bluuebunny/crossref_metadata_2025 huggingface dataset:
Rds and Rih Mediate Hypersensitive Cell Death Independent of Gene-for-Gene Resistance to the Oat Crown Rust Pathogen Puccinia coronata f. sp. avenae
Authors: Yu Gong-Xin , Braun Ed , Wise Roger P.
Published: May 2007

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.
Yellow Lupine Cyclophilin Transcripts Are Highly Accumulated in the Nodule Meristem Zone
Authors: Nuc Katarzyna , Nuc Przemysław , Słomski Ryszard
Published: May 2007

Cyclophilin (CyP) is one of the enzymes that act as peptidylprolyl cis-trans isomerases (EC 5.2.1.8). The cDNA and an intronless gene coding for cytosolic CyP have been isolated from yellow lupine. The deduced amino acid sequence of the characterized open reading frame shows approximately 80% homology with cytosolic CyP from other organisms. Southern blots of genomic DNA indicate that there is a small family of genes for CyP-related genes in the yellow lupine genome. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs. The amount of CyP transcripts is dramatically increased in root nodules. In situ hybridization experiments indicate that CyP transcripts are localized mainly in meristematic tissues, with the highest level observed in the nodule meristem zone. The promoter of the sequenced gene contains 5′ AAAGAT 3′ and AT-rich motifs that are characteristic for some nodulin promoters.
The rkp-3 Gene Region of Sinorhizobium meliloti Rm41 Contains Strain-Specific Genes that Determine K Antigen Structure
Authors: Kiss Ernő , Kereszt Attila , Barta Fatime , Stephens Samuel , Reuhs Bradley L. , Kondorosi Ádám , Putnoky Péter
Published: May 2007

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.
ngl9: A Third MADS Box Gene Expressed in Alfalfa Root Nodules
Authors: Zucchero Joseph C. , Caspi Michal , Dunn Kathleen
Published: May 2007

Expression of MADS box genes has previously been localized to the infected cells of alfalfa (Medicago sativa) root nodules. These genes represent the first putative transcription factors to be identified in nodules and are hypothesized to be involved in a signal transduction pathway initiated by the intracellular bacterium. The eventual activation of specific target genes defines pertinent characteristics of this nitrogen-fixing differentiated cell. In this study, we identify a third nodule MADS box gene, ngl9, and demonstrate that the DNA-binding activity of its protein product is dependent on the presence of a second MADS box protein, NMH7. Despite previous results to the contrary, both genes are expressed in the early stages of flower development, further strengthening the premise that nodule developmental programming may capitalize upon existing developmental cascades.
PTK1, a Mitogen-Activated-Protein Kinase Gene, Is Required for Conidiation, Appressorium Formation, and Pathogenicity of Pyrenophora teres on Barley
Authors: Ruiz-Roldán M. Carmen , Maier Frank J. , Schäfer Wilhelm
Published: May 2007

Mitogen-activated protein kinases (MAPKs) are a group of protein kinases that execute a wide variety of roles in cellular signal transduction pathways such as osmoregulation, cell wall biosynthesis, growth, and differentiation. A polymerase chain reaction (PCR) with degenerate primers based on conserved regions of known MAPKs was used to clone the MAPK gene PTK1 from the leaf pathogen Pyrenophora teres (anamorph Drechslera teres), the causal agent of net blotch of barley (Hordeum vulgare L.). The predicted amino acid sequence shows high homology with MAPKs from other phytopathogenic fungi. The gene is present in the genome as a single copy. PTK1 is expressed during in vitro growth on complete medium, under conidiation-inducing conditions and during infection of barley leaves, as shown by reverse transcription-PCR studies. In order to assess the role of PTK1 in the life cycle of P. teres, targeted gene disruption was conducted. Mutants carrying an interrupted copy of the gene were deficient in conidiation, did not form appressoria on glass surfaces or on barley leaves, lost their ability to infect barley leaves, and could not colonize host tissues following artificial wounding.
Characterization of Small GTPases Cdc42 and Rac and the Relationship Between Cdc42 and Actin Cytoskeleton in Vegetative and Ectomycorrhizal Hyphae of Suillus bovinus
Authors: Gorfer Markus , Tarkka Mika T. , Hanif Mubashir , Pardo Alejandro G. , Laitiainen Erja , Raudaskoski Marjatta
Published: May 2007

