bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical Mapping of the Magnaporthe grisea AVR1-MARA Locus Reveals the Virulent Allele Contains Two Deletions
Authors: Mandel M. Alejandra , Crouch V. Wayne , Gunawardena Uvini P. , Harper Travis M. , Orbach Marc J.
Published: May 2007

The avirulence genes that have been identified in Magna-porthe grisea exhibit varying degrees of stability in infection assays. AVR1-MARA is considered one of the stable avirulence genes. In an effort to understand this stability, we analyzed the AVR1-MARA locus by physical mapping and chromosome walking. By walking toward AVR1-MARA from restriction fragment length polymorphism markers on both sides of the locus, we isolated sequences that are inseparable from AVR1-MARA, but we were unable to clone the complete locus. In contrast, the virulent locus avr1-MARA was isolated easily. A detailed comparative map of the two loci was constructed that identifies two deletions in the virulent locus, suggesting that virulence may be due to partial or complete deletion of the gene. Physical mapping also revealed that one progeny strain from a cross between avirulent and virulent parents appears to have spontaneously mutated to virulence.
Starvation-Induced Genes of the Tomato Pathogen Cladosporium fulvum Are Also Induced During Growth In Planta
Authors: Coleman Mark , Henricot Beatrice , Arnau Jose , Oliver Richard P.
Published: May 2007

The pathogenicity of fungal pathogens is presumably dependent on genes that are expressed during infection. In order to isolate such genes from the tomato pathogen Cla-dosporium fulvum, and to test the hypothesis that starvation-induced genes are also plant induced, a cDNA library was prepared from mycelia grown in a defined medium and then transferred to a starvation medium. The library was then screened with cDNA probes prepared from starved and replete fungal mycelium. Five unique, differentially expressed cDNAs were isolated from 1,000 clones screened. Northern (RNA) hybridization confirmed that all five were starvation induced. Interestingly, all five were also found to be plant induced. The identity of two of the clones was indicated by partial DNA sequencing as alcohol and aldehyde dehydrogenase. The observed correlation between starvation induction and plant induction is discussed.
Cloning and Characterization of Two Phosphate Transporters from Medicago truncatula Roots: Regulation in Response to Phosphate and to Colonization by Arbuscular Mycorrhizal (AM) Fungi
Authors: Liu Henry , Trieu Anthony T. , Blaylock Laura A. , Harrison Maria J.
Published: May 2007

Most vascular plants can acquire phosphate from the environment either directly, via the roots, or indirectly, via a fungal symbiont that invades the cortical cells of the root. Here we have identified two cDNA clones (MtPT1 and MtPT2) encoding phosphate transporters from a mycorrhizal root cDNA library (Medicago truncatula/Glomus versiforme). The cDNAs represent M. truncatula genes and the encoded proteins share identity with high-affinity phosphate transporters from Arabidopsis, potato, yeast, Neurospora crassa, and an arbuscular mycorrhizal (AM) fungus, G. versiforme. The function of the protein encoded by MtPT1 was confirmed by complementation of a yeast phosphate transport mutant (pho84). The Km of the MtPT1 transporter in this system is 192 μM. MtPT1 and MtPT2 transcripts are present in roots and transcript levels increase in response to phosphate starvation. MtPT transcripts were not detected in leaves. Following colonization of the roots by the AM fungus G. versiforme, both MtPT1 and MtPT2 transcript levels decrease significantly. Down-regulation of phosphate starvation-inducible genes in mycorrhizal roots appears to be a common occurrence and a homologue of a phosphate starvation-inducible purple acid phosphatase is also down-regulated in the mycorrhizal roots. The functional characteristics and expression patterns of the MtPT transporters are consistent with a role in the acquisition of phosphate from the environment but suggest that they may not be involved in phosphate uptake at the symbiotic interface in mycorrhizal roots.
Use of Green Fluorescent Protein to Detect Expression of nif Genes of Azoarcus sp. BH72, a Grass-Associated Diazotroph, on Rice Roots
Authors: Egener Tanja , Hurek Thomas , Reinhold-Hurek Barbara
Published: May 2007

A gfp (green fluorescent protein) cassette for transcriptional fusions has been developed to study gene expression in Azoarcus sp. BH72 in association with plant roots. The bacteria expressed nitrogenase genes (nifHDK) in the rhi-zosphere, on root tips, and in epidermal cells of rice seedlings. Green fluorescent protein fusions also visualized promoter activity of single cells in soil.
The Endopolygalacturonase Gene Bcpg1 Is Required for Full Virulence of Botrytis cinerea
Authors: Have Arjen ten , Mulder Wietse , Visser Jaap , van Kan Jan A. L.
Published: May 2007

