bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation of fixN-Reiterated Genes in Rhizobium etli by a Novel fixL—fixK Cascade
Authors: Girard Lourdes , Brom Susana , Dávalos Araceli , López Oswaldo , Soberón Mario , Romero David
Published: May 2007

Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation.
Chromosomal Insertion of Phenazine-1-Carboxylic Acid Biosynthetic Pathway Enhances Efficacy of Damping-off Disease Control by Pseudomonas fluorescens
Authors: Timms-Wilson T. M. , Ellis R. J. , Renwick A. , Rhodes D. J. , Mavrodi D. V. , Weller D. M. , Thomashow L. S. , Bailey M. J.
Published: May 2007

A disarmed Tn5 vector (pUT∷Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P. fluorescens∷phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.
NPR1 Differentially Interacts with Members of the TGA/OBF Family of Transcription Factors That Bind an Element of the PR-1 Gene Required for Induction by Salicylic Acid
Authors: Zhou Jun-Ma , Trifa Youssef , Silva Herman , Pontier Dominique , Lam Eric , Shah Jyoti , Klessig Daniel F.
Published: May 2007

NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway leading to the induction of defense genes, such as the pathogenesis-related (PR)-1 gene, and enhanced disease resistance. Using a yeast two-hybrid screen, we identified several NPR1-interacting proteins (NIPs). Two of these NIPs are members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors; this family has been implicated in the activation of SA-responsive genes, including PR-1. Six TGA family members were tested and shown to differentially interact with NPR1: TGA2 and TGA3 showed strong affinity for NPR1; TGA5 and TGA6 exhibited weaker affinity; and TGA1 and TGA4 displayed little or no detectable interaction with NPR1, respectively. Interestingly, the amino-termini of these factors were found to decrease their stability in yeast and differentially affect their apparent affinity toward NPR1. The interacting regions on NPR1 and the TGA factors were also defined. Each of four point mutations in NPR1 that disrupt SA signaling in Arabidopsis completely blocked interaction of NPR1 with TGA2 and TGA3. TGA2 and TGA3 were also found to bind the SA-responsive element of the Arabidopsis PR-1 promoter. These results directly link NPR1 to SA-induced PR-1 expression through members of the TGA family of transcription factors.
Virus Recovery Is Induced in Brome mosaic virus p2 Transgenic Plants Showing Synchronous Complementation and RNA-2-Specific Silencing
Authors: Iyer Lakshminarayan M. , Hall Timothy C.
Published: May 2007

Nicotiana benthamiana plants expressing Brome mosaic virus (BMV) p2 protein complemented replication of RNAs1+3 but, surprisingly, supported little or no replication of RNA-2. Despite this, the p2 transgenic plants were able to support systemic migration of RNAs-1 and -3. Kinetic analyses showed identical degradation rates for RNAs-2 and -3, greatly detracting from the concept of an induction of an RNA-2-specific degradation system. Deletion analysis identified a 200-nucleotide sequence that may contribute to silencing in a context-specific manner. When (R)1 progeny of a severely silencing p2 transgenic line were tested for virus resistance, three different classes of reactions were observed. In class 1 and class 3 plants, the virus moved systemically and showed various extents of RNA-2 silencing. However, in class 2 plants, there was a stochastic onset of post-transcriptional silencing in the systemic leaves that was reminiscent of virus recovery. Plants showing recovery tended to have a greater number of transgene loci than did those exhibiting componentspecific silencing. The induction of silencing did not appear to be dependent solely on the combined steady state levels of the transgene and viral RNA. Some plants transformed with a p2 frameshift construct showed a complete silencing phenotype, but none showed RNA-2-specific silencing. While the relationship between the two types of silencing remains unclear, we speculate that our observations reflect early events in the induction of virus recovery.
Transgenic Plants Expressing Geminivirus Movement Proteins: Abnormal Phenotypes and Delayed Infection by Tomato mottle virus in Transgenic Tomatoes Expressing the Bean dwarf mosaic virus BV1 or BC1 Proteins
Authors: Hou Yu-Ming , Sanders Rick , Ursin Virgina M. , Gilbertson Robert L.
Published: May 2007

Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from Bean dwarf mosaic virus (BDMV) were generated and examined for phenotypic effects and resistance to Tomato mottle virus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes, compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth, and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3′ region of the gene, which resulted in expression of a truncated BC1 protein. Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection, compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of viral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.
Involvement of Diamine Oxidase and Peroxidase in Insolubilization of the Extracellular Matrix: Implications for Pea Nodule Initiation by Rhizobium leguminosarum
Authors: Wisniewski J.-P. , Rathbun E. A. , Knox J. P. , Brewin N. J.
Published: May 2007

Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhy-droxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. le-guminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.
Evidence Against a Direct Antimicrobial Role of H2O2 in the Infection of Plants by Erwinia chrysanthemi
Authors: Miguel Eugenio , Poza-Carrión César , López-Solanilla Emilia , Aguilar Isabel , Llama-Palacios Arancha , García-Olmedo Francisco , Rodríguez-Palenzuela Pablo
Published: May 2007

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Es-cherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.
Evidence that the cAMP Pathway Controls Emergence of Both Primary and Appressorial Germ Tubes of Barley Powdery Mildew
Authors: Kinane Julia , Dalvin Sussie , Bindslev Lene , Hall Alison , Gurr Sarah , Oliver Richard
Published: May 2007

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.
P42 Movement Protein of Beet necrotic yellow vein virus Is Targeted by the Movement Proteins P13 and P15 to Punctate Bodies Associated with Plasmodesmata
Authors: Erhardt M. , Morant M. , Ritzenthaler C. , Stussi-Garaud C. , Guilley H. , Richards K. , Jonard G. , Bouzoubaa S. , Gilmer D.
Published: May 2007

Cell-to-cell movement of Beet necrotic yellow vein virus (BNYVV) is driven by a set of three movement proteins—P42, P13, and P15—organized into a triple gene block (TGB) on viral RNA 2. The first TGB protein, P42, has been fused to the green fluorescent protein (GFP) and fusion proteins between P42 and GFP were expressed from a BNYVV RNA 3-based replicon during virus infection. GFP-P42, in which the GFP was fused to the P42 N terminus, could drive viral cell-to-cell movement when the copy of the P42 gene on RNA 2 was disabled but the C-terminal fusion P42-GFP could not. Confocal microscopy of epidermal cells of Chenopodium quinoa near the leading edge of the infection revealed that GFP-P42 localized to punctate bodies apposed to the cell wall whereas free GFP, expressed from the replicon, was distributed uniformly throughout the cytoplasm. The punctate bodies sometimes appeared to traverse the cell wall or to form pairs of disconnected bodies on each side. The punctate bodies co-localized with callose, indicating that they are associated with plasmodesmata-rich regions such as pit fields. Point mutations in P42 that inhibited its ability to drive cell-to-cell movement also inhibited GFP-P42 punctate body formation. GFP-P42 punctate body formation was dependent on expression of P13 and P15 during the infection, indicating that these proteins act together or sequentially to localize P42 to the plasmodesmata.
avrPto Enhances Growth and Necrosis Caused by Pseudomonas syringae pv. tomato in Tomato Lines Lacking Either Pto or Prf
Authors: Chang Jeff H. , Rathjen John P. , Bernal Adriana J. , Staskawicz Brian J. , Michelmore Richard W.
Published: May 2007

AvrPto was introduced into three tomato genotypes with two biotic agents to study its role in compatible interactions. avrPto enhanced the capacity of the Pseudomonas syringae pv. tomato strain T1 to induce necrotic symptoms on tomato plants that lacked either Pto or Prf genes. The enhanced necrosis correlated with a small increase in bacterial growth. In planta expression of avrPto in isolation did not elicit necrosis in the absence of a functional Prf gene.
The Pre-Symbiotic Growth of Arbuscular Mycorrhizal Fungi Is Induced by a Branching Factor Partially Purified from Plant Root Exudates
Authors: Buee M. , Rossignol M. , Jauneau A. , Ranjeva R. , Bécard G.
Published: May 2007

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.
Localized Expression of Cathepsin B-like Sequences from Root Nodules of Pea (Pisum sativum)
Authors: Vincent Jason L. , Dahiya Preeti , Brewin Nicholas J.
Published: May 2007

Cathepsin B is an ancient family of eukaryotic cysteine proteases. We describe PsCat1, a plant cathepsin B-like transcript, identified as an expressed sequence in Rhizobium-induced, nitrogen-fixing root nodules of pea. In situ hybridization studies in root nodules showed strong, extremely localized expression of PsCat1 in individual cells associated with the central infected tissue. Restriction fragment polymorphism mapping of the PsCat1 locus in pea shows no correlation with existing mutant lines defective in symbiosis.
Identification and Characterization of a Hexapeptide with Activity Against Phytopathogenic Fungi That Cause Postharvest Decay in Fruits
Authors: López-García Belén , González-Candelas Luis , Pérez-Payá Enrique , Marcos Jose F.
Published: May 2007

A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH 2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicillium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 μM and 50% inhibitory concentration (IC50) of 30 to 40 μM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicillium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues.
Positive and Negative Regulation of Salicylic Acid-Dependent Cell Death and Pathogen Resistance in Arabidopsis lsd6 and ssi1 Mutants
Authors: Greenberg Jean T.
Published: May 2007

Salicylic acid (SA) is a key defense molecule in higher plants that is required for resistance to diverse pathogens. A number of mutants of Arabidopsis with elevated resistance to pathogens and constitutive activation of defenserelated genes and cell death have been shown to require SA for all of their phenotypes. These mutants potentially identify interesting regulatory genes that control diverse SA responses. When dominant mutations confer SA-dependent phenotypes, it is important to know the genetic basis of dominance in order to draw conclusions on the possible mechanisms of action of the genes identified. Here I characterize the basis of the dominant phenotypes conferred by the ssi1 and lsd6 mutations. I show that ssi1 is haploinsufficient, while lsd6 is a gain-of-function mutation. Thus, SA-dependent responses are under both negative and positive regulation.
External pH: An Environmental Signal That Helps to Rationalize pel Gene Duplication in Erwinia chrysanthemi
Authors: Nachin Laurence , Barras Frédéric
Published: May 2007

The phytopathogenic bacterium Erwinia chrysanthemi produces five major pectate lyases that are key virulence factors in soft-rot disease development. Using transcriptional fusions, we studied the regulation of pelA, pelD, and pelE gene expression as a function of variation of the external pH. pelA and pelD were expressed when bacteria were grown in an acidic medium while pelE was transcribed only in basic medium. Using phenol red, we observed that, in chicory leaves, pH value of infected tissue varies from acidic to basic. Taken together, these findings are discussed in the context of a model unifying both catalysis and regulation to account for pel gene evolution. In particular, we propose that the three isoenzymes are produced sequentially during the infection process.
Insertion of the LINE Retrotransposon MGL Causes a Conidiophore Pattern Mutation in Magnaporthe grisea
Authors: Nishimura Marie , Hayashi Nagao , Jwa Nam-Soo , Lau Gee W. , Hamer John E. , Hasebe Akira
Published: May 2007

We obtained three Magnaporthe grisea morphological mutants that had the LINE transposon MGL inserted into the ACR1 locus. Sequence analysis revealed that ACR1 is homologous to medA, a developmental regulator of Aspergillus nidulans conidiation. These results demonstrated that MGL elements could transpose and cause insertional mutagenesis in M. grisea.
cDNA-AFLP Display for the Isolation of Peronospora parasitica Genes Expressed During Infection in Arabidopsis thaliana
Authors: van der Biezen Erik A. , Juwana Hedyani , Parker Jane E. , Jones Jonathan D. G.
Published: May 2007

To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.
Characterization of a Tomato Protein Kinase Gene Induced by Infection by Potato spindle tuber viroid
Authors: Hammond Rosemarie W. , Zhao Yan
Published: May 2007

Viroids—covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides—are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases.
CgDN3: An Essential Pathogenicity Gene ofColletotrichum gloeosporioidesNecessary to Avert a Hypersensitive-Like Response in the HostStylosanthes guianensis
Authors: Stephenson Sally-Anne , Hatfield Jodie , Rusu Anca G. , Maclean Donald J. , Manners John M.
Published: May 2007

A gene of Colletotrichum gloeosporioides that is induced by nitrogen starvation in axenic culture and is expressed at the early stages of infection of the host Stylosanthes guianensis has been identified and its role in pathogenicity tested. The sequence of this gene, named CgDN3, indicated that it encodes a protein of 74 amino acids that contains a predicted 18 amino acid signal sequence for secretion of a basic 54 amino acid mature protein with weak homology to an internal region of plant wall-associated receptor kinases. Mutants of C. gloeosporioides were produced by homologous recombination in which part of the coding sequence and promoter region of the CgDN3 gene was replaced with a hygromycin-resistance gene cassette. Mutations in the CgDN3 gene were confirmed in two independent transformants and Northern (RNA) analysis demonstrated the disrupted CgDN3 gene was not expressed. The mutants had faster mycelial growth rates in vitro but produced spores that germinated to form appressoria normally on the leaf surface. However, the CgDN3 mutants were unable to infect and reproduce on intact host leaves. Microscopic analysis revealed small clusters of necrotic host cells at inoculation sites on leaves, suggesting that these mutants elicited a localized, host hypersensitive-like response. The mutants were able to grow necrotrophically and reproduce on leaves when conidia were inoculated directly onto wound sites. The putative promoter region of the CgDN3 gene was fused to a gene encoding a modified jellyfish green fluorescent protein and introduced into the fungus. Following inoculation, strong expression of green fluorescent protein was observed in primary infection vesicles in infected epidermal cells with weaker expression evident in hyphae growing within infected leaf tissue. These findings indicate that CgDN3 encodes a novel pathogenicity determinant associated with the biotrophic phase of primary infection and required to avert a hypersensitive-like response by a compatible host.
Early Symbiotic Responses Induced by Sinorhizobium meliloti ilvC Mutants in Alfalfa
Authors: López Juan Carlos , Grasso Daniel H. , Frugier Florian , Crespi Martín D. , Aguilar O. Mario
Published: May 2007

