Functional Study Of LxAR Effectors MoLLE1?MoLLE2?MoLLE3 In Magaporthe Oryzae

Magaporthe oryzae is a plant pathogenic fungus that causes a severe disease,affecting rice production and casusing economic loss each year.A series of effector proteins are reported to play important roles in the interaction between rice and rice blast.The further studies on effectors trend to help explore the mechanisms of this process.In this study,99 putative LxAR proteins in M.oryzae(MoLLEs)were predicted by bioinfomatic tools.The coding sequence of each MoLLE was constructed into a 35S promoter conducted vector by Gateway cloning.Then the agrobacterial-mediated transient expression system in Nicotiana benthamiana was applied to screen the proteins which were able to influence the plant immunity,inducing hypersensitive reaction(HR)in plant or inhibiting HR generated by mouse BAX protein.Among then,MoLLE1,an unknown protein happened to induce HR in N.benthamiana with full length,but suppress BAX induced HR without signal peptide.Conversely,MoLLE2 stimulated HR in N.benthamiana without signal peptide while inhibited HR with full length.The other protein,MoLLE3 presented to hamper HR without signal peptide.The domain truncation assay showed that N-and C-terminal region were both required for MoLLE1 to induce and inhibit HR in planta as well as the C-terminal region in MoLLE2.The site-directed mutantation in LxAR Motif of these three proteins failed to induce or inhibit HR in N.benthamiana,indicating that LxAR may be functional in the way as RxLR in oomycete effectors.The subcellular localization tests also found that all of these three MoLLEs were able to translocalize from cytomembrane to nucleus,suggesting that functional behaviors of MoLLEs were possibly due to the cellular localization transition.The genetic analysis,such as gene deletion and over-expression,was used to detect the potential roles of MoLLEs in M.oryzae.The knockout and overexpression of MoLLE1 or MoLLE3 both reduced conidiation but failed to alter the colony Morphogenesis significantly,as well as MoLLE2 over-expression mutants.The rice leaves of susceptible rice CO39 inoculated with spores of deletion or overexpression of MoLLE1 displayed lesion earlier than the wild type M.oryzae GUY11,demonstrating that it was likely to act as a switch during the infection.Over-expreesion of MoLLE2 enhanced the pathogenicity,being opposite to that of MoLLE3 on rice leaves.In summary,these three MoLLEs may have certain roles in manipulating plant immunity and involve in the infectious process of rice blast.