Botrytis cinerea, a fungus that causes diseases in over 200 plant species, secretes a number of endopolygalacturonases that have been suggested to be involved in pathogenesis. However, so far the corresponding genes have not been isolated from this fungus. We cloned Bcpg1, encoding endopolygalacturonase, with the pgaII gene from Aspergillus niger as a heterologous probe. The Bcpg1gene is expressed to similar levels in liquid cultures of B. cinerea containing either 1% polygalacturonic acid or 1% sucrose, and is expressed during infection of tomato leaves. The Bcpg1 gene was eliminated by partial gene replacement, and the resulting mutants were tested for virulence on tomato leaves and fruits, as well as on apple fruits. Although the mutants were still pathogenic and displayed similar primary infections when compared with control strains, a significant decrease in secondary infection, i.e., growth of the lesion beyond the inoculation spot, was observed on all three host tissues. These results indicate that the Bcpg1 gene is required for full virulence.
Characterization of Type IV Pilus Genes in Pseudomonas syringae pv. tomato DC3000
Authors: Roine Elina , Raineri Deanna M. , Romantschuk Martin , Wilson Mark , Nunn David N.
Published: May 2007

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage φ6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only φ6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage φ6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.
A Metalloprotease from Xanthomonas campestris That Specifically Degrades Proline/Hydroxyproline-Rich Glycoproteins of the Plant Extracellular Matrix
Authors: Dow J. Maxwell , Davies Huw A. , Daniels Michael J.
Published: May 2007

Culture supernatants of Xanthomonas campestris pv. campestris contain an enzymic activity capable of degrading gp120, a proline-rich glycoprotein associated with the extracellular matrix of the vascular bundles in petioles of turnip (Brassica campestris). This activity did not reside in any of the three previously characterized proteases of X. campestris pv. campestris that were identified by their action against the model substrate β-casein. The novel enzyme was purified by ion-exchange and size-exclusion high-performance liquid chromatography (HPLC). The enzyme, which has no activity against β-casein, is active against some plant glycoproteins of the hydroxyproline-rich class such as extensin from potato and tomato and gpS-3, a glycoprotein induced in B. campestris petioles by wounding. Other hydroxyproline-rich glycoproteins, such as the solanaceous lectins, were not substrates however. Studies of the products released upon degradation of tomato extensin suggested that the degradative mechanism was proteolysis. Inhibitor studies suggested that the enzyme was a zinc-requiring metalloprotease. Extracellular matrix glycoproteins of the proline-rich and hydroxy-proline-rich classes have been implicated in plant resistance to microbial attack, hence their degradation by X. campestris pv. campestris may have considerable significance for black rot pathogenesis.
Tobacco Class I and II Catalases Are Differentially Expressed During Elicitor-Induced Hypersensitive Cell Death and Localized Acquired Resistance
Authors: Dorey Stéphan , Baillieul Fabienne , Saindrenan Patrick , Fritig Bernard , Kauffmann Serge
Published: May 2007

Expression of tobacco class I (CAT1) and class II (CAT2) catalases was analyzed in leaves reacting hypersensitively to tobacco mosaic virus (TMV) or to a fungal glycoprotein elicitor. In TMV-infected plants, Cat1 transcript levels declined rapidly while Cat2 transcripts accumulated strongly. The spatial and temporal changes in catalase transcripts, proteins, and activity during the hypersensitive reaction (HR) were further investigated in tobacco leaves infiltrated with a glycoprotein elicitor. Two functionally different zones were discriminated: the infiltrated tissue in which cells undergo the HR, called the HR-zone 1; and the surrounding tissue showing strong induced defense responses, called the LAR (Localized Acquired Resistance)- zone 2. Levels of Cat1 and Cat2 mRNA and proteins and catalase activity decreased in the HR-zone 1. In the LAR-zone 2, Cat1 transcripts became rapidly undetectable, but levels of Cat2 mRNA and protein and catalase activity increased. Catalase expression in elicitorinfiltrated leaves reflected that in TMV-infected leaves. A strong rise in hydrogen peroxide occurred in the HR-zone 1 and paralleled the CAT activity decline. No H2O2increase was measured in the LAR-zone 2. There was no correlation between salicylic acid levels and catalase activity. Modulation of catalase activity in tobacco leaves undergoing the HR appeared predominantly supported by changes in catalase transcripts and proteins. We have shown that neither H2O2nor salicylic acid can be the primary mobile signal diffusing from the HR-zone 1 to the LAR-zone 2 and inducing CAT2 expression. Furthermore, the signaling pathway responsible for decreased CAT2 expression in the HR-zone 1 does not involve reactive oxygen intermediates.
Gene Activation by Cytoplasmic Acidification in Suspension-Cultured Rice Cells in Response to the Potent Elicitor, N-Acetylchitoheptaose
Authors: He Dao-Yao , Yazaki Yoshiaki , Nishizawa Yoko , Takai Ryota , Yamada Kosumi , Sakano Katsuhiro , Shibuya Naoto , Minami Eiichi
Published: May 2007

