Genome-wide Analysis Of A Transposon Inserted Library Of A Rice Model Pathogenic Bacterium Xanthomonas Oryzae Pv. Oryzicola

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is becoming an important disease in rice. Construction of a mutant library is a fundamental approach to new genes and functional genomics of a plant pathogen. To genome-widely mine pathogenesis-related genes of the pathogen, a Tn5 transposon-mediated mutation library of the pathogen was produced. Twenty five thousand transformants were generated, appropriately corresponding to 5X ORF coverage of the genome. Stability, randomicy and saturation assays for this Tn5-mediated mutant library indicated that the library is of high quality.Every mutant of this library was inoculated into rice and tobacco individually and respectively. In planta pathogenicity assay revealed that 253 mutants reduced virulence in rice, but triggered hypersepsitive response (HR) in tobacco,29 mutants reduced virulence in rice, but did not elicited HR in tobacco,20 mutants did not display pathogenicity in rice and HR in tobacco, and that 7 mutants were non-pathogenic in rice, but still trigger HR in tobacco. In addition, we identified 21 white color mutants lost pigment production when grown on NA medium,12 extracellular polysaccharide deficient mutants,11 growth enhanced mutants and 12 extracellular protease deficient mutants. The above suggests that the library would facilitate identification of pathogenicity-related genes as well as functional genomics in Xooc-rice pathosystem.To rapidly and accurately identify a Tn5 insertion position in a corresponding gene in each mutant of this library of X. oryzae pv. oryzicola, thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue were adopted. The results suggest that TAIL-PCR and Tn5 transposon rescue are effective tools for isolating flanking frangments near Tn5 insertion position of a target gene and can be used coordinatively and cooperatively.To determine what genes were insterted by Tn5 transposon in 12 extrocellular protease deficient mutants, we identified Tn5 inserted positions in each of these mutants by using TAIL-PCR and Tn5 transposon rescue and found that any mutation in components of type â…¡secretion machine, cAMP regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme, or extracellular protease completely or partially led to the loss of extracellular protease activities and deduced-virulence in rice. Ectopic expression of ecpA in Escherichia coli demonstrated its extracellular protease activity. Reverse transcriptional polymerase chain reaction (RT-PCR) showed that the expression of the ecpA gene was induced in planta in all the mutants except the mutant AecpA. Genetic complementation of the mutant with a functional ecpA gene restored not only extracellular protease activity, but virulence and in planta growth. Intriguingly, the extrogenous expression of the ecpA gene did not conferred the extracellular protease activity to the vascular bacterium X. oryzae pv. oryzae, the causual agent of bacterial blight in rice. Based on our results, we hypothese that the ecpA gene product is a tissue-specific effector for X. o. pv. oryzicola pathogenesis in rice while it is a pseudogene in X. o. pv. oryzae.