bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Screening Yields a New β-1,4-Endoglucanase Gene from Heterodera glycines that May be the Product of Recent Gene Duplication
Authors: Yan Yitang , Smant Geert , Davis Eric
Published: May 2007

Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a β-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical “HG-eng-2-like” clones had considerable differences in the 5′ untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5′ flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.
Molecular Cloning of a cDNA Encoding an Amphid-Secreted Putative Avirulence Protein from the Root-Knot NematodeMeloidogyne incognita
Authors: Semblat Jean-Philippe , Rosso Marie-Noëlle , Hussey Richard S. , Abad Pierre , Castagnone-Sereno Philippe
Published: May 2007

Amplified fragment length polymorphism fingerprinting of three pairs of Meloidogyne incognita near-isogenic lines (NILs) was used to identify markers differential between nematode genotypes avirulent or virulent against the tomato Mi resistance gene. One of these sequences, present only in the avirulent lines, was used as a probe to screen a cDNA library from second-stage juveniles (J2s) and allowed cloning of a cDNA encoding a secretory protein. The putative full-length cDNA, named map-1, encoded a 458 amino acid (aa) protein containing a predictive N-terminal secretion signal peptide. The MAP-1 sequence did not show any significant similarity to proteins deposited in databases. The internal part of the protein, however, was characterized by highly conserved repetitive motives of 58 or 13 aa. Reverse transcription polymerase chain reaction (RT-PCR) experiments confirmed that map-1 expression was different between avirulent and virulent NILs. In PCR reactions, map-1-related sequences were amplified only in nematode populations belonging to the three species against which the Mi gene confers resistance: M. arenaria, M. incognita, and M. javanica. Polyclonal antibodies raised against a synthetic peptide deduced from the MAP-1 sequence strongly labeled J2 amphidial secretions in immunofluorescence microscopy assays, suggesting that MAP-1 may be involved in the early steps of recognition between (resistant) plants and (avirulent) nematodes.
Oxidative Burst in Alfalfa-Sinorhizobium meliloti Symbiotic Interaction
Authors: Santos Renata , Hérouart Didier , Sigaud Samuel , Touati Danièle , Puppo Alain
Published: May 2007

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.
Activation of an EDS1-Mediated R-Gene Pathway in the snc1 Mutant Leads to Constitutive, NPR1-Independent Pathogen Resistance
Authors: Li Xin , Clarke Joseph D. , Zhang Yuelin , Dong Xinnian
Published: May 2007

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.
Proper Regulation of Cyclic AMP-Dependent Protein Kinase Is Required for Growth, Conidiation, and Appressorium Function in the Anthracnose Fungus Colletotrichum lagenarium
Authors: Takano Yoshitaka , Komeda Kenichi , Kojima Kaihei , Okuno Tetsuro
Published: May 2007

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpk1-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.
Selection of a Plant-Bacterium Pair as a Novel Tool for Rhizostimulation of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria
Authors: Kuiper Irene , Bloemberg Guido V. , Lugtenberg Ben J. J.
Published: May 2007

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.
Identification of Putative Parasitism Genes Expressed in the Esophageal Gland Cells of the Soybean Cyst Nematode Heterodera glycines
Authors: Gao Bingli , Allen R. , Maier Tom , Davis Eric L. , Baum Thomas J. , Hussey Richard S.
Published: May 2007

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.
Novel Genomic Locus with Atypical G+C Content that Is Required for Extracellular Polysaccharide Production and Virulence in Xanthomonas oryzae pv. oryzae
Authors: Dharmapuri Sridhar , Yashitola J. , Vishnupriya M. R. , Sonti Ramesh V.
Published: May 2007

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 51.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.
Mutational Analysis of β-Glucanase Genes from the Plant-Pathogenic Fungus Cochliobolus carbonum
Authors: Kim Hoon , Ahn Joong-Hoon , Görlach Jenifer M. , Caprari Claudio , Scott-Craig John S. , Walton Jonathan D.
Published: May 2007

Two new β-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-β1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (β1,3-β1,4) glucanase with low activity against β1,4-glucan and no activity against β1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total β1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on β1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.
Activity of Nitric Oxide Is Dependent On, But Is Partially Required for Function of, Salicylic Acid in the Signaling Pathway in Tobacco Systemic Acquired Resistance
Authors: Song Fengming , Goodman Robert M.
Published: May 2007

