Functional Characterization Of Sugar Transporters Of MoHXT2 And MoHXT3 In Magnaporthe Grisea

Magnaporthe oryzae is a pathogen fungal that causes rice blast disease and has a great threaten to food security.Understanding the biological characteristics,pathogenic mechanism and the interaction between the host of the rice blast fungus will provide a basis for formulating new and effective strategies for the control of the disease Experiments have shown that sugar transporters are specifically inducing expressed in the early stage of the infection process of rice blast.There were 67 sugar transporters identified in M.oryzae genome in silico.MoHXT2 and MoHXT3 were selected for functional analysis based on the phylogenetic tree analysis.MoHXT2 and MoHXT3 genes were knocked out by homologous recombination repair（HDR）system mediated by CRISPR/Cas9,and the mutants ofΔMohxt2 andΔMohxt3 were obtained respectively.The wild-type and mutant strains were inoculated on CM medium supplemented with different carbon sources（glucose,galactose,mannose,fructose）to compare their melanin production ability.It was observed that theΔMohxt2strains were totally lost the ability of produce melanin when the cultured on the four mediums.However,theΔMohxt3 strains were showed white mycelium on glucose and mannose containing CM medium,while show white and grey mycelium in galactose and fructose CM medium.The implication was that MoHXT2 and MoHXT3 have different utilization efficiency and affinity for different sugars.Rice leaves inoculatedΔMohxt2 strain could not form lesion produced as the wild strain A60 did,while theΔMohxt3 strain just produced lesions fewer and smaller than the A60 did.These observations showed that MoHXT2 and MoHXT3play a key role in the formation and germination of rice blast fungus spores,germination and its pathogenicity.Three sites Met145,Lys421 and Ala89 were selected in site mutant experiment according to the results of molecular docking analysis between the MoHXT2 and its substrates.The transport function for each substrate of the three mutant genotypes,MoHXT2^M145K,MoHXT2^K421A and MoHXT2^A89R were analysis both in yeast and electrophysiological experiments in Xenopus laevis oocyte.The results show that Met145 site of MoHXT2 plays critical role in sugar transport function,while Lys421 mainly determines the substrate specificity of MoHXT2Transcriptome analysis showed that genes involved in the metabolic process were significantly affected theΔMohxt2 andΔMohxt3 strains.MoHXT2 and MoHXT3 regulated the biosynthetic pathway in M.grisea metabolism and affect the morphogenesis of fungal membranes.Knocking out of MoHXT2 can upregulated the expression of MoHXT3,however,defected in MoHXT3 does not affect the expression of MoHXT2,indicating that MoHXT2 may be located upstream of MoHXT3.