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Apoplastic Extracts from a Transgenic Wheat Line Exhibiting Lesion-Mimic Phenotype Have Multiple Pathogenesis-Related Proteins That Are Antifungal

December 2004 , Volume 17 , Number  12
Pages  1,306 - 1,317

Ajith Anand , 1 , 2 Zhentian Lei , 2 Lloyd W. Sumner , 2 Kirankumar S. Mysore , 2 Yasuyuki Arakane , 1 William W. Bockus , 3 and Subbaratnam Muthukrishnan 1

1Department of Biochemistry, Kansas State University, Manhattan 66506, U.S.A.; 2Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, U.S.A.; 3Department of Plant Pathology, Kansas State University, Manhattan 66506, U.S.A.

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Accepted 9 August 2004.

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic β-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 β-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 β-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.

Additional keywords: antifungal assay, antifungal proteins, programmed cell death.

© 2004 The American Phytopathological Society