The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and͞or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the Nterminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADPribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrcencoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE Pto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.Pseudomonas syringae is a host-specific bacterial pathogen of plants whose various pathovars cause necrotic lesions on leaves or fruits of susceptible plants and elicit the hypersensitive response (HR), a defense-associated localized programmed cell death, in resistant plants. P. syringae interactions with plants depend on the hrp (for "hypersensitive response and pathogenicity") gene cluster, which encodes a type III secretion system (TTSS) or Hrp system that injects bacterial proteins into plant cells (1). Similar TTSSs exist in a variety of other gram-negative plant and animal pathogens (13, 28). The P. syringae proteins injected into host cells by the Hrp system are primarily known as effectors or Avr (avirulence) or Hop (Hrp-dependent outer protein) proteins. Hop proteins may contribute to P. syringae virulence by suppressing plant immunity. For example, AvrB, AvrRpm1...

SummaryDuring infection of its hosts, Salmonella enterica serovar Typhimurium (S. typhimurium) enters the epithelial cells of the small intestine. This process requires a number of invasion genes encoded on Salmonella pathogenicity island 1 (SPI1), a 40 kb stretch of DNA located near minute 63 of the S. typhimurium chromosome. Expression of S. typhimurium SPI1 invasion genes is activated by the transcription factor HilA. hilA is tightly regulated in response to many environmental conditions, including oxygen, osmolarity and pH. Regulation of hilA expression may serve to limit invasion gene expression to the appropriate times during Salmonella infection. We have mapped the transcription start site of hilA and identified regions of the promoter that are required for the repression of hilA expression by conditions unfavourable for Salmonella invasion. We have also identified two SPI1-encoded genes, hilC and hilD, that can independently derepress hilA expression. HilC and HilD are both members of the AraC/XylS family of transcriptional regulators. A mutation in hilD significantly reduces the ability of S. typhimurium to enter tissue culture cells, whereas a mutation in hilC only modestly affects Salmonella invasion. Based on these results, we have updated our model of Salmonella SPI1 invasion gene regulation. We also speculate on the possible significance of this model for Salmonella pathogenesis.

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.

During infection, Salmonella enterica serovar Typhimurium colonizes the small intestine of its hosts. This process requires a type III secretion system encoded by several genes on Salmonella pathogenicity island 1 (SPI1), a 40 kb region of DNA near centisome 63 of the Salmonella chromosome. SPI1 gene expression is controlled by a complex regulatory cascade. HilA, a member of the OmpR/ToxR family of transcriptional regulators, directly activates the expression of two SPI1 operons encoding type III apparatus components. hilA transcription is repressed by many environmental conditions and regulatory mutations. This repression requires an upstream repressing sequence (URS) located between −314 and −68 relative to the hilA transcription start site. The repressing activity of the URS is counteracted by two AraC/XylS family members named HilC and HilD. We show that HilC and HilD bind directly to the hilA promoter region in vitro. We also provide evidence that HilC and HilD bind to the same or overlapping sites within the URS. Our data are consistent with a model in which HilC and HilD derepress hilA expression by binding directly to the URS and counteracting its repressing effect in vivo.

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that translocates virulence effector proteins into host cells via a type III secretion system (T3SS). Many effector-encoding hypersensitive response and pathogenicity (Hrp) outer protein (hop) genes have been identified previously in DC3000 using bioinformatic methods based on Hrp promoter sequences and characteristic N-terminal amino acid patterns that are associated with T3SS substrates. To approach completion of the Hop/effector inventory in DC3000, 44 additional candidates were tested by the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter assay; 10 of the high-probability candidates were confirmed as T3SS substrates. Several previously predicted hop genes were tested for their ability to be expressed in an HrpL-dependent manner in culture or to be expressed in planta. The data indicate that DC3000 harbors 53 hop/avr genes and pseudogenes (encoding both injected effectors and T3SS substrates that probably are released to the apoplast); 33 of these genes are likely functional in DC3000, 12 are nonfunctional members of valid Hop families, and 8 are less certain regarding their production at functional levels. Growth of DC3000 in tomato and Arabidopsis Col-0 was not impaired by constitutive expression of repaired versions of two hops that were disrupted naturally by transposable elements or of hop genes that are naturally cryptic. In summary, DC3000 carries a complex mixture of active and inactive hop genes, and the hop genes in P. syringae can be identified efficiently by bioinformatic methods; however, a precise inventory of the subset of Hops that are important in pathogenesis awaits more knowledge based on mutant phenotypes and functions within plants.

hilA encodes an activator of Salmonella enterica serovar Typhimurium virulence genes and is transcriptionally modulated by environmental conditions. We show that H-NS represses hilA under low-osmolarity conditions. H-NS, HU, and Fis also appear to affect the derepression of hilA by HilD. Modulation of hilA by counteracting repressing and derepressing mechanisms may allow Salmonella serovar Typhimurium to regulate its virulence genes in response to different situations in vivo.

SummarySalmonella typhimurium is a Gram-negative enteric pathogen that can infect intestinal epithelial cells and induce inflammation of the intestinal mucosa. These processes are mediated by a type III secretion system (TTSS), which is encoded on Salmonella pathogenicity island 1 (SPI1). Previous studies showed that four SPI1-encoded transcriptional regulators, HilD, HilC, HilA and InvF, act in an ordered fashion to coordinately activate expression of the SPI1 TTSS. HilD and HilC derepress hilA transcription. HilA activates invF as well as SPI1 genes that encode components of the TTS apparatus. InvF then activates genes that encode proteins secreted by the SPI1 TTS apparatus. In this scheme, HilD and HilC indirectly activate expression of the SPI1 TTS apparatus and its secreted substrates by affecting hilA expression. Here, we report that HilD and HilC can also activate expression of a subset of SPI1 genes independently of HilA. Our studies show that HilD and HilC activate transcription of invF from a promoter that is far upstream of its HilA-dependent promoter. This activation is most probably through direct binding of HilD and HilC to sequences upstream and downstream of this alternative HilA-independent promoter. We conclude that HilD and HilC have a second role in SPI1 gene regulation that is separate from their role in co-ordinating expression of the SPI1 TTSS through hilA .