A CysB regulator positively regulates cysteine synthesis, expression of type III secretion system genes, and pathogenicity in Ralstonia solanacearum.

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.

TRAPPC13 Is a Novel Target of Mesorhizobium amorphae Type III Secretion System Effector NopP.

Similar to pathogenic bacteria, rhizobia can inject effector proteins into host cells directly to promote infection via the type III secretion system (T3SS). Nodulation outer protein P (NopP), a specific T3SS effector of rhizobia, plays different roles in the establishment of multiple rhizobia-legume symbiotic systems. Mesorhizobium amorphae CCNWGS0123 (GS0123), which infects Robinia pseudoacacia specifically, secretes several T3SS effectors, including NopP. Here, we demonstrate that NopP is secreted through T3SS-I of GS0123 during the early stages of infection, and its deficiency decreases nodule nitrogenase activity of R. pseudoacacia nodules. A trafficking protein particle complex subunit 13-like protein (TRAPPC13) has been identified as a NopP target protein in R. pseudoacacia roots by screening a yeast two-hybrid library. The physical interaction between NopP and TRAPPC13 is verified by bimolecular fluorescence complementation and coimmunoprecipitation assays. In addition, subcellular localization analysis reveals that both NopP and its target, TRAPPC13, are colocalized on the plasma membrane. Compared with GS0123-inoculated R. pseudoacacia roots, some genes associated with cell wall remodeling and plant innate immunity down-regulated in ΔnopP-inoculated roots at 36 h postinoculation. The results suggest that NopP in M. amorphae CCNWGS0123 acts in multiple processes in R. pseudoacacia during the early stages of infection, and TRAPPC13 could participate in the process as a NopP target.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Proteomic Analysis of Potato Responding to the Invasion of Ralstonia solanacearum UW551 and Its Type III Secretion System Mutant.

The infection of potato with Ralstonia solanacearum UW551 gives rise to bacterial wilt disease via colonization of roots. The type III secretion system (T3SS) is a determinant factor for the pathogenicity of R. solanacearum. To fully understand perturbations in potato by R. solanacearum type III effectors(T3Es), we used proteomics to measure differences in potato root protein abundance after inoculation with R. solanacearum UW551 and the T3SS mutant (UW551△HrcV). We identified 21 differentially accumulated proteins. Compared with inoculation with UW551△HrcV, 10 proteins showed significantly lower abundance in potato roots after inoculation with UW551, indicating that those proteins were significantly downregulated by T3Es during the invasion. To identify their functions in immunity, we silenced those genes in Nicotiana benthamiana and tested the resistance of the silenced plants to the pathogen. Results showed that miraculin, HBP2, and TOM20 contribute to immunity to R. solanacearum. In contrast, PP1 contributes to susceptibility. Notably, none of four downregulated proteins (HBP2, PP1, HSP22, and TOM20) were downregulated at the transcriptional level, suggesting that they were significantly downregulated at the posttranscriptional level. We further coexpressed those four proteins with 33 core T3Es. To our surprise, multiple effectors were able to significantly decrease the studied protein abundances. In conclusion, our data showed that T3Es of R. solanacearum could subvert potato root immune-related proteins in a redundant manner.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Nod factor- and type III secretion system-dependent manner for Robinia pseudoacacia to establish symbiosis with Mesorhizobium amorphae CCNWGS0123.

Under nitrogen-limiting conditions, symbiotic nodulation promotes the growth of legume plants via the fixation of atmospheric nitrogen to ammonia by rhizobia in root nodules. The rhizobial Nod factor (NF) and type III secretion system (T3SS) are two key signaling pathways for establishing the legume-rhizobium symbiosis. However, whether NF signaling is involved in the nodulation of Robinia pseudoacacia and Mesorhizobium amorphae CCNWGS0123, and its symbiotic differences compared with T3SS signaling remain unclear. Therefore, to elucidate the function of NF signaling in nodulation, we mutated nodC in M. amorphae CCNWGS0123, which aborted NF synthesis. Compared with the plants inoculated with the wild type strain, the plants inoculated with the NF-deficient strain exhibited shorter shoots with etiolated leaves. These phenotypic characteristics were similar to those of the plants inoculated with the T3SS-deficient strain, which served as a Nod- (non-effective nodulation) control. The plants inoculated with both the NF- and T3SS-deficient strains formed massive root hair swellings, but no normal infection threads were detected. Sections of the nodules showed that inoculation with the NF- and T3SS-deficient strains induced small, white bumps without any rhizobia inside. Analyzing the accumulation of 6 plant hormones and the expression of 10 plant genes indicated that the NF- and T3SS-deficient strains activated plant defense reactions while suppressing plant symbiotic signaling during the perception and nodulation processes. The requirement for NF signaling appeared to be conserved in two other leguminous trees that can establish symbiosis with M. amorphae CCNWGS0123. In contrast, the function of the T3SS might differ among species, even within the same subfamily (Faboideae). Overall, this work demonstrated that nodulation of R. pseudoacacia and M. amorphae CCNWGS0123 was both NF and T3SS dependent.

A compound isolated from Alpinia officinarum Hance. inhibits swarming motility of Pseudomonas aeruginosa and down regulates virulence genes.

AIM: The study was aimed at purifying the active principle from Alpinia officinarum rhizomes responsible for inhibition of swarming motility of Pseudomonas aeruginosa and analysing the mechanism of action. METHODS AND RESULTS: The active compound from methanol extract of A. officinarum was purified by silica gel column chromatography followed by elution from Amberlite resin. The compound 1-(3,5-dihydroxyphenyl)-2-(methylamino)ethan-1-one, inhibited swarming motility at 12·5 µg ml-1 . This inhibition was independent of rhamnolipid production. Real-time PCR analysis showed significant down-regulation of virulence-associated genes including T3SS exoS, exoT and flagella master regulator fleQ. CONCLUSIONS: The compound from A. officinarum inhibited swarming motility and significantly down-regulated the expression of type III secretory system effector genes exoS and exoT and flagellar master regulator fleQ genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The study identifies a potent swarming inhibitory compound from the common medicinal plant A. officinarum and reinstates the potential of plant-derived compounds in tackling virulence properties of pathogenic bacteria.

A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency.

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

Type III secretion inhibitors for the management of bacterial plant diseases.

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds-including molecules effective against human pathogens-for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down-regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases.