bluuebunny/crossref_metadata_2025 huggingface dataset:
A Sinorhizobium meliloti Lipopolysaccharide Mutant Induces Effective Nodules on the Host Plant Medicago sativa (Alfalfa) but Fails to Establish a Symbiosis with Medicago truncatula
Authors: Niehaus K. , Lagares A. , Pühler A.
Published: May 2007

The specific Sinorhizobium meliloti lipopolysaccharide (LPS) mutant Rm6963 (A. Lagares, G. Caetano Anolles, K. Niehaus, J. Lorenzen, H. D. Ljunggren, A. Puhler, and G. Favelukes, J. Bacteriol. 174:5941-5952, 1992) was shown to be mutated in a region corresponding to a cloned 5-kb SstI DNA fragment that was able to complement the lpsB and lpsC mutants of S. meliloti described by Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989). Sodium dodecyl sulfate polyacryla-mide electrophoresis revealed that the LPS-I and LPS-II fractions of the LPS mutant Rm6963 were shifted to lower molecular weights. While the majority of the Medicago spp. tested established an effective symbiosis with both the S. meliloti wild-type Rm2011 and the LPS mutant Rm6963, the latter induced ineffective nodules on M. truncatula. A light- and electron-microscopic analysis of the ineffective M. truncatula root nodules revealed that the bacteria were released from the infection threads but failed to colonize the plant cells effectively. The plant cytoplasm was filled with numerous vesicles, probably the result of a disturbed bacteroid development. Sections of ineffective M. truncatula root nodules induced by the LPS mutant Rm6963 showed brown, necrotic cells within the central nodule tissue that autofluoresced when viewed under UV light. These observations are best explained by a plant defense response. Evidently, the rhizobial LPS plays a role in plant-microbe signaling during the formation of M. truncatula nodules.
Symbiotic GeneSym31Controls the Presence of a Lectinlike Glycoprotein in the Symbiosome Compartment of Nitrogen-Fixing Pea Nodules
Authors: Dahiya Preeti , Sherrier D. Janine , Kardailsky Igor V. , Borisov Alexey Y. , Brewin Nicholas J.
Published: May 2007

The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as inclusion bodies in the vacuolar compartment of infected host cells but it was apparently absent from the symbiosome compartment. The results suggest an aberrant vesicle targeting pathway during symbiosome development in this mutant.
Flavanone 3-hydroxylase (F3H) Expression and Flavonoid Localization in Nodules of Three Legume Plants Reveal Distinct Tissue Specificities
Authors: Charrier Benedicte , Trinh Hanh , Poirier Simone , Kondorosi Adam , Ratet Pascal
Published: May 2007

A fusion of the promoter of the Medicago sativa flavanone-3-hydroxylase gene to the gus reporter gene was introduced into three leguminous plants, M. truncatula, M. sativa subsp. varia, and Vicia hirsuta. Expression of this fusion was detected in the parenchyma cells surrounding the vascular tissues in the nodules of the Medicago spp. plants, correlating with the presence of flavonoid compounds detected in situ in these cells. In V. hirsuta nodules, the fusion was expressed in the meristematic region, where high accumulation of flavonoids could also be detected. Our results indicate that flavonoids of the dihydroflavonol pathway are not found in the same tissues of the nodules in these three species, where they may play different roles. In light of these results, the possible roles of the F3H products in this organ are discussed.
Snakin-1, a Peptide from Potato That Is Active Against Plant Pathogens
Authors: Segura Ana , Moreno Manuel , Madueño Francisco , Molina Antonio , García-Olmedo Francisco
Published: May 2007

A new type of antimicrobial peptide, snakin-1 (SN1), has been isolated from potato tubers and found to be active, at concentrations <10 μM, against bacterial and fungal pathogens from potato and other plant species. The action of SN1 and potato defensin PTH1 was synergistic against the bacterium Clavibacter michiganensis subsp. sepedonicus and additive against the fungus Botrytis cinerea. Snakin-1 causes aggregation of both gram-positive and gram-negative bacteria. The peptide has 63 amino acid residues (Mr6,922), 12 of which are cysteines, and is unrelated to any previously isolated protein, although it is homologous to amino acid sequences deduced from cloned cDNAs that encode gibberellin-inducible mRNAs and has some sequence motifs in common with kistrin and other hemotoxic snake venoms. A degenerate oligonucleotide probe based on the internal sequence CCEECKC has been used to clone an SN1 cDNA. With the cDNA used as probe, one copy of the StSN1 gene per haploid genome has been estimated and expression of the gene has been detected in tubers, stems, axillary buds, and young floral buds. Expression levels in petals and carpels from fully developed flowers were much higher than in sepals and stamens. The expression pattern of gene StSN1 suggests that protein SN1 may be a component of constitutive defense barriers, especially those of storage and reproductive plant organs.
The PecT Repressor Coregulates Synthesis of Exopolysaccharides and Virulence Factors in Erwinia chrysanthemi
Authors: Condemine Guy , Castillo Arnaud , Passeri Fabrice , Enard Corine
Published: May 2007

