Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea

Abstract

Important for the lifestyle and survival of every organism is the ability to respond to changing environmental conditions. The necrotrophic plant pathogen Botrytis cinerea triggers an oxidative burst in the course of plant infection and therefore needs efficient signal transduction to cope with this stress. The factors involved in this process and their precise roles are still not well known. Here, we show that the transcription factor Bap1 and the response regulator (RR) B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B. cinerea; both have a major influence on the regulation of classical OSR genes. A yeast-one-hybrid (Y1H) approach proved direct binding to the promoters of gsh1 and grx1 by Bap1 and of glr1 by BcSkn7. While the function of Bap1 is restricted to the regulation of oxidative stress, analyses of ⌬bcskn7 mutants revealed functions beyond the OSR. Involvement of BcSkn7 in development and virulence could be demonstrated, indicated by reduced vegetative growth, impaired formation of reproductive structures, and reduced infection cushion-mediated penetration of the host by the mutants. Furthermore, ⌬bcskn7 mutants were highly sensitive to oxidative, osmotic, and cell wall stress. Analyses of ⌬bap1 bcskn7 double mutants indicated that loss of BcSkn7 uncovers an underlying phenotype of Bap1. In contrast to Saccharomyces cerevisiae, the ortholog of the glutathione peroxidase Gpx3p is not required for nuclear translocation of Bap1. The presented results contribute to the understanding of the OSR in B. cinerea and prove that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.

BcSUN1, a B. cinerea SUN-Family Protein, Is Involved in Virulence

“…Production of reactive oxygen species (ROS) by fungal strains was assayed according to Viefhues et al (2015) with minor modifications. Briefly, YGG-agar solid medium was overlaid with cellophane, inoculated with mycelium plugs, incubated for 4 days, and finally used to harvest 6 mg of fresh mycelium that was then placed at the bottom of a well in a microtiter (96 wells) plate.…”

“…Then 250 μl of a 3,3′-Diaminobenzidine (DAB) solution (0.5 mg/ml DAB in 100 mM citric acid, pH 3.7) were added to cover the mycelium, and the plate was incubated for 1.5 h in the dark and visually evaluated. Positive and negative controls were done as in Viefhues et al (2015). Production of ROS during infection was assayed in tobacco leaves inoculated with 5-μl droplets of a conidial suspension (5 × 10 5 conidia/ml in TGGK solution).…”

DHN melanin biosynthesis in the plant pathogenic fungus Botrytis cinerea is based on two developmentally regulated key enzyme (PKS)‐encoding genes

“…It is widely believed that the dark pigmentation of conidia and sclerotia is due to the accumulation of DHN melanin (Zeun and Buchenauer, 1985;Bell and Wheeler, 1986;Doss et al, 2003). Mutants producing increased amounts of a dark pigment and expression studies of some predicted melanogenic genes have been described (Schamber et al, 2010;Liu et al, 2011;2013;Yang et al, 2013;Viefhues et al, 2015); however, the involvement of the analyzed genes in melanogenesis was not shown. Recently, Zhang et al (2015) reported on two genes required for conidial melanogenesis.…”

Reactive Oxygen Species in the Botrytis – Host Interaction

“…Moreover, Bap1 and BcSkn7 share several target genes, including Bctrx1 , Bctrr1 , Bcgpx3 , and Bcgrx1 and thus a coordinated regulation of Bap1 and BcSkn7 is possible. Nevertheless, the role of BcSkn7 in pathogenicity is only minor (Yang et al 2014 ;Viefhues et al 2015 ). Besides the previously mentioned transcription factors, the Cys2His2 zinc fi nger protein Msn2/4 could play a role in ROS response in concert with Bap1 and Skn7.…”

“…Glutathione peroxidase for example can convert H 2 O 2 to H 2 O upon oxidation of glutathione. However, investigations of the glutathione peroxidase BcGpx3 showed neither involvement in pathogenicity nor in response to oxidative stress (Viefhues et al 2015 ). Because of the numerous ROS detoxifying enzymes, the effect of a single deletion can be masked in most cases.…”