A Genome-Wide Mutant Screen Identifies XopN and XopX as Core Type III Effectors Required for Peach Infection by Xanthomonas arboricola pv. pruni

Xanthomonas arboricola pv. pruni causes bacterial spot in peaches, a major disease affecting global Prunus production. Despite its economic significance, the virulence mechanisms that enable X. arboricola pv. pruni to colonize peach tissues and induce characteristic necrotic symptoms remain poorly understood. To identify key virulence determinants, a robust and reliable detached-leaf inoculation system was developed, and a genome-wide forward genetic screen of 2400 Tn5 mutants was conducted. A total of 34 mutants with consistently reduced virulence were identified, representing diverse functional categories including secretion systems, nutrient acquisition, primary metabolism, and regulatory pathways. The most prominent findings were the repeated identification of independent mutants in two type III effector genes, xopN and xopX, highlighting these effectors as central and nonredundant contributors to symptom induction. Mutants in the type III secretion system translocon-associated gene hrpF also showed virulence defects. Additional mutants affecting phosphate uptake (pstS), ammonium transport, and vitamin B6 biosynthesis (pdxA, serC) revealed metabolic requirements essential for in planta fitness. Notably, several mutants reached bacterial population levels comparable to the wild-type isolate but produced little or no symptoms, indicating that bacterial multiplication and symptom development are not necessarily linked. This study provides the first comprehensive genome-wide functional screen of X. arboricola pv. pruni virulence and establishes a framework for dissecting infection mechanisms. The essential factors identified here, particularly XopN, XopX, and key metabolic pathways, represent promising targets for future anti-virulence strategies to manage bacterial spot disease. Characterizing the specific functions of each virulence factor identified in this study will be an important focus of future work.