Erwinia chrysanthemi 3937 synthesizes an exopolysaccharide (EPS) composed of rhamnose, galactose, and galacturonic acid. Fourteen transcriptional fusions in genes required for EPS synthesis, named eps, were obtained by Tn5-B21 mutagenesis. Eleven of them are clustered on the chromosome and are repressed by PecT, a regulator of pectate lyase synthesis. In addition, expression of these fusions is repressed by the catabolite regulatory protein, CRP, and induced in low osmolarity medium. The three other mutations are located in genes that are not regulated by pecT. A 13-kb DNA fragment containing pecT-regulated eps genes has been cloned. All the genes identified on this fragment are transcribed in the same orientation and could form a large operon. The promoter region of this operon has been sequenced. It contains a JUMP-start sequence, a sequence required for the expression of polysaccharide-associated operons. E. chrysanthemi 3937 produces a systemic soft rot on its host Saintpaulia ionantha. An eps mutant was less efficient than the wild-type strain in initiating a maceration symptom, suggesting that production of EPS is required for the full expression of the E. chrysanthemi virulence.
Tomatinase from Fusarium oxysporum f. sp. lycopersici Defines a New Class of Saponinases
Authors: Roldán-Arjona Teresa , Pérez-Espinosa Alonso , Ruiz-Rubio Manuel
Published: May 2007

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate α-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have over-expressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves α-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by α-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.
CFP, the Putative Cercosporin Transporter of Cercospora kikuchii, Is Required for Wild Type Cercosporin Production, Resistance, and Virulence on Soybean
Authors: Callahan Terrence M. , Rose Mark S. , Meade Maura J. , Ehrenshaft Marilyn , Upchurch Robert G.
Published: May 2007

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.
Defense Genes Are Differentially Induced by a Mycorrhizal Fungus and Rhizobium sp. in Wild-Type and Symbiosis-Defective Pea Genotypes
Authors: Ruiz-Lozano Juan M. , Roussel Hélène , Gianinazzi Silvio , Gianinazzi-Pearson Vivienne
Published: May 2007

Mycorrhiza-resistant and non-nodulating pea mutants provide a model system for identifying common genes regulated during the early events in mycorrhiza and nodule establishment. Inoculation of pea roots with Glomus mosseae or Rhizobium leguminosarum can induce overex-pression of seven defense-related genes (pI 206, pI 49, pI 176, PR 10, basic A1-chitinase, transcinnamic acid 4-hydroxylase, chalcone isomerase), depending on the plant genotype and the time point of interaction between the plant and the microsymbiont. Expression of the pI 206 gene is closely correlated with appressorium formation by the mycorrhizal fungus on both mutant and wild-type pea roots. The gene is also induced by the pathogen Aphanomyces euteiches. Transcript accumulation was higher in mutant than in wild-type genotypes for five and six of the studied genes during early stages of root interactions with G. mosseae and R. leguminosarum, respectively, and this is discussed in relation to the symbiotic-defective phenotype of Myc-1Nod¯ pea. The early induction of similar plant defense genes in response to arbuscular mycorrhizal fungi and rhizobia reinforces the hypothesis of common regulatory steps in both root symbioses.
Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?
Authors: Canto Tomas , Palukaitis Peter
Published: May 2007

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.
Comparison of Erysiphe cichoracearum and E. cruciferarum and a Survey of 360 Arabidopsis thaliana Accessions for Resistance to These Two Powdery Mildew Pathogens
Authors: Adam L. , Ellwood S. , Wilson I. , Saenz G. , Xiao S. , Oliver R. P. , Turner J. G. , Somerville S.
Published: May 2007

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 × Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.
A Cytochrome P450 Gene Is Differentially Expressed in Compatible and Incompatible Interactions Between Pepper (Capsicum annuum) and the Anthracnose Fungus,Colletotrichum gloeosporioides
Authors: Oh Boung-Jun , Ko Moon Kyung , Kim Young Soon , Kim Kwang Sang , Kostenyuk Igor , Kee Hye Kyung
Published: May 2007

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of Pep-CYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.
A Cysteine Protease Gene Is Expressed Early in Resistant Potato Interactions with Phytophthora infestans
Authors: Avrova Anna O. , Stewart Helen E. , De Jong Walter , Heilbronn Jacqueline , Lyon Gary D. , Birch Paul R. J.
Published: May 2007

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.
A Novel 53-kDa Nodulin of the Symbiosome Membrane of Soybean Nodules, Controlled by Bradyrhizobium japonicum
Authors: Winzer Till , Bairl Andrea , Linder Monica , Linder Dietmar , Werner Dietrich , Müller Peter
Published: May 2007

A nodule-specific 53-kDa protein (GmNOD53b) of the symbiosome membrane from soybean was isolated and its LysC digestion products were microsequenced. cDNA clones of this novel nodulin, obtained from cDNA library screening with an RT-PCR (reverse-transcriptase polymerase chain reaction)-generated hybridization probe exhibited no homology to proteins identified so far. The expression of GmNOD53b coincides with the onset of nitrogen fixation. Therefore, it is a late nodulin. Among other changes, the GmNOD53b is significantly reduced in nodules infected with the Bradyrhizobium japonicum mutant 184 on the protein level as well as on the level of mRNA expression, compared with the wild-type infected nodules. The reduction of GmNOD53b mRNA is related to an inactivation of the sipF gene in B. japonicum 184, coding for a functionally active signal peptidase.
T-DNA Tagging of Nodulation- and Root-Related Genes in Lotus japonicus: Expression Patterns and Potential for Promoter Trapping and Insertional Mutagenesis
Authors: Martirani Luca , Stiller Jiri , Mirabella Rossana , Alfano Flora , Lamberti Alessandro , Radutoiu Simona E. , Iaccarino Maurizio , Gresshoff Peter M. , Chiurazzi Maurizio
Published: May 2007

High-efficiency transformation of the autogamous diploid legume Lotus japonicus by means of Agrobacterium rhizogenes was used to develop plant lines expressing a promoter-less gusA gene in a nodulation- or lateral root-associated manner. The approach exploits the putatively preferential integration of T-DNA into actively transcribed regions, thereby providing an enrichment for gene tagging events associated with the quickly assayable activation of a gusA promoter-less construct. Taking advantage of this enrichment and selection strategy, a T-DNA tagging program was initiated and screening for β-glucuronidase (GUS) activity was performed on root clones isolated after transformation with a gusA-promoter-less binary vector. The aim of this approach is the identification of genes involved in nodule formation induced by Mesorhizobium loti, lateral root organogenesis, and the eventual isolation of corresponding mutants. A large collection (220) of GUS-positive transformants showing a variety of expression patterns in different regions of roots and nodules was obtained; a preliminary molecular characterization of these plants is presented.
New Advances in Understanding the Molecular Biology of Plant/Potyvirus Interactions
Authors: Revers Frédéric , Le Gall Olivier , Candresse Thierry , Maule Andrew J.
Published: May 2007

In recent years, researchers have adopted many new technologies to help understand potyvirus pathogenesis. Their findings have illuminated key aspects of the interactions between the host and the virus, and between the virus and its aphid vector. This review focuses on advances in our understanding of the molecular determinants of systemic infection, symptom expression, aphid and seed transmission, and natural and engineered resistance to potyviruses. Very recent developments in the area of post-transcriptional gene silencing indicate not only that the process is fundamental to engineered resistance, but may also underlie many aspects of the biology of plant viruses.
The Interaction of Lipodepsipeptide Toxins fromPseudomonas syringaepv.syringaewith Biological and Model Membranes: A Comparison of Syringotoxin, Syringomycin, and Two Syringopeptins
Authors: Serra Mauro Dalla , Fagiuoli Giulia , Nordera Paola , Bernhart Ivonne , Volpe Claudio Della , Di Giorgio Domenico , Ballio Alessandro , Menestrina Gianfranco
Published: May 2007

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2(SRE), 1.5 nm2(SP22A), and 1.3 nm2(SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.
Conductive Properties and Gating of Channels Formed by Syringopeptin 25A, a Bioactive Lipodepsipeptide fromPseudomonas syringaepv.syringae, in Planar Lipid Membranes
Authors: Serra Mauro Dalla , Bernhart Ivonne , Nordera Paola , Di Giorgio Domenico , Ballio Alessandro , Menestrina Gianfranco
Published: May 2007

