bluuebunny/crossref_metadata_2025 huggingface dataset:
The Coat Protein Is Required for the Elicitation of the Capsicum L2 Gene-Mediated Resistance Against the Tobamoviruses
Authors: de la Cruz A. , López L. , Tenllado F. , Díaz-Ruíz J. R. , Sanz A. I. , Vaquero C. , Serra M. T. , García-Luque I.
Published: May 2007

In Capsicum, the resistance against tobamoviruses conferred by the L2 gene is effective against all but one of the known tobamoviruses. Pepper mild mottle virus (PMMoV) is the only virus which escapes its action. To identify the viral factors affecting induction of the hypersensitive reaction (HR) mediated by the Capsicum spp. L2 resistance gene, we have constructed chimeric viral genomes between paprika mild mottle virus (PaMMV) (a virus able to induce the HR) and PMMoV. A hybrid virus with the PaMMV coat protein gene substituted in the PMMoV-S sequences was able to elicit the HR in Capsicum frutescens (L2L2) plants. These data indicate that the sequences that affect induction of the HR mediated by the L2 resistance gene reside in the coat protein gene. Furthermore, a mutant that codes for a truncated coat protein was able to systemically spread in these plants. Thus, the elicitation of the host response requires the coat protein and not the RNA.
Identification of a High Affinity Binding Site for Lipo-oligosaccharidic NodRm Factors in the Microsomal Fraction of Medicago Cell Suspension Cultures
Authors: Niebel Andreas , Bono Jean-Jacques , Ranjeva Raoul , Cullimore Julie V.
Published: May 2007

Protease-sensitive binding sites for a 35S-labeled ligand corresponding to the major lipo-oligosaccharidic symbiotic signal of Rhizobium meliloti (NodRm factor), have been identified in the microsomal fraction of Medicago varia cell suspension culture extracts. Binding was reversible and saturable and tetra-N-acetyl chitotetraose was a poor competitor of NodRm binding. Scatchard analysis suggests the presence of a high affinity binding site, termed Nod factor binding site two (NFBS2), with a Kd of 1.9 nM, and perhaps a second site with an affinity (Kd of 70 nM) similar to that of a site (NFBS1) previously characterized in Medicago truncatula root extracts.
Defense Response in Slash Pine: Chitosan Treatment Alters the Abundance of Specific mRNAs
Authors: Mason Mary E. , Davis John M.
Published: May 2007

We used differential display to identify chitosan responsive cDNAs in slash pine cell cultures. Two clones that showed increased mRNA abundance had sequence similarity to genes with roles in major plant defense responses, clone 18 to cinnamic acid 4-hydroxylase, and clone 30 to chitinase.
Cloning and Expression of the Cutinase A Gene of Botrytis cinerea
Authors: van der Vlugt-Bergmans C. J. B. , Wagemakers C. A. M. , van Kan J. A. L.
Published: May 2007

Cutinase of Botrytis cinerea has been suggested to play an important role in penetration of host tissues. A protein fraction with cutin hydrolyzing activity was purified from culture filtrates of B. cinerea induced for cutinase activity. An 18-kDa protein in this fraction was identified as cutinase and the corresponding gene cutA was cloned. The gene is present in a single copy in the genome of B. cinerea strain SAS56 and its predicted amino acid sequence shows significant homology (31 to 35% identity) to other fungal cutinases. RNA blot analysis showed that cutA mRNA is induced in vitro by the cutin monomer 16-hydroxy-hexadecanoic acid and repressed by glucose. The expression of cutA during infection of tomato leaves is low during early phases of infection, but high when the fungus has colonized the leaf and starts to sporulate.
Cutinase A of Botrytis cinerea is Expressed, but not Essential, During Penetration of Gerbera and Tomato
Authors: van Kan J. A. L. , van 't Klooster J. W. , Wagemakers C. A. M. , Dees D. C. T. , van der Vlugt-Bergmans C. J. B.
Published: May 2007

The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.
MsEnod12A Expression Is Linked to Meristematic Activity During Development of Indeterminate and Determinate Nodules and Roots
Authors: Bauer Petra , Poirier Simone , Ratet Pascal , Kondorosi Adam
Published: May 2007

