Preliminary Studies On The Antimicrobial Mechanism Of Phenazine-l-carboxylic Acid (PCA) On Fungi By Screening Mutant Library Of Saccharomyces Cerevisiae

Biopesticides are used to control most plant diseases caused by pathogenic fungi. Shenqinmycin is a new type of green biopesticides, which is isolated from fluorescent pseudomonas M18. The key effective component of shenqinmycin is Phenazine-1-carboxylic acid (PCA). Shenqinmycin has the advantage of its broad-spectrum, high efficiency, low toxicity and good environmental compatibility. However, by now, the suppression mechanism of PC A on pathogenic fungi is still poorly understood.As a model organism, yeast has the similarity with other eukaryotes in basic physiological processes. We use yeast mutant library to explore the possible suppression mechanism of PC A on pathogenic fungi. Firstly, the most optimal PC A concentration (semi-lethal dose) was determined. Secondly, the yeast gene mutant library was screened for their sensitivity or resistance to PCA with the optimal PCA concentration. Thirdly, the reliability of yeast cells to PCA response was verified with repeated experiments. Finally, the screened mutants were grouped according to their functions and the possible suppression mechanism of PCA to yeast growth was proposed. PCA may hurt or kill yeast through the structure and synthesis of cell wall and cell membranes, redox system, cellular metabolic pathways, MAPK signal transduction pathway, protein sorting and transport vesicles, autophagy, cell polarity and morphogenesis.Based on the yeast library screen result, the mutation effects of a series of phospholipid synthesis genes on autophagy and vacuole morphology were investigated. The results show that:1) Mutations of genes encoding phospholipid synthesis enzymes had no effect on autophagy.2) Mutations in CDS1, but not other phospholipid synthesis genes resulted in fragmented vacuoles.3) Overexpression of CDS1 can restore fragmented vacuoles caused by CDS1 mutation.4) CDS1 mutant is more sensitive than wild-type to PCA treatment in growth. We hypothesized that fragmented vacuoles of yeast mutant may not affect the process of autophagy, but affect its response to extracellular environment, such as PCA treatment.This study further investigated the mutation effect of a series of V-ATPase related genes on autophagy and vacuole morphology, as some of them are also sensitive to PCA treatment. The results show that:1) VMA6, VMA9 and VPH2 mutation have an effect on autophagy. The fluorescent phenotype of GFP-Atg8 and RFP-Apel, the degradation of GFP-Atg8 and the maturation of prApel are all abnormal.2) VMA4, VMA13 or VPH1 mutation have no effect on autophagy.3) The autophagy situation in other mutants is unclear as RFP-Apel displays multiple dots and the maturation of prApel is blocked. However, the degradation of GFP-Atg8 is not blocked.4) The morphology of vacuole in VMA4, VMA13 and VPH1 mutants is normal. However, other mutants show defects in vacuolar morphology with fragmented vacuoles.5) Most mutants of V-ATPase are sensitive to PCA.In summary, We conclude that not only normal vacuolar morphology and function but also autophagy plays an important role in the resistance to PCA stress. The obtained information will provide useful reference for further study of yeast to PCA treatment in the future.