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.
Mutation in a Gene Required for Lipopolysaccharide and Enterobacterial Common Antigen Biosynthesis Affects Virulence in the Plant Pathogen Erwinia carotovora subsp. atroseptica
Authors: Toth I. K. , Thorpe C. J. , Bentley S. D. , Mulholland V. , Hyman L. J. , Perombelon M. C. M. , Salmond G. P. C.
Published: May 2007

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.
Expression of a Single, Host-Specific, Bacterial Pathogenicity Gene in Plant Cells Elicits Division, Enlargement, and Cell Death
Authors: Duan Y. P. , Castañeda A. , Zhao G. , Erdos G. , Gabriel D. W.
Published: May 2007

A fundamental question about microbial pathogens is how they elicit host-specific symptoms. We report here that expression of a single gene from a plant-pathogenic bacterium in plant cells elicits host-specific symptoms diagnostic of the disease caused by the pathogen. Expression of pthA from Xanthomonas citri in citrus cells is sufficient to cause division, enlargement, and death of host cells. Since elicitation of these symptoms depends on a functional type III protein secretion system in X. citri, we deduce that the PthA protein is a specific plant signal, its site of action is inside the plant cell, and it is a major determinant of host range.
dnaK and the Heat Stress Response of Pseudomonas syringae pv. glycinea
Authors: Keith Lisa M. W. , Partridge James E. , Bender Carol L.
Published: May 2007

The dnaK gene from Pseudomonas syringae pv. glycinea PG4180 was cloned and sequenced. The dnaK coding region was 1,917 bp and contained a putative σ32 heat shock promoter 86 bp upstream of the translational start site. grpE, another heat shock gene, was found immediately upstream of the putative dnaK promoter. The predicted amino acid sequence of dnaK showed relatedness to the ATPase and substrate binding domains commonly found in heat shock proteins, as well as the highly conserved signature sequence motifs belonging to the Hsp70 protein family. Furthermore, the PG4180 dnaK gene complemented an Escherichia coli dnaK mutant for growth at temperatures above 37°C, indicating that a fully functional dnaK homologue had been cloned from P. syringae pv. glycinea. All attempts to eliminate dnaK function by insertion mutagenesis failed, possibly because DnaK performs essential functions in P. syringae pv. glycinea. Expression of dnaK in P. syringae pv. glycinea PG4180 was investigated by constructing dnaK∷uidA transcriptional fusions; expression of dnaK increased markedly when cells were preincubated at 18°C and then shifted to 35°C. An anti-DnaK monoclonal antibody was used to detect DnaK; in P. syringae pv. glycinea race 4, DnaK levels followed cell density during a 6-h incubation at 26°C. When cells were shifted from 26°C to either 32 or 38°C, DnaK levels increased transiently, and then decreased rapidly. Although the cells continued to grow when incubated at 32°C, growth was not supported at 38°C. Our results indicate that P. syringae pv. glycinea responds to heat shock by producing DnaK, but DnaK does not aid in acclimation to sustained elevated temperatures.
The Response Regulator ExpM Is Essential for the Virulence of Erwinia carotovora subsp. carotovora and Acts Negatively on the Sigma Factor RpoS (σS)
Authors: Andersson Robert A. , Palva E. Tapio , Pirhonen Minna
Published: May 2007

The main virulence factors of Erwinia carotovora subsp. carotovora, the secreted, extracellular cell-wall-degrading enzymes, are controlled by several regulatory mechanisms. We have isolated transposon mutants with reduced virulence on tobacco. One of these mutants, with a mutation in a gene designated expM, was characterized in this study. This mutant produces slightly reduced amounts of extracellular enzymes in vitro and the secretion of the enzymes is also affected. The expM wild-type allele was cloned together with an upstream gene, designated expL, that has an unknown function. The expM gene was sequenced and found to encode a protein with similarity to the RssB/SprE protein of Escherichia coli and the MviA protein of Salmonella typhimurium. These proteins belong to a new type of two-component response regulators that negatively regulate the stability of the Sigma factor RpoS (σS) at the protein level. The results of this study suggest that ExpM has a similar function in E. carotovora subsp. carotovora. We also provide evidence that the overproduction of RpoS in the expM mutant is an important factor for the reduced virulence phenotype and that it partly causes the observed phenotype seen in vitro. However, an expM/rpoS double mutant is still affected in secretion of extracellular enzymes, suggesting that ExpM in addition to RpoS also acts on other targets.
Infection of Barley by Brome Mosaic Virus Is Restricted Predominantly to Cells in and Associated with Veins through a Temperature-Dependent Mechanism
Authors: Ding Xin Shun , Flasinski Stanislaw , Nelson Richard S.
Published: May 2007

