Biological Function Analysis Of The Aspartate Semialdehyde Dehydrogenase (ASADH) In Magnaporthe Oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating ricediseases that seriously restricts rice production all over the world. Rice blast occurredby "rice blast fungus-rice interaction which process is extremely complex. We can’tfully grasp the disease-resistant mechanism from the single aspect of host resistance.So it is importantly scientific value and practical significance to understand molecularmechanisms of the growth development and pathogenic process in M.oryzae.The research material obtained from our laboratory had built T-DNA insertionalmutant library of M.oryzae, and had identified a pathogenicity-defective mutantATMT13that are sensitive to hydrogen peroxide by screening this mutant library.Wegot flanking sequence of T-DNA insertional site by TAIL-PCR, then to studyMoASADH function in the growth、development and pathogenic process ofM.oryzaeby gene knockout and complementary mutation, these results are showed as follows:1.The identification of ATMT13mutant T-DNA insertional locus geneWe got gene sequence on the T-DNA insertional site right side of about900bpgenome DNA sequenceby TAIL-PCR,sequence sequenced and analysis revealed theT-DNA in the mutant disrupted775bp downstream away from the predicted geneMGG₀3051.6.The gene encoding aspartate-β-semialdehyde dehydrogenase（ASADH）, located in Chromosomal â…¦.The predicted ORF of this gene was1503bp, composed of two extrons and two introns, encoding363amino acids.2. The analysis of ΔMoASADH phenotypesThe mutant ΔMoASADH compared with wild type:1) The colony colour of wildtype is dark brown,ΔMoASADH shows fawn which colony color becomes shallowerthan wild type. It suggested MoASADH had an impact on the synthesis of melanin inrice blast.2) After different medium added exogenous MoASADHmetabolic product-lysine, threonine, methionine, isoleucine, the color of hyphae is dark, and become the similar with wild type.It suggested after recovering the function of MoASADHgene,the synthesis of melanin gradually restored.3)The conidiation of the ΔMoASADHmutant was much less than wild type which was about a quarter of wild type,conidium germination rate is less than1/2of the wild type. It indicates MoASADHmay related to conidium formation and differentiation.4)On thehydrophobicmembrane,formation and differentiation of appressorium is lag,30.83%of the appressorium reduce melanin content. These appressorium can’t formpenetration peg and hyphae extension limited in onion epidermal cells.5) Comparedwith wild type, the hyphae growth rate of theΔMoASADH mutant is a little slower,andhyphae curl at the end. It suggests the infection of hyphae may be blocked andpathogenicity defect.6)Under the hydrogen peroxide, congo red, SDS,Sorbitol, NaClstress conditions, The mutantΔMoASADH only showed highly hypersensitivity toH2O2.7) TheΔMoASADH mutant showed a significant reduction in virulence on riceleaves.3. The analysis of ΔMoasadh/ASADH phenotypesComplementary mutation is consistent with the wild-type strains in the colonycolor, growth speed, conidiation, appressorium morphology, pathogenicity.Thephenotype of complementary mutation recovery prove MoASADH gene regulate thesetraits.In summary, MoASADH gene played an essential role on colony growth,conidiation, appressorium formation and development, melanin anabolism,pathogenicity.