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.
The Rhizobium etli argC Gene Is Essential for Arginine Biosynthesis and Nodulation of Phaseolus vulgaris
Authors: Ferraioli Simona , Taté Rosarita , Caputo Emilia , Lamberti Alessandro , Riccio Anna , Patriarca Eduardo J.
Published: May 2007

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-γ-glutamylphosphate reductase activity. Unless ar-ginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oli-gosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.
Transient Accumulation of Jasmonic Acid During the Synchronized Hypersensitive Cell Death in Tobacco mosaic virus-Infected Tobacco Leaves
Authors: Seo Shigemi , Seto Hideharu , Yamakawa Hiromoto , Ohashi Yuko
Published: May 2007

Jasmonic acid (JA) transiently accumulated during temperature-dependent synchronous necrotic lesion formation in Tobacco mosaic virus-infected tobacco leaves. The accumulation of JA was preceded by activation of a tobacco mitogen-activated protein kinase, WIPK, which functions upstream of JA in wound signal transduction pathways.
Elicitin Genes Expressed In Vitro by Certain Tobacco Isolates of Phytophthora parasitica Are Down Regulated During Compatible Interactions
Authors: Colas Virginie , Conrod Sandrine , Venard Paul , Keller Harald , Ricci Pierre , Panabières Franck
Published: May 2007

Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parA1 is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parA1 expression during compatible interactions, whichever host plant is tested. Conversely, one elicitin-producing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parA1 in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen.
The Nodulation Protein NodG Shows the Enzymatic Activity of an 3-Oxoacyl-Acyl Carrier Protein Reductase
Authors: López-Lara Isabel M. , Geiger Otto
Published: May 2007

The acyl carrier protein NodF is required for the synthesis of unusual polyunsaturated fatty acids that confer specificity to lipochitin oligosaccharide nodulation (Nod) factors of Rhizobium leguminosarum. In this study, homogeneous NodF protein was used as a ligand to identify proteins of R. leguminosarum that specifically interact with NodF and presumably are involved in the biosynthesis or transfer of the unusual fatty acids. The N-terminal amino acid sequence of a 29-kDa protein that interacts strongly with NodF revealed high similarity to NodG of Rhizobium sp. N33 and to NodG of Sinorhizobium meliloti. We cloned and sequenced the gene coding for the NodG-like protein of R. leguminosarum and found it to be the product of the constitutively expressed gene fabG. FabG is the 3-oxoacyl-acyl carrier protein reductase that catalyzes the first reduction step in each cycle of fatty acid elongation. FabG of R. leguminosarum and NodG of Rhizobium sp. N33 were expressed in Escherichia coli. In both cases, the purified protein showed 3-oxoacyl-acyl carrier protein reductase activity in vitro. Therefore, NodG has the same biochemical function as FabG, and the high degree of similarity at the protein and DNA level suggest that nodG is a duplication of the housekeeping gene fabG.
Relative Effects on Virulence of Mutations in the sap, pel, and hrp Loci of Erwinia chrysanthemi
Authors: López-Solanilla Emilia , Llama-Palacios Arancha , Collmer Alan , García-Olmedo Francisco , Rodríguez-Palenzuela Pablo
Published: May 2007

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel¯), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.
Induction and Signaling of an Apoptotic Response Typified by DNA Laddering in the Defense Response of Oats to Infection and Elicitors
Authors: Tada Yasuomi , Hata Shingo , Takata Yoko , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki
Published: May 2007

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.
A Viral Double-Stranded RNA Up Regulates the Fungal Virulence ofNectria radicicola
Authors: Ahn II-Pyung , Lee Yong-Hwan
Published: May 2007

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.
A Novel Jasmonic Acid-Inducible Rice myb Gene Associates with Fungal Infection and Host Cell Death
Authors: Lee Min-Woo , Qi Min , Yang Yinong
Published: May 2007