Results from previous cytological studies on barley (Hordeum vulgare) infected with brome mosaic virus (BMV) indicated that this virus can infect and accumulate to high levels in mesophyll and other cell types within the leaves. Through immunocytochemistry and in situ hybridization, we have determined that BMV infection in barley is restricted predominantly to cells within and associated with vasculature when plants are grown at 24/20°C (day/night). This tissue restriction can be fully overcome by growing infected plants at 34°C for 2 h. Our results also indicate that BMV is likely to start systemic infection of young, uninoculated leaves in barley before spreading into all longitudinal veins in inoculated leaves. Possible barrier(s) to BMV movement between vascular bundle sheath cells and mesophyll cells, and the relationship between virus and photoassimilate transport through longitudinal and transverse veins are discussed.
Antifungal Activity of a Plant Cystatin
Authors: Pernas Mónica , López-Solanilla Emilia , Sánchez-Monge Rosa , Salcedo Gabriel , Rodríguez-Palenzuela Pablo
Published: May 2007

Purified chestnut cystatin inhibited the growth of the phytopathogenic fungi Botrytis cinerea, Colletotrichum graminicola, and Septoria nodorum, but not that of the saprophyte Trichoderma viride. Furthermore, the cystatin strongly affected the protease activity of B. cinerea but had no effect on the protease activity of T. viride. These results suggest that chestnut cystatin contributes to plant defense against phytopathogenic fungi.
Relationship Between Localized Acquired Resistance (LAR) and the Hypersensitive Response (HR): HR Is Necessary for LAR to Occur and Salicylic Acid Is Not Sufficient to Trigger LAR
Authors: Costet Laurent , Cordelier Sylvain , Dorey Stéphan , Baillieul Fabienne , Fritig Bernard , Kauffmann Serge
Published: May 2007

In tobacco plants reacting hypersensitively to pathogen infection, localized acquired resistance (LAR) is induced in a sharp zone surrounding hypersensitive response (HR) lesions. Using a fungal glycoprotein inducing HR and LAR when infiltrated at 50 nM into tobacco leaves, we have shown previously that a plant signal(s) is released by HR cells and diffuses to induce LAR. Here we address two questions: does LAR occur when HR is not induced, and is salicylic acid the (or one of the) mobile LAR signal? We found that application to tobacco leaves of 0.25 nM glycoprotein triggered defense responses without HR and without an H2O2 burst. The analyzed responses include changes in expression of O-methyltransferase (OMT), 3-hydroxy-3-methylglutarylCoA reductase, pathogenesis-related (PR) proteins, and changes in levels of the signal salicylic acid. No defense responses and no increased resistance to tobacco mosaic virus infection were found beyond the elicitor-infiltrated tissue, providing strong evidence that there is no LAR without HR. Treatments of NahG tobacco leaves with 50 nM elicitor induced the HR and, in the sharp zone surrounding the HR lesion, a strong activation of OMT and of basic PR proteins, but not of acidic PR-1 proteins. This indicates that a signal different from salicylic acid is diffusing.
Association of the Movement Protein of Alfalfa Mosaic Virus with the Endoplasmic Reticulum and Its Trafficking in Epidermal Cells of Onion Bulb Scales
Authors: Huang Mei , Zhang Lee
Published: May 2007

