Cloning And Functional Analysis Of Mnh6 And Mtp1 Genes In Rice Blast Fungus, Magnaporthe Grisea

Rice blast is a severe disease to harm rice and the rice blast fungus is a well-known ascomycete Magnaporthe grisea. This fungal pathogen has been used as a primary model for elucidating various aspects of the pathogen-host interaction with its host. And the results to research the molecular basis of this disease not only help us to understand the pathogen-host plant interaction, but also promote to exploit new bactericide. Identification of pathogenicity-relative genes is a quick and effective step to study the molecular basis of this disease. A nonhistone 6 (MNH6) and a type-III integral membrane protein (MTP1) are two unknown proteins in functions of rice blast fungus. Until now genes, mnh6 and mtp1, or their homologous proteins were studied in rice blast fungus or other filamentous fungi. In this paper, mnh6 and mtp1 genes in M. grisea were cloned and their roles in growth, development and pathogenicity were partly analyzed and identified by targeted gene replacement.The results are showed as following:1. cDNA fragments of mnh6 and mtp1 genes were cloned from M. grisea appressorium cDNA library and full length DNA fragments of these genes were cloned from genomic DNA by LD-PCR. 2. The structures and functions of MNH6 and MTP1 were analyzed and predicted by using several biological soft wares. MNH6 is a nonhistone (DNA combining protein), belonging to HMGB protein family. MTP1 is a type-III integral membrane protein, having eight transmembrane domains.3. Knockout mutants of mnh6 or mtp1 were obtained separately by gene replacement vector construction and fungal transformation. And knockout mutants were identified in DNA and RNA level using PCR, Southern Blot, RT-PCR, and real-time RT-PCR.4. Null mutantΔmnh6 produced appeared offwhite hyphae, showed reduction in growth, conidiation and appressorium formation, and also showed reduction in appressorium turgor pressure. And the hyphae were more readily wettable by a solution containing SDS and EDTA and more easily digested by fungal cell-wall-digesting enzymes. The appressoria produced byΔmnh6 mutants showed diminished host penetration and the infectious hyphae ofΔmnh6 mutants showed reduced in planta growth. Targeted deletion of mnh6 resulted in the greatly reduced virulence capacity to barley and rice CO39 in infection assays.5. MNH6-GFP fusion protein was observed mainly in nucleolus. And this implied MNH6 protein mainly functions in nucleolus. mnh6 plays significant roles in the all-different development events of the fungus globally during its disease cycle such as vegetative growth, conidiation, appressorium formation, penetration, infectious growth and conidiation in diseased tissues in M. grisea. The pleiotropic effects on fungal morphogenesis exhibited byΔmnh6 mutant suggested that mnh6 is required for effective pathogenicity and completing the disease cycle of rice blast fungus.6. Null mutantΔmtp1 showed reduction in conidiation, delayed in germination and appressorium formation, and its hyphae showed blacker than wild type strain. But mutants showed no differences in growth, appressorium turgor, and virulence capacity to barley and rice CO39.7. GFP controlled by mtp1 promotor was expressed mainly in hyphae and spores, less expressed in appressoria. mtp1 has no visible effects on the growth of rive blast fungus. However, it has significant effects on conidiation, conidium germination of the fungus. And, mtp1 is dispensable for pathogenicity of M. grisea.