Functional Analysis Of OsMAPKKK43 In Rice Resistance To Bacterial Blight And BR Signaling

Rice is one of the most important food crops in China,and bacterial blight caused by Xanthomonas oryzae pv.oryzae(Xoo)seriously affects the yield and quality of rice.MAPK(Mitogen-activated proein kinase)cascades play key roles in plant disease resistance signal transduction,but whether and how MAPK cascades regulate responses to rice bacterial diseases largely remain open questions.By scanning the Rice Mutant Database,we identified a T-DNA insertion mutant,osmapkkk43,which could negatively confer the broad-spectrum resistance to Xoo.By LC-MS/MS analysis,we found that S434 and T438 residues were critical for the kinase activity of OsMAPKKK43.By yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC)assays,we also found that OsMAPKKK43 could regulate its own kinase activity by forming homologous dimer via its N-terminal domain.To investigate the function of OsMAPKKK43,we performed split-LUC assay,semi-in vivo pull-down assay and in vitro phosphorylation analysis,and found that OsMAPKKK43 could interact with OsMAPKK4-and phosphorylate OsMAPKK4-in vitro and in vivo.Genetic and biochemical analysis showed that OsMAPKK4-OsMAPK6 cascade positively triggered the resistance to Xoo and was downstream of OsMAPKKK43.To further investigate the mechanism of OsMAPKK4 regulated by OsMAPKKK43,we performed in vitro phosphorylation assay and LC-MS/MS analysis,and we found that OsMAPKKK43 mainly phosphorylates the T34 residue in the N terminal domain of OsMAPKK4.In vitro and in vivo analyses indicated that OsMAPKKK43 is likely to negatively regulate the protein stability of OsMAPKK4 via phosphorylation of the T34 site of OsMAPKK4.The subsequent Y2 H analysis and split-LUC assay showed that the T34 phosphorylation could promote the interaction between OsMAPKK4 and two 14-3-3proteins,OsGFa and OsGFb.In this study,we also found that the OsMAPKK4 activated OsMAPK6 could feedback phosphorylate the S8,T126 and T129 residues located in the N-terminal domain of OsMAPKKK43 in vitro,and OsMAPKK4 activated OsMAPK6 could also feedback phosphorylate the T23 and T75 residues located in the N-terminal domain of OsMAPKKK4 in vitro.osmapkkk43,osmapkk4 and osmapk6 mutants all show obvious developmental phenotypes of brassinosteroid(BR)signaling activation or inactivation,and thus we investigated the relationship between BR signaling pathway and the OsMAPKKK43-mediated OsMAPKK4-OsMAPK6 cascade.The results showed that overexpressing OsMAPKKK43 or knocking out OsMAPKK4 in OsGSK2 RNAi plants both could suppress the BR signaling activated phenotype of OsGSK2 RNAi plants.Meanwhile,the osmapkkk43 dlt double mutant show similar plant architecture with dlt mutant.These results suggest that the OsMAPKKK43-mediated OsMAPKK4-OsMAPK6 cascade is involved in rice BR signaling pathway,and is downstream of OsGSK2 and upstream of DLT.We also found that OsGSK2 negatively confer broad-spectrum resistance to Xoo and Xoc in rice.Interestingly,OsGSK2 could phosphorylate both OsMAPKKK43 and OsMAPKK4 via in vitro phosphorylation assay.These results suggest that the OsMAPKKK43-mediated OsMAPKK4-OsMAPK6 cascade could be involved in BR signaling and defen response in rice.These findings reveal the important functions and molecular mechanisms of MAPK cascade in regulating immune response and development in rice,and broaden the understanding of MAPK cascade signal transduction and disease resistance signaling pathways in plants.