N-Acetylchitoheptaose strongly induces a set of defense reactions in suspension-cultured rice cells including cytoplasmic acidification (K. Kuchitsu, Y. Yazaki, K. Sakano, and N. Shibuya, Plant Cell Physiol. 38:1012-1018, 1997) and the accumulation of mRNAs for two rapidly activated genes, EL2 and EL3 (E. Minami, K. Kuchitsu, D.-Y. He, H. Kouchi, N. Midoh, Y. Ohtsuki, and N. Shibuya, Plant Cell Physiol. 37:563-567, 1996), as well as phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase. Treatment of cells with propionic acid resulted in the accumulation of the mRNAs for EL2, EL3, and PAL in a manner similar to the accumulation induced by N-acetylchitoheptaose. Concomitantly, there was a rapid decrease in the cytoplasmic pH as detected with in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Interestingly, K-252a, a potent inhibitor of Ser/Thr protein kinases, strongly inhibited gene induction by N-acetylchitoheptaose, but showed much less inhibition of gene induction caused by propionic acid. Calyculin A, a protein phosphatase inhibitor, induced mRNA accumulations for EL2, EL3 and PAL, with concomitant acidification of the cytoplasm. On the other hand, chitinase and β-glucanase mRNA levels did not change after addition of propionic acid or calyculin A. Treatment of the cells with propionic acid did not induce the production of reactive oxygen species. These results strongly suggest that cytoplasmic acidification at the early stage of elicitor action could be a key step in the signal transduction events leading to the expression of elicitor-responsive genes. A hypothetical model of elicitor signal pathway is proposed based on these results.
Flavonoids Synthesized in Cortical Cells During Nodule Initiation Are Early Developmental Markers in White Clover
Authors: Mathesius Ulrike , Bayliss Cathy , Weinman Jeremy J. , Schlaman Helmi R. M. , Spaink Herman P. , Rolfe Barry G. , McCully Margaret E. , Djordjevic Michael A.
Published: May 2007

We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated β-glucuronidase (GUS) transgene (CHS3:gusA) was up-regulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4′-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a nodulating Rhizo-bium strain and by clover-specific lipo-chitinoligosac-charides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule or-ganogenesis in clover and may have direct roles in nodule formation.
Pepper Mild Mottle Virus Coat Protein Alone Can Elicit theCapsicumspp.L3Gene-Mediated Resistance
Authors: Gilardi P. , García-Luque I. , Serra M. T.
Published: May 2007

The pepper mild mottle virus (PMMoV-S) (an L3hypersensitive response [HR]-inducer strain) coat protein was expressed in Capsicum chinense (L3L3) plants with the heterologous potato virus X (PVX)-based expression system. The chimeric virus was localized in the inoculated leaves and induced the HR, thus indicating that the tobamoviral sequences that affect induction of the HR conferred by the L3resistance gene reside in the coat protein gene. Furthermore, transient expression of the PMMoV-S coat protein in C. chinense leaves by biolistic co-bombardment with a plasmid expressing the β-glucuronidase (GUS) gene leads to the induction of cell death and expression of host defense genes. Thus, the coat protein of PMMoV-S is the elicitor of the Capsicum spp. L3resistance gene-mediated HR.
Involvement of Salicylic Acid in the Establishment of the Rhizobium meliloti-Alfalfa Symbiosis
Authors: Martínez-Abarca F. , Herrera-Cervera J. A. , Bueno P. , Sanjuan J. , Bisseling T. , Olivares J.
Published: May 2007

