Modulation of the Redox Climate and Phosphatase Signaling in a Necrotroph: an Axis for Inter- and Intra-cellular Communication that Regulates Development and Pathogenicity

The long-term goals of our research are to understand the regulation of sclerotial development and pathogenicity in S. sclerotiorum. The focus in this project is on the elucidation of the signaling events and environmental cues that contribute to broad pathogenic success of S. sclerotiorum. In this proposal, we have taken advantage of the recent conceptual (ROS/PPs signaling) and technical (genome sequence availability and gene inactivation possibilities) developments to address the following questions, as appear in our research goals stated below, specifically concerning the involvement of REDOX signaling and protein dephosphorylation in the regulation of hyphal/sclerotial development and pathogenicity of S. sclerotiorum. Our stated specific objectives were to progress our understanding of the following questions: (i) Which ROS species affect S. sclerotiorum development and pathogenicity? (ii) In what manner do PPs affect S. sclerotiorum development and pathogenicity? (iii) Are PPs affected by ROS production and does PP activity affect ROS production and SMK1? (iv) How does Sclerotinia modulate the redox environment in both host and pathogen? While addressing these questions, our main findings include the identification and characterization the NADPH oxidase (NOX) family in S. sclerotiorum. Silencing of Ssnox1 indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of Ssnox2 resulted in limited sclerotial development but remained fully pathogenic. Interestingly, we found a consistent correlation with Ssnox1(involved with pathogenicity) and oxalate levels. This same observation was also noted with Sssod1. Thus, fungal enzymes involved in oxidative stress tolerance,when inactivated, also exhibit reduced OA levels. We have also shown that protein phosphatases (specifically PP2A and PTP1) are involved in morphogenesis and pathogenesis of S. sclerotiorum, demonstrating the regulatory role of these key proteins in the mentioned processes. While probing the redox environment and host-pathogen interactions we determined that oxalic acid is an elicitor of plant programmed cell death during S. sclerotiorum disease development and that oxalic acid suppresses host defense via manipulation of the host redox environment. During the course of this project we also contributed to the progress of understanding S. sclerotiorum function and the manipulation of this fungus by establishing an efficient gene replacement and direct hyphal transformation protocols in S. sclerotiorum. Lastly, both PIs were involved in thegenomic analysis of this necrotrophic fungal pathogen (along with Botrytis cinerea). Our results have been published in 11 papers (including joint papers and refereed reviews) and have set the basis for a continuum towards a better understanding and eventual control of this important pathogen (with implications to other fungal-host systems as well).