Endogenous signal molecules such as jasmonic acid (JA) and salicylic acid (SA) play an important role in induced resistance against pathogen infection and insect herbivory. In rice seedlings, JA is an effective inducer of systemic acquired resistance (SAR) against infection of blast fungus (Pyricularia grisea). To gain further insights into JA-mediated defense signaling pathways, we isolated and characterized a pathogen- and JA-induced rice gene (JAmyb) that encodes a Myb transcription factor. The JAmyb gene was induced within 1 day after fungal infection in resistant and susceptible interactions prior to lesion formation. Unlike most defense-related genes that are activated faster and stronger in resistant interactions, JAmyb induction by blast fungus is much higher in susceptible interactions, accompanied by large lesions and extensive tissue damage. Significant induction of JAmyb also was observed during cell death and lesion formation in certain lesion mimic mutants. Interestingly, JAmyb was activated rapidly by JA or wounding, independent of de novo protein synthesis, but not by other endogenous signal molecules such as SA and abscisic acid or SAR inducers such as benzothiadiazole and probenazole. We used SA-deficient transgenic plants to further demonstrate that depletion of SA in rice did not abolish but rather enhanced blast-induced JAmyb expression. These results suggest that JAmyb is related closely to host cell death and is involved in the JA-mediated, SA-independent signaling pathways in rice.
The ABC Transporter BcatrB Affects the Sensitivity of Botrytis cinerea to the Phytoalexin Resveratrol and the Fungicide Fenpiclonil
Authors: Schoonbeek H. , Del Sorbo G. , De Waard M. A.
Published: May 2007

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection against plant defense compounds and fungicides by ATP-driven efflux mechanisms. To test this hypothesis, we cloned BcatrB, an ABC transporter-encoding gene from B. cinerea. This gene encodes a 1,439 amino acid protein with nucleotide binding fold (NBF) and transmembrane (TM) domains in a [NBF-TM6]2 topology. The amino acid sequence has 31 to 67% identity with ABC transporters from various fungi. The expression of BcatrB is up regulated by treatment of B. cinerea germlings with the grapevine phytoalexin resveratrol and the fungicide fenpiclonil. BcatrB replacement mutants are not affected in saprophytic growth on different media but are more sensitive to resveratrol and fenpiclonil than the parental isolate. Furthermore, virulence of ΔBcatrB mutants on grapevine leaves was slightly reduced. These results indicate that BcatrB is a determinant in sensitivity of B. cinerea to plant defense compounds and fungicides.
The Plant Pathogen Rhodococcus fascians Colonizes the Exterior and Interior of the Aerial Parts of Plants
Authors: Cornelis Karen , Ritsema Tita , Nijsse Jaap , Holsters Marcelle , Goethals Koen , Jaziri Mondher
Published: May 2007

Rhodococcus fascians is a plant-pathogenic bacterium that causes malformations on aerial plant parts, whereby leafy galls occur at axillary meristems. The colonization behavior on Nicotiana tabacum and Arabidopsis thaliana plants was examined. Independent of the infection methods, R. fascians extensively colonized the plant surface where the bacteria were surrounded by a slime layer. R. fascians caused the collapse of epidermal cells and penetrated intercellularly into the plant tissues. The onset of symptom development preceded the extensive colonization of the interior. The meristematic regions induced by pathogenic strain D188 were surrounded by bacteria. The nonpathogenic strain, D188-5, colonized the exterior of the plant equally well, but the linear plasmid (pFiD188) seemed to be involved in the penetration efficiency and colonization of tobacco tissues.
Medicago truncatula ENOD11: A Novel RPRP-Encoding Early Nodulin Gene Expressed During Mycorrhization in Arbuscule-Containing Cells
Authors: Journet Etienne-Pascal , El-Gachtouli Naima , Vernoud Vanessa , de Billy Françoise , Pichon Magalie , Dedieu Annie , Arnould Christine , Morandi Dominique , Barker David G. , Gianinazzi-Pearson Vivienne
Published: May 2007

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.
Induction of Hydroxycinnamoyl-Tyramine Conjugates in Pepper by Xanthomonas campestris, a Plant Defense Response Activated by hrp Gene-Dependent and hrp Gene-Independent Mechanisms
Authors: Newman Mari-Anne , von Roepenack-Lahaye Edda , Parr Adrian , Daniels Michael J. , Dow J. Maxwell
Published: May 2007