Inoculation of alfalfa plants with either incompatible Rhizobium or a Rhizobium mutant blocked in Nod factor synthesis led to an accumulation of salicylic acid in roots, in contrast to plants inoculated with a wild-type (compatible) R. meliloti strain. When salicylic acid was exogenously applied prior to inoculation of alfalfa plants with either purified Nod factor or compatible Rhizobium strains, a significant inhibition of nodule primordia formation and a reduction of the number of emerging nodules, respectively, as well as a delay in nodule visualization, were observed. These results suggest an involvement of Rhizobium-synthesized Nod factors in the inhibition of salicylic acid-mediated defense in legumes.
Auxin Production Is a Common Feature of Most Pathovars of Pseudomonas syringae
Authors: Glickmann Eric , Gardan Louis , Jacquet Sylvie , Hussain Shafik , Elasri Miena , Petit Annik , Dessaux Yves
Published: May 2007

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.
Cloning, Expression, and Role in Pathogenicity of pg1 Encoding the Major Extracellular Endopolygalacturonase of the Vascular Wilt Pathogen Fusarium oxysporum
Authors: Di Pietro Antonio , Roncero M. Isabel G.
Published: May 2007

pg1 encoding the major in vitro extracellular endopolygalacturonase of the tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici was cloned and sequenced. The deduced mature protein had a calculated molecular mass of 35.5 kDa and a pI of 6.2, and showed significant similarity with other fungal endoPGs. pg1 mRNA was induced in vitro by citrus pectin, tomato vascular tissue, 0.1% D-galacturonic acid, and polygalacturonic acid, and repressed by 1% D-galacturonic acid and 1% glucose. Reverse transcription-polymerase chain reaction revealed pg1 expression in roots and lower stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Three naturally occurring F. oxysporum f. sp. melonis isolates deficient in PG1 were transformed with the cloned gene. The PG1 enzyme secreted by the transformants had the same molecular mass, pI, and glycosylation pattern as those of the donor isolate. Polygalacturonase activity in cultures of transformants grown in vitro on citrus pectin and on melon plants, but not on glucose, increased 10- to 20-fold, compared with the PG1-deficient wild-type isolate, whereas mycelial dry weight increased two- to threefold. Transformants exhibited the same degree of virulence toward susceptible muskmelon cultivars as the wild-type isolate and were avirulent on a resistant cultivar.
Carbon Allocation in Ectomycorrhizas: Identification and Expression Analysis of an Amanita muscaria Monosaccharide Transporter
Authors: Nehls Uwe , Wiese Joachim , Guttenberger Martin , Hampp Rüdiger
Published: May 2007

Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants. An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus. As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters. A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction. This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A. muscaria mycorrhizal cDNA library. The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter. The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system. AmMst1 was constitutively expressed in fungal hyphae under all growth conditions. Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold. We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.
A Transcriptional Regulator of the LuxR-UhpA Family, FlcA, Controls Flocculation and Wheat Root Surface Colonization by Azospirillum brasilense Sp7
Authors: Pereg-Gerk Lily , Paquelin Annick , Gounon Pierre , Kennedy Ivan R. , Elmerich Claudine
Published: May 2007

Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UhpA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.
Heterologous Expression of Septoria lycopersici Tomatinase in Cladosporium fulvum: Effects on Compatible and Incompatible Interactions with Tomato Seedlings
Authors: Melton Rachel E. , Flegg Lynda M. , Brown James K. M. , Oliver Richard P. , Daniels Michael J. , Osbourn Anne E.
Published: May 2007

The anti-fungal, steroidal, glycoalkaloid saponin, α-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to α-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify α-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to α-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, α-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.
A Novel Rust Resistance Gene in Maize Showing Overdominance
Authors: Delaney Donna E. , Webb Craig A. , Hulbert Scot H.
Published: May 2007

The Rp8 rust resistance locus of maize is unique in that only Rp8-A/Rp8-B heterozygotes provide resistance and homozygotes are susceptible. A third allele, Rp8-C, provides no resistance in homozygotes or heterozygous combinations. Rp8 maps to the long arm of chromosome 6, near the restriction fragment length polymorphism marker umc21.
Identification of R-Gene Homologous DNA Fragments Genetically Linked to Disease Resistance Loci in Arabidopsis thaliana
Authors: Aarts Mark G. M. , Lintel Hekkert Bas te , Holub Eric B. , Beynon Jim L. , Stiekema Willem J. , Pereira Andy
Published: May 2007