To study the intercellular trafficking potential of alfalfa mosaic virus movement protein (AlMV MP) and the cellular structures that may assist in this activity, the AlMV MP gene was fused with the green fluorescent protein (GFP) gene and delivered into epidermal cells of onion bulb scales and Nicotiana tabacum (BY-2) suspension culture cells by biolistic gene bombardment. Its subcellular distribution as well as intracellular and intercellular trafficking in epidermal cells of onion bulb scales and in insect cells were systematically studied. Cells expressing the MP-GFP showed a fluorescent filamentous network in the cell cortex and fluorescent irregular bodies scattered in the cytoplasm and present in the cell wall. By 44 h post transfection with the MP-GFP construct, 56% of fluorescent sites consisted of clustered cells, whereas the fluorescent sites from cells bombarded with the free GFP construct consisted of isolated cells. Time course experiments demonstrated that the MP-GFP was able to move into adjacent cells from a primarily transfected cell. Optical serial sectioning studies suggested that the MP-GFP could target to the plasmodesmata, and move through them into neighboring cells. Furthermore, detailed fluorescence microscopy analyses of both tobacco and onion cells showed that the MP-GFP co-localized with the endoplasmic reticulum (ER). Subcellular fractionation of insect cells and immunoblotting analysis revealed that the MP-GFP behaves as an integral membrane protein. These results indicate that the MP-GFP is associated with the ER and suggest that the ER association may be important for intracellular and intercellular movement of the AlMV MP.
Endophytic Expression of nif Genes of Azoarcus sp. Strain BH72 in Rice Roots
Authors: Egener Tanja , Hurek Thomas , Reinhold-Hurek Barbara
Published: May 2007

Transcriptional fusions of gusA and gfp to the nifH gene as well as immunogold labeling with antibodies against the iron protein of nitrogenase revealed high nitrogenase gene expression levels of the endophyte Azoarcus sp. strain BH72 inside infected rice roots (Oryza sativa) in the laboratory. Thus, environmental conditions inside rice roots are permissive for endophytic nitrogen fixation in bacterial microcolonies in the aerenchyma.
Potato mop-top virus RNA Can Move Long Distance in the Absence of Coat Protein: Evidence from Resistant, Transgenic Plants
Authors: McGeachy Kara D. , Barker Hugh
Published: May 2007

Transgenic expression of a translatable version of the Potato mop-top virus (PMTV) coat protein (CP) gene (encoded by RNA 3) in Nicotiana benthamiana prevented production of symptoms and infective virus particles. RNAs 1 and 2 accumulated in inoculated and systemic leaves but, apart from small amounts of CP transgene RNA transcript, no genomic-length RNA 3 was found. Crude leaf extracts from inoculated plants were not infective. However, when RNA extracts from such transgenic plants were inoculated to nontransgenic N. benthamiana and N. clevelandii, RNA 1 and RNA 2 replicated in systemic leaves of both species in the absence of RNA 3 and virus particles, but symptoms did not develop. We suggest that the triple-gene block proteins of PMTV (encoded by RNA 2) represent a class of long-distance RNA movement factors.
N-Deacetylation ofSinorhizobium melilotiNod Factors Increases Their Stability in theMedicago sativaRhizosphere and Decreases Their Biological Activity
Authors: Staehelin Christian , Schultze Michael , Tokuyasu Ken , Poinsot Véréna , Promé Jean-Claude , Kondorosi Éva , Kondorosi Ád´m
Published: May 2007

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16:2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.
Reduced Virulence Caused by Meiotic Instability of the TOX2 Chromosome of the Maize Pathogen Cochliobolus carbonum
Authors: Pitkin John W. , Nikolskaya Anastasia , Ahn Joong-Hoon , Walton Jonathan D.
Published: May 2007

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.
Molecular Analysis of Thermoregulation of Phaseolotoxin-Resistant Ornithine Carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola
Authors: Rowley Karla B. , Xu Ronghui , Patil Suresh S.
Published: May 2007

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudo-monas syringae pv. phaseolicola at 18°C but not at 28°C. At 28°C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28°C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28°C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.
A Potato Gene Encoding a WRKY-like Transcription Factor Is Induced in Interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and Is Coregulated with Class I Endochitinase Expression
Authors: Dellagi Alia , Heilbronn Jacqueline , Avrova Anna O. , Montesano Marcos , Palva E. Tapio , Stewart Helen E. , Toth Ian K. , Cooke David E. L. , Lyon Gary D. , Birch Paul R. J.
Published: May 2007

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h post-inoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.
Bromovirus Movement Protein Conditions for the Host Specificity of Virus Movement Through the Vascular System and Affects Pathogenicity in Cowpea
Authors: Fujita Yasunari , Fujita Miki , Mise Kazuyuki , Kobori Takashi , Osaki Takeshi , Furusawa Iwao
Published: May 2007

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vas-culature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.
Agroinfiltration of Cauliflower mosaic virus Gene VI Elicits Hypersensitive Response in Nicotiana Species
Authors: Palanichelvam Karuppaiah , Cole Anthony B. , Shababi Monir , Schoelz James E.
Published: May 2007

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.