Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In addition to the previously identified albicidin biosynthesis gene ciuster mutations, 33 new loci were identified in which insertions were correlated with reduced pathogenicity. These insertions affected genes predicted to encode proteins involved in a variety of functions, inciuding exopolysaccharide and lipopolysaccharide biosynthesis, fatty acid biosynthesis, regulatory and cell signaling, and secretion systems. Several of these have been associated with virulence in other bacterial plant pathogens that invade the xylem. However, sorne loci were identified that are predicted to encode previously unrecognized and apparently essential pathogenicity factors, at least for sugarcane leaf scald, inciuding an OmpA family outer membrane protein. Five independent TnS insertions in OmpA locus XALc_0557 of X albilineans strain XaFL07-1 produced no or very few leaf symptoms. These mutants produced albicidin in vitro and were able to multiply in sugarcane leaf tissue to levels similar to the wild-type