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus. Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes. Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome contains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRac1p is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity. In the predicted amino acid sequences of SbRac1p and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These domain structures suggest that in S. bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals. SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place. Cdc42p and actin were localized at the tips of S. bovinus vegetative hyphae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae. The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.
Mutational Analysis of the Arabidopsis RPS2 Disease Resistance Gene and the Corresponding Pseudomonas syringae avrRpt2 Avirulence Gene
Authors: Axtell Michael J. , McNellis Timothy W. , Mudgett Mary Beth , Hsu Caroline S. , Staskawicz Brian J.
Published: May 2007

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.
Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall
Authors: Brown Ian R. , Mansfield John W. , Taira Suvi , Roine Elina , Romantschuk Martin
Published: May 2007

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.
The C-terminal Dilysine Motif for Targeting to the Endoplasmic Reticulum Is Not Required for Cf-9 Function
Authors: Van der Hoorn Renier A. L. , Van der Ploeg Anke , de Wit Pierre J. G. M. , Joosten Matthieu H. A. J.
Published: May 2007

The tomato resistance gene Cf-9 encodes a membrane-anchored, receptor-like protein that mediates specific recognition of the extracellular elicitor protein AVR9 of Cladosporium fulvum. The C-terminal dilysine motif (KKRY) of Cf-9 suggests that the protein resides in the endoplasmic reticulum. Previously, two conflicting reports on the subcellular location of Cf-9 were published. Here we show that the AARY mutant version of Cf-9 is still functional in mediating AVR9 recognition, suggesting that functional Cf-9 resides in the plasma membrane. The data presented here and in reports by others can be explained by masking the dilysine signal of Cf-9 with other proteins.
A Bacterial Artificial Chromosome Library of Lotus japonicus Constructed in an Agrobacterium tumefaciens-Transformable Vector
Authors: Men Artem E. , Meksem Khalid , Kassem My Abdelmajid , Lohar Dasharath , Stiller Jiri , Lightfoot David , Gresshoff Peter M.
Published: May 2007

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.
Stable RK2-Derived Cloning Vectors for the Analysis of Gene Expression and Gene Function in Gram-Negative Bacteria
Authors: Dombrecht Bruno , Vanderleyden Jos , Michiels Jan
Published: May 2007

The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported. Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp. NGR234. The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences. Vector derivatives with the constitutive nptII promoter or a promoter-less gusA gene are suitable for the study of gene function or regulation in bacteria.
Functional Studies of the Bacterial Avirulence Protein AvrPto by Mutational Analysis
Authors: Chang Jeff H. , Tobias Christian M. , Staskawicz Brian J. , Michelmore Richard W.
Published: May 2007

Pseudomonas syringae pathovars expressing avrPto are avirulent on plants expressing the resistance gene Pto. Over 85 mutants of avrPto were generated with multiple strategies, and several assays were used to characterize AvrPto function. Only a core of 95 amino acids of the 164 residues was sufficient for binding Pto in the yeast two-hybrid system. Only nine of 65 mutant proteins of AvrPto with amino acid substitutions, created in planta and in vitro, did not interact with Pto in the Gal4 yeast two-hybrid system, suggesting that AvrPto can tolerate many nonconservative substitutions and still interact with Pto. These nine and 12 additional substitution mutants of AvrPto were characterized further. The ability to elicit a hypersensitive response and the effect on pathogenesis in planta for these 21 mutants of AvrPto were strongly correlated with recognition by Pto in the yeast two-hybrid system. Analyses of two proteins with substitutions H54P or D52G/L65P indicated that these residues may be required for delivery into the host cell and protein stability in the bacterial cytoplasm, respectively. The mutants that no longer interacted with Pto and had modified activities in planta were predicted to have changes in their secondary structure.
Most AAL Toxin-Sensitive Nicotiana Species are Resistant to the Tomato Fungal Pathogen Alternaria alternata f. sp. lycopersici
Authors: Brandwagt Bas F. , Kneppers Tarcies J. A. , Van der Weerden Gerard M. , Nijkamp H. John J. , Hille Jacques
Published: May 2007

