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Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a cochaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.

The type III effector protein encoded by avirulence gene B (AvrB) is delivered into plant cells by pathogenic strains of Pseudomonas syringae. There, it localizes to the plasma membrane and triggers immunity mediated by the Arabidopsis coiled-coil (CC)-nucleotide binding (NB)-leucine-rich repeat (LRR) disease resistance protein RPM1. The sequence unrelated type III effector avirulence protein encoded by avirulence gene Rpm1 (AvrRpm1) also activates RPM1. AvrB contributes to virulence after delivery from P. syringae in leaves of susceptible soybean plants, and AvrRpm1 does the same in Arabidopsis rpm1 plants. Conditional overexpression of AvrB in rpm1 plants results in leaf chlorosis. In a genetic screen for mutants that lack AvrB-dependent chlorosis in an rpm1 background, we isolated TAO1 (target of AvrB operation), which encodes a Toll-IL-1 receptor (TIR)-NB-LRR disease resistance protein. In rpm1 plants, TAO1 function results in the expression of the pathogenesis-related protein 1 (PR-1) gene, suggestive of a defense response. In RPM1 plants, TAO1 contributes to disease resistance in response to Pto (P. syringae pathovars tomato) DC3000(avrB), but not against Pto DC3000(avrRpm1). The tao1-5 mutant allele, a stop mutation in the LRR domain of TAO1, posttranscriptionally suppresses RPM1 accumulation. These data provide evidence of genetically separable disease resistance responses to AvrB and AvrRpm1 in ARABIDOPSIS: AvrB activates both RPM1, a CC-NB-LRR protein, and TAO1, a TIR-NB-LRR protein. These NB-LRR proteins then act additively to generate a full disease resistance response to P. syringae expressing this type III effector.

Plants recognize microbes via specific pattern recognition receptors that are activated by microbe-associated molecular patterns (MAMPs), resulting in MAMP-triggered immunity (MTI). Successful pathogens bypass MTI in genetically diverse hosts via deployment of effectors (virulence factors) that inhibit MTI responses, leading to pathogen proliferation. Plant pathogenic bacteria like Pseudomonas syringae utilize a type III secretion system to deliver effectors into cells. These effectors can contribute to pathogen virulence or elicit disease resistance, depending upon the host plant genotype. In disease resistant genotypes, intracellular immune receptors, typically belonging to the nucleotide binding leucine-rich repeat family of proteins, perceive bacterial effector(s) and initiate downstream defense responses (effector triggered immunity) that include the hypersensitive response, and transcriptional re-programming leading to various cellular outputs that collectively halt pathogen growth. Nucleotide binding leucine-rich repeat sensors can be indirectly activated via perturbation of a host protein acting as an effector target. AvrRpm1 is a P. syringae type III effector. Upon secretion into the host cell, AvrRpm1 is acylated by host enzymes and directed to the plasma membrane, where it contributes to virulence. This is correlated with phosphorylation of Arabidopsis RIN4 in vivo. RIN4 is a negative regulator of MAMP-triggered immunity, and its modification in the presence of four diverse type III effectors, including AvrRpm1, likely enhances this RIN4 regulatory function. The RPM1 nucleotide binding leucine-rich repeat sensor perceives RIN4 perturbation in disease resistant plants, leading to a successful immune response. Here, demonstrate that AvrRpm1 has a fold homologous to the catalytic domain of poly(ADP-ribosyl) polymerase. Site-directed mutagenesis of each residue in the putative catalytic triad, His63-Tyr122-Asp185 of AvrRpm1, results in loss of both AvrRpm1-dependent virulence and AvrRpm1-mediated activation of RPM1, but, surprisingly, causes a gain of function: the ability to activate the RPS2 nucleotide binding leucine-rich repeat sensor.