During development of nodules and roots, the alfalfa early nodulin gene MsEnod12A is expressed adjacent to the meristem. Using transgenic alfalfa carrying a MsEnod12A promoter-gusA fusion, we investigated the regulation of MsEnod12A expression in mature nodules and roots by GUS assays and reverse transcription-PCR. We found that in alfalfa indeterminate nodules induced by various Fix- Rhizobium meliloti mutants and in spontaneous nodules devoid of rhizobia, MsEnod12A was expressed at the distal end when a persistent meristem was present. However, this gene was not expressed when the meristem was lacking in nodules arrested in development. The MsEnod12A-gusA fusion was introduced into Lotus corniculatus plants that form determinate nodules devoid of a persistent meristem. Using these plants we found MsEnod12A-gusA expression only in young nodules and a disappearance in mature nodules. Moreover, when alfalfa roots were treated with auxins a lateral band of MsEnod12A expression was observed surrounding the club-shaped root apex and coinciding with induced lateral meristematic activities. Thus, in all cases MsEnod12A expression was associated with meristematic activities, suggesting that MsEnod12A plays a role in the differentiation processes of nodule and root cells and that it may serve as molecular tool for analyzing meristem establishment during nodule development.
Characterization of a

S

alicylic

A

cid-

I

nsensitive Mutant (sai1) ofArabidopsis thaliana, Identified in a Selective Screen Utilizing the SA-Inducible Expression of thetms2Gene
Authors: Shah Jyoti , Tsui Frank , Klessig Daniel F.
Published: May 2007

Salicylic acid (SA) plays an important signaling role in the resistance of many plants to pathogen invasion. Increases in endogenous SA levels have been associated with the hypersensitive response as well as systemic acquired resistance (SAR). SA also induces the expression of a subset of the pathogenesis-related (PR) genes. However, relatively little is known about the events occurring subsequent to SA accumulation during a resistance response. In order to identify mutations in components of the SA signal transduction pathway, we have developed a genetic screen in Arabidopsis thaliana that utilizes the Agrobacterium tumefaciens tms2 gene as a counter-selectable marker. SA-inducible expression of the tms2 gene from the tobacco PR-1a promoter confers sensitivity to α-naphthalene acetamide (α-NAM), resulting in inhibition of root growth in germinating transgenic Arabidopsis seedlings. Mutants in which root growth is insensitive to α-NAM have been selected from this PR-1a:tms2 transgenic line with the expectation that a subset will lack a regulatory component downstream of SA. The sai1 mutant so identified expressed neither the PR-1a:tms2 transgene nor the endogenous Arabidopsis PR-1, PR-2, and PR-5 genes in response to SA. These genes also were not induced in sai1 by 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), two chemical inducers of SAR. As expected of a mutation acting downstream of SA, sai1 plants accumulate SA and its glucoside in response to infection with an avirulent pathogen and are more susceptible to this avirulent pathogen than the wild-type parent. sai1 is allelic to npr1, a previously identified SA-noninducible mutation. The recessive nature of the noninducible sai1 mutation suggests that the wild-type SAI1 gene acts as a positive regulator in the SA signal transduction pathway.
Cel1, Probably Encoding a Cellobiohydrolase Lacking the Substrate Binding Domain, Is Expressed in the Initial Infection Phase of Claviceps purpurea on Secale cereale
Authors: Müller Ulrike , Tenberge Klaus B. , Oeser Birgitt , Tudzynski Paul
Published: May 2007

At the host-pathogen interface of hyphae penetrating host cell walls in the rye ovary, a lack of cellulase-gold labeling of β-1,4-glucan in host cell walls indicates that enzymatic degradation of cellulose might be an important factor during the infection of rye by Claviceps purpurea. Using cbhI from Trichoderma reesei as a probe, a putative cellulase gene (cel1) was isolated from a genomic library of the C. purpurea strain T5. The coding region of 1,616 bp contains two introns and a putative signal peptidase cleavage site, leaving a coding capacity of 437 amino acids for the mature protein. The derived amino acid sequence shares significant homology with other fungal cellobiohydrolases and lacks the substrate binding domain. Expression analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) shows that cel1 is induced during the first days of infection of rye by C. purpurea. It may be involved in the penetration and degradation of host cell walls by depolymerizing plant β-1,4-glucan and, therefore, play a role in the infection process.
Effects of Rhizobium tropici, R. etli, and R. leguminosarum bv. phaseoli on nod Gene-Inducing Flavonoids in Root Exudates of Phaseolus vulgaris
Authors: Bolaños-Vásquez Maria Cristina , Werner Dietrich
Published: May 2007

