Molecular Mechanism Of MEL-SHMT1 Module Regulating Plant Immunity And Broad-Spectrum Resistance

Rice(Oryza sativa L.)provides a staple food source for more than half of the global population.Ensuring rice production is of great significance to national food security.Plant diseases seriously affect rice production as a result of interfering the growth and development during the process of rice maturation.Rice stripe disease caused by rice stripe virus(RSV)is one of the most devastating plant rice viruses and causes enormous rice yield losses.Currently,breeding the resistant varieties is the most economical and effective method to control crop diseases.Therefore,mining new broad-spectrum resistance(BSR)genes and deciphering their regulatory mechanisms are key to control rice stripe disease and other crop diseases.BSR genes could mediate many defense responses,such as epigenetic regulation,ubiquitination-mediated protein degradation,etc.So far,research on how plant ubiquitination modulate RSV infection has not been reported.Hence,we try to screen BSR E3 ligases,which determine substrate recognition in ubiquitination that regulate the RSV infection and analyzed its regulatory mechanism to provide theoretical basis for controling crop diseases.Through the analysis of RNA-seq results of RSV infection on Nicotiana benthamiana at 10 dpi,we identified an E3 ligase Niben101Scf01611g03014 that is remarkable up-regualted in response to RSV infection and is mainly located in nucleus and microtubule,and we name this gene as NbMEL(Nicotiana benthamiana microtubule-associated E3 ligase).NbMEL is highly homologous to N-terminus of human MEKK1 by sequence homology analysis.However,the function of NbMEL in plants has not been reported.The results of RSV challenging showed that overexpression of NbMEL could inhibit RSV infection,but knock out of NbMEL promotes RSV infection,which means E3 ligase NbMEL negatively regulates RSV infection.According to human MEKK1 could mediate human immune responses and induce apoptosis,we try to explore whether NbMEL could regulate plant immune responses.We found that OE-NbMEL could induce plant immune responses,including accumulation of ROS,activation of MAPK pathway,up-regulation of pathogenesisrelated(PR)gene,while KO-Nbmel leads to the opposite results.Furthermore,we demonstrated that NbMEL could respond to and regulate Botrytis cinerea infection.These results indicated that NbMEL is a BSR gene that regulate plant immune responses.In order to explore the regulation mechanism of NbMEL,we used NbMEL as bait to screen the yeast two-hybrid(Y2H)library and found that NbMEL can interact with Nicotiana benthamiana serine hydroxymethyltransferase 1(NbSHMT1)by Y2 H assay,GST pull down assay and Bimolecular fluorescent complimentary(Bi FC)assay.Furthermore,we demonstrated that NbMEL could promote NbSHMT1 polyubiquitination and degradation through the 26 S proteasome pathway.Then,whether NbSHMT1 could regulate BSR mediated by plant immune responses was further investigated.We found that KO-Nbshmt1 could induce plant immune responses,including accumulation of ROS,activation of MAPK pathway,upregulation of pathogenesis-related(PR)gene,which indicate NbSHMT1 could regulate plant immune responses.Furthermore,the results of RSV or B.cinerea challenging showed that NbSHMT1 could respond to RSV or B.cinerea infection and KO-Nbshmt1 inhibits RSV or B.cinerea infection.These results demonstrated that NbMELNbSHMT1 module could respond to and regulate RSV or B.cinerea infection.We further found that NbMEL(207-229aa)determines the interaction between NbMEL and NbSHMT1 by constructing NbMEL truncated mutant and conservation analysis of amino acids,and NbMEL 216 to 219 amino acids are highly conserved in all plants that containing MEL homologues.216 to 219 amino acids of NbMEL was then named as YφNL motif.Furthermore,we found that the ability of NbMEL(m YφNL)mutants to interact with NbSHMT1 was significantly attenuated,indicating that NbMEL recognizes NbSHMT1 through the YφNL motif.In addition,we found that the ability of NbMEL(m YφNL)mutant to induce polyubiquitination and degradation of NbSHMT1 was significantly weakened,hence the ability to induce plant immune responses was also significantly reduced.On the other hand,we found that NbMEL interacts with itself.Moreover,NbMEL(m SWIM)mutant could not interact with itself,while NbMEL(H179Y)and NbMEL(m YφNL)mutants still interact with itself,which means NbMEL interact with itself through the SWIM domain.Furthermore,we demonstrated that NbMEL forms NbMEL dimer through the intermolecular disulfide bond within the SWIM domain by reducing agent dithiothreitol(DTT)and NbMEL(m SWIM)mutant could not interact with NbSHMT1.Moreover,NbMEL(m SWIM)mutant could not promote the polyubiquitination and degradation of NbSHMT1 and could not induce plant immune responses.These results demonstrated that NbMEL forms homodimer through the SWIM domain,to recognize substrate NbSHMT1 through YφNL motif,which promotes poly-ubiquitination and degradation of NbSHMT1 through the 26 S proteasome pathway,thereby inducing plant immune responses.Through sequence homology analysis and multiple sequence alignment of NbMEL,we found that MEL is highly conserved in 28 plant species.Moreover,we try to explore whether the MEL-SHMT1 module could regulate immune responses and BSR against pathogens in rice.We found that OsMEL,which has E3 ligase activity,located in nucleus and microtubule,and OsSHMT1 located in mitochondria.Furthermore,we demonstrated that OsMEL could interact with OsSHMT1 and promote OsSHMT1poly-ubiquitination and degradation through the 26 S proteasome pathway.The results of DAB staining showed that OE-OsMEL and KO-Osshmt1 could induce ROS accumulation.Moreover,OE-OsMEL induced the up-regulation of PR gene expression;while KO-Osmel and OE-OsSHMT1 lead to the opposite results.These results demonstrated that the OsMEL-OsSHMT1 module can regulate the immune responses of rice.Next,we tested whether OsMEL-OsSHMT1 module could regulate BSR against devastating rice diseases,such as rice blast,rice bacterial blight or rice stripe disease.The results of Magnaporthe oryzae,Xanthomonas oryzae pv.oryzae and RSV challenging showed that OE-OsMEL could inhibit M.oryzae,Xoo.or RSV infection,while KO-Osmel or OE-OsSHMT1 could promote M.oryzae,Xoo.or RSV infection.These results demonstrated that OsMEL-OsSHMT1 module could regulate BSR against rice diseases.In summary,we identified and demonstrated for the first time the function of a new conserved BSR gene MEL in plants,and demonstrated that the MEL-SHMT1 module confers broad spectrum resistance of monocot and dicot plants against multiple pathogens by regulating plant immune responses.This study provides theoretical basis for using the BSR gene MEL to breed resistant varieties.