One defense mechanism used by plant cells is the release of reactive oxygen species (ROS), produced in part by a NOX (NADPH oxidase) complex whose catalytic subunit buy Nutlin-3a shares sequence homology with mammalian NOX enzymes. The plant’s oxidative burst is thought to inhibit the progress of the invader. Furthermore, ROS provide a signal to promote programmed death of neighboring cells, a hallmark of the hypersensitive response (HR). The complete picture is more complex, because ROS also provide signals in addition to those for the HR (Torres & Dangl, 2005). Necrotrophic fungal pathogens that kill host tissue appear to thrive in an oxidant environment,
as shown for the gray mold pathogen Botrytis cinerea (Govrin & Levine, 2000). They produce their own ROS in addition to those originating from the host (see Heller & Tudzynski, 2011). To establish infection, the pathogen must be able to cope
with oxidative stress. Cochliobolus heterostrophus, a necrotrophic foliar pathogen of maize, counteracts oxidative stress by several pathways. The redox-sensitive Selleck Venetoclax transcription factor ChAP1 is responsible for induction of a set of genes with predicted functions in detoxifying ROS, for example glutathione reductase (GLR1) and thioredoxin (TRX2); loss-of-function mutants in ChAP1 are hypersensitive to oxidants (Lev et al., 2005). Loss of the stress-activated MAPK ChHog1, its upstream two-component system response regulator Ssk1, and the response regulator Skn7 also result in hypersensitivity to oxidants (Izumitsu et al., 2007; Igbaria et al., 2008; Oide et al., 2010). Although Δchap1 and Δskn7 mutants are sensitive to oxidants in culture, no difference
in virulence to maize was reported (Lev et al., 2005; Oide et al., 2010). If the pathways mediated by these two transcription factors act in an additive, rather than sequential manner, a double mutant would be expected to show a more severe phenotype than either single mutant. Two independent stress response pathways would, in this way, act together to provide tolerance to oxidants. To address this question, we generated double mutants in which the coding sequences of both ChAP1 and Skn7 were replaced by selectable antibiotic resistance markers and tested their virulence and tolerance to stresses. Bay 11-7085 Wild-type C4 (MAT1-2 Tox1+), Δchap1 and Δskn7 strains of C. heterostrophus were described previously (Turgeon et al., 1987; Lev et al., 2005; Oide et al., 2010). Standard growth medium was complete xylose medium (CMX, see Turgeon et al., 2010). The Δchap1-Δskn7 mutant was constructed starting with Δskn7. Linear DNA for double-crossover integration was amplified using the split-marker method (Catlett et al., 2003). A linear DNA construct was made, consisting of the neomycin selectable marker flanked on both sides with ChAP1 UTR`s, targeting the integration to the ChAP1 locus in the Δskn7 genome.