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PubMed2003

Natural selection for polymorphism in the disease resistance gene Rps2 of Arabidopsis thaliana.

Pathogen resistance is an ecologically important phenotype increasingly well understood at the molecular genetic level. In this article, we examine levels of avrRpt2-dependent resistance and Rps2 locus DNA sequence variability in a worldwide sample of 27 accessions of Arabidopsis thaliana. The rooted parsimony tree of Rps2 sequences drawn from a diverse set of ecotypes includes a deep bifurcation separating major resistance and susceptibility clades of alleles. We find evidence for selection maintaining these alleles and identify the N-terminal part of the leucine-rich repeat region as a probable target of selection. Additional protein variants are found within the two major clades and correlate well with measurable differences among ecotypes in resistance to the avirulence gene avrRpt2 of the pathogen Pseudomonas syringae. Long-lived polymorphisms have been observed for other resistance genes of A. thaliana; the Rps2 data suggest that the long-term maintenance of phenotypic variation in resistance genes may be a general phenomenon and are consistent with diversifying selection acting in concert with selection to maintain variation.

Arabidopsis↗
PubMed2000

Induction of Ltp (lipid transfer protein) and Pal (phenylalanine ammonia-lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae.

The expression of a lipid transfer protein (LTP) gene is regulated in Oryza sativa roots in response to colonization by the mycorrhizal fungus Glomus mosseae. Transcript levels increased when the fungus forms appressoria and penetrates the root epidermis and decreased at the onset of the intercellular colonization of the root cortex. The analysis of histochemical GUS staining in transgenic rice plants carrying the Ltp/Gus construct confirm the induction of LTP: gene associated with fungal appressoria formation and penetration area. The induction of Ltp gene expression coincided in time with a transient increase in the expression of a phenylalanine ammonia-lyase (PAL:) gene and a transient accumulation of salicylic acid (SA) in the mycorrhizal roots. The expression of LTP: and PAL: was induced in rice roots after treatment with SA and Pseudomonas syringae indicating that both genes could be implicated in the plant defence response. The exogenous application of SA to rice interacting with the mycorrhizal fungus did not affect appressoria formation but, instead, resulted in a transient delay of root mycorrhization. Nevertheless, although LTP: maintained a prolonged SA-induced expression level, mycorrhizal formation could still proceed.

Antigens, Plant↗
PubMed2004

Elemental sulphur as an induced antifungal substance in plant defence.

Man's oldest fungicide has probably long functioned in this role in plants, as a natural component of induced antifungal defence. Elemental sulphur (S(0)) is the only inorganic phytoalexin and the only phytoalexin produced by so many different taxa. S(0) (detected by GC-MS as (32)S(8)) is produced in representative species of Sterculiaceae (cocoa), Solanaceae (tomato, tobacco), Malvaceae (cotton), and Leguminosae (French bean) in response to xylem-invading fungal and bacterial pathogens. Production was more rapid and intensive in disease-resistant genotypes. Gene expression for S(0) production may be xylem-specific as S(0) was not present in leaves of six species undergoing hypersensitivity to Pseudomonas syringae. Anomalously, high constitutive S(0) levels occurred in leaves of Arabidopsis and Brassica oleracea. S(0) was highly toxic (ED(50) 1-3 microg ml(-1)) to many fungal pathogens representing ascomycetes, basidiomycetes, and deuteromycetes, but not to an oomycete, Phytophthora, or to bacteria. Levels in cocoa and tomato xylem and Arabidopsis leaves were potentially inhibitory, but in other interactions were below theoretically toxic concentrations. However, S(0) accumulation is highly localized, suggesting that the element is produced in sufficient amounts, at the right time and place to be effective. SEM-EDX revealed S in tomato and cocoa xylem walls, xylem parenchyma, and vascular gels and tyloses, all sites appropriate to counter vascular pathogenic Verticillium dahliae. Transient increases in sulphate, glutathione and cysteine occurred in tomato xylem. The sulphate may reflect the over-expression of sulphate transporters, but the thiols might be possible precursors. Analysis of differential gene expression should reveal what may be a novel biosynthetic pathway of S(0) formation in eukaryotes.

Antifungal Agents↗
PubMed2006

Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid.

