The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of Xoo. Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, hpa1, and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity (hrp) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

We previously identified a plant-derived phenolic compound ortho-coumaric acid (OCA) as an inhibitor of type III secretion system (T3SS) of Xanthomonas oryzae pv. oryzae (Xoo), the pathogen causing bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. However, the molecular mechanisms by which OCA suppresses T3SS and the transcriptional responses to the OCA treatments in Xoo remains unclear. The present study conducted the RNA-seq-based transcriptomic analysis to reveal changes in gene expression in Xoo in response to 30 min, 1 h, 3 h, and 6 h of OCA treatment. Results showed that OCA significantly inhibited the expression of T3SS genes after 30 min, and the inhibition also existed after 1 h, 3 h, and 6 h. After treatment for 30 min, membrane proteins in the functional category of cellular process was the predominant group affected, indicating that Xoo was in the early stress stage. Over time, more differentially-expressed genes (DEGs) gathered in the functional category of biological process. Analysis of common DEGs at all four of time points revealed the core elements of Xoo during the response to OCA treatment. Notable, a multidrug transporter cluster that consisted of a MarR-family protein (PXO_RS13760), a multidrug RND transporter (PXO_RS13755), a multidrug transporter (PXO_RS13750), and an MFS transporter (PXO_RS13745) were significantly up-regulated at all four of the time points. Although these three transporter genes were not upregulated by OCA in the PXO_RS13760 deletion mutant, the deficiency of PXO_RS13760 in Xoo did not affect T3SS transcript, and OCA still had the ability to inhibit the expression of T3SS in the mutant, suggesting that the MarR-family protein was involved in bacterial responses to OCA, but not direct OCA inhibition of T3SS in Xoo. We analyzed the transcriptome of Xoo during OCA treatment at both early and late stages, which revealed the landscape of Xoo responses to OCA at the whole-genome transcription level. A multidrug transporter cluster was identified to be involved in the response process, but had no direct relation to T3SS in Xoo.

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D⁴⁰³ residue of the I site in GGDEF motif and E⁴¹¹ residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99^A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E⁴¹¹ residue but not D⁴⁰³ residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D⁴⁰³ or E⁴¹¹ did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.