Type III effector proteins (T3Es) of many Gram-negative pathogenic bacteria manipulate highly conserved cellular processes, indicating conservation in virulence mechanisms during the infection of hosts of divergent evolutionary origin. In order to identify conserved effector functions, we used a cross-kingdom approach in which we expressed selected T3Es from the mammalian pathogen Salmonella enterica in leaves of Nicotiana benthamiana and searched for possible virulence or avirulence phenotypes. We show that the T3E SseF of S. enterica triggers hypersensitive response (HR)-like symptoms, a hallmark of effector-triggered immunity in plants, either when transiently expressed in leaves of N. benthamiana by Agrobacterium tumefaciens infiltration or when delivered by Xanthomonas campestris pv vesicatoria (Xcv) through the type III secretion system. The ability of SseF to elicit HR-like symptoms was lost upon silencing of suppressor of G2 allele of skp1 (SGT1), indicating that the S. enterica T3E is probably recognized by an R protein in N. benthamiana. Xcv translocating an AvrRpt2–SseF fusion protein was restricted in multiplication within leaves of N. benthamiana. Bacterial growth was not impaired but symptom development was rather accelerated in a compatible interaction with susceptible pepper (Capsicum annuum) plants. We conclude that the S. enterica T3E SseF is probably recognized by the plant immune system in N. benthamiana, resulting in effector-triggered immunity.

Many bacterial plant pathogens require the type III secretion system (T3SS) and its effector proteins (T3SEs) to invade and extract nutrients from their hosts successfully. While the molecular function of this system is being studied intensively, we know comparatively little about the evolutionary and ecological pressures governing its fate over time, and even less about the detailed mechanisms underlying and driving complex T3SS-mediated coevolutionary dynamics. In this review we summarize our current understanding of how host-pathogen interactions evolve, with a particular focus on the T3SS of bacterial plant pathogens. We explore the evolutionary origins of the T3SS relative to the closely related flagellar system, and investigate the evolutionary pressures on this secretion and translocation apparatus. We examine the evolutionary forces acting on T3SEs, and compare the support for vertical descent with modification of these virulence-associated systems (pathoadaptation) vs horizontal gene transfer. We address the evolutionary origins of T3SEs from the perspective of both the evolutionary mechanisms that generate new effectors, and the mobile elements that may be the source of novel genetic material. Finally, we propose a number of questions raised by these studies, which may serve to guide our thinking about these complex processes.

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.

This paper highlights insights gained from fully sequenced bacterial pathogen genomes that are of particular relevance to plant biologists. It describes the range of bacterial phytopathogens and their lifestyles in plants, lessons gained from type III effector repertoires (a focus of much study during this period), major insights arising from each of the phytopathogen groups with completely sequenced genomes, and future challenges.

The enteropathogenic and enterohemorrhagic NleB proteins as well as the SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian -linked -acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed cross-reactivity with the mammalian -linked -acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases.

The aim of this study was to determine the prevalence of type III effector proteins (ExoS, ExoU, ExoT and ExoY)-encoding genes among clonally unrelated nosocomial Pseudomonas aeruginosa strains and to analyze their distribution in respect to the infection site and antimicrobial resistance. Polymerase chain reaction-based detection of the genes was performed on 176 non-duplicate P. aeruginosa isolates from three University hospitals in Sofia, previously genotyped by random amplified polymorphic DNA technique. The prevalence of the studied genes was as follows: exoS –61.9%, exoU –32.4%, exoT –100%, and exoY –85.8%. The part of P. aeruginosa strains harboring either the exoS (54.0%) or the exoU (23.8%) gene was higher ( P  < 0.001) than that of isolates containing both genes (8.5%). The gene dissemination varied according to the infection localization. The exoU gene manifested a higher spread ( P  < 0.001) among multidrug-resistant (MDR) than in non-MDR strains (42.6 vs 18.7%). In conclusion, the P. aeruginosa type III secretion system is present in nearly all studied isolates but the individual isolates from distinct infection sites differ in their effector genotypes. The ubiquity of type III effector proteins-encoding genes among clinical isolates is consistent with an important role for this system in P. aeruginosa pathogenesis.

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. Translocated type III effector proteins from Gram-negative plant-pathogenic bacteria promote bacterial virulence by interfering with defense responses, signaling pathways, protein degradation, gene expression or the formation of the cytoskeleton.