The phytopathogenic fungus Alternaria alternata f. sp. lycopersici produces AAL toxins required to colonize susceptible tomato (Lycopersicon esculentum) plants. AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which are toxic for some plant species and mammalian cell lines. Insensitivity of tomato to SAMs is determined by the Alternaria stem canker gene 1 (Asc-1), and sensitivity is associated with a mutated Asc-1. We show that SAM-sensitive species occur at a low frequency in the Nicotiana genus and that candidate Asc-1 homologs are still present in those species. In Nicotiana spp., SAM-sensitivity and insensitivity also is mediated by a single codominant locus, suggesting that SAM-sensitive genotypes are host for A. alternata f. sp. lycopersici. Nicotiana umbratica plants homozygous for SAM-sensitivity are indeed susceptible to A. alternata f. sp. lycopersici. In contrast, SAM-sensitive genotypes of Nicotiana spegazzinii, Nicotiana acuminata var. acuminata, Nicotiana bonariensis, and Nicotiana langsdorffii are resistant to A. alternata f. sp. lycopersici infection concomitant with localized cell death. Additional (nonhost) resistance mechanisms to A. alternata f. sp. lycopersici that are not based on an insensitivity to SAMs are proposed to be present in Nicotiana species.
Signal Peptide-Selection of cDNA Cloned Directly from the Esophageal Gland Cells of the Soybean Cyst NematodeHeterodera glycines
Authors: Wang Xiaohong , Allen Rex , Ding Xiongfei , Goellner Melissa , Maier Tom , de Boer Jan M. , Baum Thomas J. , Hussey Richard S. , Davis Eric L.
Published: May 2007

Secretions from the esophageal gland cells of plantparasitic nematodes play critical roles in the nematodeparasitic cycle. A novel method to isolate cDNA encoding putative nematode secretory proteins was developed that utilizes mRNA for reverse transcription-polymerase chain reaction derived from microaspiration of the esophageal gland cell contents of parasitic stages of the soybean cyst nematode Heterodera glycines. The resulting H. glycines gland cell cDNA was cloned into the pRK18 vector, and plasmid DNA was transformed into a mutated yeast host for specific selection of cDNA inserts that encode proteins with functional signal peptides. Of the 223 cDNA clones recovered from selection in yeast, 97% of the clones encoded a predicted signal peptide. Fourteen unique cDNA clones hybridized to genomic DNA of H. glycines on Southern blots and, among them, nine cDNA clones encoded putative extracellular proteins, as predicted by PSORT II computer analysis. Four cDNA clones hybridized to transcripts within the dorsal esophageal gland cell of parasitic stages of H. glycines, and in situ hybridization within H. glycines was not detected for eight cDNA clones. The protocol provides a direct means to isolate potential plant-parasitic nematode esophageal gland secretory protein genes.
A Purine-Related Metabolite Negatively Regulates fixNOQP Expression in Sinorhizobium meliloti by Modulation of fixK Expression
Authors: Soberón Mario , Morera Claudia , Kondorosi Adam , Lopez Oswaldo , Miranda Juan
Published: May 2007

5-aminoimidazole-4-carboxamide nucleotide (AICAR) is a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli. In this work, the effect of AICAriboside (AICAr), the precursor of AICAR on the expression of the Sinorhizobium meliloti fixNOQP operon encoding the symbiotic terminal oxidase cbb3, was analyzed. AICAr reduced the microaerobic induction levels of fixN-lacZ and fixT-lacZ gene fusions 18- and sevenfold respectively, and both genes were activated by the transcriptional activator FixK. A fixK-lacZ fusion presented 14-fold-reduced induction levels in microaerobic cell cultures in the presence of AICAr. AICAr also reduced three-fold the microaerobic expression levels of the nifA-lacZ fusion, whose expression as well as that of fixK is controlled by the two-component system FixL-FixJ. In contrast, AICAr had no effect on the expression levels of a hemA-lacZ fusion. These data suggest that AICAr prevents fixNOQP induction by the inhibition of fixK transcription.
Novel Constructions to Enable the Integration of Genes into the Agrobacterium tumefaciens C58 Chromosome
Authors: Lee Lan-Ying , Humara Jaime M. , Gelvin Stanton B.
Published: May 2007