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC¯ background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC¯ strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.
Inhibition of Fungal Appressorium Formation by Pepper (Capsicum annuum) Esterase
Authors: Kim Young Soon , Lee Hyun Hwa , Ko Moon Kyung , Song Chae Eun , Bae Cheol-Yong , Lee Yong Hwan , Oh Boung-Jun
Published: May 2007

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.
The Antioxidants of Legume Nodule Mitochondria
Authors: Iturbe-Ormaetxe Iñaki , Matamoros Manuel A. , Rubio Maria C. , Dalton David A. , Becana Manuel
Published: May 2007

The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender × Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehy-droascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.
Wheat Puroindolines Enhance Fungal Disease Resistance in Transgenic Rice
Authors: Krishnamurthy Konduru , Balconi Carlotta , Sherwood John E. , Giroux Michael J.
Published: May 2007

Antimicrobial peptides play a role in the immune systems of animals and plants by limiting pathogen infection and growth. The puroindolines, endosperm-specific proteins involved in wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties. Rice, the most widely used cereal crop worldwide, normally does not contain puroindolines. Transgenic rice plants that constitutively express the puroindoline genes pinA and/or pinB throughout the plants were produced. PIN extracts of leaves from the transgenic plants reduced in vitro growth of Magnaporthe grisea and Rhizoctonia solani, two major fungal pathogens of rice, by 35 to 50%. Transgenic rice expressing pinA and/or pinB showed significantly increased tolerance to M. grisea (rice blast), with a 29 to 54% reduction in symptoms, and R. solani (sheath blight), with an 11 to 22% reduction in symptoms. Puroindolines are effective in vivo in antifungal proteins and could be valuable new tools in the control of a wide range of fungal pathogens of crop plants.
Ectopic Expression of an Arabidopsis Calmodulin-Like Domain Protein Kinase-Enhanced NADPH Oxidase Activity and Oxidative Burst in Tomato Protoplasts
Authors: Xing Tim , Wang Xiao-Jing , Malik Kamal , Miki Brian L.
Published: May 2007

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect. tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.
The Bradyrhizobium japonicum hsfA Gene Exhibits a Unique Developmental Expression Pattern in Cowpea Nodules
Authors: Oh Hye-Sook , Son Ora , Chun Jong-Yoon , Stacey Gary , Lee Myung-Sok , Min Kyung-Hee , Song Eun-Sook , Cheon Choong-III
Published: May 2007

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the β-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5′ of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5′ of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.
Guidelines for Genetic Nomenclature and Community Governance for the Model Legume Medicago truncatula
Authors: VandenBosch Kathryn A. , Frugoli Julia
Published: May 2007

At the 2nd Medicago meeting (a satellite of the 1999 IS-MPMI meeting in Amsterdam), investigators perceived a need for standardization of genetic nomenclature in Medicago truncatula, due to the rapid growth of research on this species in the past few years. Establishment of such standards grew out of discussions begun at this meeting and continued electronically throughout the M. truncatula community. The proposed standards presented here are the consensus results of those discussions. In addition to standards for gene nomenclature, a method for community governance and a website for cataloging gene names and submitting new ones are presented. The purpose of implementing these guidelines is to help maintain consistency in the literature, to avoid redundancy, to contribute to the accuracy of databases, and, in general, to aid the international collaborations that have made M. truncatula a model system for legume biology.
Production of Antimicrobial Defensin in Nicotiana benthamiana with a Potato Virus X Vector
Authors: Saitoh Hiromasa , Kiba Akinori , Nishihara Masahiro , Yamamura Saburo , Suzuki Kazumi , Terauchi Ryohei
Published: May 2007

