Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

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Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

The complete general secretory pathway in gram-negative bacteria

Many plant bacteriologists, if not all, feel that their particular microbe should appear in any list of the most important bacterial plant pathogens. However, to our knowledge, no such list exists. The aim of this review was to survey all bacterial pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate the bacterial pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 458 votes from the international community, and allowed the construction of a Top 10 bacterial plant pathogen list. The list includes, in rank order: (1) Pseudomonas syringae pathovars; (2) Ralstonia solanacearum; (3) Agrobacterium tumefaciens; (4) Xanthomonas oryzae pv. oryzae; (5) Xanthomonas campestris pathovars; (6) Xanthomonas axonopodis pathovars; (7) Erwinia amylovora; (8) Xylella fastidiosa; (9) Dickeya (dadantii and solani); (10) Pectobacterium carotovorum (and Pectobacterium atrosepticum). Bacteria garnering honourable mentions for just missing out on the Top 10 include Clavibacter michiganensis (michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus. This review article presents a short section on each bacterium in the Top 10 list and its importance, with the intention of initiating discussion and debate amongst the plant bacteriology community, as well as laying down a benchmark. It will be interesting to see, in future years, how perceptions change and which bacterial pathogens enter and leave the Top 10.

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Top 10 plant pathogenic bacteria in molecular plant pathology

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1 , the ethylene response mutant etr1 , and the SAR regulatory mutant npr1 , we demonstrate that signal transduction leading to P. fluorescens WCS417r‐mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid‐nonaccumulating NahG plants. Moreover, methyl jasmonate‐induced protection was blocked in jar1 , etr1 , and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate‐induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

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A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis

The phytotoxin coronatine and the plant growth regulator methyl jasmonate (MeJA) caused similar growth-inhibitory effects on Arabidopsis seedlings. To test whether these two compounds have similar action, 14 independent coi1 (coronatine-insensitive) mutants of Arabidopsis were selected. The mutants segregated as single recessive Mendelian markers, and all were alleles at the coi1 locus. All coi1 mutants were also insensitive to MeJA and were male sterile. Both coronatine and MeJA inhibited root growth, stimulated anthocyanin accumulation, and increased the level of two proteins of ~31 and ~29 kD detected in SDS-polyacrylamide gels of wild-type Arabidopsis but caused none of these effects in the coi1 mutant. Coronatine and MeJA also induced the systemic appearance of proteinase inhibitor activity in tomato. The male-sterile flowers of the coi1 mutant produced abnormal pollen and had reduced level of an ~31-kD protein, which was abundant in the wild-type flowers. A coronatine-producing strain of Pseudomonas syringae grew in leaves of wild-type Arabidopsis to a population more than 100 times greater than it reached in the coi1 mutant. We conclude that coronatine mimics the action of MeJA and that coi1 controls a step in MeJA perception/response and in flower development.

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Arabidopsis Mutants Selected for Resistance to the Phytotoxin Coronatine Are Male Sterile, Insensitive to Methyl Jasmonate, and Resistant to a Bacterial Pathogen.

A genomic library of Erwinia chrysanthemi EC16 was constructed in plasmid pHC79, and seven putative pectate lyase (PL) clones in Escherichia coli were selected on pectate agar. Six of the recombinant cosmids contained a common PstI fragment of ca. 8.2 kilobases (kb). Subcloning of this fragment in either orientation into the PstI site of plasmid pBR329 resulted in E. coli transformants that produced a PL of pI 9.8 which was indistinguishable from one of two PLs produced by strain EC16. A 6.6-kilobase PstI fragment from the remaining cosmid clone caused production of an Erwinia PL of pI 8.8 when the fragment was subcloned in either orientation into plasmid pBR329 and transformed into E. coli. Selected pBR329 subclones for the 8.2- and 6.6-kilobase PstI fragments showed no similarity in their restriction maps and did not cross-hybridize. All of the E. coli cosmid clones that produced large amounts of PL also caused soft-rot of potato tubers and tuber slices, thus confirming the role of the enzymes in plant tissue maceration. The E. coli cosmid clones and plasmid pBR329 subclones produced the PLs constitutively, unlike Erwinia chrysanthemi, which made the enzymes inducibly. However, catabolite repression appeared to function in the E. coli clones, and almost all of the PL activity occurred in the periplasm and culture fluids. Thus, the Erwinia PL clones appear to contain signal peptide sequences, transcription and translation signals, and a recognition sequence for the catabolite activator protein, all of which function efficiently in E. coli.

Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species. Images

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

The molecular and genetic control of the interaction between tomato races of Xanthomonas campestris pv. vesicatoria (XcvT) and tomato was studied. Based on inoculation phenotype and analysis of in planta bacterial growth, tomato line Hawaii 7998 is resistant to XcvT race 1 75-3 but not to XcvT race 2 89-1. Two cosmid clones from a genomic library of XcvT race 1 75-3 converted the normally virulent race 2 89-1 to avirulence on Hawaii 7998. The two clones contained the previously isolated, nonhost avirulence gene avrRxv, and their activity was localized to a 2.1-kbp subclone of avrRxv. avrRxv inhibits growth of race 2 89-1 in the resistant line Hawaii 7998 and an insertional mutation in avrRxv prevents this inhibition. In addition, a dramatic increase in electrolyte leakage of leaves of Hawaii 7998 occurred after 12-hr postinfiltration with race 2 89-1 carrying avrRxv. The nucleotide sequence of avrRxv revealed one major open reading frame (ORF) that accords well with activity analysis of nested deletions. ORF 2-2 encodes a putative protein of 374 amino acids with a molecular weight of 42.1 kDa and a pI of 10.7. Inheritance of the avrRxv-specific resistance in Hawaii 7998 was studied in a total of 587 F2 individuals from crosses between Hawaii 7998 and susceptible lines. The inheritance of avrRxv-specific resistance in Hawaii 7998 appears to be governed by more than one locus.

Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998.

Cloned avirulence gene of Xanthomonas campestris pv. vesicatoria complements spontaneous race-change mutants

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

D. Dahlbeck

Papers

Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli.

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998.

Cloned avirulence gene of Xanthomonas campestris pv. vesicatoria complements spontaneous race-change mutants

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.