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.