Regulation Of STK1 Gene On Appressorium Development And Osmotic Stress And Construction Of Celluar-localized Vector Of Setosphaeria Turcica

Northern Leaf Blight of Corn, one of the most important fungal diseases of corn in the world, can cause enormous losses in the blast area. The previous researches indicated STK1(Setosphaeria turcica mitogen activated protein kinase 1) gene in Setosphaeria turcica regulated the fungal condiation, HT-toxin activity and pathogenicity, but the function of the gene on the appressorium development, osmotic stress regulation and the intracellular location of STK1 protein in S. turcica remained unclear.The wild-type isolate 01-23 and its STK1 gene-knocked mutant STM-35 were tested to induce the appressorium on glass plate, onion epidermis and corn leaf surfaces. The results revealed that STK1 gene had no regulation on the appressorium defferentiation, but it played an important role in appressorium shape and appressorium development by regulating the synthesis of glycerol and lipid. The results theoretically explained STK1 gene controlled the pathogenicity by affecting appressorium development. In order to study the location of STK1 in the fungal cells, we had constructed an expression vector ready for Agrobacterium tumefaciens-mediated transformation and fluorescence detecton. The vector had been confirmed by the PCR, double enzyme digestion, sequencing and gene expression analysis in yeast tansformants.Both 01-23 and STM-35 were able to produce appressorium on glass plate, epidermis and maize leaves. The wild-typle 01-23 produced only two kinds of appressoria, single appressorium and multiplicative appressorium. But STM-35 mutant could produce another four typies of appressoria: single-branch ramus, two-branch ramus, multiple-branch ramus, and O-like ramus. It proved that STK1 gene had relation with the shape change of fungal appressorium.The oil red O lipid stainning method and K2I/K2 glycerol staining one were developed in dyeing the lipid and glycerol in conidia, hyphal and appressoriom of S. turcica. The hyphal color of isolate 01-23 was darker than that of STM-35, suggesting that STK1 gene regulated anabolism of hyphal lipid and glycerol. The distribution of lipid and glycogen in development of appressorium was also studied by red oil O staining and KI/I2 staining. The lipid and glycerol were first abundant in the conidia of isolate 01-23 and then translocated into the appressorium. Large lipid deposits and glycolgen were observed within appressoria before lipid and glycogen degradation. The lipid and glycerol accumulation was not observed during appressorium development of STM-35. The above results had shown that STK1 gene regulated the anabolism and translocation of lipid and glycerol during appressorium development, and then affected the fungal pathogenicity.Both isolate 01-23 and STM-35 were growth on solid SPA medium with varying concentration of sorbitol to compare the differences between them in colony growth speed, colony morphism, and mycelia morphology. Under the tested sorbitol concentration of 1.0～2.0 mol/L, the colony growth speed of isolate 01-23 decreaced significantly with the increasing of sorbitol concentration, but 1.0 mol/L sorbitol could activate the growth of STM-35, and the effect was weaken as the increase of sorbitol concentration. Treated with 2.0 mol/L sorbitol, the activation effect disappeared. The increase of sorbitol supplement affected the mycelia modality of isolate 01-23 and mutant STM-35. The myceliun color of isolate 01-23 became light, and lots of grains and interspaces appeared in its hypha because of shrinkage of bioplasms. The mycelium color of STM-35 was getting light gradually, and lots of grains also appeared in hypha but interspaces were not found. The above results supported that STK1 gene regulated the mycelia growth and morphology under hyperosmotic stress.Two expression vectors pCAMBIA 1300-PGN and pCAMBIA 1300-PSGN had been constructed by recombinant PCR. Both plasmids were identified by PCR, enzyme digestion and base sequencing. The pasmids were transformated into yeast GS115 with Agrobacterium tumefaciens-mediated transformation (ATMT) system. The expressed products were obviously detected by fluorescence microscope analysis. The results proved that the plasmids could be used in complementation of the mutant and STK1 protein intracellular localization.