Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.     

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram‐negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ^Xoo) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ^Xoo does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca²⁺ coordination shell of a ribose‐binding site and the spacious active site in XopQ^Xoo, we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ^Xoo. Here, we report the crystal structure of XopQ^Xoo in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca²⁺ concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ^Xoo with its ribosyl end tethered to the Ca²⁺ coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand‐induced conformational changes. These data showed that XopQ^Xoo is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ^Xoo. Proteins 2014; 82:2910–2914. © 2014 Wiley Periodicals, Inc.     

Eggplant and related species are promising genetic resources to dissect the plant immune response to Pseudomonas syringae and Xanthomonas euvesicatoria and to identify new resistance determinants

The apparent lack of durability of many resistance (R) genes highlights the need for the constant identification of new genetic sources of resistance for the breeding of new disease‐resistant crop cultivars. To this end, we screened a collection of accessions of eggplant and close relatives for resistance against Pseudomonas syringae pv. tomato (Pto) and Xanthomonas euvesicatoria (Xeu), foliar plant pathogens of many solanaceous crops. Both pathogens caused substantial disease on most genotypes of eggplant and its relatives. Promisingly, however, some of the genotypes were fully or partially resistant to either of the pathogens, suggesting the presence of effective resistance determinants in these genotypes. Segregation of resistance to the growth of Xeu following infiltration in F2 progeny from a cross of a resistant and susceptible genotype suggests that resistance to Xeu is inherited as a multigenic trait. With regard to Pto, a mutant strain lacking all 28 functional type III secreted effectors, and a Pseudomonas fluorescens strain expressing a P. syringae type III secretion system (T3SS), both elicit a strong cell death response on most eggplant lines. Several genotypes thus appear to harbour a mechanism for the direct recognition of a component of the T3SS. Therefore, eggplant and its close relatives are promising resources to unravel novel aspects of plant immunity and to identify new candidate R genes that could be employed in other Solanaceae in which Xeu and Pto cause agriculturally relevant diseases.     

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire‐resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram‐negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease‐resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E‐deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C‐terminal truncated HopAZ1 abolished HopAZ1‐dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

细菌三型分泌系统效应蛋白转运的研究进展

三型分泌系统(Type 3 secretion system,T3SS)作为存在于革兰氏阴性菌中的分泌系统之一,对革兰氏阴性菌的致病有重要作用。T3SS的致病作用体现在T3SS能直接将效应蛋白转运至宿主细胞,进而通过效应蛋白调控细胞的一系列通路,促进细菌定殖于细胞。而效应蛋白的转运受到两方面因素的调控,一方面是效应蛋白本身的信号序列,另一方面是T3SS相关蛋白的辅助。本文围绕近年来T3SS的构成、效应蛋白转运机制方面的最新进展进行概要综述。     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.     

Functional dissection of SseF, a type III effector protein involved in positioning the salmonella-containing vacuole

Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.     

十字花科黑腐病菌一个新的III型效应物的鉴定

【目的】在全基因组范围内鉴定十字花科黑腐病菌Xcc 8004中新的Ⅲ型效应物(type Ⅲ secreted effector,T3SE)基因。【方法】通过构建与AvrBs1_59-445整合的Tn5转座子系统,进行文库筛选。在avrBs1缺失突变体背景下生成突变文库,在辣椒ECW-10R上进一步筛选。【结果】大规模HR测定筛选出7个具有明显过敏反应(hypersensitive response,HR)的克隆。通过质粒拯救和测序发现除了插入已知T3SE基因的3个突变体外,还鉴定了一个位于XC_0438和XC_0439之间且在Xcc 8004中未注释的新基因。结合生物信息学,我们将其命名为一个新的基因XC_0438a。易位实验证实XC_0438a信号区可引导报告蛋白AvrBs1的分泌和易位,并诱导辣椒ECW-10R产生HR反应。β-葡萄糖醛酸酶(β-glucuronidase,GUS)活性测定,XC_0438a在基本培养基中诱导表达,并由关键调控蛋白HrpG和HrpX激活。然而,在我们的实验条件下,XC_0438a对Xcc的致病力没有显著的贡献。【结论】我们鉴定了一种新的依赖于Ⅲ型分泌系统的效应基因XC_0438a。     

Suppression of type III effector secretion by polymers

Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa. We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.     

Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.     

Structural insights into caspase ADPR deacylization catalyzed by a bacterial effector and host calmodulin

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