title: Eplt4 proteinaceous elicitor produced in Pichia pastoris has a protective effect against Cercosporidium sofinum infections of soybean leaves.
abstract: A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.
authors.affiliation: Department of Life Science and Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Song
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wu
authors.forename: Margaret
authors.initials: M
authors.lastname: Odeph
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Liu
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Howlett
authors.forename: Shuang
authors.initials: S
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.forename: Lin
authors.initials: L
authors.lastname: Yao
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Qian
authors.initials: Q
authors.lastname: Yang
mesh_terms: D015153:Blotting, Western; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D003904:Mitosporic Fungi / Q000187:drug effects* / Q000472:pathogenicity*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000382:microbiology*; D013025:Glycine max / Q000382:microbiology*
chemical_list: D005656:Fungal Proteins