| Most plant diseases are caused by pathogenic fungi. Currently, chemical pesticides are widely used to control plant diseases although biopesticides are occasionally used. However, as time passes, the drawbacks of using chemical pesticides gradually exposed, such as pesticide residues, difficult to degrade and harmful to humans and animals. Biopesticides, because of their deriving from nature, lower human and animal toxicity and strong bactericidal specificity, catchs more and more public attention. Phenazine-1-carboxylic acid (PCA) isolated from microorganisms is the main ingredient of a new type of green biopesticides, shenqinmycin, which is catching more and more attention because of its broad-spectrum, high efficiency and low toxicity. However, by now, the pathogen suppression mechanism of PCA on fungi is still poorly understood.In this study, budding yeast Saccharomyces cerevisiae is used to determine the possible fungal suppression mechanism of PCA by testing the response of multiple physiological processes after PCA treatment, and pathogenic yeast Candida albicans is used in some parts to confirm some of the conclusions.The results show that, high concentration of PCA (80μg/ml) could completely inhibit the growth of Saccharomyces cerevisiae and Candida albicans, if not kill them. We further examined the expression levels of various genes through microarray with semi-lethal dose of PCA (40μg/ml) for 2 hours treatment in Saccharomyces cerevisiae cells. We found that PCA affected a variety of physiological processes in Saccharomyces cerevisiae cells. According to the microarray results and research focus in this lab, we examined the effects of PCA on vesicle transport and autophagy. By examining the intracellular localization and degradation of some key proteins in these two pathways by fluorescence observation and immunoblotting under certain amount of PCA treatment, we found the following main results:1) PCA affected the localization and recycling of vesicle transport v-SNARE protein Sncl. GFP-Sncl formed a circle and multiple dot structures with the cirle of GFP-Sncl localizing to the endoplasmic reticulum (ER) and multiple dots in the ER and cytoplasm under PCA treatment; 2) PCA affected the localization of small GTPase Ypt6 which regulates vesicle trafficking. Ypt6 was mislocalized to the ER under PCA treatment; 3) endoplasmic reticulum locating protein Pmp2-LRKR also formed multiple dot structures under PCA treatment; 4) PCA affected localization and transport of autophagy protein Atg8, Atg9 and Apel and the degradation of GFP-Atg8 and maturation of Apel; 5) the anti-fungi antibiotic Hygromycin B inhibited growth of yeast cells but had no effect on Atg8 localization; 6) PCA treatment also affected the localization of GFP and CFP proteins, which formed multiple dot structures; 7) PCA did not influence the nuclear localization of Htbl-GFP or Htbl-CFP when they exist alone in the yeast cells. These findings indicate that PCA works on proteins existed outside the nucleus, either in the cytoplasm or the ER. We suggest that PCA may affect intracellular protein synthesis and transport, thus affecting the normal physiological activity and even death of Saccharomyces cerevisiae cells.This study preliminarily investigated the mechanism of PCA on suppresson of yeast. PCA treatment significantly affect vesicle trafficking and autophagy in yeast. The obtained information will provide useful reference for further study of the antifungal mechanism for PCA in the future. |
