Immunity-inducing Proteins From Trichoderma Asperellum And Chaetomium Globosum And Their Functional Analysis

Biocontrol agents inhibit the pathogens by a variety of mechanisms, one ofwhich is to induce the defense responses of the host plants, and elicit their local orsystemic acquired resistance (SAR). Current researches mainly focus on theinteractions between biocontrol agents and pathogens or pathogens and plants.However, there is very few study focuses on the relation between biocontrol agentsand plant. In order to explore biocontrol related genes, cDNA libraries of themycelium from both Trichoderma asperellum T4and Chaetomium globosum W7were constructed in our laboratory. By sequencing and bioinformatics analysis ofthese two libraries, the gene which encodes the immunity-inducing protein in thesetwo organisms were found and designated as EplT4and EplW7respectively. Therelatively low(65.22%) protein sequence identity of these two genes’ productssuggests possible structural and functional differences between them that need to beinvestigated separately.In order to get plenty of proteins for functional studies of EplT4and EplW7,both genes were cloned and expressed in Pichia pastoris GS115. In order tofacilitate subsequent protein purification, the N terminal signal peptides of bothproteins were replaced by a signal peptide of the Saccharomyces cerevisiaealpha-factor. A6×His-tag was also added in the C terminus. P. pastoristransformants Ppa-EplT4and Ppa-EplW7were obtained by G418antibioticscreening. The transformants were further validated by PCR and Western blot.Response Surface Methodology was used to improve the yield of recombinantproteins. The yields of recombinant EplT4and recombinant EplW7were37.70mg/L and17.80mg/L respectively at the optimized conditions. A highest yield of55.30mg/L was obtained when Ppa-EplT4was induced in the fermenter. Therecombinant proteins were purified by Ni-NTA affinity chromatography and gelfiltration and EplT4reached20mg/L. Recombinant EplT4forms dimers and thedimer formation is concentration dependent.The monomer of purified EplT4was applied to soybean for inductionexperiments. The results showed that EplT4monomer can up-regulate the level ofthe following pathogen related proteins: beta-1-3-glucan enzyme, chitinase III-A,cysteine protease inhibitor and peroxidase. EplT4also induced the production ofH2O2and the accumulation of phenolic compounds in soybean leaves, which are theearly cellular events in plant defense responses. EplT4was able to protect soybeanleaves from the infection by Cercosporidium sofinum (Hara). These resultsindicated that EplT4monomer can enhance disease resistance of soybean to C. sofinum (Hara).To investigate the function of immunity-induceing proteins in fungi, Eplover-expression strain and gene disruption mutants of T. asperellum and C.globosum were obtained by Agrobacterium-mediated method. Epl genes have littleeffects on these strains’ morphology, physiology, and their ability to recognize andinvade pathogen hosts. EplT4can affect the hydrophobicity of T. asperellum T4hyphae but not their spores. EplW7had no effect on the hydrophobicity of either thehyphae or the spores of C. globosum. The deletion of Epl genes did not affect theircolonization on plant roots.The wild-type, Epl over-expression and gene disruption strains of T.asperellum T4was applied to soybean to induce its disease resistance. Both thewild-type and Epl over-expression strain were able to upregulate most of thepathogen related protein genes tested, and enhance the disease resistance ofsoybean to pathogenic fungi. The Epl gene disruption strain lost its elicitor activity.T. asperellum T4can induce both the local and systemic resistance of soybean. Theinduction tests of C. globosum W7showed similar results as the T. asperellum T4tests, with differences in Epl’s ability in inducing some of the pathogen relatedprotein genes. Deletion of EplW7gene made C. globosum partially lost its elicitoractivity, which suggests it may contain other factors that also contribute to itselicitor activity.Furthermore, yeast one-hybrid system was used to study the regulationmechanism of EplT4and EplW7. cDNA libraries of both T. asperellum T4and C.globosum W7were constructed, and the promoters of each Epl gene were insertedinto pHIS2plasmid. The pHIS2-promoter plasmid and cDNA library were thencotransformed into S. cerevisiae Y187strain along with the pGADT7-Rec2plasmid.Five regulatory factor candidates of EplT4were found. They are hypotheticalprotein I, Phosphatase PSR1, signal recognition particle receptor alpha subunit,hypothetical protein II and glucosamine-phosphate N-acetyltransferase. Fourregulatory factor candidates of EplW7were found. They are WD repeatdomain-containing protein, palmitoyltransferase-like protein and two hypotheticalproteins.