Xanthomonas oryzea pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, which is a significant threat to many of rice-growing regions. The type III secretion system (T3SS) is an essential virulence factor in Xoo. Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium. The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo. Thus, an efficient and reliable genetic tool system is required for detection of the T3SS proteins. In this study, we constructed a protein expression vector pH3-flag based on the backbone of pHM1, a most widely used vector in Xoo strains, especially a model strain PXO99^A. This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site (MCS), containing a modified pUC18 polylinker, and Flag as a C-terminal tag. The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression. We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo. This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Plant-pathogenic Xanthomonas infects a wide variety of host plants and causes many devastating diseases on crops.  Transcription activator-like effectors (TALEs) are delivered by a type III secretion system (T3SS) of Xanthomonas into plant nuclei to directly bind specific DNA sequences (TAL effector-binding elements, EBEs) on either strand of host target genes with an unique modular DNA-binding domain and to bidirectionally drive host gene transcription.  The target genes in plants consist of host susceptibility (S) genes promoting disease (ETS) and resistance (R) genes triggering defense (ETI).  Here we generally summarized the discovery of TALEs in Xanthomonas species, their functions in bacterial pathogenicity in plants and their target genes in different host plants, and then focused on the newly revealed modes of protein action in triggering or suppressing plant defense.

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. 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 hypersensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III 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.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant RΔimp was constructed in the wild-type RS105. The RΔimp mutant was significantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufficiently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deficiencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was significantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also significantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Xanthomonas oryzae pv. oryzicola (Xoc) causes a destructive bacterial leaf streak disease in rice. Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice. A mutant, Mxoc1679, screened from our previous Tn5-tagged mutant library for Xoc strain RS105, showed reduced virulence in rice. In this mutant, a gene named as Xoryp_08180 was disrupted by Tn5 insertion. Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp. Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice, a delayed hypersensitive response (HR) in non-host tobacco, and a decrease in extracellular protease activity. The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans. In addition, the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition. These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production, HR induction and full virulence of Xoc.

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