This paper reports on the identification and characterization of a new ATP-binding cassette (ABC) transporter which was identified as a salicylic acid-induced gene from soybean (Glycine max cv. Williams 82) in a subtractive suppression hybridization approach. A fragment of an ABC-transporter gene was used to isolate a full-length cDNA clone for this gene with a length of 4750 bp. The encoded protein has a length of 1447 amino acids and is composed of two similar repeat units typical of full-size ABC transporters. The sequence displays a close relationship to plant pleiotropic drug resistance (PDR)-type transporters and, on a homology basis, clusters together with the Arabidopsis thaliana PDR12 gene, suggesting GmPDR12 as a name for the gene isolated from soybean. GmPDR12 is rapidly responsive to salicylic acid and methyl jasmonate. The mRNA starts to accumulate 30 min after the addition of the signalling compounds. Salicylic acid is required for the execution of the hypersensitive reaction in soybean cell suspension cultures inoculated with Pseudomonas syringae pv. glycinea. It has been demonstrated previously that salicylic acid can be substituted by a variety of functional analogues of salicylic acid. All of these compounds lead to a strong and rapid transcriptional activation of GmPDR12, suggesting a shared signalling pathway.

ATP-Binding Cassette Transporters↗
PubMed1986

Conserved repeats in diverged ice nucleation structural genes from two species of Pseudomonas.

Sequence analysis shows that an ice nucleation gene (inaW) from Pseudomonas fluorescens is related to the inaZ gene of Pseudomonas syringae. The two genes have diverged by many amino acid substitutions, and have effectively randomized the third bases of homologous codons. By reference to their potential for change, it is shown that certain conserved features must have been maintained by selection pressure. In particular, their conservation of internal sequence repetition, with three orders of repeat periodicity in each gene, suggests that the pattern of repetition is significant to the gene products' function. We propose models for the structure of the gene products in which each order of periodicity would be required for the nucleation function.

Bacterial Proteins↗
PubMed1986

Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci.

The nucleotide sequence of a Pseudomonas trans-zeatin producing gene (ptz) from the pCK1 plasmid of Pseudomonas syringae pv. savastanoi strain 1006 has been determined. This gene confers upon E. coli the ability to synthesize and secrete several cytokinins including trans-zeatin, iso-pentenyladenine and their respective N9-ribosyl derivatives. Sequence analysis indicates an open reading frame encoding a protein of 234 amino acids with a molecular weight of 26,816. Significant sequence homology is found between ptz and both the tzs and tmr genes from Agrobacterium tumefaciens. The results suggest a close relationship between the cytokinin biosynthetic pathways in P. savastanoi and A. tumefaciens.

Amino Acid Sequence↗
PubMed2004

PeerGAD: a peer-review-based and community-centric web application for viewing and annotating prokaryotic genome sequences.

PeerGAD is a web-based database-driven application that allows community-wide peer-reviewed annotation of prokaryotic genome sequences. The application was developed to support the annotation of the Pseudomonas syringae pv. tomato strain DC3000 genome sequence and is easily portable to other genome sequence annotation projects. PeerGAD incorporates several innovative design and operation features and accepts annotations pertaining to gene naming, role classification, gene translation and annotation derivation. The annotator tool in PeerGAD is built around a genome browser that offers users the ability to search and navigate the genome sequence. Because the application encourages annotation of the genome sequence directly by researchers and relies on peer review, it circumvents the need for an annotation curator while providing added value to the annotation data. Support for the Gene Ontology vocabulary, a structured and controlled vocabulary used in classification of gene roles, is emphasized throughout the system. Here we present the underlying concepts integral to the functionality of PeerGAD.

Computational Biology↗
PubMed1999

Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect.

Sarcotoxin IA is a bactericidal peptide of 39 amino acids found in the common flesh fly, Sarcophaga peregrina. Many agronomically important bacteria in Japan are killed by this peptide at sub-micro molar levels, and the growth of tobacco and rice suspension cultured cells is not inhibited with less than 25 microM. Transgenic tobacco plants which overexpress the peptide, i.e. over 250 pmol per gram of fresh leaf, under the control of a high expression constitutive promoter showed enhanced resistance to the pathogens for wild fire disease (Pseudomonas syringae pv. tabaci) and bacterial soft rot disease (Erwinia carotovora subsp. carotovora).

Animals↗
PubMed2000

Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme.

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.

Blotting, Southern↗
PubMed2002

A pathogen-responsive cDNA from potato encodes a protein with homology to a phosphate starvation-induced phosphatase.

Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv. maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks. By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease. The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato. Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene.

Amino Acid Sequence↗
PubMed2005

The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain.

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.

Amino Acid Sequence↗
PubMed2005

Microarray analysis of the gene expression profile induced by the endophytic plant growth-promoting rhizobacteria, Pseudomonas fluorescens FPT9601-T5 in Arabidopsis.

Pseudomonas fluorescens FPT9601-T5 was originally identified as an endophytic plant growth-promoting rhizobacteria (PGPR) on tomato. To perform a molecular dissecttion of physiological and biochemical changes occurring in the host triggered by P. fluorescens FPT9601-T5 colonization, the model plant Arabidopsis was used in this study. Root colonization of Arabidopsis with P. fluorescens FPT9601-T5 promoted plant growth later than three weeks after inoculation and partially suppressed disease symptoms caused by Pseudomonas syringae pv. tomato DC3000, indicating that P. fluorescens FPT9601-T5 acted as a PGPR on Arabidopsis. To obtain a global view on transcript modification during the Arabidopsis-FPT9601-T5 interaction, we performed microarray analysis using Affymetrix Genechip probe arrays representing approximately 22,800 genes. The results showed that 95 and 105 genes were up- or down-regulated, respectively, more than twofold in FPT9601-T5-treated Arabidopsis plants as compared with control plants. Those up-regulated included genes involved in metabolism, signal transduction, and stress response. Noteworthy, upon FPT9601-T5 colonization, putative auxin-regulated genes and nodulin-like genes were up-regulated, and some ethylene-responsive genes were down-regulated. Our results suggest that P. fluorescens FPT9601-T5 triggered plant responses in a manner similar to known PGPR and, at least in some aspects, to rhizobia.

Arabidopsis↗
PubMed2005

Identification of arabidopsis loci required for susceptibility to the downy mildew pathogen Hyaloperonospora parasitica.

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.

Arabidopsis↗
PubMed2006

Regulation of the type III secretion system in phytopathogenic bacteria.

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.

Gene Expression Regulation, Bacterial↗
PubMedDate not supplied

Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868.

Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide. They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB. The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene. Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp. savastanoi. The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868. We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site. The deletion was caused during the second transposition or by later recombination between the two IS868 copies. Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes. Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution.

Agrobacterium tumefaciens↗
PubMedDate not supplied

Differential expression of tomato proteinase inhibitor I and II genes during bacterial pathogen invasion and wounding.

Expression of proteinase inhibitor I and II genes was investigated during infection by Pseudomonas syringae pv. tomato, the causal agent of bacterial speck disease in tomato. Inoculation of leaves with P. s. pv. tomato of two inbred tomato lines that are resistant and susceptible to the pathogen resulted in the accumulation of proteinase inhibitor I and II mRNAs in this organ. Our data showed that in the lines used in this study, proteinase inhibitor II mRNAs accumulated in leaves to higher levels than proteinase inhibitor I mRNA in response to P. s. pv. tomato infection and wounding. Proteinase inhibitor II mRNAs accumulated more rapidly in disease-resistant than in disease-susceptible plants. Proteinase inhibitor I mRNAs were first detected in the disease-susceptible line during infection and wounding. In contrast to wounding, the systemic induction of these genes during pathogen ingression was limited. These data show that the plant proteinase inhibitors constitute one of the components of the plant defense system that are induced in response to bacterial pathogen invasion.

Gene Expression Regulation↗
PubMedDate not supplied

Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava.

Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel- mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsicum↗
PubMedDate not supplied

Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens.

The plant hormone ethylene has been hypothesized to play roles both in disease resistance and in disease susceptibility. These processes were examined by using isogenic virulent and avirulent bacterial pathogens and mutants of Arabidopsis thaliana that were altered in ethylene physiology. Ethylene-insensitive ein1 and ein2 mutants of Arabidopsis were resistant to Pseudomonas syringae pv. tomato made avirulent by the addition of the cloned avirulence genes avrRpt2, avrRpm1, or avrB; this suggests that ethylene is not required for active resistance against avirulent bacteria. In a second set of experiments, susceptibility was monitored with virulent P. s. pv. tomato, P. s. pv. maculicola, or Xanthomonas campestris pv. campestris strains. Wild-type Arabidopsis and ein1 mutants were susceptible to these strains, but ein2 mutants developed only minimal disease symptoms. Despite these reduced symptoms, virulent P. s. pv. tomato grew extensively within ein2 leaves. The Pseudomonas phytotoxin coronatine induces ethylene biosynthesis and diseaselike symptoms on many plant species, but the reduced symptomology of ein2 mutants could not be attributed to insensitivity to coronatine. The enhanced disease tolerance of ein2 plants suggests that ethylene may mediate pathogen-induced damage, but the absence of tolerance in ein1 mutants has yet to be explained.

Drug Resistance↗