We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer. One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPα plasmid. The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert. A second plasmid also contains a β-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization). A third plasmid contains, in addition to the lacZ, lacIq, and β-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A. tumefaciens. To test this system, we introduced a wild-type virD2 gene into the A. tumefaciens chromosome at the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A. tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.
Mutation of an Arginine Biosynthesis Gene Causes Reduced Pathogenicity in Fusarium oxysporum f. sp. melonis
Authors: Namiki Fumio , Matsunaga Michiko , Okuda Mitsuru , Inoue Iori , Nishi Kazufumi , Fujita Yoshikatsu , Tsuge Takashi
Published: May 2007

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95–1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95–1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.
Genetic Mapping and Functional Analysis of the Tomato Bs4 Locus Governing Recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 Protein
Authors: Ballvora Agim , Pierre Michéle , van den Ackerveken Guido , Schornack Sebastian , Rossier Ombeline , Ganal Martin , Lahaye Thomas , Bonas Ulla
Published: May 2007

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4- linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.
Polymorphism of the Polyketide Synthase Gene phlD in Biocontrol Fluorescent Pseudomonads Producing 2,4-Diacetylphloroglucinol and Comparison of PhlD with Plant Polyketide Synthases
Authors: Ramette Alban , Moënne-Loccoz Yvan , Défago Geneviève
Published: May 2007

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.
Molecular Characterization of a Subtilase from the Vascular Wilt Fungus Fusarium oxysporum
Authors: Pietro Antonio Di , Huertas-González M. Dolores , Gutierrez-Corona J. Felix , Martínez-Cadena Guadalupe , Méglecz Emese , Roncero M. Isabel G.
Published: May 2007

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici, whose predicted amino acid sequence shows significant homology with subtilisin-like fungal proteinases. Prt1 is a single-copy gene, and its structure is highly conserved among different formae speciales of F. oxysporum. Prt1 is expressed constitutively at low levels during growth on different carbon and nitrogen sources and strongly induced in medium containing collagen and glucose. As shown by reverse transcription-polymerase chain reaction and fluorescence microscopy of F. oxysporum strains carrying a prt1-promoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F. oxysporum strains transformed with an expression vector containing the prt1 coding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extracellular subtilase activity that resolved into a single peak of pI 4.0 upon isoelectric focusing. The active fraction produced two clearing bands of 29 and 32 kDa in sodium dodecyl sulfate gels containing gelatin. Targeted inactivation of prt1 in F. oxysporum f. sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.
Agrobacterium rhizogenes-Transformed Roots of Medicago truncatula for the Study of Nitrogen-Fixing and Endomycorrhizal Symbiotic Associations
Authors: Boisson-Dernier Aurélien , Chabaud Mireille , Garcia Fernand , Bécard Guillaume , Rosenberg Charles , Barker David G.
Published: May 2007

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous “composite plants.” In addition, we found that kanamycin can be used to select for the co-transformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter p MtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbus-cules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.
Transposon Mutagenesis of Xylella fastidiosa by Electroporation of Tn5 Synaptic Complexes
Authors: Guilhabert Magalie R. , Hoffman Les M. , Mills David A. , Kirkpatrick Bruce C.
Published: May 2007

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 × 103 and 4 × 103 Tn5 mutants per μg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.
Endopolygalacturonase Is Essential for Citrus Black Rot Caused byAlternaria citribut Not Brown Spot Caused byAlternaria alternata
Authors: Isshiki Atsunori , Akimitsu Kazuya , Yamamoto Mikihiro , Yamamoto Hiroyuki
Published: May 2007

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.
The Disruption of a Gene Encoding a Putative Arylesterase Impairs Pyruvate Dehydrogenase Complex Activity and Nitrogen Fixation in Sinorhizobium meliloti
Authors: Soto María José , Sanjuan Juan , Olivares José
Published: May 2007

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the tri-chloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3′ end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.
The Rhizobium GstI Protein Reduces the NH4+ Assimilation Capacity of Rhizobium leguminosarum
Authors: Tatè Rosarita , Mandrich Luigi , Spinosa Maria R. , Riccio Anna , Lamberti Alessandro , Iaccarino Maurizio , Patriarca Eduardo J.
Published: May 2007

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.
Cell Biological Changes of Outer Cortical Root Cells in Early Determinate Nodulation
Authors: van Spronsen Paulina C. , Grønlund Mette , Bras Cristina Pacios , Spaink Herman P. , Kijne Jan W.
Published: May 2007

