Function And Mechanism Of TgSWO From T.Guizhouense NJAU 4742

Trichoderma spp.,a plant growth promoting fungi(PGPF),is widely found in agricultural ecosystems.It can invade and colonize in plant roots,and greatly promote the growth and development of plant roots.In the past decades,most studies on the growth promoting mechanism of Trichoderma spp.were focused on the small molecular substances,e.g.,IAA,secondary products and organic acids which are produced by Trichoderma spp.,while the direct effect of Trichoderma spp.on plant roots were overlooked.In this study,it was indicated that TgS WO secreted by T.guizhouense NJAU 4742 could effectively promote the growth and development of the cucumner root indirectly.The facilitated effect of TgS WO protein on cucumber root growth was explored by knockout and overexpression of Tgswo gene and heterologous expression of TgSWO domain deletion protein.AFM,XPS,FT-IR were used to clarify the mechanism of TgSWO protein swelling on cellulose mediated by YoaJ domain.Finally,from proteomic perspective,we confirmed that Tgswo was an important factor affecting the extracellular cellulase system of T.a guizhouense NJAU 4742.The main results of this study are listed as follows:1)According to the pot experiments,when organic fertilizers with NJAU 4742 were added in cucumber cultivation,the fresh weight of root increased by 13.5%,and the height and fresh weight of shoot increased by 37.6%and 28.8%,respectively,incomparison with those without NJAU 4742.The effects of T.guizhouense NJAU 4742 on root growth and the response of transcription level of Tgswo gene were investigated by simulating root cellulose composition.The results show that the leaf area and root fresh weight of cucumber with NJAU 4742 increased by 86.6%and 40.3%,the transcription level of Tgswo gene increased by 15 folds,respectively,than those without with NJAU 4742.By comparing the ability of cellulose utilization and root colonization of T.guizhouense between Tgswo knockout mutant(KO1 and KO2)and overexpression mutant(OE1 and OE2),we confirm that Tgswo participated in the identification of NJAU 4742 with cucumber root,and can promote the growth of cucumber root.2)The main structural domain of TgSWO protein was predicted by NCBI database.The results show that TgSWO protein contained two cellulose binding domains which existed in CBD and YoaJ domains,respectively.Then we compare the root colonization ability of TgSWO and △CBD and △YoaJ proteins,and the results indicated that TgSWO and △CBD proteins can significantly promote the colonization of NJAU 4742 on cucumber root which highlight the importance of YoaJ domain to TgSWO protein.3)The effects of TgSWO(20μM),△CBD and △YoaJ protein on root morphogenesis and cell structure of cucumber were further observed,and the results confirm the vital roles of YoaJ domain in TgSWO mediated cell wall relaxation.Yoaj domain could dissolve the connection of cellulose microfibrils in cell wall or pectin combination by physicochemical actions,and induce swelling of the root cell walls.The enlargement of cells and intercellular space could promote the occurrence and number-increasing of lateral roots.Furthermore,it would promote the root crown to produce large amounts of mucilage,adsorbe more Trichoderma spores colonizing in the root,which promotes the growth of cucumber root indirectly.By far,a completely different root growth mechanism from IAA mediated lateral root development was put forward.In addition,the growth of cucumber was analyzed by combining TgSWO protein pretreatment with Tgswo knockout mutant.The results show that the fresh weight of cucumber root and seedling increased significantly by 30.1%and 18.8%,respectively,than those of the Tgswo treatment.4)The activity of TgSWO protein(0.2 mg/mL)and its domain deletion proteins(△CBD and △YoaJ protein)on cellulose substrates with different crystallinity and the adsorption results of amorphous cellulose and crystalline cellulose showed that the swelling effect of TgSWO protein and △CBD protein on cellulose is related to its crystallinity.By analyzing the synergistic effect of TgSWO and cellulase single enzyme,the effect of TgSWO protein on cellulose crystal zone was determined.The destructive ability of TgSWO and △CBD protein on crystalline cellulose crystal zone was directly characterized by AFM and XRD methods.In addition,XPS analysis of carbon and oxygen in crystalline cellulose reveals that TgSWO and △CBD protein destroyed the hydrogen bond of cellulose molecules.Finally,the OH stretching vibration mode of crystalline cellulose molecule was further analyzed by FT-IR spectrum.It was clarified that TgSWO protein and △CBD protein promoted the breaking of hydrogen bonds between cellulose molecules and water molecules and the association of new hydrogen bonds between water molecules and cellulose molecules.These results indicatthat TgSWO is mainly mediated by YoaJ domain.YoaJ domain could promote the cleavage of hydrogen bonds between cellulose molecules,which will contribute to the dissociation of cellulose chain structure and promote the transformation of crystalline cellulose into amorphous.Subsequently,the water molecules enter into the amorphous region of cellulose and result in the swelling of cellulose.5)The secretory components of NJAU 4742,Tgswo knockout and overexpression mutants were analyzed by proteomic analysis,and the result highlishts the important regulatory role of Tgswo in extracellular cellulase system of T.guizhouense NJAU 4742.In addition,proteomic analysis showed that the expression of cellulase in OE mutant is significantly up-regulated,especially for endocellulase.In conclusion,our study reveals that TgSWO could not only directly destroy the structure of crystalline cellulose,but also degrade lignocellulose and effectively depolymerize the complex network of lignocellulose by adjusting the composition of cellulase system of NJAU 4742.