Novel Candidate Virulence Factors in Rice Pathogen Xanthomonas oryzae pv. oryzicola as Revealed by Mutational Analysis

Abstract

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...

Ketoglutarate Transport Protein KgtP Is Secreted through the Type III Secretion System and Contributes to Virulence in Xanthomonas oryzae pv. oryzae

“…IR24, susceptible to BB, 2 months old) using leaf clipping for lesion length measurement or to rice seedlings (2 weeks old) for evaluating water-soaked symptoms. All plants were maintained in a greenhouse as described previously (58). Plant phenotypes were scored 24 h postinoculation for the HR in tobacco, 3 days postinoculation (dpi) for water-soaked symptoms, and 14 dpi for lesion length.…”

In vitro Antimicrobial Assay of Actinomycetes in Rice AgainstXanthomonas oryzae pv. oryzicola and as Potential Plant Growth Promoter

“…oryzicola (Xoc) is the causal pathogen for Bacterial Leaf Streak (BLS) disease, which is one of the important diseases in the tropical and sub-tropical rice cultivation areas of Asia (Xie and Mew 1998;Chen et al 2006). BLS is prevalent in Asia and certain regions of Africa, where the yield loss can reach up to 17%, depending on the rice variety and climatic conditions (Wang et al 2007). To-date, no reports are available on the efficacy of actinomycetes against Xoc in rice.…”

Requirement of the Lipopolysaccharide O-Chain Biosynthesis Gene wxocB for Type III Secretion and Virulence of Xanthomonas oryzae pv. Oryzicola

“…We demonstrated previously that T3S (hrp) mutants of X. oryzae pv. oryzicola BLS303 are nonvirulent (3,30). Further studies comparing plant defense responses to a T3S mutant and a T3S wxocB double mutant, for example, might distinguish whether interplay between defense elicitation by LPS and defense inhibition and possibly other actions by type III effectors determines wild-type virulence.…”

“…A library of transposon insertion mutants of BLS303 (3) was screened for nonmucoid colonies on GYE agar. Insertions were mapped as described previously (3). An isolate with a single forward-oriented insertion 584 bp from the start codon of the 1,455-bp gumD gene was selected and designated BLS303gumD…”

“…Previously, we reported the isolation of a transposon insertion mutant of X. oryzae pv. oryzicola strain BLS303, designated TN38C10, that shows markedly reduced virulence (3). TN38C10 carries a single transposon insertion in the wxocB gene and is here referred to as BLS303wxocB Ϫ .…”