Inoculation of pepper leaves, Capsicum annuum cv. Early Calwonder ECW 10R, with strains of Xanthomonas campestris led to an accumulation of the phenolic conjugates feruloyltyramine (FT) and p-coumaroyltyramine (CT) 24 h postinoculation in nonhost- and gene-for-gene-determined incompatible interactions with X. campestris pv. campestris and X. campestris pv. vesicatoria, respectively. In contrast, neither compound was detected in compatible interactions with X. campestris pv. vesicatoria. The accumulation of FT and CT was preceded by an increase in the extractable activity of tyrosine decarboxylase as well as increases in the transcription of genes encoding phenylalanine ammonia-lyase and tyramine hydroxycinnamoyl transferase. No such changes were detected in compatible interactions. Very rapid accumulation of FT and CT occurred (4 h postinoculation) in pepper in response to a X. campestris pv. campestris mutant carrying a deletion of the hrp gene cluster. In contrast, hrp mutants of X. campestris pv. vesicatoria failed to elicit the production of FT and CT. These observations suggest the existence of hrp gene-dependent and -independent activation mechanisms of a defense response involving hydroxycinnamoyltyramines.
Control of exuT Activity for Galacturonate Transport by the Negative Regulator ExuR in Erwinia chrysanthemi EC16
Authors: Valmeekam Venugopal , Loh Yen-Lin , San Francisco Michael J. D.
Published: May 2007

The negative regulatory protein ExuR in Erwinia chrysanthemi regulates expression of the galacturonate uptake (exuT) and utilization (uxaA, uxaB, uxaC) genes. We cloned and determined the nucleotide sequence of the exuR gene from E. chrysanthemi EC16. Analysis of the deduced amino acid sequence indicates that this protein possesses a helix-turn-helix motif and belongs to the GntR family of transcriptional repressors. Northern blot analysis and studies with transcriptional fusions of exuT in wild-type and exuR mutant backgrounds indicate that exuT transcription is deregulated in the exuR strain in vivo and in planta. [14C]-galacturonic acid uptake was constitutively high under inducing and noninducing conditions in the exuR mutant. Maximal exuT transcription activity was observed within 8 h of bacterial inoculation into potato tubers, well before any visible symptoms of disease were detected. This suggests that ExuT transport activity in E. chrysanthemi is important in the early stages of disease development.
Mutants of Tobacco mosaic virus with Temperature-Sensitive Coat Proteins Induce Heat Shock Response in Tobacco Leaves
Authors: Jockusch Harald , Wiegand Christiane , Mersch Birgit , Rajes Daniela
Published: May 2007

We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30°C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30°C. After 4 to 6 days, HSP70 was also induced at 23°C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.
Investigation of myo-Inositol Catabolism in Rhizobium leguminosarum bv. viciae and Its Effect on Nodulation Competitiveness
Authors: Fry Judith , Wood Martin , Poole Philip S.
Published: May 2007

Three discrete loci required for growth on myo-inositol in Rhizobium leguminosarum bv. viciae have been characterized. Two of these are catabolic loci that code for malonate semialdehyde dehydrogenase (iolA) and malonate semialdehyde dehydrogenase (iolD). IolD is part of a possible operon, iolDEB, although the functions of IolE and IolB are unknown. The third locus, int, codes for an ABC transport system that is highly specific for myo-inositol. LacZ analysis showed that mutation of iolD, which codes for one of the last steps in the catabolic pathway, prevents increased transcription of the entire pathway. It is likely that the pathway is induced by an end product of catabolism rather than myo-inositol itself. Mutants in any of the loci nodulated peas (Pisum sativum) and vetch (Vicia sativa) at the same rate as the wild type. Acetylene reduction rates and plant dry weights also were the same in the mutants and wild type, indicating no defects in nitrogen fixation. When wild-type 3841 was coinoculated onto vetch plants with either catabolic mutant iolD (RU360) or iolA (RU361), however, >95% of the nodules were solely infected with the wild type. The competitive advantage of the wild type was unaffected, even when the mutants were at 100-fold excess. The myo-inositol transport mutant (RU1487), which grows slowly on myo-inositol, was only slightly less competitive than the wild type. The nodulation advantage of the wild type was not the result of superior growth in the rhizosphere. Instead, it appears that the wild type may displace the mutants early on in the infection and nodulation process, suggesting an important role for myo-inositol catabolism.
MucR and MucS Activate exp Genes Transcription and Galactoglucan Production in Sinorhizobium meliloti EFB1
Authors: Lloret Javier , Martín Marta , Oruezabal Roke I. , Bonilla Ildefonso , Rivilla Rafael
Published: May 2007