Flavonoids play an important role as signal molecules in the early stages of the legume-Rhizobium symbiosis. The flavonoid content in root exudates of individual seedlings of Phaseolus vulgaris cv. Rab39 was determined by a sensitive method that combines absorption onto cellulose acetate filter strips with separation, identification, and quantification of individual compounds by high-performance liquid chromatography/diode-array detector and gas chromatography-mass spectroscopy analyses. We identified six flavonoids, daidzein, coumestrol, naringenin, genistein, liquiritigenin, and isoliquiritigenin, the latter two of which had not previously been found in bean root exudate. Biological activities were demonstrated by induction of β-galactosidase activity in a Rhizobium leguminosarum bv. phaseoli strain containing a nodC::lacZ fusion controlled by the nodD1 gene. Root exudate of common bean that had been inoculated with symbiotic R. etli, R. leguminosarum bv. phaseoli, or R. tropici contained more of the isoflavonoid daidzein, and the flavonoids naringenin, liquiritigenin, and isoliquiritigenin, than did exudate of plants that had not been inoculated. The relative concentrations of the signals depended on the nature of the bacterium. While daidzein, coumestrol, and naringenin were the dominant compounds in exudates of infected as well as uninfected seedlings, the daidzein and naringenin concentrations in particular were markedly enhanced after inoculation by all strains.
Identification of a Pathogenicity Locus, rpfA, in Erwinia carotovora subsp. carotovora That Encodes a Two-Component Sensor-Regulator Protein
Authors: Frederick Reid D. , Chiu Jiliang , Bennetzen Jeffrey L. , Handa Autar K.
Published: May 2007

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.
A Putative Amino Acid Transporter Is Specifically Expressed in Haustoria of the Rust Fungus Uromyces fabae
Authors: Hahn Matthias , Neef Ulrike , Struck Christine , Göttfert Michael , Mendgen Kurt
Published: May 2007

A cDNA library constructed from haustoria of the rust fungus Uromyces fabae was screened for clones that are differentially expressed in haustoria. One family of cDNAs (in planta-induced gene 2 [PIG2]) was isolated and found to encode a protein with high homologies to fungal amino acid transporters. A cDNA clone containing the complete coding region of PIG2 and the corresponding genomic clone were isolated and sequenced, revealing the presence of 17 introns in the PIG2 gene. Expression of PIG2 mRNA appeared to be restricted to haustoria. With antibodies raised against synthetic peptides, the PIG2-encoded protein was found in membrane fractions of isolated haustoria but not of germinated rust spores. With immunofluo-rescence microscopy, the putative amino acid transporter was localized to plasma membranes of the haustorial bodies, but not detected in the haustorial neck, haustorial mother cells, or intercellular fungal hyphae growing within infected leaf tissue. These data present for the first time molecular evidence that the rust haustorium plays a special role in the uptake of nutrients from an infected host cell.
Two Distinct Uricase II (Nodulin 35) Genes Are Differentially Expressed in Soybean Plants
Authors: Takane Ken-ichi , Tajima Shigeyuki , Kouchi Hiroshi
Published: May 2007

Nodule-specific uricase (uricase II) is a homotetramer of a 33-kDa polypeptide, nodulin 35, and plays a key role in the assimilation of nitrogen fixed by microsymbionts in most legumes that have determinate nodules. We have isolated two distinct genes, UR2 and UR9, that encode for nodulin 35 from a soybean genomic library. Their corresponding cDNAs were also isolated from a nodule cDNA library. UR2 and UR9 both encode for 309 amino acid proteins with 12 amino acid differences. The expression of these two genes in various organs of soybean was examined by reverse transcription-polymerase chain reaction with primers specific to each cDNA sequences. Expression of UR9 was almost specific in root nodules, although it was expressed in roots, primary leaves, and developing seed at very low levels. In contrast, the UR2 transcripts were present in almost all plant organs at low levels, but no enhancement of the expression was observed in nodules. Thus, UR9 behaves as a nodulin gene, whereas UR2 is a nonsymbiotic uricase II gene. The sequences of their potential promoter regions share high homology within regions up to about 400 bp upstream from the translation initiation sites. These results suggest that symbiotic and nonsymbiotic uricase II genes diverged by gene duplication and that relatively small alterations in the promoter sequence enable the nodule-specific expression.
Nht1, a Transposable Element Cloned from a Dispensable Chromosome inNectria haematococca
Authors: Enkerli Jürg , Bhatt Garima , Covert Sarah F.
Published: May 2007

Certain isolates of the plant-pathogenic fungus Nectria haematococca mating population VI (MPVI) contain dispensable chromosomes that are unstable during sexual reproduction. Several of these chromosomes carry genes for phytoalexin detoxification and thus contribute to the pathogenic potential of this organism. A repeated DNA sequence, Nht1, was cloned from one of these dispensable chromosomes in N. haematococca MPVI. One copy of the repeated element (Nht1A) was completely sequenced. It is 2,198 bp long and it possesses incomplete inverted terminal repeats (ITRs) at each end. Nht1B, a partially sequenced copy of Nht1, has complete ITRs. Nht1A appears to contain 2 introns and encodes a protein of 550 amino acids that is highly similar to the protein encoded by the Fusarium oxysporum transposon, Fot1. Due to the presence of ITRs, its repeated nature, and its similarity to Fot1, we conclude that Nht1 is a transposable element. Within North American N. haematococca MPVI populations, Nht1 is distributed discontinuously. Its copy number in different field isolates varies from zero to approximately 100 copies per genome. The Nht1A source isolate is estimated to contain nine to 11 copies of Nht1; at least six are on the chromosome from which Nht1A was cloned.
Salicylic Acid Produced by Serratia marcescens 90-166 Is Not the Primary Determinant of Induced Systemic Resistance in Cucumber or Tobacco
Authors: Press C. M. , Wilson M. , Tuzun S. , Kloepper J. W.
Published: May 2007

