The Functional Study Of MoNLP Family In Magnaporthe Oryzae

The non-host resistance(NHR)is exhibited by the natural phenomenon that all members of a plant species show resistance against all isolates of a plant pathogen.NHR is considered durable and effective against a broad range of races of the non-adapted pathogen species.However,it is still not clear how the immune system of non-host plants recognizes pathogens and triggers defense responses.Nicotiana benthamiana is a non-host for Magnaporthe oryzae,and therefore,in this study,we use N.benthamiana and M oryzae as the model system to investigate the incompatible response.We tried to construct a genomic library of M oryzae with activation tagging method to find the clones that could induce the plant immune response.Subsequently,we constructed a genomic library of M.oryzae with modified binary vector,and screened candidate clones on N.benthamiana by Agrobacterium-mediated transient expression.Meanwhile,we analyzed the effect of the 35S promoter and enhancer repeats on the activation of fungal gene expression in planta.The results showed that 35S promoter was essential and more important than the enhancer for fungal gene activation in transient assay.We thus screened about 10,000 clones,from which we identified 2 clones that induced prominent cell death in N.benthamiana.These results demonstrated that the functional genomics-based approach developed in this study is feasible and effective for identification of potential elicitor genes from pathogens,and it will facilitate current studies on genetic dissection of NHR.Through analysis of nucleotide and amino acid sequence,we identified the candidate elicitors as MoNLP4 and OSBP2,which belong to necrosis-and ethylene-inducing protein 1(Nepl)-like proteins(NLPs)and oxysterol binding protein(OSBP)family,respectively.Transient expression of four OSBPs in N.benthamiana could induce cell death and oxidative burst.Meanwhile,we found that the oxysterol_BP domain of the protein was the minimal domain capable of inducing the cell death.The transcript levels of OSBP4 were induced during the infection of rice plants,which indicated that OSBP4 might be involved in the interaction between M oryzae and rice plants.However,more experiments were needed to demonstrate the elicitor activity of OSBPs.Besides,Arabidopsis transgenic lines that conditionally overexpress MoNLP4 or OSBP2 under the control of an estradiol-inducible promoter were generated.Seeds of these transgenic lines were subjected to mutagenesis with EMS,and the resulting M2 seeds were collected for genetic screening for mutants deficient in elicitor-triggered immunity.NLPs are a class of microbe-associated molecular patterns widely distributed across diverse groups of plant-associated microorganisms.In spite of the cytotoxic activity in dicot plants,the role of most NLPs in the virulence of plant pathogens is still largely unknown.We showed that transient expression of three MoNLPs induced cell death and the production of reactive oxygen species in N.benthamiana,and the expression of MoNLPs was induced during infection of susceptible rice plants.To further investigate the biological role of the MoNLP family,a marker-free gene replacement vector was developed and used to knock out the whole family in M oryzae.Results showed no significant difference in disease levels caused by wild type and the quadruple AMoNLP mutant strains.Likewise,the sporulation and radial growth of the two strains were similar under various unfavorable cultural conditions including malnutrition and abiotic stresses.These observations demonstrated that the MoNLP family is dispensable for the fungal tolerance to the tested adverse cultural conditions,and more importantly,for the virulence of blast fungus on susceptible rice plants.To investigate the factors that induce the expression of MoNLP family,the transcript levels of individual MoNLP gene were analyzed in mycelia grown in various culture media.The results showed that the transcript levels of different MoNLPs varied significantly in these growth conditions.The expression of MoNLP1 could be induced in MM and Czapek Dox medium,and that of MoNLP4 was induced in several media.Meanwhile,we did not observe the induction of MoNLP2 and MoNLP3 in these media at 3 days after inoculation,but the expression of MoNLP2 was induced at a late stage of culture in CM medium.These results showed that the functional diversification of MoNLPs may exist in M oryzae.We subsequently determined the factors that induced the expression of MoNLP1 in MM medium,and found that osmotic stress is one of the factors which was able to induce the transcript levels of MoNLP1,and trace elements,as well as MgSO4,further enhanced the induction of MoNLP1 under this condition.