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Signaling pathways coordinating the alkaline pH response confer resistance to the hevein-type plant antimicrobial peptide Pn-AMP1 in Saccharomyces cerevisiae

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Abstract

Main conclusion

Genome-wide screening of Saccharomyces cerevisiae revealed that signaling pathways related to the alkaline pH stress contribute to resistance to plant antimicrobial peptide, Pn-AMP1.

Plant antimicrobial peptides (AMPs) are considered to be promising candidates for controlling phytopathogens. Pn-AMP1 is a hevein-type plant AMP that shows potent and broad-spectrum antifungal activity. Genome-wide chemogenomic screening was performed using heterozygous and homozygous diploid deletion pools of Saccharomyces cerevisiae as a chemogenetic model system to identify genes whose deletion conferred enhanced sensitivity to Pn-AMP1. This assay identified 44 deletion strains with fitness defects in the presence of Pn-AMP1. Strong fitness defects were observed in strains with deletions of genes encoding components of several pathways and complex known to participate in the adaptive response to alkaline pH stress, including the cell wall integrity (CWI), calcineurin/Crz1, Rim101, SNF1 pathways and endosomal sorting complex required for transport (ESCRT complex). Gene ontology (GO) enrichment analysis of these genes revealed that the most highly overrepresented GO term was “cellular response to alkaline pH”. We found that 32 of the 44 deletion strains tested (72 %) showed significant growth defects compared with their wild type at alkaline pH. Furthermore, 9 deletion strains (20 %) exhibited enhanced sensitivity to Pn-AMP1 at ambient pH compared to acidic pH. Although several hundred plant AMPs have been reported, their modes of action remain largely uncharacterized. This study demonstrates that the signaling pathways that coordinate the adaptive response to alkaline pH also confer resistance to a hevein-type plant AMP in S. cerevisiae. Our findings have broad implications for the design of novel and potent antifungal agents.

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Abbreviations

AMP:

Antimicrobial peptide

GO:

Gene ontology

CWI:

Cell wall integrity

ESCRT:

Endosomal sorting complex required for transport

MIC:

Minimum inhibitory concentration

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Acknowledgments

This project was supported by a grant from the National Institute of Agricultural Sciences (PJ01086903), RDA, Korea.

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Authors and Affiliations

  1. Division of Applied Life Science and IALS, Gyeongsang National University, Jinju, 660-701, Republic of Korea

    Youngho Kwon & Youn-Sig Kwak

  2. University of British Columbia, Pharmaceutical Sciences, Vancouver, BC, Canada

    Jennifer Chiang, Grant Tran, Guri Giaever & Corey Nislow

  3. National Academy of Agricultural Sciences, Rural Development Administration, Jeonju, 560-500, Republic of Korea

    Bum-Soo Hahn

  4. Division of Science Education and Institute of Science Education, Chonbuk National University, Jeonju, 761-756, Republic of Korea

    Ja-Choon Koo

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  1. Youngho Kwon
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425_2016_2579_MOESM1_ESM.jpg

Fig. S1 No significant changes in Pn-AMP1 sensitivity or resistance in mutant strains carrying deletion of single or multiple potassium transporters. Exponentially growing cells including wild type (WT), Δtrk1, Δtok1, Δena1, and Δnha1 were diluted and grown in 0.5X YPD broth buffered to pH 5.8, 7.5 and 8.3 in the presence or absence of Pn-AMP1. The Δslt2 was included as a control strain sensitive to Pn-AMP1. After 24 h of incubation at 30 °C, growth curves were obtained by measuring the optical density. a The MIC50 values of Pn-AMP1 were calculated from the dose–response growth curves. Error bars represent the standard error of the mean of three replicates. b Representative microplate images of growth inhibition assay. The turbid and clear wells indicate fungal growth and lack thereof, respectively (JPEG 346 kb)

425_2016_2579_MOESM2_ESM.jpg

Fig. S2 Effect of alkaline pH on Pn-AMP1 sensitivity of phytopathogenic fungi. Phytopathogenic fungi including Botritis cinerea, Fusarium fujikuroi, Penicillium digitatum and Rhizoctonia solani were obtained from the Korean Agricultural Culture Collection (KACC, South Korea) and used to isolated fungal spores as described previously (Koo et al. 1998). Potato dextrose broth (PDB) medium was buffered with Mes and Tris to a final concentration of 50 mM as described in Materials and methods. An inoculum of 104 fungal spores was diluted in 100 μl of 0.5X PDB medium at pH 5.8, 7.5 or 8.3, supplemented with various concentrations of Pn-AMP1 or water, in a 96-well microplate. Growth was measured at 600 nm after incubation for 2 days at 26 °C. The MIC50 of Pn-AMP1 was determined from the growth response curve. a The MIC50 values of Pn-AMP1 were calculated from the dose–response growth curves. Error bars represent the standard error of the mean of three replicates. b Representative microplate images of the growth inhibition assay. The turbid and clear wells indicate fungal growth and lack thereof, respectively (JPEG 757 kb)

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Kwon, Y., Chiang, J., Tran, G. et al. Signaling pathways coordinating the alkaline pH response confer resistance to the hevein-type plant antimicrobial peptide Pn-AMP1 in Saccharomyces cerevisiae . Planta 244, 1229–1240 (2016). https://doi.org/10.1007/s00425-016-2579-2

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