DNA MOLECULES AND POLYPEPTIDES OF \u3ci\u3ePSEUDOMONAS SYRINGAE\u3c/i\u3e HRP PATHOGENICITY ISLAND AND THEIR USES: U.S. Patent No. US 7,102,059 B2

One aspect of the present invention relates to isolated nucleic acid molecules (i) encoding proteins or polypeptides of Pseudomonas CEL and EEL genomic regions, (ii) nucleic acid molecules which hybridize thereto under stringent conditions, or (iii) nucleic acid molecules that include a nucleotide sequence which is complementary to the nucleic acid molecules of (i) and (ii). Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. Another aspect relates to the isolated proteins or polypeptides and compositions containing the same. The nucleic acid molecules and proteins of the present invention can be used to imparting disease resistance to a plant, making a plant hypersusceptible to colonization by nonpathogenic bacteria, causing eukaryotic cell death, and treating cancerous conditions

Common and contrasting themes in host cell-targeted effectors from bacterial, fungal, oomycete and nematode plant symbionts described using the Gene Ontology

A wide diversity of plant-associated symbionts, including microbes, produce proteins that can enter host cells, or are injected into host cells in order to modify the physiology of the host to promote colonization. These molecules, termed effectors, commonly target the host defense signaling pathways in order to suppress the defense response. Others target the gene expression machinery or trigger specific modifications to host morphology or physiology that promote the nutrition and proliferation of the symbiont. When recognized by the host's surveillance machinery, which includes cognate resistance (R) gene products, defense responses are engaged to restrict pathogen proliferation. Effectors from diverse symbionts may be delivered into plant cells via varied mechanisms, including whole organism cellular entry (viruses, some bacteria and fungi), type III and IV secretion (in bacteria), physical injection (nematodes and insects) and protein translocation signal sequences (oomycetes and fungi). This mini-review will summarize both similarities and differences in effectors and effector delivery systems found in diverse plant-associated symbionts as well as how these are described with Plant-Associated Microbe Gene Ontology (PAMGO) terms

Gene Ontology annotation highlights shared and divergent pathogenic strategies of type III effector proteins deployed by the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic Escherichia coli strains

Genome-informed identification and characterization of Type III effector repertoires in various bacterial strains and species is revealing important insights into the critical roles that these proteins play in the pathogenic strategies of diverse bacteria. However, non-systematic discipline-specific approaches to their annotation impede analysis of the accumulating wealth of data and inhibit easy communication of findings among researchers working on different experimental systems. The development of Gene Ontology (GO) terms to capture biological processes occurring during the interaction between organisms creates a common language that facilitates cross-genome analyses. The application of these terms to annotate type III effector genes in different bacterial species – the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic strains of Escherichia coli – illustrates how GO can effectively describe fundamental similarities and differences among different gene products deployed as part of diverse pathogenic strategies. In depth descriptions of the GO annotations for P. syringae pv tomato DC3000 effector AvrPtoB and the E. coli effector Tir are described, with special emphasis given to GO capability for capturing information about interacting proteins and taxa. GO-highlighted similarities in biological process and molecular function for effectors from additional pathosystems are also discussed

PSEUDOMONAS AVR AND HOP PROTEINS, THEIR ENCODING NUCLEIC ACIDS, AND USE THEREOF

One aspect of the present invention relates to isolated nucleic acid molecules encoding avirulence proteins or polypeptides of Pseudomonas syringae pv. syringae DC 3000, or nucleic acid moleculues which are complementary thereto. Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. Another aspect relates to the isolated proteins or polypeptides and compositions containing the same. The various nucleic acid molecules and proteins of the present invention can be used to impart disease resistance to a plant, make a plant hypersusceptible to colonization by nonpathogenic bacteria, modify a metabolic pathway in a cell, cause eukaryotic cell death and treat a cancerous condition, as well as inhibit programmed cell death

NUCLEC ACDS ENCODING PSEUDOMONAS HOP PROTEINS AND USE THEREOF

The present invention relates to isolated nucleic acid mol ecules encoding a type III—secreted bacterial protein capable of modifying a cell death pathway in a plant cell. One aspect of the present invention involves an isolated nucleic acid molecule having a nucleotide sequence that encodes the HopPtol)2 protein of Pseudomonas syringae pv. syringae DC 3000. Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. The nucleic acid mol ecules of the present invention can be used to impart disease resistance to a plant and to make a plant hypersusceptible to colonization by nonpathogenic bacteria

NUCLEC ACDS ENCODING PSEUDOMONAS HOP PROTEINS AND USE THEREOF

The present invention relates to isolated nucleic acid mol ecules encoding a type III—secreted bacterial protein capable of modifying a cell death pathway in a plant cell. One aspect of the present invention involves an isolated nucleic acid molecule having a nucleotide sequence that encodes the HopPtol)2 protein of Pseudomonas syringae pv. syringae DC 3000. Expression vectors, host cells, and transgenic plants which include the DNA molecules of the present invention are also disclosed. The nucleic acid mol ecules of the present invention can be used to impart disease resistance to a plant and to make a plant hypersusceptible to colonization by nonpathogenic bacteria

\u3ci\u3ePseudomonas syringae\u3c/i\u3e Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

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 AvrPphEPto 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

The \u3ci\u3ePseudomonas syringae\u3c/i\u3e pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DhrpZ::nptII or hrpW::VSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall

Relative Effects on Virulence of Mutations in the sap, pel, and hrp Loci of Erwinia chrysanthemi

We constructed strains of Erwinia chrysanthemi EC16 with múltiple mutations involving three virulence systems in this bacterium, namelypel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the peí (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel- Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive Índices of strains BT105, UM1005 (Peí"), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indícate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in peí and sap cannot

The \u3ci\u3ePseudomonas syringae\u3c/i\u3e pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DhrpZ::nptII or hrpW::VSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall