| Oomycetes represent a unique group of pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damages.The plant Phytophthora pathogens usually cause destructive diseases.Investigating the interaction mechanisms of plants and Phytophthora will be useful for better disease control.During compatible interaction,pathogens recruit plant susceptibility factors to help penetration,plant immunity inhibition,and expansion.For the hosts of pathogens,susceptibility is the norm.Investigating the mechanism of the susceptibility factors helps us to understand the occurrence of plant disease and to develop new materials in disease resistance.Phytophthora parasitica is the representative pathogen in Phytophthora,and its wellstudied pathosystem is helpful to explore the molecular mechanism of plant-Phytophthora interactions.An Arabidopsis thaliana T-DNA insertion mutant with resistance to P.parasitica was previously identified in our laboratory.The gene with T-DNA is named Raf36,which encodes a Raf-like mitogen-activated protein kinase kinase kinase.Plant Raf-like kinases are implicated in stress responses and other life activities.In this study,we used molecular biology and genetic analyses to investigate the mechanism of Raf36 in the negative regulation of plant resistance to P.parasitica.Meanwhile,we also studied the participation of Arabidopsis Raf36 in the immune signaling pathway and the function of the homologous genes in Nicotiana benthamiana.The main contents and results are as follows:Firstly,Raf36 acts as a negative regulator of resistance to P.parasitica and P.capsici in Arabidopsis.We knocked out Raf36 by CRISPR/Cas9 in Col-0 plants and found that the knockout mutants show enhanced resistance to P.parasitica.When we complemented or overexpressed Raf36 into raf36 mutant,the susceptibility to P.parasitica was restored and enhanced.Besides,we found that Raf36 also negatively regulates resistance to another Phytophthora pathogen P.capsici but does not respond to Botrytis cinerea upon inoculation in raf36 mutant plants and Raf36 overexpression mutant plants.Secondly,the negative regulation in plant disease resistance of At Raf36 is conserved in N.benthamiana.We used virus-induced gene silencing to silence Nb Raf36 s,the homologous genes of At Raf36 in N.benthamiana and inoculated them with P.parasitica.The Nb Raf36 s gene silencing plants showed enhanced resistance to P.parasitica.Thirdly,Raf36 does not participate in ET signaling.Raf36 knockout and overexpression mutants showed no difference in triple response by ACC treatment compared with wild-type plants.And the genes in ethylene signaling pathway showed no difference between raf36 plants and the wildtype plantsFourthly,Raf36 interacts with MKK2 in planta,and the kinase activity of Raf36 is required for this interaction.Using co-IP assay and luciferase complementation image assay,we found that Raf36 interacts with MKK2 in planta.Moreover,the bimolecular fluorescence complementation assay and the subcellular localization assay showed that Raf36 and MKK2 could co-localize in the cytoplasm of N.benthamiana leaves.Then,we found that the kinase domain-containing C-terminal region and the 185-206 amino acids sequences in the Nterminal domain of Raf36 are required for this interaction by the yeast two-hybrid system.Fifthly,MKK2 positively regulates Arabidopsis resistance to P.parasitica.Plants became more susceptible to P.parasitica when we knocked out MKK2 in Arabidopsis,and the resistance was rescued by complementing MKK2 in mkk2 plants.Additionally,the overexpression of MKK2 significantly enhanced plant resistance to P.parasitica.Sixthly,MKK2 contributes to the resistance offered by raf36 mutant to P.parasitica.We generated mkk2 raf36 double knockout mutant,the subsequent P.parasitica infection showed that the double knockout mutant showed enhanced susceptibility to raf36 mutant,indicating the upstream role of Raf36 in the MKK2-containing signaling pathway.We found that the kinase activity of Raf36 and MKK2 are required for their functions in plant immunity through transient expression of the kinase-dead mutants in N.benthamiana leaves upon P.parasitica infection.Also,through transiently expressing At MPK6 and its kinase-dead mutant At MPK6 MR in N.benthamiana leaves and inoculating them with P.parasitica,we found that At MPK6 positively regulates resistance to P.parasitica,and the resistance mediated by At MPK6 is associated with its kinase activity.In summary,our results confirmed that the previously screened Arabidopsis Raf-like kinase Raf36 negatively regulates plant resistance to Phytophthora pathogens and interacts with MKK2 in planta.The interactor MKK2 positively regulates plant resistance to P.parasitica and works downstream of Raf36.These results provide evidence for Raf36 as a susceptibility factor for resistance offered by the knockout mutation,and lay the groundwork for further study on the molecular mechanism of plant Raf-like kinases in disease resistance regulation. |
