A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp.citri

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citrisubsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper- sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type Ill secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp, citri.