Disease resistance in plants is a desirable economic trait. A number of disease resistance genes from various plant species have been cloned so far. The gene products of some of these can be distinguished by the presence of an N-terminal nucleotide binding site and a C-terminal stretch of leucine-rich repeats. Although these gene products are structurally related, the DNA sequences are poorly conserved. Only parts of the nucleotide binding site share enough DNA identity to design primers for polymerase chain reaction amplification of related DNA sequences. Such primers were used to amplify different resistance-gene-like (RGL) DNA fragments from Arabidopsis thaliana accessions Landsberg erecta and Columbia. Almost all cloned DNA fragments were genetically closely linked with known disease resistance loci. Most RGL fragments were found in a clustered or dispersed multi-copy sequence organization, supporting the supposed correlation of RGL sequences and disease resistance loci.
The exuT Gene of Erwinia chrysanthemi EC16: Nucleotide Sequence, Expression, Localization, and Relevance of the Gene Product
Authors: Haseloff B. J. , Freeman T. L. , Valmeekam V. , Melkus M. W. , Oner F. , Valachovic M. S. , San Francisco M. J. D.
Published: May 2007

Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall. In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration. Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E. chrysanthemi. The GalUA uptake gene (exuT) was cloned and sequenced. Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da. This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile. Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein. This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis. Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA. A targeted mutation in the exuT gene affected the utilization of GalUA as a sole carbon source for growth. Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16.
Characterization of a Pepper Mild Mottle Tobamovirus Strain Capable of Overcoming the L3 Gene-Mediated Resistance, Distinct from the Resistance-Breaking Italian Isolate
Authors: Tsuda Shinya , Kirita Masanobu , Watanabe Yuichiro
Published: May 2007

Green pepper plants with the L3 resistance gene usually develop necrotic lesions on leaves infected with a Japanese strain of pepper mild mottle tobamovirus (PMMoV-J). A recently discovered strain, PMMoV-Ij, has the ability to overcome L3 resistance. Phytopathological responses of a variety of plant species to PMMoV-J and PMMoV-Ij were determined and the coat protein (CP) sequence comparisons revealed both amino acids 43 and 50 of PMMoV-Ij were unique. This led us to believe that substitutions at these residues would enable PMMoV-J to overcome L3 resistance. This was confirmed by Western blot (immunoblot) detection of PMMoV-J containing both point mutations in upper uninoculated leaves of resistant plants. Computer models suggest the critical residues in overcoming resistance lie in CP regions that putatively interact with other subunits. These results contribute to our understanding of the virus's ability to circumvent plant resistance.
The Coat Protein of Cucumber Mosaic Virus Is a Host Range Determinant for Infection of Maize
Authors: Ryu Ki Hyun , Kim Chung-Ho , Palukaitis Peter
Published: May 2007

Infection of maize by the Fny strain of cucumber mosaic virus (CMV) and resistance against infection by the M strain of CMV were mapped to the coat protein gene on RNA 3 of CMV, using biologically active cDNA clones of Fny-CMV RNAs 1, 2, and 3 and RNAs 2 and 3 of M-CMV, as well as chimeras constructed between cDNA clones of M-CMV and Fny-CMV RNA 3. Changes in the coat protein gene of M-CMV RNA 3 at both positions 129 (Leu to Pro) and 162 (Thr to Ala) were required to overcome the resistance against M-CMV in maize. Resistance to M-CMV in maize was correlated with an inability to detect virus accumulation in the inoculated leaves. Since the coat protein of CMV is involved in virus movement, but not virus replication, the data suggest that the resistance in maize to M-CMV is due to the inability of the M-CMV coat protein to promote the cell-to-cell movement of CMV in maize.
Two Distinct Mechanisms of Transgenic Resistance Mediated by Groundnut Rosette Virus Satellite RNA Sequences
Authors: Taliansky M. E. , Ryabov E. V. , Robinson D. J.
Published: May 2007

Transformation of Nicotiana benthamiana with full-length sequences of a mild variant of the groundnut rosette virus (GRV) satellite RNA (sat-RNA) yielded plants that did not produce symptoms when inoculated with GRV and a virulent sat-RNA. Two different resistance mechanisms operated in different transformed lines. In the first, plants contained high levels of transcript RNA, and replication of both sat-RNA and GRV genomic RNA was inhibited. This mechanism is analogous to the down-regulation of GRV genomic and sat-RNA replication in infections containing the mild sat-RNA, and indeed infection of sat-RNA transgenic plants with GRV was shown to lead to liberation of unit-length sat-RNA from transgene transcripts. In the second resistance mechanism, plants contained low transcript RNA levels, and replication of sat-RNA but not of GRV genomic RNA was inhibited. These plants were also resistant to infection by potato virus X derivatives containing GRV sat-RNA sequences. This mechanism is an example of homology-dependent gene silencing or cosuppression. Resistant plants were also produced by transformation with sequences representing only the 5′ terminal one-third of the mild sat-RNA; the mechanism of resistance in these plants was of the cosuppression type.
Symbiosis-Specific Expression of Two Medicago truncatula Nodulin Genes, MtN1 and MtN13, Encoding Products Homologous to Plant Defense Proteins
Authors: Gamas Pascal , de Billy Françoise , Truchet Georges
Published: May 2007

