Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice. Transposon-mediated mutational analysis of the pathogen with a quantitative assay revealed candidate virulence factors including genes involved in the pathogenesis of other phytopathogenic bacteria, virulence factors of animal pathogens, and genes not previously associated with virulence.Bacterial leaf streak is an important disease of rice (Oryza sativa) for which control measures are limited (22). In particular, no simply inherited gene for resistance to the disease has been reported. The disease is caused by Xanthomonas oryzae pv. oryzicola, a member of the gamma subdivision of the class Proteobacteria. The pathogen enters through leaf stomata or wounds and colonizes the parenchyma apoplast, causing interveinal lesions that appear water soaked initially and then develop into translucent, yellow-to-white streaks. Leaf streak is prevalent in Asia and parts of Africa, where it can decrease yield by as much as 30%. In the United States, the pathogen is quarantined and has been designated a select agent under the Agricultural Bioterrorism Act of 2002. To lay the groundwork for disease prevention and control strategies based on interference with bacterial virulence, transposon-mediated mutational analysis of X. oryzae pv. oryzicola was carried out to identify candidate virulence factors.Identification and characterization of reduced-virulence mutants. Strain BLS303 of X. oryzae pv. oryzicola (C. VeraCruz, International Rice Research Institute) was mutagenized by using the EZ::TN ϽR6K␥ori/KAN-2Ͼ Tn5 insertion kit (Epicentre Biotechnologies), which generates random, stable insertions. BLS303 cells were transformed by electroporation as described previously (30). Insertion mutants were selected on glucose yeast extract agar (18) containing 25 g/ml kanamycin and then cultured overnight in liquid glucose yeast extract with kanamycin. Cells were washed twice and resuspended in sterile water to an optical density at 600 nm of 0.5 and used to spot infiltrate, in duplicate, leaves of 4-week-old rice plants of Indica variety IRBB10 with a needleless syringe. Plants were grown and maintained in a PGC-105 growth chamber (1,000 mol/m 2 /s; Percival Scientific, Inc., Perry, IA) under a cycle of 12 h of light at 28°C and 12 h of dark at 25°C with relative humidity at 75 to 80%. Symptoms were observed after 4 days. Ten thousand mutants were screened. For 153 mutants, symptoms appeared reduced relative to the wild type or were absent. These were characterized further with a more stringent quantitative assay (Fig. 1). In this assay, leaves of 8-week-old rice plants were inoculated with a mutant on one side of the midrib and the wild type directly opposite on the other. For each mutant, five replicate, paired inoculations on each of two leaves were made. After 10 days, lesion lengths were measured for each paired inoculation, and a paired, two-tailed Student t test was performed across all replicates. By this test, 21 mutants were conf...