A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Enterohemorrhagic Escherichia coli O157:H7 antigens produced in transgenic lettuce effective as an oral vaccine in mice.

KEY MESSAGE: Transgene with recombination sites to address biosafety concerns engineered into lettuce to produce EspB and γ-intimin C280 for oral vaccination against EHEC O157:H7. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen where ruminant farm animals, mainly bovine, serve as reservoirs. Bovine vaccination has been used to prevent disease outbreaks, and the current method relies on vaccines subcutaneously injected three times per year. Since EHEC O157:H7 colonizes mucosal surfaces, an oral vaccine that produces an IgA response could be more convenient. Here, we report on oral vaccination against EHEC O157:H7 in mice orally gavaged with transgenic lettuce that produces EHEC O157:H7 antigens EspB and γ-intimin C280. Younger leaves accumulated a higher concentration of antigens; and in unexpanded leaves of 30-day-old T2 plants, EspB and γ-intimin C280 were up to 32 and 51 µg/g fresh weight, respectively. Mice orally gavaged with lettuce powders containing < 3 µg antigens for 6 days showed a mucosal immune response with reduced colonization of EHEC O157:H7. This suggests that the transgenic lettuce has potential to be used for bovine vaccination. To promote the biosafety of crop plants producing medically relevant proteins, recombination sites were built into our transgenic lines that would permit optional marker removal by Cre-lox recombination, as well as transgene deletion in pollen by CinH-RS2 recombination. The ability to upgrade the transgenic lettuce by stacking additional antigen genes or replacing older genes with newer versions would also be possible through the combined use of Bxb-att and Cre-lox recombination systems.

The Methyltransferase HemK Regulates the Virulence and Nutrient Utilization of the Phytopathogenic Bacterium Xanthomonas citri Subsp. citri.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), seriously affects fruit quality and yield, leading to significant economic losses around the world. Understanding the mechanism of Xcc virulence is important for the effective control of Xcc infection. In this report, we investigate the role of a protein named HemK in the regulation of the virulence traits of Xcc. The hemK gene was deleted in the Xcc jx-6 background, and the ΔhemK mutant phenotypically displayed significantly decreased motility, biofilm formation, extracellular enzymes, and polysaccharides production, as well as increased sensitivity to oxidative stress and high temperatures. In accordance with the role of HemK in the regulation of a variety of virulence-associated phenotypes, the deletion of hemK resulted in reduced virulence on citrus plants as well as a compromised hypersensitive response on a non-host plant, Nicotiana benthamiana. These results indicated that HemK is required for the virulence of Xcc. To characterize the regulatory effect of hemK deletion on gene expression, RNA sequencing analysis was conducted using the wild-type Xcc jx-6 strain and its isogenic ΔhemK mutant strain, grown in XVM2 medium. Comparative transcriptome analysis of these two strains revealed that hemK deletion specifically changed the expression of several virulence-related genes associated with the bacterial secretion system, chemotaxis, and quorum sensing, and the expression of various genes related to nutrient utilization including amino acid metabolism, carbohydrate metabolism, and energy metabolism. In conclusion, our results indicate that HemK plays an essential role in virulence, the regulation of virulence factor synthesis, and the nutrient utilization of Xcc.

A cyclic di-GMP-binding adaptor protein interacts with a N5-glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both in vitro and in vivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

Transcriptome profile analysis of Indian mustard (Brassica juncea L.) during seed germination reveals the drought stress-induced genes associated with energy, hormone, and phenylpropanoid pathways.

Indian mustard (Brassica juncea L. Czern and Coss) is an important oil and vegetable crop frequently affected by seasonal drought stress during seed germination, which retards plant growth and causes yield loss considerably. However, the gene networks regulating responses to drought stress in leafy Indian mustard remain elusive. Here, we elucidated the underlying gene networks and pathways of drought response in leafy Indian mustard using next-generation transcriptomic techniques. Phenotypic analysis showed that the drought-tolerant leafy Indian mustard cv. 'WeiLiang' (WL) had a higher germination rate, antioxidant capacity, and better growth performance than the drought-sensitive cv. 'ShuiDong' (SD). Transcriptome analysis identified differentially expressed genes (DEGs) in both cultivars under drought stress during four germination time points (i.e., 0, 12, 24, and 36 h); most of which were classified as drought-responsive, seed germination, and dormancy-related genes. In the Kyoto Encyclopedia of Genes and Genome (KEGG) analyses, three main pathways (i.e., starch and sucrose metabolism, phenylpropanoid biosynthesis, and plant hormone signal transduction) were unveiled involved in response to drought stress during seed germination. Furthermore, Weighted Gene Co-expression Network Analysis (WGCNA) identified several hub genes (novel.12726, novel.1856, BjuB027900, BjuA003402, BjuA021578, BjuA005565, BjuB006596, novel.12977, and BjuA033308) associated with seed germination and drought stress in leafy Indian mustard. Taken together, these findings deepen our understanding of the gene networks for drought responses during seed germination in leafy Indian mustard and provide potential target genes for the genetic improvement of drought tolerance in this crop.