Showing papers by "Alan Collmer published in 1997"

The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death.

Abstract: The ability of plant pathogenic bacteria to deliver deathtriggering proteins to the interior of plant cells was revealed in a rapid succession of papers in 1996 that transformed our concepts of bacterial plant pathogenicity. The breakthrough came with the convergence of work on Hrp systems and Avr proteins, an understanding of which requires an introduction to the most prevalent bacterial pathogens of plants, the cardinal importance of the Hrp pathway, and the paradoxical phenotype associated with avr genes. Plant pathogenic bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia cause diverse, and sometimes devastating, diseases in many different plants, but they all share three characteristics: they colonize the intercellular spaces of plants, they are capable of killing plant cells, and they possess hrp genes. Many of these pathogens are host specific. In host plants, they produce various symptoms after several days of multiplication, whereas in nonhost plants, they trigger the hypersensitive response (HR), a rapid, defense-associated, programmed death of plant cells at the site of invasion (21, 43). With inoculum levels typically encountered in natural environments, the HR produces individual dead plant cells that are scattered within successfully defended healthy tissue (71). However, experimental infiltration of high inoculum levels (.10 bacterial cells/ml) results in macroscopically observable death of the entire infiltrated tissue, usually within 24 h (42). Pioneer screens for random transposon mutants with impaired plant interactions yielded a prevalent class that was designated Hrp, that is, deficient in both HR elicitation in nonhost plant species and pathogenicity (and parasitic growth) in host species (49, 56). This complete loss of pathogenic behavior results from mutation of any one of several hrp genes, which largely encode components of a type III protein secretion system (73). Because the capacity to elicit the HR is a convenient marker for the capacity to be pathogenic and these two abilities have a common genetic basis, the “simple” problem of HR elicitation is being studied as an entry to the larger problem of pathogenesis. A key part of the HR puzzle is that HR elicitation and the resulting limitation in host range can occur if the pathogen possesses any one of many possible avr (avirulence) genes that interact with corresponding R (resistance) genes in the host plant. Such “gene-for-gene” interactions result in recognition of the bacterium and the triggering of plant defenses. For example, Pseudomonas syringae pv. glycinea is one of over 40 P. syringae pathovars differing largely in host range among plant species and is subdivided into races on the basis of their interactions with genetically distinct cultivars of its host, soybean. Those race-cultivar interactions involving matching bacterial avr and plant R genes result in the HR and avirulence, i.e.; failure of the bacterium to produce disease. The R genes encode components of a parasite surveillance system and are crossed into crops from wild relatives by plant breeders for disease control. avr genes are identified and cloned on the basis of the avirulence they confer on virulent races in appropriate test plants (39, 69). In most cases, it is not clear why plant pathogens carry avr genes that betray them to host defenses but new insights into this question are discussed below. Both hrp and avr genes were originally defined on the basis of the phenotypes they confer on bacteria interacting with plants. Molecular studies have revealed a functional relationship between the products of these two classes of genes and an underlying similarity with a key virulence system of several animal pathogens. Yersinia, Salmonella, and Shigella spp. transfer virulence effector proteins directly into animal cells via the type III pathway (16, 17, 62, 67, 84). Similarly, plant pathogens use the Hrp type III pathway to transfer Avr effector proteins to the interior of plant cells. The genetic dissection of type III secretion systems is just beginning, and little is known of the mechanisms of protein translocation. In this review, we will describe (i) the recently completed inventory of genes directing type III secretion in plant pathogens and new insights into type III secretion mechanisms gained from research with Hrp systems, (ii) two classes of proteins (harpins and pilins) that are secreted by the Hrp type III pathway when plant pathogens are grown in media that mimic plant intercellular fluids, (iii) evidence that Avr proteins are delivered by the Hrp pathway directly to the interior of plant cells, and (iv) a resulting new paradigm for bacterial plant pathogenicity. The focus will be on quite recent work, and readers are referred to other reviews for a classic introduction to the HR phenomenon (43), earlier investigations of the Hrp system (11), avr genes (20, 46), and a wider perspective on bacterial virulence systems and plant responses (2).

Altered localization of HrpZ in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria.