The rhizobacterial strain Serratia marcescens 90–66 mediates induced systemic resistance (ISR) to fungal, bacterial, and viral pathogens. It was determined that strain 90–166 produced salicylic acid (SA), using the salicylateresponsive reporter plasmid pUTK21. High-pressure liquid chromatography analysis of culture extracts confirmedthe production of SA in broth culture. Mini-Tn5phoA mutants, which did not produce detectable amounts of SA, retained ISR activity in cucumber against the fungal pathogen Colletotrichum orbiculare. Strain 90–166 induced disease resistance to Pseudomonas syringae pv. tabaci in wild-type Xanthi-nc and transgenic NahG-10 tobacco expressing salicylate hydroxylase. Increasing ferric iron concentrations in vitro reduced SA production below detectable limits, and increasing ferric iron concentration in planta, applied as a root drench, significantly reduced the level of ISR observed in cucumber to C. orbiculare. An ISR¯ mutant (90-166-2882) still produced SA. The results of this study indicate that SA produced by 90–166 is not the primary bacterial determinant of ISR and that this bacterial-mediated ISR system is affected by iron concentration.
Endopolygalacturonase Genes fromColletotrichum lindemuthianum:Cloning ofCLPG2and Comparison of Its Expression to That ofCLPG1During Saprophytic and Parasitic Growth of the Fungus
Authors: Centis Sylvie , Guillas Isabelle , Séjalon Nathalie , Esquerré-Tugayé Marie-Thérèse , Dumas Bernard
Published: May 2007

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.
Rapid, Plateau-like Increases in Intracellular Free Calcium Are Associated with Nod-Factor—Induced Root-Hair Deformation
Authors: Gehring Christoph A. , Irving Helen R. , Kabbara Akram A. , Parish Roger W. , Boukli Nawal M. , Broughton William J.
Published: May 2007

Rhizobia excrete variously substituted lipo-oligosaccha-ride Nod factors into the legume rhizosphere. Homologous legumes respond to these signals through deformation of the root hairs and the development of nodulation foci in the root cortex. Cellular events in root hairs from the susceptible zone of nearly mature root hairs were studied in root segments loaded with the calcium indicators Fura-2 or Fluo-3. Application of 10-9 M Nod factors of the broad-host-range Rhizobium sp. NGR234 to the homologous legume Vigna unguiculata resulted, within seconds, in plateau-like increases in intracellular free calcium ([Ca2+]i) in the root hairs and root epidermal cells. Nod factors of R. meliloti at 10-9 M caused equally rapid increases in [Ca2+]i in the root hairs and epidermal cells of the nonhost V. unguiculata, and also induced root-hair deformation. The chitin tetramer, N-N′-N″-N′″-tetracetylchitotetraose, which represents the backbone of Nod factors, induced neither root-hair deformation nor changes in [Ca2+]i in V. unguiculata. Root hairs and epidermal cells of the nonlegume non-host Arabidopsis thaliana showed neither [Ca2+]i increases nor root-hair deformation in response to both factors.
Extracellular Polysaccharides from Xanthomonas axonopodis pv. manihotis Interact with Cassava Cell Walls During Pathogenesis
Authors: Boher B. , Nicole M. , Potin M. , Geiger J. P.
Published: May 2007

The location of lipopolysaccharides produced by Xanthomonas axonopodis pv. manihotis during pathogenesis on cassava (Manihot esculenta) was determined by fluorescence and electron microscopy immunolabeling with monoclonal antibodies. During the early stages of infection, pathogen lipopolysaccharides were detected on the outer surface of the bacterial envelope and in areas of the plant middle lamellae in the vicinity of the pathogen. Later in the infection process, lipopolysaccharide-specific antibodies bound to areas where the plant cell wall was heavily degraded. Lipopolysaccharides were not detected in the fibrillar matrix filling intercellular spaces of infected cassava leaves. Monoclonal antibodies specific for the exopolysaccharide xanthan side chain labeled the bacteria, the fibrillar matrix, and portions of the host cell wall. The association of Xanthomonas lipopolysaccharides with host cell walls during plant infection is consistent with a role of these bacterial extracellular polysaccharides in the infection process.
Assignment of Amino Acid Residues of the AVR9 Peptide ofCladosporium fulvumThat Determine Elicitor Activity
Authors: Kooman-Gersmann Miriam , Vogelsang Ralph , Hoogendijk Erwin C. M. , De Wit Pierre J. G. M.
Published: May 2007

