Nickolas J. Panopoulos

Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria

Genes of plant-pathogenic bacteria controlling hypersensitive response (HR) elicitation and pathogenesis were designated ‘hrp’ by Lindgren et al. in 1986 (J Bacteriol 168: 512–522). hrp genes have been characterized in several species of the four major genera of Gramnegative plant pathogens, Erwinia, Pseudomonas, Ralstonia (a new proposed genus including Pseudomonas solanacearum) and Xanthomonas. To date, hrp genes have been found mainly in large clusters, and they have been shown to be conserved physically and, in many cases, functionally among different bacteria. Hybridization studies and genetic analyses have revealed the presence of functional hrp genes even in species that are not typically observed to elicit an HR, such as Erwinia chrysanthemi and Erwinia stewartii, suggesting that hrp genes may be common to all Gram-negative plant pathogens, possibly excluding Agrobacterium spp. Current knowledge of hrp genes has been reviewed by Bonas (1994, Curr Top Microbiol Immunol 192: 79–98) and by Van Gijsegem et al. (1995, In Pathogenesis and Host–Parasite Specificity in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Basis. Volume 1. (Kohmoto et al., eds); Oxford: Pergamon Press, pp. 273–292). The nucleotide sequences of four hrp gene clusters, those of Ralstonia solanacearum (previously P. solanacearum) (Genin et al., 1992, Mol Microbiol 6: 3065–3076; Gough et al., 1992, Mol Plant–Microbe Interact 5: 384–389; Gough et al., 1993, Mol Gen Genet 239: 378–392; Van Gijsegem et al., 1995, Mol Microbiol 15: 1095–1114), Erwinia amylovora (Bogdanove et al., 1996, J Bacteriol 178: 1720– 1730; Wei and Beer, 1993, J Bacteriol 175: 7958–7967; Wei and Beer, 1995, J Bacteriol 177: 6201–6210; Wei et al., 1992, Science 257: 85–88; S. V. Beer, unpublished), Pseudomonas syringae pv. syringae (Huang et al., 1992, J Bacteriol 174: 6878–6885; Huang et al., 1993, Mol Plant–Microbe Interact 6: 515–520; Huang et al., 1995, Mol Plant–Microbe Interact 8: 733–746; Lidell and Hutcheson, 1994, Mol Plant–Microbe Interact 7: 488–497; Preston et al., 1995, Mol Plant–Microbe Interact 8: 717–732; Xiao et al., 1994, J Bacteriol 176: 1025–1036), and Xanthomonas campestris pv. vesicatoria (Fenselau et al., 1992, Mol Plant–Microbe Interact 5: 390–396; Fenselau and Bonas, 1995, Mol Plant–Microbe Interact 8: 845–854; U. Bonas, unpublished), have been largely determined. These clusters each contain more than twenty genes, many of which encode components of a novel proteinsecretion pathway designated ‘type III’. It has been shown directly that various extracellular proteins involved in pathogenesis and defence elicitation by plantpathogenic bacteria utilize this pathway (Arlat et al., 1994, EMBO J 13: 543–553; He et al., 1993, Cell 73: 1255–1266; Wei and Beer, 1993, ibid.), and the pathway is known to function in the export of virulence factors from the animal pathogens Salmonella typhimurium, Shigella flexneri, and Yersinia entercolitica, Yersinia pestis, and Yersinia pseudotuberculosis (for reviews, see Salmond and Reeves, 1993, Trends Biochem Sci 18: 7–12; and Van Gijsegem et al., 1993, Trends Microbiol 1: 175– 180). Nine type III secretion genes are conserved among all four of the plant pathogens listed above and among the animal pathogens. Based on sequence analysis and some experimental evidence, they are believed to encode one outer-membrane protein, one outer-membrane-associated lipoprotein, five inner-membrane proteins, and two cytoplasmic proteins, one of which is a putative ATPase. All of the predicted gene products, except the outer-membrane protein, show significant similarity to components of the flagellar biogenesis complex (for reviews see Blair, 1995, Annu Rev Microbiol 49: 489–522; and Bischoff and Ordal, 1992, Mol Microbiol 6: 23–28). We herein refer to the hrp-encoded type III pathway as the ‘Hrp pathway’. Because hrp genes have been characterized independently in diverse plant-pathogenic bacteria, hrp gene nomenclature differs in different species, and it is not always consistent even within the same organism. Different designations are used for homologous genes, and, even worse, the same designation is used for different genes in different organisms. For example, hrpI of E. amylovora is homologous with hrpC2 of X. campestris pv. vesicatoria and hrpO of R. solanacearum, and the homologue in P. syringae pv. syringae appears in the literature both as hrpI and as hrpJ2. Also, ‘hrpN ’ in R. solanacearum designates a secretion-pathway gene, whereas in E. amylovora, ‘hrpN ’ designates the gene encoding the elicitor harpin. Furthermore, in many bacteria the number of known hrp genes approaches 26. In anticipation of exhausting the alphabet, some authors chose to designate hrp genes with a letter and a number, creating the potential for confusion of distinct genes with alleles of the same gene. For hrp gene researchers, the current nomenclature is at best inconvenient; for other scientists, it is bewildering. Another problem exists: accumulation of knowledge about the structure of hrp loci has outpaced the accumulation of Molecular Microbiology (1996) 20(3), 681–683

Conserved features of type III secretion

Type III secretion systems (TTSSs) are essential mediators of the interaction of many Gram‐negative bacteria with human, animal or plant hosts. Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens. Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data. Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum. Recent comparative genomic analysis, bioinformatic genome mining and genome‐wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria. Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries. Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge.

Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae

Chemical or biological synthesis of plant secondary metabolites has attracted increasing interest due to their proven or assumed beneficial properties and health promoting effects. Resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and the flavonols kaempferol and quercetin have been shown to possess high nutritional and agricultural value. Four metabolically engineered yeast strains harboring plasmids with heterologous genes for enzymes involved in the biosynthesis of these compounds from phenylalanine have been constructed. Time course analyses of precursor utilization and end-product accumulation were carried out establishing the production of 0.29-0.31 mg/L of trans-resveratrol, 8.9-15.6 mg/L of naringenin, 0.1-7.7 mg/L of genistein, 0.9-4.6 mg/L of kaempferol and 0.26-0.38 mg/L of quercetin in defined media under optimal growth conditions. The recombinant yeast strains can be used further for the construction of improved flavonoid- and stilbenoid-overproducers.

The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit

The right part of the hrp cluster of Pseudomonas syringae pv. phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR. In this study we determined the sequence of hrpR and analysed the functional organization of the two genes. HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two‐component regulatory system. This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv. syringae. The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product. DNA‐protein binding studies and site‐directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site. This HrpR binding site has mapped in a fragment which is located 378–609 nucleotides upstream of the hrpS transcription start site. The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical ‐12/‐24 promoter motif.

Playing the Harp : Evolution of Our Understanding of hrp/hrc Genes

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.

Engineered Polyamine Catabolism Preinduces Tolerance of Tobacco to Bacteria and Oomycetes

Polyamine oxidase (PAO) catalyzes the oxidative catabolism of spermidine and spermine, generating hydrogen peroxide. In wild-type tobacco (Nicotiana tabacum ‘Xanthi’) plants, infection by the compatible pathogen Pseudomonas syringae pv tabaci resulted in increased PAO gene and corresponding PAO enzyme activities; polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine was excreted into the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher hydrogen peroxide accumulation. Moreover, plants overexpressing PAO showed preinduced disease tolerance against the biotrophic bacterium P. syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in a specific apoplast-localized plant-pathogen interaction, which also signals parallel defense responses, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of plant pathogens.

Melon ascorbate oxidase: cloning of a multigene family, induction during fruit development and repression by wounding.

A small family of at least four genes encoding melon ascorbate oxidase (AO) has been identified and three members of it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymes homologous to ascorbate oxidases from other plants and similar to other multicopper oxidases. We describe detailed molecular studies addressing melon AO expression during organ specific differentiation, fruit development and ripening, and in response to wounding. In particular, AO transcript accumulation was induced in ovaries and the outer mesocarp of mature preclimacteric melon fruits, before the expression of genes encoding the necessary enzymatic activities for ethylene biosynthesis. On the other hand, AO was not expressed in late stages of fruit ripening and was repressed in wounded fruits. The role of ethylene in transcriptional regulation of AO is discussed.

Elicitation of hypersensitive cell death by extracellularly targeted HrpZPsph produced in planta.

The ability of the Pseudomonas syringae pv. phaseolicola harpin (HrpZPsph) to elicit hypersensitive response was investigated in three Nicotiana genotypes. The hrpZPsph gene was placed under chemical regulation (tetracycline induction) in TetR+ Nicotiana tabacum cv. Wisconsin 38 (W38) or was transiently expressed in N. benthamiana following infection with a PVX-derived vector and in three Nicotiana genotypes by agroinfiltration. The constructs were designed to express either the canonical form of harpin (HrpZPsph) or an N-terminally extended version of the protein carrying the signal peptide portion of the tobacco pathogenesis-related protein PR1a (SP-HrpZPsph). Stable transformants of N. tabacum cv. W38 did not develop necrosis upon induction with tetracycline, probably as a result of insufficient harpin accumulation. In contrast, N. benthamiana plants infected with the PVX constructs produced high concentrations of harpin in biologically active form, but only those expressing the secretable form of harpin developed necrotic symptoms. These symptoms were less severe than those caused by PVX::avrPto; however, they were accompanied by induction of hsr203J, a hypersensitive response-specific gene transcript. These results suggest that the plant cellular receptor(s) for harpin is extracellular.

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Abstract Plasmid pAS8Tc s rep -1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas . The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.