Two Medicago truncatula nodulin genes putatively encoding proteins structurally related to two classes of proteins commonly associated with plant defense reactions have been characterized. MtN1 is homologous to two small, cysteine-rich, pathogen-inducible proteins from pea (pI39 and pI230), whereas MtN13 is closely related to the PR10 family of pathogenesis-related proteins. We show that neither MtN1 nor MtN13 is induced in leaves in response to pathogenic bacteria, and that both are exclusively expressed during nodulation. In situ hybridization experiments as well as Northern (RNA) studies of interactions between M. truncatula and either wild-type Rhizobium meliloti or mutants deficient in infection establish that MtN1 is associated with the infection process, while MtN13 represents the first specific marker described for the nodule outer cortex. Possible roles for MtN1 and MtN13 are discussed. We also present the identification of another member of the PR10 family, designated as MtPR10-1, whose regulation is strikingly different from that observed for MtN13, being constitutively expressed in roots and pathogen-inducible in leaves.
Expression of the Tomato Leaf Curl Geminivirus C4 Gene Produces Viruslike Symptoms in Transgenic Plants
Authors: Krake L. R. , Rezaian M. A. , Dry I. B.
Published: May 2007

Transgenic tobacco and tomato plants expressing the C4 gene from tomato leaf curl geminivirus (TLCV), under the control of the cauliflower mosaic virus 35S promoter, developed virus-disease-like phenotypes. Plants transformed with a frame-shift version of the C4 gene construct appeared normal but produced levels of transcript similar to those of plants expressing the C4 gene. Thus, the abnormal plant phenotypes result from translation of the C4 gene. These results support the hypothesis that the C4 open reading frame of TLCV encodes a protein involved in the development of disease symptoms during viral infection.
Expression of Maize and Fungal Nitrate Reductase Genes in Arbuscular Mycorrhiza
Authors: Kaldorf Michael , Schmelzer Elmon , Bothe Hermann
Published: May 2007

The role of arbuscular mycorrhizal (AM) fungi in assisting their host plant in nitrate assimilation was studied. With polymerase chain reaction technology, part of the gene coding for the nitrate reductase (NR) apoprotein from either the AM fungus Glomus intraradices or from maize was specifically amplified and subsequently cloned and sequenced. Northern (RNA) blot analysis with these probes indicated that the mRNA level of the maize gene was lower in roots and shoots of mycorrhizal plants than in noncolonized controls, whereas the fungal gene was transcribed in roots of AM plants. The specific NR activity of leaves was significantly lower in AM-colonized maize than in the controls. Nitrite formation catalyzed by NR was mainly NADPH-dependent in roots of AM-colonized plants but not in those of the controls, which is consistent with the fact that NRs of fungi preferentially utilize NADPH as reductant. The fungal NR mRNA was detected in arbuscules but not in vesicles by in situ RNA hybridization experiments. This appears to be the first demonstration of differential formation of transcripts of a gene coding for the same function in both symbiotic partners.
Suppression of Tobacco Basic Chitinase Gene Expression in Response to Colonization by the Arbuscular Mycorrhizal Fungus Glomus intraradices
Authors: David Rakefet , Itzhaki Hanan , Ginzberg Idit , Gafni Yedidya , Galili Gad , Kapulnik Yoram
Published: May 2007

A differentially displayed cDNA clone (MD17) was isolated from tobacco roots (Nicotiana tabacum cv. Xanthi-nc) infected with the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The isolated DNA fragment exhibited a reduced level of expression in response to AM establishment and 90% identity with the 3′ noncoding sequence of two basic chitinases (EC 3.2.1.14) from N. tabacum. Northern (RNA) blots and Western blots (immunoblots), probed with tobacco basic chitinase gene-specific probe and polyclonal antibodies raised against the chitinase enzyme, yielded hybridization patterns similar to those of MD17. Moreover, the up-regulation of the 32-kDa basic chitinase gene expression in tobacco roots by (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was less effective in mycorrhizal roots than in nonmycorrhizal controls. Suppression of endogenous basic chitinase (32-kDa) expression was also observed in transgenic mycorrhizal plants that constitutively express the 34-kDa basic chitinase A isoform. When plants were grown with an increased phosphate supply, no suppression of the 32-kDa basic chitinase was obtained. These findings indicate that during the colonization and establishment of G. intraradices in tobacco roots, expression of the basic chitinase gene is down-regulated at the mRNA level.
Cutinase and Other Lipolytic Esterases Protect Bean Leaves from Infection byRhizoctonia solani
Authors: Parker Diana M. , Köller Wolfram
Published: May 2007

