Complementary roles of EPS, T3SS and Expansin for virulence of , the causative agent of cucurbit wilt.

(Smith) is a recently emerged plant pathogen that causes severe economic losses in cucurbit crops in temperate Eastern North America. is xylem restricted, and virulence is thought to be related to Exopolysaccharides (EPS) and biofilm formation, which occlude the passage of sap in xylem vessels and causes systemic wilt. However, the role of EPS and biofilm formation, and their contribution to disease in relation to other virulence loci are unknown. Here, we use deletion mutants to explore the roles of EPS, Hrp Type III secretion system (Hrp T3SS) and Expansin in plant colonization and virulence. Then, we quantify the expression of the genes encoding these factors during infection. Our results show that Exopolysaccharides are essential for survival in host plants, while Hrp T3SS and Expansin are dispensable for survival but needed for systemic wilt symptom development. EPS and Hrp T3SS display contrasting expression patterns in the plant, reflecting their relevance in different stages of the infection. Finally, we show that expression of the and T3SS operons is downregulated in mildly increased temperatures, suggesting a link between expression of these virulence factors and geographic restriction of to temperate regions. Our work highlights how virulence is a complex trait where several loci are coordinated during infection. These results further shed light into the relationship between virulence factors and the ecology of this pathosystem, which will be essential for developing sustainable management strategies for this emerging pathogen.