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX∷Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX∷Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the “hydrophobic β-loop” region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.
Mobilization, Cloning, and Sequence Determination of a Plasmid-Encoded Polygalacturonase from a Phytopathogenic Burkholderia (Pseudomonas) cepacia
Authors: Gonzalez Carlos F. , Pettit Elizabeth A. , Valadez Victoria A. , Provin Ellen M.
Published: May 2007

Phytopathogenic strains of Burkholderia cepacia (synonym Pseudomonas cepacia) produce endopolygalacturonase, whereas strains of clinical and soil origin do not. Growth of a phytopathogenic strain (ATCC25416) at elevated temperatures resulted in nonpectolytic derivatives that were either cured of a resident plasmid or contained a plasmid of reduced mass. The resident 200-kb plasmid (pPEC320) in strain ATCC25416 was tagged with Tn5-Mob. The pPEC320∷Tn5-Mob (pPEC321) plasmid was mobilized in B. cepacia strains of soil and clinical origin. Transconjugants containing pPEC321 expressed the endopolygalac-turonase and showed differential activity on plant tissue. No evidence for self-transfer of pPEC320 or the tagged derivative was observed. A 2.85-kb cloned fragment from pPEC320 containing the plasmid-borne pehA gene was sequenced and compared to the pehA gene from Erwinia carotovora subsp. carotovora and Ralstonia solanacearum and the polygalacturonase sequence from Lycopersicon esculentum.
Polygenic Resistance of Pepper to Potyviruses Consists of a Combination of Isolate-Specific and Broad-Spectrum Quantitative Trait Loci
Authors: Caranta Carole , Lefebvre Véronique , Palloix Alain
Published: May 2007

At least six potyviruses infect pepper crops around the world. Only monogenic resistance with isolate-specific effects or broad-spectrum effects against potyviruses has been identified. Quantitative trait loci (QTLs) for resistance to two potato virus Y (PVY) isolates and to potyvirus E have been mapped in a doubled-haploid progeny from the hybrid between the line Perennial with resistance factors against several potyviruses and the line Yolo Wonder, with 172 DNA markers. Eleven chromosomal regions were found to be associated with quantitative resistance to PVY and potyvirus E by both analysis of variance and non-parametric tests. This resistance results from the association of a major-effect QTL and several minor-effect QTLs. Two minor-effect QTLs originated from the susceptible parent. In addition to additive-effect QTLs, interactions between QTLs were also identified. Based on their spectrum of action, we discriminated QTLs with PVY isolate-or potyvirus-specific effect and loci involved in the resistance to several distinct potyviruses. The locations of QTLs were compared with loci having known qualitative effects against potyviruses; QTLs for PVY and potyvirus E resistance were detected in the vicinity of the pvr2 and pvr6 loci. The origin of the broad-spectrum resistance against potyviruses and the relationships between major genes and QTLs are discussed.
Evaluation of the Role of RecA Protein in Plant Virulence with recA Mutants of Xanthomonas campestris pv. campestris
Authors: Martínez Socorro , Martínez-Salazar Jaime , Camas Alberto , Sánchez Rosalba , Soberón-Chávez Gloria
Published: May 2007

Xanthomonas campestris pv. campestris NRRL B1459 recA mutants were isolated by recombination with an interrupted Rhizobium etli recA gene and selection of double recombinants. The mutants were impaired in homologous genetic recombination and in DNA repair as judged by their sensitivity to methyl-methane-sulfonate and to UV irradiation; these defects are complemented in trans by the R. etli recA gene. Virulence of X. campestris pv. campestris NRRL B1459 to cabbage is considerably diminished by the recA mutation. The recA mutation is not correlated with the frequency of occurrence of a genetic rearrangement that affects chemotaxis, plant virulence, and xanthan gum production.
A Single Amino Acid Substitution in the Virus-Encoded Replicase of Tomato Mosaic Tobamovirus Alters Host Specificity
Authors: Hamamoto Hiroshi , Watanabe Yuichiro , Kamada Hiroshi , Okada Yoshimi
Published: May 2007