Abstract: Pseudomonas syringae pv. syringae 61 (Pss61) secretes the HrpZ harpin by a type III protein secretion pathway encoded by a cluster of hrp (hypersensitive response and pathogenicity) and hrc genes. The nine hrc genes represent a subset of hrp genes that are also conserved in the type III virulence protein secretion systems of animal pathogenic Yersinia, Shigella, and Salmonella spp. The hrpJ and hrpU operons contain seven hrc genes (counting hrcQ(A) and hrcQ(B) as one gene), all with additional homologs involved in flagellar biogenesis and secretion, and five of which encode predicted inner membrane proteins. The hrpC and hrpZ operons encode HrcC and HrcJ, respectively, which are associated with the outer membrane. Interposon mutants affected in all of the hrc genes in the hrpJ and hrpU operons and TnphoA-induced hrcC and hrcJ mutants were assayed for altered localization of HrpZ in mid-log-phase cultures by immunoblotting sodium dodecyl sulfate-polyacrylamide gels that were run with various cell fractions. The hrpJ and hrpU operon mutants revealed a novel phenotype of partially reduced accumulation of HrpZ in the total culture (despite wild-type levels of hrpZ operon transcription), all of which was cell bound and equivalent in level to that of cell-bound HrpZ in the wild type. The hrcC and hrcJ mutant cultures accumulated the same total amount of HrpZ as the wild type, but the HrpZ was cell bound. Among all the strains tested, only the hrcC mutant accumulated significant amounts of HrpZ in the periplasm, as indicated by selective release through spheroplasting. Analysis of nonpolar mutations in the hrpU and hrpC operons support the results obtained with polar mutations. These observations indicate that a constant pool of HrpZ is maintained in the cytoplasm of Pss61 despite secretion deficiencies, that the hrpJ and hrpU operons encode an alternative to the Sec (general protein export) pathway for translocation across the inner membrane, that genes in the hrpC operon are necessary for translocation across the outer membrane, and that the Pss61 Hrp system permits study of two genetically distinguishable stages in type III protein secretion.

Evidence That the Pseudomonas syringae pv. syringae hrp-Linked hrmA Gene Encodes an Avr-Like Protein That Acts in an hrp-Dependent Manner Within Tobacco Cells

Abstract: A 25-kb DNA region, previously cloned from Pseudomonas syringae pv. syringae 61 in cosmid pHIR11, enables non-pathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum). hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR. DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein. DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P. syringae pv. syringae B728a, P. syringae pv. tabaci 11528, and P. syringae pv. glycinea race 4 U1, but present in P. syringae pv. tomato DC3000. A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P. syringae pv. syringa...

Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.

Abstract: DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.

Molecular cloning, characterization, and mutagenesis of a pel gene from Pseudomonas syringae pv. lachrymans encoding a member of the Erwinia chrysanthemi pelADE family of pectate lyases

Abstract: The pelS gene from Pseudomonas syringae pv. lachrymans 859 was cloned by heterologous expression in nonpectolytic P. syringae pv. syringae BUVS1, using genomic DNA libraries constructed with two novel broad-host-range cosmid vectors, pCPP34 and pCPP47. Screening of P. syringae pv. syringae transconjugants for the ability to pit pectate media at pH 6.0 and 8.5 yielded several overlapping clones of the same DNA region. Ultrathin-layer isoelectric focusing gels, activity-stained with diagnostically buffered substrate overlays, revealed that this region encoded a single pectate lyase (PelS) with a pI of 9.4. pelS was subcloned from cosmid pCPP5020 and sequenced, revealing it to encode a member of the Erwinia chrysanthemi PelADE family, with highest similarity to Pseudomonas viridiflava PelV. A pelS probe hybridized at high stringency in DNA gel blots with total DNA from P. syringae pv. lachrymans strains 859 and Pla5, P. syringae pv. tabaci, P. syringae pv. phaseolicola, P. syringae pv. glycinea, P. fluorescens (marginalis), P. viridiflava, and Xanthomonas campestris pv. campestris, but not with P. syringae pv. pisi, P. syringae pv. syringae, P. syringae pv. tomato, P. syringae pv. papulans, E. chrysanthemi, or Ralstonia (Pseudomonas or Burkholderia) solanacearum. The PelS sequence revealed an N-terminal signal peptide, whose processing in Escherichia coli was confirmed by protein sequence analysis. PelS was similar to E. chrysanthemi PelE in its substrate preference and ability to reduce the viscosity of pectate and to macerate potato tuber tissue. A pelS:: omega Kmr mutation was marker-exchanged into P. syringae pv. lachrymans Pla5, pelS was also subcloned into the broad-host-range expression vector pML122 under control of the vector nptII promoter, and then transformed into P. syringae pv. lachrymans Pla5 to produce a strain overproducing PelS. Necrotic lesions developed in cotyledons following inoculation with all of the P. syringae pv. lachrymans Pla5 derivatives, regardless of their Pel phenotype. However, only cotyledons infected with pelS+ strains showed evidence of maceration and yielded Pel activity upon extraction. In contrast, pelS+ P. syringae pv. syringae BUVS1(pCPP5020) produced no symptoms in cucumber cotyledons. Thus, PelS in P. syringae pv. lachrymans appears to alter the final symptoms in infected cucumber cotyledons without contributing to pathogenicity or altering host range.