bluuebunny/crossref_metadata_2025 huggingface dataset:
Mating-Type Genes from Asexual Phytopathogenic AscomycetesFusarium oxysporumandAlternaria alternata
Authors: Arie Tsutomu , Kaneko Isao , Yoshida Takanobu , Noguchi Masami , Nomura Yoshikuni , Yamaguchi Isamu
Published: May 2007

Mating-type (MAT) loci were cloned from two asexual (mitosporic) phytopathogenic ascomycetes, Fusarium oxysporum (a pyrenomycete) and Alternaria alternata (a loculoascomycete), by a polymerase chain reaction (PCR)-based strategy. The conserved high mobility group (HMG) box domain found in the MAT1-2-1 protein was used as a starting point for cloning and sequencing the entire MAT1-2 idiomorph plus flanking regions. Primer pairs designed to both flanking regions were used to amplify the opposite MAT1-1 idiomorph. The MAT1-1 and MAT1-2 idiomorphs were approximately 4.6 and 3.8 kb in F. oxysporum and approximately 1.9 and 2.2 kb in A. alternata, respectively. In both species, the MAT1-1 idiomorph contains at least one gene that encodes a protein with a putative alpha box domain and the MAT1-2 idiomorph contains one gene that encodes a protein with a putative HMG box domain. MAT-specific primers were used to assess the mating type of F. oxysporum and A. alternata field isolates by PCR. MAT genes from A. alternata were expressed. The A. alternata genes were confirmed to be functional in a close sexual relative, Cochliobolus heterostrophus, by heterologous expression.
Restriction Enzyme-Mediated Integration Used to Produce Pathogenicity Mutants of Colletotrichum graminicola
Authors: Thon M. R. , Nuckles E. M. , Vaillancourt L. J.
Published: May 2007

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.
Elicitation of Hypersensitive Cell Death by Extracellularly Targeted HrpZPsph Produced In Planta
Authors: Tampakaki Anastasia P. , Panopoulos Nickolas J.
Published: May 2007

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.
Beticolins, Nonpeptidic, Polycyclic Molecules Produced by the Phytopathogenic Fungus Cercospora beticola, as a New Family of Ion Channel-Forming Toxins
Authors: Goudet Cyril , Milat Marie-Louise , Sentenac Hervé , Thibaud Jean-Baptiste
Published: May 2007

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Véry, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H.Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planarlipid bilayers. All beticolins were able to form ionchannels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.
Mapping of Avirulence Genes in the Rice Blast Fungus, Magnaporthe grisea, with RFLP and RAPD Markers
Authors: Dioh Waly , Tharreau Didier , Notteghem Jean Loup , Orbach Marc , Lebrun Marc-Henri
Published: May 2007

Three genetically independent avirulence genes, AVR1-Irat7, AVR1-MedNoï, and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoï and AVR1-Ku86 were closely linked to te-lomere RFLPs such as marker TelG (6 cM from AVR1-MedNoï) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.
Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria
Authors: Heeb Stephan , Itoh Yoshifumi , Nishijyo Takayuki , Schnider Ursula , Keel Christoph , Wade Julie , Walsh Ultan , O'Gara Fergal , Haas Dieter
Published: May 2007

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.
Is a Fully Established Arbuscular Mycorrhizal Symbiosis Required for Bioprotection of Pisum sativum Roots against Aphanomyces euteiches?
Authors: Slezack Sophie , Dumas-Gaudot Eliane , Paynot Michel , Gianinazzi Silvio
Published: May 2007

Bioprotection of pea roots against Aphanomyces euteiches by the arbuscular mycorrhizal fungus G. mosseae was demonstrated to depend on a fully established symbiosis. This was related with induction of mycorrrhiza-related chitinolytic enzymes. Possible mechanisms implicated in bioprotection are discussed.
The hrpB and hrpG Regulatory Genes of Ralstonia solanacearum Are Required for Different Stages of the Tomato Root Infection Process
Authors: Vasse Jacques , Genin Stéphane , Frey Pascal , Boucher Christian , Brito Belen
Published: May 2007

hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.
Inheritance of Partial Resistance AgainstColletotrichum lindemuthianuminPhaseolus vulgarisand Co-localization of Quantitative Trait Loci with Genes Involved in Specific Resistance
Authors: Geffroy Valérie , Sévignac Mireille , De Oliveira Julio C. F. , Fouilloux Guy , Skroch Paul , Thoquet Philippe , Gepts Paul , Langin Thierry , Dron Michel
Published: May 2007

Anthracnose, one of the most important diseases of common bean (Phaseolus vulgaris), is caused by the fungus Colletotrichum lindemuthianum. A “candidate gene” approach was used to map anthracnose resistance quantitative trait loci (QTL). Candidate genes included genes for both pathogen recognition (resistance genes and resistance gene analogs [RGAs]) and general plant defense (defense response genes). Two strains of C. lindemuthianum, identified in a world collection of 177 strains, displayed a reproducible and differential aggressiveness toward BAT93 and JaloEEP558, two parental lines of P. vulgaris representing the two major gene pools of this crop. A reliable test was developed to score partial resistance in aerial organs of the plant (stem, leaf, petiole) under controlled growth chamber conditions. BAT93 was more resistant than Jalo-EEP558 regardless of the organ or strain tested. With a recombinant inbred line (RIL) population derived from a cross between these two parental lines, 10 QTL were located on a genetic map harboring 143 markers, including known defense response genes, anthracnose-specific resistance genes, and RGAs. Eight of the QTL displayed isolate specificity. Two were co-localized with known defense genes (phenylalanine ammonia-lyase and hydroxyproline-rich glycoprotein) and three with anthracnose-specific resistance genes and/or RGAs. Interestingly, two QTL, with different allelic contribution, mapped on linkage group B4 in a 5.0 cM interval containing Andean and Mesoamerican specific resistance genes against C. lindemuthianum and 11 polymorphic fragments revealed with a RGA probe. The possible relationship between genes underlying specific and partial resistance is discussed.
The Colletotrichum lagenarium MAP Kinase Gene CMK1 Regulates Diverse Aspects of Fungal Pathogenesis
Authors: Takano Yoshitaka , Kikuchi Taisei , Kubo Yasuyuki , Hamer John E. , Mise Kazuyuki , Furusawa Iwao
Published: May 2007

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.
Quorum Sensing in the Plant Pathogen Erwinia carotovora subsp. carotovora: The Role of expREcc
Authors: Andersson Robert A. , Eriksson Anders R. B. , Heikinheimo Riikka , Mäe Andres , Pirhonen Minna , Kõiv Viia , Hyytiäinen Heidi , Tuikkala Anne , Palva E. Tapio
Published: May 2007

The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-l-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expREcc, is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpREcc protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii(formerly Erwinia stewartii subsp. stewartii) and to the ExpREch protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expREcc gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpREcc were probably mediated by changes in OHHL levels. Inactivation of expREcc resulted in increased OHHL levels during early logarithmic growth. In addition, over-expression of expREcc caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpREcc. ExpREcc did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.
Mutants of Xanthomonas oryzae pv. oryzae Deficient in General Secretory Pathway Are Virulence Deficient and Unable to Secrete Xylanase
Authors: Ray Suvendra K. , Rajeshwari R. , Sonti Ramesh V.
Published: May 2007

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Trans-poson mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with trans-poson-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space.
Recessive and Dominant Genes Interfere with the Vascular Transport of Potato virus A in Diploid Potatoes
Authors: Hämäläinen Jaana H. , Kekarainen Tuija , Gebhardt Christiane , Watanabe Kazuo N. , Valkonen Jari P. T.
Published: May 2007

Resistance to Potato virus A (PVA) was examined in a diploid cross involving Solanum tuberosum subsp. andigena as a resistance source. Hypersensitive resistance (HR) to PVA cosegregated with extreme resistance (ER) to Potato virus Y conferred by the dominant gene Ryadg on chromosome XI. Hence, HR to PVA was controlled by a novel, dominant resistance gene closely linked to Ryadg, or Ryadg recognized both viruses but conferred a different type of resistance to each virus. The HR prevented systemic infection with PVA following mechanical inoculation but not following graft inoculation. Another, recessive gene, ra, that may be linked or even allelic with Ryadg fully blocked vascular transport of PVA in graft-inoculated plants. Hence, a possibility exists that the genes for the three types of resistance to potyviruses may reside at the same, resistance gene-rich chromosome region syntenic in solanaceous species and might be related. The gene ra acted against all of the three PVA strains tested and, therefore, the avirulence determinants could not be mapped. However, also, PVA strain-specific resistance was found in the progeny. It was overcome by mutations introduced into the viral genome-linked protein and the helper component proteinase and/or the coat protein.
A Two-Component Regulatory System, pehR-pehS, Controls Endopolygalacturonase Production and Virulence in the Plant Pathogen Erwinia carotovora subsp. carotovora
Authors: Flego Diana , Marits Reet , Eriksson Anders R. B. , Kõiv Viia , Karlsson Maj-Brit , Heikinheimo Riikka , Palva E. Tapio
Published: May 2007

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.
Comparison of the Hypersensitive Response Induced by the Tomato Cf-4 and Cf-9 Genes in Nicotiana spp.
Authors: Thomas Colwyn M. , Tang Saijun , Hammond-Kosack Kim , Jones Jonathan D. G.
Published: May 2007

We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato. We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N. tabacum and N. benthamiana). The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9. We also compared HR induction with Agrobacterium-mediated transient expression. The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9. Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco.
Sinorhizobium meliloti nfe (Nodulation Formation Efficiency) Genes Exhibit Temporal and Spatial Expression Patterns Similar to Those of Genes Involved in Symbiotic Nitrogen Fixation
Authors: García-Rodríguez Fernando M. , Toro Nicolás
Published: May 2007

The nfe genes (nfeA, nfeB, and nfeD) are involved in the nodulation efficiency and competitiveness of the Sinorhizobium meliloti strain GR4 on alfalfa roots. The nfeA and nfeB genes are preceded by functional nif consensus sequences and NifA binding motifs. Here, we determined the temporal and spatial expression patterns of the nfe genes in symbiosis with alfalfa. Translational fusions of the nfe promoters with the gusA gene and reverse transcription-polymerase chain reaction analyses indicate that they are expressed and translated within mature nitrogen-fixing nodules and not during early steps of nodule development. Within the nodules the three nfe genes exhibit a spatial expression pattern similar to that of genes involved in symbiotic nitrogen fixation. We show that nfeB and nfeD genes are expressed not only from their own promoters but also from the upstream nfe promoter sequences. Furthermore, with the use of specific antibodies the NfeB and NfeD proteins were detected within the root nodule bac-teroid fraction. Finally, NfeB was inmunolocalized in the bacteroid cell membrane whereas NfeD was detected in the bacteroid cytoplasm.
A Cluster of Mutations Disrupt the Avirulence but Not the Virulence Function of AvrPto
Authors: Shan Libo , He Ping , Zhou Jian-Min , Tang Xiaoyan
Published: May 2007

avrPto in Pseudomonas syringae pv. tomato encodes an avirulence protein that triggers race-specific resistance in tomato plants carrying Pto. The AvrPto protein is secreted from P. syringae pv. tomato to plant cells through the type III secretion pathway and activates race-specific resistance by a direct interaction with the Pto protein. Here we report that avrPto enhances the virulence of P. syringae pv. tomato in a strain-dependent manner in tomato plants lacking Pto. To determine whether the virulence function can be structurally separated from the avirulence function, we examined the virulence activity of a group of AvrPto mutants that carry single amino acid substitutions and lack the avirulence activity on tomato plants. Three mutants that were clustered in the center of AvrPto exhibited virulence activity in tomato plants with or without Pto. The rest of the mutations abolished the virulence. The identification of these mutants suggested that the aviru-lence function of AvrPto can be structurally separated from the virulence function.
Disruption of a Gene Essential for Sulfoquinovosyldiacylglycerol Biosynthesis in Sinorhizobium meliloti Has No Detectable Effect on Root Nodule Symbiosis
Authors: Weissenmayer Barbara , Geiger Otto , Benning Christoph
Published: May 2007

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3′ of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sul-folipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.
Genetic Mapping of the Tsw Locus for Resistance to the Tospovirus Tomato spotted wilt virus in Capsicum spp. and Its Relationship to the Sw-5 Gene for Resistance to the Same Pathogen in Tomato
Authors: Jahn Molly , Paran Ilan , Hoffmann Katrin , Radwanski Elaine R. , Livingstone Kevin D. , Grube Rebecca C. , Aftergoot Ester , Lapidot Moshe , Moyer James
Published: May 2007

The Tsw gene conferring dominant resistance to the Tospo-virus Tomato spotted wilt virus (TSWV) in Capsicum spp. has been tagged with a random amplified polymorphic DNA marker and mapped to the distal portion of chromosome 10. No mapped homologues of Sw-5, a phenotypically similar dominant TSWV resistance gene in tomato, map to this region in C. annuum, although a number of Sw-5 homologues are found at corresponding positions in pepper and tomato. The relationship between Tsw and Sw-5 was also examined through genetic studies of TSWV. The capacity of TSWV-A to overcome the Tsw gene in pepper and the Sw-5 gene in tomato maps to different TSWV genome segments. Therefore, despite phenotypic and genetic similarities of resistance in tomato and pepper, we infer that distinct viral gene products control the outcome of infection in plants carrying Sw-5 and Tsw, and that these loci do not appear to share a recent common evolutionary ancestor.
Entry of Rhizobium leguminosarum bv. viciae into Root Hairs Requires Minimal Nod Factor Specificity, but Subsequent Infection Thread Growth Requires nodO or nodE
Authors: Walker Simon A. , Downie J. Allan
Published: May 2007

Using various mutant strains of Rhizobium leguminosarum bv. viciae, we have investigated the role of nodO in stimulating infection thread development in vetch and pea. Analysis of R. leguminosarum bv. viciae nodE and nodO mutants revealed no significant difference from the wild-type infection phenotype. Conversely, an R. leguminosarum bv. viciae nodE nodO double mutant was severely impaired in its ability to form normal infection threads. This strain displayed a number of novel infection-related events, including intracellular accumulations of bacteria at the base of root hairs, distended and enlarged infection threads, and reversed threads growing up root hairs. Since normal infection was seen in a nodE mutant, nodO must suppress these abnormal infection phenomena. A deletion mutant, retaining only the nodD and nodABCIJ genes, also formed intracellular accumulations at the base of root hairs. Addition of R. leguminosarum bv. viciae nodO could alleviate this phenotype and restore some infection thread formation, although these threads appeared to be abnormal. Exogenous application of R. leguminosarum bv. viciae Nod factors could not alleviate the aberrant infection phenotype. Our results show that the most basic Nod factor structure can allow bacterial entry into the root hair, and that nodO can promote subsequent infection thread development.
The ORF8 Gene Product of Agrobacterium rhizogenes TL-DNA Has Tryptophan 2-Monooxygenase Activity
Authors: Lemcke Kai , Prinsen Els , van Onckelen Harry , Schmülling Thomas
Published: May 2007

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.
Nod Factors of Rhizobium leguminosarum bv. viciae and Their Fucosylated Derivatives Stimulate a Nod Factor Cleaving Activity in Pea Roots and Are Hydrolyzed In Vitro by Plant Chitinases at Different Rates
Authors: Ovtsyna Alexandra O. , Schultze Michael , Tikhonovich Igor A. , Spaink Herman P. , Kondorosi Éva , Kondorosi Ádám , Staehelin Christian
Published: May 2007

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Non-modified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.
Inhibition of Fungal and Bacterial Plant Pathogens by Synthetic Peptides: In Vitro Growth Inhibition, Interaction Between Peptides and Inhibition of Disease Progression
Authors: Ali Gul Shad , Reddy A. S. N.
Published: May 2007

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 μM and by 100% at 20 μM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 μM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.
Lotus japonicus Contains Two Distinct ENOD40 Genes That Are Expressed in Symbiotic, Nonsymbiotic, and Embryonic Tissues
Authors: Flemetakis Emmanouil , Kavroulakis Nektarios , Quaedvlieg Nicolette E. M. , Spaink Herman P. , Dimou Maria , Roussis Andreas , Katinakis Panagiotis
Published: May 2007

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.
Modulations in Gene Expression and Mapping of Genes Associated with Cyst Nematode Infection of Soybean
Authors: Vaghchhipawala Zarir , Bassüner Ronald , Clayton Kathryn , Lewers Kimberley , Shoemaker Randy , Mackenzie Sally
Published: May 2007

Infection of the soybean root by the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) induces a well-documented, yet poorly understood, response by the host plant. The plant response, involving the differentiation of a feeding structure, or “syncytium,” facilitates the feeding and reproduction of the nematode to the detriment of the host. We used a genetic system involving a single dominant soybean gene conferring susceptibility to an inbred nematode strain, VL1, to characterize the nematode-host interaction in susceptible line PI 89008. The restriction fragment length polymorphism marker pB053, shown to map to a major SCN resistance locus, cosegregates with resistance among F2 progeny from the PI 89008 × PI 88287 cross. Cytological examination of the infection process confirmed that syncytium development in this genetic system is similar to that reported by others who used noninbred nematode lines. Our study of infected root tissue in the susceptible line PI 89008 revealed a number of genes enhanced in expression. Among these are catalase, cyclin, elongation factor 1α, β-1,3-endoglucanase, hydroxymethylglutaryl coenzyme A reductase, heat shock protein 70, late embryonic abundant protein 14, and formylglycinamidine ribonucleotide synthase, all of which we have genetically positioned on the public linkage map of soybean. Formylglycinamidine ribonucleotide synthase was found to be tightly linked with a major quantitative trait locus for SCN resistance. Our observations are consistent with the hypothesis proposed by others that feeding site development involves the dramatic modulation of gene expression relative to surrounding root cells.
Cloning and Characterization of a Bean Udp-Glucosyltransferase cDNA Expressed During Plant-Bacterial Interactions
Authors: Sullivan T. A. , Jakobek J. L. , Lindgren P. B.
Published: May 2007

A cDNA clone, which corresponds to an RNA transcript that accumulates in bean during the hypersensitive reaction, was isolated and designated Hra25 (for hypersensitive reaction associated). Hra25 is predicted to encode a UDP-glucosyltransferase. Northern analysis was used to study Hra25 transcript accumulation in bean in response to incompatible and compatible strains of Pseudomonas syringae, an Hrp¯ mutant, and wounding. Our data suggest that the Hra25 transcript is activated in response to specific avr-derived signals as well as non-avr, general signals.
Azorhizobium caulinodans ORS571 Colonizes the Xylem of Arabidopsis thaliana
Authors: Stone Philip J. , O'Callaghan Kenneth J. , Davey Michael R. , Cocking Edward C.
Published: May 2007

Improved conditions were used for the aseptic growth of Arabidopsis thaliana to investigate whether xylem colonization of A. thaliana by Azorhizobium caulinodans ORS571 might occur. When seedlings were inoculated with ORS571 (pXLGD4) tagged with the lacZ reporter gene, nearly all of the plants showed blue regions of ORS571 colonization at lateral root cracks (LRC). The flavonoids naringenin and liquiritigenin significantly stimulated colonization of LRC by ORS571. Blue bands of ORS571 (pXLGD4) bacteria were observed histochemically in the xylem of intact roots of inoculated plants. Detailed microscopic analysis of sections of primary and lateral roots from inoculated A. thaliana confirmed xylem colonization. Xylem colonization also occurred with an ORS571 nodC mutant deficient in nodulation factors. There was no significant difference in the percentage of plants with xylem colonization or in the mean length of xylem colonized per plant between plants inoculated with either ORS571 (pXLGD4) or ORS571::nodC (pXLGD4), with or without naringenin.
Construction of a Derivative of Agrobacterium tumefaciens C58 That Does Not Mutate to Tetracycline Resistance
Authors: Luo Zhao-Qing , Clemente Thomas E. , Farrand Stephen K.
Published: May 2007

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.
A Phosphate Transporter Gene from the Extra-Radical Mycelium of an Arbuscular Mycorrhizal Fungus Glomus intraradices Is Regulated in Response to Phosphate in the Environment
Authors: Maldonado-Mendoza Ignacio E. , Dewbre Gary R. , Harrison Maria J.
Published: May 2007

The majority of vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. These symbioses, termed arbuscular mycorrhizas, are mutually beneficial, and the fungus delivers phosphate to the plant while receiving carbon. In these symbioses, phosphate uptake by the arbuscular mycorrhizal fungus is the first step in the process of phosphate transport to the plant. Previously, we cloned a phosphate transporter gene involved in this process. Here, we analyze the expression and regulation of a phosphate transporter gene (GiPT) in the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices during mycorrhizal association with carrot or Medicago truncatula roots. These analyses reveal that GiPT expression is regulated in response to phosphate concentrations in the environment surrounding the extra-radical hyphae and modulated by the overall phosphate status of the mycorrhiza. Phosphate concentrations, typical of those found in the soil solution, result in expression of GiPT. These data imply that G. intraradices can perceive phosphate levels in the external environment but also suggest the presence of an internal phosphate sensing mechanism.
Genetic Organization of the Pantoea stewartii subsp. stewartii hrp Gene Cluster and Sequence Analysis of the hrpA, hrpC, hrpN, and wtsE Operons
Authors: Frederick Reid D. , Ahmad Musharaf , Majerczak Doris R. , Arroyo-Rodríguez Angel S. , Manulis Shulamit , Coplin David L.
Published: May 2007

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.
The Role of G Protein Alpha Subunits in the Infection Process of the Gray Mold Fungus Botrytis cinerea
Authors: Gronover Christian Schulze , Kasulke Daniela , Tudzynski Paul , Tudzynski Bettina
Published: May 2007

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding α subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the Gαi class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.
Differential Regulation of Gene Expression in the Obligate Biotrophic Interaction of Uromyces fabae with Its Host Vicia faba
Authors: Wirsel Stefan G. R. , Voegele Ralf T. , Mendgen Kurt W.
Published: May 2007

Classical analysis of obligate biotrophic fungi revealed changes of enzyme activities or the concentration of metabolites in infected areas. However, due to the intricate integration of host and parasite metabolism, it was not possible to delineate the individual contributions of the two organisms. Here, we used reverse-transcription-polymerase chain reaction to monitor expression of genes from the rust fungus Uromyces fabae and its host Vicia faba. We focused on genes relevant for amino acid and sugar uptake and metabolism in both organisms. In the fungus, mRNA for plasma membrane ATPase was detected in spores and all infection structures. Two genes for fungal amino acid transporters showed dissimilar regulation. Transcripts for one were detected during all developmental stages, whereas those of the other appeared to be under developmental control. The latter result was also obtained for the so far only hexose transporter known from U. fabae and for one gene of the thiamine biosynthesis pathway. In the host plant, transcripts for two ATPases analyzed generally declined upon infection. Sucrose synthase expression increased in leaves, but decreased in roots. Transcript levels of glucose and sucrose transporter genes appeared unchanged. Markers for amino acid metabolism did not show a uniform trend: transcripts for asparagine synthetase increased, whereas those for two amino acid transporters either decreased or increased. Our analyses revealed that not only expression of genes in the immediate vicinity of the primary infection site is altered, but infection also influences transcription of certain genes in remote organs, like stems and roots. This demonstrates alterations in the source-sink relationships.
Complementation of the Magnaporthe grisea ΔcpkA Mutation by the Blumeria graminis PKA-c Gene: Functional Genetic Analysis of an Obligate Plant Pathogen
Authors: Bindslev Lene , Kershaw Michael J. , Talbot Nicholas J. , Oliver Richard P.
Published: May 2007

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a ΔcpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea ΔcpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the ΔcpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.