When grown under standard conditions, Sinorhizobium meliloti EFB1 simultaneously produces two acidic exopolysaccharides, succinoglycan and galactoglucan, yielding very mucoid colonies. In this strain, MucR is essential for galactoglucan synthesis. A mutation in the mucS gene resulted in less mucoid colonies than in the wild-type EFB1. This mucS¯ strain was complemented to the wild-type phenotype by the cloned mucS gene, indicating that mucS is necessary for a wild-type level of galactoglucan production. Reverse transcription-polymerase chain reaction analysis of exp genes, which encode the pathway for galactoglucan production, in EFB1 and in the mutants affected in mucS, mucR, and both genes simultaneously, showed that MucS is a transcriptional activator of the exp genes but does not affect its own transcription. Furthermore, MucR is necessary for mucS transcriptional activation. As introduction of a cloned mucS gene in a mucR¯ strain yielded colonies less mucoid than the wild type, MucR could also activate exp genes transcription through other pathways. Deletion analysis of the expE promoter showed a region important for transcription and MucS activation. This region, containing a palindrome, is present in the putative expA, expC, expD, and expE promoters but not in the mucS promoter, suggesting that it is the target for MucS. A mucR¯mucS¯ mutant, which does not produce galactoglucan, was impaired in competitive nodulation of alfalfa in soil microcosms, indicating another possible role for this exopolysaccharide in symbiosis.
Chemically Induced Virus Resistance in Arabidopsis thaliana Is Independent of Pathogenesis-Related Protein Expression and the NPR1 Gene
Authors: Wong Chui Eng , Carson Rachael A. J. , Carr John P.
Published: May 2007

Salicylic acid (SA) treatment triggers inhibition of replication or movement of several positive-sense RNA plant viruses in tobacco. This resistance can also be stimulated by nonlethal concentrations of cyanide and antimycin A (AA) without triggering induction of pathogenesis-related PR-1 protein genes. In two ecotypes of Arabidopsis thaliana (Columbia and Nössen), SA-induced resistance to a tobamovirus, Turnip vein clearing virus (TVCV), was also induced by nonlethal concentrations of cyanide and AA without concomitant induction of PR-1 gene expression. Furthermore, chemically induced resistance to TVCV, as well as the induction of the plant mitochondrial alternative oxidase (a potential target for the chemicals), was independent of NPR1, a gene that plays a key role downstream of SA in the induction of PR proteins. The chemically induced resistance to TVCV appeared to be due to inhibition of replication at the site of inoculation. Taken together, these results show that in Arabidopsis, as in tobacco, resistance to viruses can be induced via a distinct branch of the defensive signal transduction pathway. This suggests that the existence of this virus-specific branch may be widespread among plants.
Multiple Domains Within the Cauliflower mosaic virus Gene VI Product Interact with the Full-Length Protein
Authors: Li Yongzhong , Leisner Scott M.
Published: May 2007

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral propagation. It is likely that at least some of these functions require P6 self-association. The work described here was performed to confirm that P6 self-associates and to identify domains involved in this interaction. Yeast two-hybrid analyses indicated that full-length P6 self-associates and that this interaction is specific. Additional analyses indicated that at least four independent domains bind to full-length P6. When a central domain (termed domain D3) was removed, these interactions were abolished. However, this deleted P6 was able to bind to the full-length wild-type protein and to isolated domain D3. Viruses lacking domain D3 were incapable of producing a systemic infection. Isolated domain D3 was capable of binding to at least two of the other domains but was unable to self-associate. This suggests that domain D3 facilitates P6 self-association by binding to the other domains but not itself. The presence of multiple domains involved in P6 self-association may help explain the ability of this protein to form the intracellular inclusions characteristic of caulimoviruses.
Ethylene Insensitivity Impairs Resistance to Soilborne Pathogens in Tobacco and Arabidopsis thaliana
Authors: Geraats Bart P. J. , Bakker Peter A. H. M. , van Loon L. C.
Published: May 2007

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.
Potato spindle tuber viroid Strains of Different Pathogenicity Induces and Suppresses Expression of Common and Unique Genes in Infected Tomato
Authors: Itaya Asuka , Matsuda Yoshie , Gonzales Robert A. , Nelson Richard S. , Ding Biao
Published: May 2007

