Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, Vol. 99, No. 6 (Supplement), 2009 S111 sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. Xa produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in Xa, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. We used Tn5 (transposome) mutagenesis in an attempt to identify additional Xa pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 780 independently derived Tn5 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 biosynthetic gene cluster mutations, four new Tn5 mutants were identified that produced no or very few leaf symptoms. These mutants produced albicidin in vitro but did not efficiently colonize sugarcane stalks. The transposon insertion site of all four mutants was found to be located in Orf XALc_0557 of the Xa genome. This gene is predicted to encode an OmpA family outer membrane protein, a previously unrecognized and apparently essential pathogenicity factor in Xa.