title: Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal.
abstract: A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan 66506, USA. aanand@noble.org
authors.forename: Ajith
authors.initials: A
authors.lastname: Anand
authors.forename: Zhentian
authors.initials: Z
authors.lastname: Lei
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Sumner
authors.forename: Kirankumar S
authors.initials: KS
authors.lastname: Mysore
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Arakane
authors.forename: William W
authors.initials: WW
authors.lastname: Bockus
authors.forename: Subbaratnam
authors.initials: S
authors.lastname: Muthukrishnan
mesh_terms: D005658:Fungi / Q000187:drug effects*; D018506:Gene Expression Regulation, Plant; D025301:Hyphae / Q000187:drug effects; D010641:Phenotype; D010935:Plant Diseases; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D030821:Plants, Genetically Modified; D013172:Spores, Fungal / Q000187:drug effects; D014908:Triticum / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins