicated time points in the susceptible and resistant accessions upon Magnaporthe oryzae infection,and identified an amount of s RNAs that differentially responsive to M.oryzae.Osa-mi R167d is one of them.However,whether and how Osa-mi R167d has the function in rice immune response is unclear.In addition,ectopic expressing RPW8.1 in rice enhances rice immunity against rice blast.However,how RPW8.1-activated defense is controlled remains unclear.Therefore,we use rice and Arabidopsis,focus on mechanism of Osa-mi R167d and RPW8.1 in regulating plant immune response.MiR167 belongs to a conserved mi RNA family in plants.In previous studies,the degradation sequencing and RACE experiments confirmed that Osa-mi R167d has seven target genes,including four auxin response factor(ARF)genes.Here,we firstly examined the expression pattern of Osa-mi R167d in the susceptible accession Lijiangxin tuan heigu(LTH)and the resistance accession IRBLkm-Ts upon infection of the M.oryzae.We collected the samples at the indicated time points upon M.oryzae infection in the susceptible and resistant accessions,then total RNAs were extracted and used for confirmation of the deep sequencing results.The results showed that the expression of Osa?mi R167d was significantly suppressed in a resistant accession at and after 24?hour post inoculation(hpi),however,its expression was significantly increased at 24 hpi in the susceptible accession upon M.oryzae infection and no difference in other time points compare with wildtype.These data indicated that Osa?mi R167d is differentially responsive to M.oryzae.Therefore,it is worthwhile to study the function of Osa?mi R167d in rice immunity against M.oryzae.Therefore,we used the genomic DNA of kasalath as a template and generated the construct that overexpressing Osa-mi R167d which is driven by 35S promoter.Then,we constructed transgenic plants overexpressing Osa?mi R167d(OX167d)under the Kasalath background.We selected two independent lines that showed significantly increased accumulation of Osa-mi R167d for resistance assay.The results showed that overexpressing Osa-mi R167d results in more susceptible to the rice blast.We also examined the infection process of M.oryzae with the GFP?tagged strain Zhong-8?10?14(GZ8)on the leaf sheath of wild?type and OX167d lines,the results showed that overexpression of Osa?mi R167d facilitates M.oryzae invasion.Moreover,overexpression of Osa?mi R167d can compromise the expression of defense?and cell death?related genes,production of H₂O₂ and accumulation of JA.Target mimicry is a well?established technology that is exploited to investigate the function of mi RNA.It can inhibit the function of mi RNAs on their target genes.Therefore,to confirm the function of Osa?mi R167d in the resistance against rice blast,we made the construct expressing the target mimicry of Osa?mi R167d(named MIM167d)which is driven by 35S promoter.Finally,we generated transgenic plants overexpressing MIM167d under Kasalath background.Upon inoculation the M.oryzae,MIM167d transgenic lines show smaller lesions than control.These results suggest that blocking Osa?mi R167d via target mimicry may inhibit M.oryzae invasion.Moreover,overexpression of MIM167d can enhance the expression of defense-and cell death-related genes,production of H₂O₂ and accumulation of JA.In order to further study the function of Osa-mi R167d in rice immunity against rice blast,we analyzed the target genes of Osa-mi R167 and examined the expression patterns of its target genes in the susceptible accession LTH and the resistance accession IRBLkm-Ts upon infection of the M.oryzae.The results showed that all the Osa?mi R167d target genes were differentially expressed upon M.oryzae infection.In a previous study,ARF12 acts as a target gene of Osa?mi R167,which positively regulates Os GH3?2 that is involved in the production of cellular free IAA by its IAA?conjugating activity in rice.In addition,Os GH3?2 mediates basal resistance to bacteria and M.oryzae in rice.The expression of ARF12 was downregulated in LTH upon infection of M.oryzae and ARF25 was upregulated at 12-72 hpi.Therefore,we focused on the ARF12 and ARF25 first.To investigate the function of ARF12 and ARF25 in rice immunity against M.oryzae,we designed target sites and generated ARF12 and ARF25 knock out mutants via CRISPR/Cas9 technology.We next assessed the disease phenotypes of these mutants via punch?inoculation with M.oryzae strains.The results showed that arf12 mutants were highly susceptible,but arf25 mutants showed no obvious difference from kasalath.Our data demonstrated that Osa?mi R167d acts as a negative regulator in rice immunity to facilitate the infection of M.oryzae via suppression of ARF12.Osa-miR167d is not only involved in the regulating the immune response of rice against the rice blast,but also in regulating the growth and development of rice.Overexpression of Osa-mi R167d resulted in shorter plant height,reduced tiller number,enhanced tiller angle,shrinkage of the leaves,increased chlorophyll content in the leaf sheath,closed flower fertilization,impacted the development of filaments,stigma and glumelle,reduced seed setting rate and 1000-grain weight;While,overexpression of MIM167d also led to shorter plant height,reduced chlorophyll content in the leaf sheath,impacted the development of filaments,stigma and glumelle,reduced seed setting rate and 1000-grain weight.Overall,Osa-mi R167d participates in regulating rice immunity against rice blast and regulates the rice growth and development.Therefore,characterization the function of Osa-mi R167d in rice is expected to provide a theoretical basis for molecular breeding of rice.Arabidopsis RESISTANCE TO POWDERY MILDEW 8.1(RPW8.1),identified from accession Ms-0,encode an atypical resistance(R)protein.In previous study,we found that ectopic expressing PRW8.1 in rice enhances rice immunity against rice blast,when expressed from its native promoter in Arabidopsis,RPW8.1 is localized to a membrane-like structure surrounding chloroplasts in the mesophyll cells to promote basal defense against different pathogens and hypersensitive response-like cell death.Also,the cell death lesions activated by expression of RPW8.1 as a transgene in Col-gl(a glabrous mutant of Col-0)are often discrete and confined.In order to understand the underlying regulatory mechanism of RPW8.1-mediated immunity,we performed yeast two-hybrid(Y2H)to screen for potential interacting proteins of RPW8.1 and found that one of the putative interactors is ACO4,which is one of the ACC oxidases converting ACC into ethylene.We provided further evidence to demonstrate that RPW8.1 interacts with and stabilizes ACO4,thus enhance ethylene production and ethylene signaling.However,the mutant of ACO4,EIN2 or EIN3 EIL1 in R1Y4 showed enhanced cell death and immunity.By construct,overexpressing EIN3 in R1Y4 showed compromised cell death and immunity,indicating that RPW8.1-mediated cell death and immunity was tightly controlled,and ethylene signaling is likely to be this regulator.Because RPW8.1 actives ethylene signaling,and ethylene response factors(ERFs)are important in ethylene signaling,thus,we try to identify that whether some of the ERF regulate RPW8.1 expression.Based on the LUC reporter system,we obtained three ERFs that regulate PRW8.1 promoter,i.e.ERF6,ERF016 and ORA59.The Y1H and EMSA demonstrated that ORA59 directly binds to a T/A-rich motif in RPW8.1 promoter.Also,the mutant of ERF6,ERF016 or ORA59 in R1Y4 showed enhanced cell death and immunity.In addition,there is a coordinated regulation between RPW8.1 and ORA59.Overall,we identified the repressor of RPW8.1 and clarified the function of ethylene signaling in RPW8.1-medicated cell death and immunity.