The results describe a novel activity of fungal cutinase, the protection of bean leaves from disease. Development of web blight symptoms on bean leaves infected with Rhizoctonia solani (AG-1) was prevented in the presence of cutinase purified from Venturia inaequalis. Instead of disease, small areas of tissue necrosis became visible, and the tissue in which the pathogen was restricted displayed strong autofluorescence beneath the inoculation sites. Mechanical wounding of the leaf surface had no effect on disease development and the permeability of the cuticle was not increased by cutinase action, indicating that surface wounding was not the cause for this novel activity of cutinase. A comparative study involving cutinase and other serine hydrolases revealed that the disease prevention resided in the lipolytic esterase activity rather than the cutinase activity. The pattern of expression of four pathogenesis-related (PR) protein genes provided no evidence for the modulation of known resistance responses of bean leaves in response to cutinase action. The protective mechanism of the esterase activity remains unknown.
In Vitro Sulfotransferase Activity of NoeE, a Nodulation Protein of Rhizobium sp. NGR234
Authors: Quesada-Vincens D. , Hanin M. , Broughton W. J. , Jabbouri S.
Published: May 2007

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium liberate morphogenetic lipochitin-oligosac-charides (Nod factors) into legume rhizospheres. Nod factors, which are synthesized by the products of rhizobial nodulation (nod) genes, vary in core length as well as in the number and type of substitutions. In Rhizobium sp. NGR234, the N-acylated pentamers of N-acetyl-D-glucosamine carry an O-methylfucose group on the reducing terminus that is substituted, on a mutually exclusive basis, with either an acetyl or a sulfuryl group. A sulfotransferase encoded by noeE is required for adjunction of activated sulfate donated by 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Here we show that when expressed in NGR234 cured of its symbiotic plasmid (= ANU265) or when purified as a fusion protein (MBP-NoeE), NoeE transfers sulfate from PAPS to fucosylated lipochitin-oligosaccharides. Enzyme assays showed that sulfotransferase activity is dependent on the presence of an acyl group (stearic and vaccenic acids were tested) since no activity was detected when fucosylated oligochitins (oligomers of two to six N-acetyl-D-glucosamine units) were used as substrates. Thus, NoeE is unique in that it is the only characterized sulfotransferase that is specific for fucosylated Nod factors. It probably acts after NodA, which acylates the amino-sugar backbone.
Salicylic Acid Accumulation in Barley Is Pathogen Specific but Not Required for Defense-Gene Activation
Authors: Vallélian-Bindschedler Laurence , Métraux Jean-Pierre , Schweizer Patrick
Published: May 2007

Barley (Hordeum vulgare) seedlings were inoculated with the biotrophic pathogen Erysiphe graminis f. sp. hordei, the biotrophic nonhost pathogen E. graminis f. sp. tritici, and the necrotrophic nonhost pathogen Pseudomonas syringae pv. syringae. The levels of free salicylic acid and of salicylic-acid conjugates remained low after infection with E. graminis f. sp. hordei or E. graminis f. sp. tritici while they increased after inoculation with P. syringae pv. syringae. Pathogenesis-related proteins PR1, PR3 (chitinase), PR5 (thaumatin-like), and PR9 (peroxidase) accumulated after inoculation with all three pathogens.
Syringolin, a Novel Peptide Elicitor from Pseudomonas syringae pv. syringae that Induces Resistance to Pyricularia oryzae in Rice
Authors: Wäspi Urs , Blanc Daniel , Winkler Tammo , Rüedi Peter , Dudler Robert
Published: May 2007

