Investigations on VELVET regulatory mutants confirm the role of host tissue acidification and secretion of proteins in the pathogenesis of Botrytis cinerea

Abstract

The Botrytis cinerea VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their molecular basis. Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed. VELVET mutants showed reduced release of citric acid, the major acid secreted by the wild-type, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation. The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion.

Comparative proteomics of osmotic signal transduction mutants inBotrytis cinereaexplain loss of pathogenicity phenotypes and highlight interaction with cAMP and Ca2+signalling pathways

“…These results highlight the strong impact of protein kinase inactivation on protein production and turnover even in the absence of external stress. This is in agreement with other proteomic studies of fungal ST mutants (Zhang, H et al, 2014;Isasa et al, 2015;Liu et al, 2018;Müller et al, 2018) and, in the case of ∆sak1, with the transcriptional results obtained by Heller and colleagues .…”

Botrytis cinerea differentially induces postharvest antioxidant responses in ‘Braeburn’ and ‘Golden Delicious’ apple fruit

“…B. cinerea like other postharvest pathogens modulate the host pH environment during infection, and low pH is conducive to gray mold development . Botrytis cinerea secretes a number of organic acids, of which citric acid appears to play an important role in its interaction with host plant tissue . Oxalic acid, another organic acid that is implicated in virulence in Sclerotinia sclerotiorum , a close relative to B. cinerea , does not appear to be involved in the gray mold‐apple interaction …”

“…42,43 Botrytis cinerea secretes a number of organic acids, of which citric acid appears to play an important role in its interaction with host plant tissue. 44 Oxalic acid, another organic acid that is implicated in virulence in Sclerotinia sclerotiorum, a close relative to B. cinerea, does not appear to be involved in the gray mold-apple interaction. 43,44 Based on the current view of the strategy of B. cinerea to deal with ROS during pathogenesis, it is reasonable to suggest that the observed increase in antioxidant enzyme activity (SOD and APX) in the susceptible apple tissues were of apple origin.…”

“…44 Oxalic acid, another organic acid that is implicated in virulence in Sclerotinia sclerotiorum, a close relative to B. cinerea, does not appear to be involved in the gray mold-apple interaction. 43,44 Based on the current view of the strategy of B. cinerea to deal with ROS during pathogenesis, it is reasonable to suggest that the observed increase in antioxidant enzyme activity (SOD and APX) in the susceptible apple tissues were of apple origin. Particularly at the last time point, both host and pathogen would have attempted to maintain cellular redox homeostasis and they would therefore have tried to activate effective antioxidant systems.…”

Hetero‐oligomeric CPN60 resembles highly symmetric group‐I chaperonin structure revealed by Cryo‐EM

“…In addition, the QconCAT protein covering all Chlamydomonas CPN60 subunits reported previously by Bai et al (2015) were allowed to migrate into an SDS-polyacrylamide gel. The proteins were stained with colloidal Coomassie G (Neuhoff et al, 1988) and bands containing CPN60 subunits and the QconCAT protein were cut out, followed by tryptic in-gel digestion, as described previously (Muller et al, 2018). Three biological replicates were analyzed with two technical replicates by microliquid chromatography tandem mass spectrometry (lLC-MS/MS) using a TripleTOF 6600 instrument coupled to an Eksigent 425 HPLC system (SCIEX, https://sciex.com) as described previously (Muller et al, 2018), with the following modifications: the HPLC gradient (buffer A -2% acetonitrile, 0.1% formic acid; buffer B -90% acetonitrile, 0.1% formic acid; flow rate of 4 ll min À1 ) ramped from 99% buffer A, 1% buffer B to 33% buffer B within 19 min, then to 50% buffer B within 3 min, followed by washing and equilibration steps.…”