Introduction of a single amino acid substitution (Gln979 to Ile) into the 130- and 180-kDa proteins of tomato mosaic tobamovirus (ToMV) rendered the mutant virtually unable to replicate in tomato cells while it replicated perfectly in tobacco cells. The mechanism of this change in host specificity is discussed.
Encapsidation of Potyviral RNA in Various Forms of Transgene Coat Protein Is Not Correlated with Resistance in Transgenic Plants
Authors: Hammond John , Dienelt Margaret M.
Published: May 2007

Transgenic plants expressing either bean yellow mosaic potyvirus or chimeric potyvirus coat protein (CP) were inoculated with various potyviruses. Antigen-coated plate, indirect enzyme-linked immunosorbent assay and immunoelectron microscopy of virus purified from transgenic plants showed that progeny virions contained from <1% to as much as 25% transgenic CP. Different levels of transcapsidation may reflect the extent of compatibility between transgene CP and the viral CP.
Region II of Rhizobium sp. NGR234 Inhibits Nodulation of Medicago sativa by R. meliloti nodIJ and nodQ1 Mutants
Authors: Rochepeau P. , Fellay R. , Jabbouri S. , Perret X. , Broughton W. J.
Published: May 2007

Rhizobium sp. NGR234 contains a plasmid-borne locus that hybridized strongly to region II of R. meliloti. Surprisingly, NGR region II completely inhibited nodulation of Medicago sativa when conjugated into either R. meliloti nodIJ or nodQ1 region II mutants. Further characterization showed that region II of NGR234 contains three putative coding open reading frames (ORFs), which are homologous to, respectively, hypothetical protein A of R. leguminosarum, hypothetical protein C of R. leguminosarum, and ORF2 of Agrobacterium tumefaciens insertion sequence (IS) 66, as well as an ORF of unknown function located downstream of nodQ1 of R. meliloti. A site-directed mutation in the IS66 homologue improved nodulation efficiency on some NGR234 hosts, but the structure and composition of the Nod-factor family produced by the mutant were unchanged. The quantity of Nod factors secreted was reduced by two-thirds, however. Insertions or deletions of the genes encoding either the hypothetical protein C or the IS66 homologue abolished the blocking effect observed in the “complementation” tests with R. meliloti region II mutants.
Targeted Disruption of a Fungal G-Protein β Subunit Gene Results in Increased Vegetative Growth but Reduced Virulence
Authors: Kasahara Shin , Nuss Donald L.
Published: May 2007

Targeted disruption of two G-protein α subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Giα subunit CPG-1 in fungal reproduction, virulence, and vegetative growth. A second Gα subunit, CPG-2, was found to be dispensable for these functions. We now report the cloning and targeted disruption of a C. parasitica G-protein β subunit gene. The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with Gβ homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae Gβ homologue STE4 product. Low stringency Southern hybridization failed to detect any related Gβ subunit genes in C. parasitica. Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C. parasitica Giα subunit gene cpg-1. These included very significant reductions in pigmentation, asexual sporulation, and virulence. In contrast to results obtained for Giα gene disruption, the reduction in virulence resulting from the disruption of a Gβ gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium. The relevance of these results to mechanisms of fungal virulence is considered.
High Resolution Genetic and Physical Mapping of Molecular Markers Linked to the Phytophthora Resistance Gene Rps1-k in Soybean
Authors: Kasuga Takao , Salimath Shanmukhaswami S. , Shi Jinrui , Gijzen Mark , Buzzell Richard I. , Bhattacharyya Madan K.
Published: May 2007

The resistance of soybean to Phytophthora root and stem rot caused by Phytophthora sojae is conferred by a series of single-dominant Rps genes. We have applied random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses to isolate molecular markers linked to Rps1-k. Five RAPD markers were identified and mapped closely to one side of Rps1-k. AFLP analysis was carried out with near isogenic lines and bulks obtained from F3 families. Twenty-seven markers were identified. Nineteen of these were specific to the resistant parent. Five AFLP markers were amplified from the susceptible parent. One of these markers, TC1, mapped at 0.07 centimorgans (cM) from the Rps1 locus. Three AFLP markers were co-dominant, and one of these, CG1, mapped at a distance of 0.06 cM from the Rps1 locus on the opposite side of the rest of the markers. Two RAPD, 17 AFLP, and 14 restriction fragment length polymorphism (RFLP) markers originating from duplicated sequences were mapped within a 3-cM map interval. These results suggest that Rps1-k is located at the end of an introgressed region. Physical mapping data indicate that the Rps1-k-flanking markers CG1 and TC1 may be located within a 125-kb chromosomal fragment.
Cell Wall-Degrading Enzymes from Erwinia carotovora Cooperate in the Salicylic Acid-Independent Induction of a Plant Defense Response
Authors: Vidal Sabina , Eriksson Anders R. B. , Montesano Marcos , Denecke Jürgen , Palva E. Tapio
Published: May 2007

The virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a large variety of plant cell wall-degrading enzymes, including several pectinases and cellulases. Treatment of tobacco plants with culture filtrates (CFs) from the pathogen (containing the secreted cell wall-degrading enzymes) induces both local and systemic expression of genes involved in plant defense response. We have characterized the contribution of individually produced cell wall-degrading enzymes from E. carotovora subsp. carotovora in their ability to trigger local and systemic induction of a defense-related gene (coding for a basic β-1,3-glucanase) as well as resistance to this pathogen in tobacco. Our results show that mainly pectic enzymes and to some extent one cellulase induce expression of the β-1,3-glucanase gene. Cellulases appear to act cooperatively in the defense gene induction when added in combination with pectic enzymes. Similarly, pectinases and cellulases cooperate in triggering systemic resistance to the pathogen. Salicylic acid (SA) does not appear to be involved in this process, as systemic resistance was induced similarly in transgenic NahG plants that overproduce a salicylate hydroxylase and cannot accumulate SA and in nontrans-formed control plants. The lack of SA requirement for the induced resistance against E. carotovora subsp. carotovora suggests the presence of a different signal transduction pathway involved in this plant-pathogen interaction.
NodFE-Dependent Fatty Acids That Lack an α-β Unsaturation Are Subject to Differential Transfer, Leading to Novel Phospholipids
Authors: Geiger Otto , Glushka John , Lugtenberg Ben J. J. , Spaink Herman P. , Thomas-Oates Jane E.
Published: May 2007

In Rhizobium leguminosarum, the nodABC and nodFEL operons are involved in the production of lipo-chitin oligosaccharide signals that mediate host specificity. A nodFE-determined, highly unsaturated C18:4 fatty acid (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule meristems and pre-infection thread structures on the host plant Vicia sativa. Of the nod genes, induction of only nodFE is sufficient to modify fatty acid biosynthesis to yield trans-2, trans-4, trans-6, cis-11-octadeca-tetraenoic acid, with an absorbance maximum of 303 nm. This unusual C18:4 fatty acid is not only found in the lipo-chitin oligosaccharides but is also associated with the phospholipids (O. Geiger, J. E. Thomas-Oates, J. Glushka, H. P. Spaink, and B. J. J. Lugtenberg, 1994, J. Biol. Chem. 269:11090-11097). Here we report that the phospholipids can contain other nodFE-derived fatty acids, a C18:3 trans-4, trans-6, cis-11-octadecatrienoic acid that has a characteristic absorption maximum at 225 nm, and a C18:2 octadecadienoic acid. Neither this C18:3 nor this C18:2 fatty acid has to date been observed attached to lipo-chitin oligosaccharides, suggesting that an as yet unknown acyl transferase (presumably NodA), responsible for the transfer of the fatty acyl chain to the glycan backbone of the lipo-chitin oligosaccharides, does not transfer all fatty acids synthesized by the action of NodFE to the lipo-chitin oligosaccharides. Rather, it must have a preference for α-β unsaturated fatty acids during transfer.
Phytophthora Resistance Through Production of a Fungal Protein Elicitor (β-Cryptogein) in Tobacco
Authors: Tepfer David , Boutteaux Catherine , Vigon Catherine , Aymes Sylvie , Perez Valérie , O'Donohue Michael J. , Huet Jean-Claude , Pernollet Jean-Claude
Published: May 2007

Transformation of tobacco with a gene encoding the fungal elicitor protein, β-cryptogein, resulted in resistance to the pathogen Phytophthora parasitica var. nicotianae. Resistance was improved when the foreign gene was in the hemizygous state, and a single amino acid substitution that reduced the necrotic effects of the protein also conferred some resistance.
Sulfation of Nod Factors via nodHPQ Is nodD Independent in Rhizobium tropici CIAT899
Authors: Folch-Mallol Jorge Luis , Manyani Hamid , Marroquí Silvia , Sousa Carolina , Vargas Carmen , Nava Noreide , Colmenero-Flores José Manuel , Quinto Carmen , Megías Manuel
Published: May 2007

A cosmid from the Rhizobium tropici CIAT899 symbiotic plasmid, containing most of the nodulation genes described in this strain, has been isolated. Although this cosmid does not carry a nodD gene, it confers ability to heterologous Rhizobium spp. to nodulate R. tropici hosts (Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala). The observed phenotype is due to constitutive expression of the nodABCSUIJ operon, which has lost its regulatory region and is expressed from a promoter present in the cloning vector. Thin-layer chromatography (TLC) analysis of the Nod factors produced by this construction shows that it is still capable of synthesizing sulfated compounds, suggesting that the nodHPQ genes are organized as an operon that is transcribed in a nodD-independent manner and is not regulated by flavonoids.

