Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

• Published in: Current genetics Vol. 47; no. 3; pp. 194 - 201
• Main Authors: Stephens, C, Harrison, S.J, Kazan, K, Smith, F.W.N, Goulter, K.C, Maclean, D.J, Manners, J.M
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01-03-2005
• Subjects: Antifungal Agents - pharmacology; antifungal properties; antimicrobial peptides; cDNA libraries; complementary DNA; cytochrome-c oxidase; DNA, Complementary; Drug Resistance, Fungal - genetics; electron transport chain; Electron Transport Complex IV - genetics; Gene Library; gene overexpression; Genes; Genes, Fungal; genetically engineered microorganisms; H-transporting ATPase; Macadamia integrifolia; mutants; Peptides; phosphoprotein phosphatase; Plant Proteins - pharmacology; Proton-Translocating ATPases - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; screening; Signal Transduction - drug effects; Signal Transduction - genetics; vacuoles; Yeast; Yeasts
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/s00294-005-0562-8

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H(+)-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.