RBOHD-dependent apoplastic oxidative burst plays a protective role against the necrotrophic fungus Botrytis cinerea

The necrotrophic fungus Botrytis cinerea rapidly kills host tissue with its secreted proteins and metabolites. Necrotrophs can trigger the hypersensitive response (HR), which is accompanied by reactive oxygen species (ROS) production, but it remains unclear whether this ROS burst promotes or restricts B. cinerea infection. We have combined Arabidopsis RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) mutant analysis, luminol-based in vivo assays, and the use of CRISPR-Cas generated B. cinerea ROS detoxification mutants to analyze the toxic effect of the ROS burst. In addition, RNA-sequencing, expression analyses and redox imaging were used to study RBOHD-dependent signaling and redox homeostasis. RBOHD mutants showed increased susceptibility to B. cinerea infection, and the apoplastic ROS burst was dependent on RBOHD. Fungal mutants confirmed the importance of SUPEROXIDE DISMUTASE 1 (SOD1) and ACTIVATOR PROTEIN 1 (AP1) for ROS detoxification and virulence. Unexpectedly, the transcriptomes of B. cinerea-infected Arabidopsis Col-0 and RBOHD mutant were highly similar, providing no evidence for a connection of extracellular ROS production and intracellular ROS-induced transcriptional signaling. Nevertheless, RBOHD activity was found to be involved in the maintenance of cellular redox homeostasis under phytotoxic stress. Our results demonstrate an exclusive and protective role of the RBOHD-dependent oxidative burst, but B. cinerea counteracts this response via different detoxification mechanisms. Overall design: Arabidopsis Col-0 and RBOHD S39A/S339A/S343A were infected with B. cinerea and mock (semisolid Gamborg medium) and incubated in a humid chamber for 48 hours. After this time, samples from at least 5 different plants were harvested and the RNA extracted. 3 different biological replicates were performed, each one consisting in at least 5 plants per genotype and 3 leaves infected per plant.