Recognition by rice plants (Oryza sativa) of the nonhost pathogen Pseudomonas syringae pv. syringae leads to an active response ultimately resulting in local acquired resistance against the rice blast fungus Pyricularia oryzae. An observable aspect of this defense response is the increased abundance of a set of transcripts. The accumulation of one of these transcripts, Pir7b, was dependent on the function of the bacterial lemA gene, which encodes part of a two-component regulatory system. This suggested that the lemA regulatory system controlled the production of an elicitor of Pir7b transcript accumulation. This elicitor, which we name syringolin, was purified to homogeneity and its structure was elucidated. Syringolin is a novel and unusual secreted peptide consisting of a 12-membered ring formed by the two non-proteinogenic amino acids 5-methyl-4-amino-2-hexenoic acid and 3,4-dehydrolysine. The α-amino group of the latter is connected by a peptide bond to a valine that in turn is linked to a second valine via a urea moiety. Application of syringolin onto rice leaves elicited the accumulation of Pir7b as well as of other defense-related transcripts and induced resistance toward P. oryzae. Thus, syringolin is one of several determinants by which rice plants can perceive the nonhost pathogen P. syringae pv. syringae.
Dual Role of Desferrioxamine in Erwinia amylovora Pathogenicity
Authors: Dellagi Alia , Brisset Marie-Noëlle , Paulin Jean-Pierre , Expert Dominique
Published: May 2007

To investigate the role of iron in Erwinia amylovora pathogenicity, virulence properties of two mutants of strain CFBP 1430 isolated by insertional mutagenesis and affected in the iron transport pathway mediated by desferrioxamine (DFO) were analyzed. One mutation (dfoA∷MudIIpR13) disrupts DFO biosynthesis. The present analysis shows that this mutation affects an open reading frame that belongs to a biosynthetic gene cluster and shares identity with the alcA gene required for synthesis of the siderophore alcaligin in Bordetella spp. A second mutation (foxR∷MudIIpR13) affects the synthesis of the ferrioxamine receptor FoxR, encoded by the foxR gene, and was shown to be transcribed into a monocistronic message. Accordingly, the foxR mutant accumulates DFO in the external medium. The growth of the mutants when supplied with various iron sources was examined; it indicates that the production of DFO and the specific transport of the DFO ferric complex are required only when iron is strongly liganded. Pathogenicity was scored after inoculation of apple seedlings and after infection of apple flowers. On seedlings, the DFO biosynthetic mutant behaved like the wild-type strain while the frequency of necrotic plants caused by the receptor mutant decreased by a factor of two to five, depending on the initial inoculum. On flowers, both mutants were strongly affected in their ability to initiate a necrotic symptom and their growth was reduced by two orders of magnitude relative to the wild-type strain. However, the virulence of the dfoA mutant varied with the inoculum concentration. Unlike the foxR mutant, the dfoA mutant only weakly induced plant cell electrolyte leakage in tobacco leaf disks. The supply with exogenous DFO, only when iron free, restored the ability to induce electrolyte leakage to the dfoA mutant and increased the leakage induced by other strains. DFO alone was not an inducer. Iron-free DFO was able to protect E. amylovora cells against lethal doses of hydrogen peroxide. The main conclusion was that production of DFO in E. amylovora during pathogenesis is not only a critical function for iron acquisition, but can play a role in the oxidative burst elicited by the bacteria.
Resistance Gene Candidates Identified by PCR with Degenerate Oligonucleotide Primers Map to Clusters of Resistance Genes in Lettuce
Authors: Shen Katherine A. , Meyers Blake C. , Islam-Faridi M. Nurul , Chin Doris B. , Stelly David M. , Michelmore Richard W.
Published: May 2007

The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.
Salicylic Acid Can Induce Resistance to Plant Virus Movement
Authors: Naylor Martin , Murphy Alex M. , Berry James O. , Carr John P.
Published: May 2007

Salicylic acid (SA) treatment has recently been reported to inhibit replication of tobacco mosaic virus (TMV) in inoculated tissue. Furthermore, resistance is induced via a novel defensive signal transduction pathway sensitive to inhibition by salicylhydroxamic acid (SHAM; S. Chivasa, A. M. Murphy, M. Naylor, and J. P. Carr, Plant Cell 9: 547–557, 1997). The goals of this study were to determine if replication of viruses other than TMV could be inhibited by SA and, if so, whether the resistance to other viruses could also be prevented by SHAM. Potato virus X (PVX) RNA accumulation in inoculated tobacco leaf tissue was reduced by SA treatment and resistance was dependent on the SHAM-sensitive signaling pathway. However, although symptoms of cucumber mosaic virus (CMV) infection were delayed in SA-treated tobacco plants, this was not due to inhibition of replication but rather to inhibition of systemic movement of the virus.14CO2-feeding experiments indicated that SA-induced interference with long-distance virus movement is not a by-product of disrupted photosynthate translocation. Significantly, SA-induced resistance to CMV was abolished by SHAM. Thus, the SHAM-sensitive signaling pathway activates both resistance mechanisms: inhibition of long-distance CMV movement and inhibition of TMV and PVX replication.