Se ha aislado un cósmido del plásmido simbiótico de Rhizobium tropici CIAT899 que contiene la mayoría de los genes de nodulación descrito para esta estirpe, menos el gen regulador nodD. La introducción de este cósmido en una estirpe curada del plásmido simbiótico de R. tropici CIAT899 permite la nodulación en las plantas ensayadas (Phaseolus vulgaris, Macroptilium atropurpureum, y Leucaena leucocephala). El fenotipo observado se debe a la expresión constitutiva del operón nodABCSUIJ bajo el promotor del gen de resistencia a la kanamicina, que lleva el vector donde se ha clonado el fragmento de ADN. Análisis por cromatografia de capa fina demuestran que esta construcción es capaz de sulfatar el extremo reductor del factor Nod. Estas evidencias sugieren que los genes nodHPQ constituyen un operón, y que su expresión es independiente del gen regulador nodD.
Aldonic Acids: A Novel Family of nod Gene Inducers of Mesorhizobium loti, Rhizobium lupini, and Sinorhizobium meliloti
Authors: Gagnon Hubert , Ibrahim Ragai K.
Published: May 2007

Molecular signals, such as flavonoids (or nonflavonoid type), nod gene-inducers, and bacterial lipochitin oligosac-charides (LCOs) act as modulators of species specificity during early stages of infection in Rhizobium spp.-legume interactions. The fact that signaling in Lupinus albus remains to be determined prompted us to investigate the flavonoid signal responsible for nod gene induction in Rhizobium lupini. A screening method was used based on the measurement of β-galactosidase activity of R. lupini strains harboring nodC::lacZ fusions in the presence of (i) authentic lupin isoflavones, (ii) carbohydrate-like inducers, and (iii) high-pressure liquid chromatography (HPLC)-fractionated lupin seed effusates and root exudates, as putative nod gene inducers. The results indicate that both erythronic and tetronic acids (4-C sugar acids) led to low but significant increases in β-galactosidase activities, compared with the controls. In addition, lupi-wighteone, a monoprenylated isoflavone, exerts a synergistic effect with the carbohydrate-like inducers, compared with other isoflavone treatments. The natural occurrence of aldonic acids in L. albus root exudates and seed effusates has been demonstrated by HPLC analysis. When tested with nodC::lacZ fusions, tetronic acid resulted in nod gene induction in Sinorhizobium meliloti. In addition, a combination of luteolin and tetronic acid promotes further increases in S. meliloti nod gene expression, as shown by β-galactosidase assays. Incorporation studies with [14C]LCO precursors confirmed the inductive role of both erythronic and tetronic acids in promoting LCO biosynthesis in R. lupini cultures, and of tetronic acid in Mesorhizobium loti and S. meliloti. Hydrolysis of the LCOs with various enzymes substantiated their putative identities. These results are discussed in relation to the impact of these unusual signal molecules on our knowledge of flavonoid signaling in Rhizobium-legume symbiosis.
Expression and Regulation of Phosphate Stress Inducible Genes in Sinorhizobium meliloti
Authors: Summers Michael L. , Elkins James G. , Elliott Brian A. , McDermott Timothy R.
Published: May 2007

Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be upregulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB¯ mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.
Production of Acyl-Homoserine Lactone Quorum-Sensing Signals by Gram-Negative Plant-Associated Bacteria
Authors: Cha Chung , Gao Ping , Chen Yu-Ching , Shaw Paul D. , Farrand Stephen K.
Published: May 2007

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorumsensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of l-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-l-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonads, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-l-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.
Mutagenesis of Putative Acylation Sites Alters Function, Localization, and Accumulation of a Giα Subunit of the Chestnut Blight Fungus Cryphonectria parasitica
Authors: Gao Shaojian , Nuss Donald L.
Published: May 2007

Targeted disruption of cpg-1, a gene encoding the G protein Giα subunit, CPG-1, in the chestnut blight fungus, Cryphonectria parasitica, results in reduced mycelial growth, reduced orange pigmentation, loss of virulence, loss of asexual sporulation, and female infertility. We report the development of a complementation system for cpg-1 null mutants and its use to evaluate the in vivo consequences of mutating conserved putative CPG-1 myristoylation (G2) and palmitoylation (C3) sites. Independent mutations of the two putative acylation sites differentially altered complex fungal biological processes, including virulence, and modified CPG-1 membrane association. Results of combined Northern (RNA) and Western (immunoblot) analysis also indicated a role for lipid modification in post-transcriptional regulation of CPG-1 accumulation.