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

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

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...

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Bibliographic Details
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
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Summary: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.
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ISSN:0172-8083
1432-0983
DOI:10.1007/s00294-005-0562-8