Functional Analysis Of Mitogen-Activated Protein Kinase Cascades In Rice Immune Response

Eukaryotic evolutionary conserved mitogen-activated protein kinase(MAPK)cascades transduce extracellular stimuli into cellular responses through sequential phosphorylation.In this three-tiered system,phosphorylated MAPK kinase kinase(MAPKKK)phosphorylates and activates downstream MAPK kinase(MAPKK),which in turn phosphorylates and activates MAPK.Plant MAPK cascades are widely involved in physiological responses,including plant growth and development,abiotic stress and immune response.Biological functions of MAPK cascades in plant innate immunity have been defined well in Arabidopsis.While in the case of rice,a monocot model plant,little was known about the function of a complete MAPK cascade,let alone how it was regulated.MPKK10.2,a MAPKK of group D,is a structural non-canonical MAPKK because it doesn’t have the typical motif of most plant MAPKKs that are phosphorylation sites of MAPKKKs.MPKK10.2-overexpressing(oe)plants showed enhanced resistance to both bacterial streak disease which caused by Xanthomonas oryzae pv.oryzicola(Xoc)and drought,whereas MPKK10.2-RNA interference(RNAi)plants had increased sensitivity to both Xoc and drought.MPKK10.2 physically interacted with MPK6 and MPK3 in rice cells and phosphorylated the two MAPKs in vivo.Transcriptionally modulating MPKK10.2 influenced MPK6 phosphorylation during rice-Xoc interaction,and MPKK10.2-oe/MPK6-RNAi double mutants showed increased sensitivity to Xoc.MPKK10.2-oe/MPK3-RNAi double mutants showed survival rates similar to those of control plants,although the survival rates of MPKK10.2 transgenic plants changed after drought stress.These results suggest that MPKK10.2 is a node involved in rice response to biotic and abiotic responses by functioning in the cross point of two MAPK cascades leading to Xoc resistance and drought tolerance.MPKKK1,a Raf-like MAPKKK,negatively regulates rice resistance to bacterial blight which caused by Xanthomonas oryzae pv.oryzae(Xoo).Immunoprecipitated protein kinase activity analysis reavealed that MPKKK1 negatively regulates MPK4activity after Xoo infection.mpkkk1/MPK4-RNAi double mutants showed increased sensitivity to Xoo compared with mpkkk1 mutant,suggesting that MPKKK1 negatively regulates MPK4 in rice-Xoo interaction.MPKK5 interacted with MPKKK1 and MPK4 in yeast and rice cells.Overexpression of MPKK5 elevated MPK4 kinase activity and enhanced rice resistance to Xoo,While MPKK5 suppressing(MPKK5-RNAi)rice showed normal MPK4 kinase activity and similar resistance to Xoo compared with wild type.mpkkk1/MPKK5-RNAi double mutants showed increased sensitivity to Xoo compared with mpkkk1 mutant.In vitro phosphorylation assay showed that MPKKK1phosphorylated MPKK5 on unknown sites,which in turn inhibited the phosphorylation of MPKK5 to MPK4.These results suggest that MPKKK1 negatively regulates MPKK5-MPK4 in rice-Xoo interaction.In vitro phosphorylation assay also showed that MPK4 feedback phosphorylated the kinase domain of MPKKK1(KD).The feedback phosphorylation site in MPKKK1identified by mass spectra located in the activation loop.MPK4 didn’t phosphorylate the mutanted KD at this site(KD~A)in vitro.Overexpressing MPKKK1~A in mpkkk1 mutant reduced the resistance to Xoo.The results suggest that MPKKK1 negatively regulates rice resistance to Xoo through inhibiting MPKK5-MPK4,while the feedback phosphorylation of MPKKK1 by MPK4 releases MPKK5-MPK4,which increases the resistance to Xoo.