A recombinant plasmid, pTXS.TH, was constructed to express the gene-encoding wasabi (Wasabia japonica) defensin with the potato virus X (PVX) vector. pTXS.TH allows the expression of defensin in the host Nicotiana benthamiana, and the defensin protein WT1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. WT1 exhibits strong antifungal activity toward the phytopathogenic fungi Magnaporthe grisea(50% inhibitory concentration [IC50] = 5 μg/ml) and Botrytis cinerea (IC50 = 20 μg/ml) but is weakly active against the phytopathogenic bacterium Pseudomonas cichorii. This virus-mediated expression system is a rapid and efficient method to produce and characterize antimicrobial proteins in plants. It is particularly useful for the study of proteins that are difficult to produce with other expression systems.
Tobamoviral Movement Protein Transiently Expressed in a Single Epidermal Cell Functions Beyond Multiple Plasmodesmata and Spreads Multicellularly in an Infection-Coupled Manner
Authors: Tamai Atsushi , Meshi Tetsuo
Published: May 2007

Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome.
The Induction of Tomato Leucine Aminopeptidase Genes (LapA) After Pseudomonas syringae pv. tomato Infection Is Primarily a Wound Response Triggered by Coronatine
Authors: Pautot Véronique , Holzer Frances M. , Chaufaux Josette , Walling Linda L.
Published: May 2007

Tomato plants constitutively express a neutral leucine aminopeptidase (LAP-N) and an acidic LAP (LAP-A) during floral development and in leaves in response to insect infestation, wounding, and Pseudomonas syringae pv. tomato infection. To assess the physiological roles of LAP-A, a LapA-antisense construct (35S:asLapA1) was introduced into tomato. The 35S:asLapA1 plants had greatly reduced or showed undetectable levels of LAP-A and LAP-N proteins in healthy and wounded leaves and during floral development. Despite the loss of these aminopeptidases, no global changes in protein profiles were noted. The 35S:asLapA1 plants also exhibited no significant alteration in floral development and did not impact the growth and development of Manduca sexta and P. syringae pv. tomato growth rates during compatible or incompatible infections. To investigate the mechanism underlying the strong induction of LapA upon P. syringae pv. tomato infection, LapA expression was monitored after infection with coronatine-producing and -deficient P. syringae pv. tomato strains. LapA RNA and activity were detected only with the coronatine-producing P. syringae pv. tomato strain. Coronatine treatment of excised shoots caused increases in RNAs for jasmonic acid (JA)-regulated wound-response genes (LapA and pin2) but did not influence expression of a JA-regulated pathogenesis-related protein gene (PR-1). These results indicated that coronatine mimicked the wound response but was insufficient to activate JA-regulated PR genes.
Immunogold Labeling of Hrp Pili of Pseudomonas syringae pv. tomato Assembled in Minimal Medium and In Planta
Authors: Hu Wenqi , Yuan Jing , Jin Qiao-Ling , Hart Patrick , He Sheng Yang
Published: May 2007

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.
Overcoming Host- and Pathogen-Mediated Resistance in Tomato and Tobacco Maps to the M RNA of Tomato spotted wilt virus
Authors: Hoffmann K. , Qiu W. P. , Moyer J. W.
Published: May 2007

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (MA). Overcoming the Sw-5 gene was linked solely to the presence of MA, to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.
Expression of the Avirulence Gene Avr9 of the Fungal Tomato Pathogen Cladosporium fulvum Is Regulated by the Global Nitrogen Response Factor NRF1
Authors: Pérez-García Alejandro , Snoeijers Sandor S. , Joosten Matthieu H. A. J. , Goosen Theo , De Wit Pierre J. G. M.
Published: May 2007

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.
The Contribution of Syringopeptin and Syringomycin to Virulence of Pseudomonas syringae pv. syringae strain B301D on the Basis of sypA and syrB1 Biosynthesis Mutant Analysis
Authors: Scholz-Schroeder Brenda K. , Hutchison Michael L. , Grgurina Ingeborg , Gross Dennis C.
Published: May 2007

Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv. syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis. The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases. Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure. Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production. The contribution of syringopeptin and syringomycin to the virulence of P. syringae pv. syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively. Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin-syringomycin double mutant was reduced 76% in virulence. These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P. syringae pv. syringae.
Alcohol Oxidase Is a Novel Pathogenicity Factor for Cladosporium fulvum, but Aldehyde Dehydrogenase Is Dispensable
Authors: Segers G. , Bradshaw N. , Archer D. , Blissett K. , Oliver R. P.
Published: May 2007

Cladosporium fulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxi-dase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.