Viroids are the smallest plant pathogens. These RNAs do not encode proteins and are not encapsidated, and yet they can replicate autonomously, move systemically, and cause diseases in infected plants. Notably, strains of a viroid with subtle differences in nucleotide sequences can cause dramatically different symptoms in infected plants. These features make viroids unique probes to investigate the role of a pathogenic RNA genome in triggering host responses. We conducted a comprehensive analysis of the differential gene expression patterns of tomato plants at various stages of infection by a mild and severe strain of Potato spindle tuber viroid (PSTVd). We also compared tomato gene expression altered by the PSTVd strains with that altered by Tobacco mosaic virus (TMV). Our analyses revealed that the two PSTVd strains altered expression of both common and unique tomato genes. These genes encode products involved in defense/stress response, cell wall structure, chloroplast function, protein metabolism, and other diverse functions. Five genes have unknown functions. Four genes are novel. The expression of some but not all of these genes was also altered by TMV infection. Our results indicate that viroids, although structurally simple, can trigger complex host responses. Further characterization of viroid-altered gene expression in a host plant should help understand viroid pathogenicity and, potentially, the mechanisms of RNA-mediated regulation of plant gene expression.
Genetic and Cytogenetic Mapping of DMI1, DMI2, and DMI3 Genes of Medicago truncatula Involved in Nod Factor Transduction, Nodulation, and Mycorrhization
Authors: Ané Jean-Michel , Lévy Julien , Thoquet Philippe , Kulikova Olga , de Billy Françoise , Penmetsa Varma , Kim Dong-Jin , Debellé Frédéric , Rosenberg Charles , Cook Douglas R. , Bisseling Ton , Huguet Thierry , Dénarié Jean
Published: May 2007

The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromosomes confirmed the location of DMI1, DMI2, and DMI3 on chromosomes 2, 5, and 8. FISH-pachytene studies revealed that the three genes are in euchromatic regions of the genome, with a ratio of genetic to cytogenetic distances between 0.8 and 1.6 cM per μm in the DMI1, DMI2, and DMI3 regions. Through grafting experiments, we showed that the genetic control of the dmi1, dmi2, and dmi3 nodulation phenotypes is determined at the root level. This means that mutants can be transformed by Agrobacterium rhizogenes to accelerate the complementation step of map-based cloning projects for DMI1, DMI2, and DMI3.
Fungal ABC Transporters and Microbial Interactions in Natural Environments
Authors: Schoonbeek Henk-jan , Raaijmakers Jos M. , De Waard Maarten A.
Published: May 2007

In natural environments, microorganisms are exposed to a wide variety of antibiotic compounds produced by competing organisms. Target organisms have evolved various mechanisms of natural resistance to these metabolites. In this study, the role of ATP-binding cassette (ABC) transporters in interactions between the plant-pathogenic fungus Botrytis cinerea and antibiotic-producing Pseudomonas bacteria was investigated in detail. We discovered that 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and phenazine-1-carboxamide (PCN), broad-spectrum antibiotics produced by Pseudomonas spp., induced expression of several ABC transporter genes in B. cinerea. Phenazines strongly induced expression of BcatrB, and ΔBcatrB mutants were significantly more sensitive to these antibiotics than their parental strain. Treatment of B. cinerea germlings with PCN strongly affected the accumulation of [14C]fludioxonil, a phenylpyrrole fungicide known to be transported by BcatrB, indicating that phenazines also are transported by BcatrB. Pseudomonas strains producing phenazines displayed a stronger antagonistic activity in vitro toward ΔBcatrB mutants than to the parental B. cinerea strain. On tomato leaves, phenazine-producing Pseudomonas strains were significantly more effective in reducing gray mold symptoms incited by a ΔBcatrB mutant than by the parental strain. We conclude that the ABC transporter BcatrB provides protection to B. cinerea in phenazine-mediated interactions with Pseudomonas spp. Collectively, these results indicate that fungal ABC transporters can play an important role in antibiotic-mediated interactions between bacteria and fungi in plant-associated environments. The implications of these findings for the implementation and sustainability of crop protection by antagonistic microorganisms are discussed.
Modulation of Defense Responses of Malus spp. During Compatible and Incompatible Interactions with Erwinia amylovora
Authors: Venisse Jean-Stéphane , Malnoy Mickaël , Faize Mohamed , Paulin Jean-Pierre , Brisset Marie-Noëlle
Published: May 2007

Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae. In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E. amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv. tabaci. Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course. This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E. amylovora, because an hrp secretion mutant did not induce such responses. However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E. amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant. This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.