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylenebiosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.
Possible Involvement of the Phloem Lectin in Long-Distance Viroid Movement
Authors: Owens R. A. , Blackburn M. , Ding B.
Published: May 2007

Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.
Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen Clavibacter michiganensis
Authors: Filippone M. Paula , Diaz-Ricci Juan C. , Castagnaro Atilio P. , Farías Ricardo N.
Published: May 2007

Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganensis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dissipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability.
Dark Green Islands in Plant Virus Infection are the Result of Posttranscriptional Gene Silencing
Authors: Moore Carolyn J. , Sutherland Paul W. , Forster Richard L. S. , Gardner Richard C. , MacDiarmid Robin M.
Published: May 2007

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.
Maize rhm1 Resistance to Bipolaris maydis Is Associated with Few Differences in Pathogenesis-Related Proteins and Global mRNA Profiles
Authors: Simmons Carl R. , Grant Susan , Altier Daniel J. , Dowd Patrick F. , Crasta Oswald , Folkerts Otto , Yalpani Nasser
Published: May 2007

The maize rhm1 mutant resists Bipolaris maydis, the causal agent of Southern corn leaf blight, by producing small necrotic lesions surrounded by chlorotic haloes. The rhm1 and wild-type lesions contain viable fungus in equal frequency, but fungal sporulation was markedly inhibited on rhm1. The levels of the pathogenesis-related (PR) proteins chitinase, PR1, and peroxidase differ little between rhm1 and wild type, with or without B. maydis inoculation. The global mRNA profiles surveyed revealed hundreds of cDNA fragments that were twofold or more induced or suppressed in rhm1 and wild-type plants following B. maydis inoculation. Nonetheless, between rhm1 and wild type, only 0.4 to 0.7% of the cDNA fragments were expressed differentially by twofold or more. Among the up-regulated genes in rhm1 was beta-glucosidase glu1, which prompted a test of whether rhm1 resistance depends upon the antimicrobial compound 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one or other hydroxamic acids whose glucosyl conjugates are preferred substrates for the Glu1 enzyme. Double mutants of rhm1 and bx1, a hydroxamic acid-deficient mutant, indicate that rhm1 resistance is hydroxamic acid independent. The rhm1 resistance presently appears to operate via a mechanism unlike those of previously described resistance genes.
Inhibition of Growth ofAspergillus flavusand Fungal α-Amylases by a Lectin-Like Protein fromLablab purpureus
Authors: Fakhoury A. M. , Woloshuk C. P.
Published: May 2007

Aspergillus flavus is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. Associated with the disease is the production of aflatoxins, which are a series of structurally related mycotoxins known to be carcinogenic. Previous research has suggested that the α-amylase of A. flavus promotes aflatoxin production in the endosperm of infected maize kernels. We report here the isolation and characterization of a 36-kDa α-amylase inhibitor from Lablab purpureus (AILP). AILP inhibited the α-amylases from several fungi but had little effect on those from animal and plant sources. The protein inhibited conidial germination and hyphal growth of A. flavus. The amino acid sequence indicated that AILP is similar to lectin members of a lectin-arcelin-α-amylase inhibitor family described in common bean and shown to be a component of plant resistance to insect pests. AILP also agglutinated papain-treated red blood cells from human and rabbit. These data indicate that AILP represents a novel variant in the lectin-arcelin-α-amylase inhibitor family of proteins having lectin-like and α-amylase inhibitory activity.
Transgenic Plants Producing the Bacterial Pheromone N-Acyl-Homoserine Lactone Exhibit Enhanced Resistance to the Bacterial Phytopathogen Erwinia carotovora
Authors: Mäe Andres , Montesano Marcos , Koiv Viia , Palva E. Tapio
Published: May 2007

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.
Cloning and Expression Analysis of Phytoplasma Protein Translocation Genes
Authors: Kakizawa Shigeyuki , Oshima Kenro , Kuboyama Tsutomu , Nishigawa Hisashi , Jung Hee-young , Sawayanagi Toshimi , Tsuchizaki Tsuneo , Miyata Shin-ichi , Ugaki Masashi , Namba Shigetou
Published: May 2007

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.
Induction of a Hypersensitive Response by Chimeric Helicase Sequences of Tobamoviruses U1 and Ob in N-Carrying Tobacco
Authors: Abbink Truus E. M. , de Vogel Johan , Bol John F. , Linthorst Huub J. M.
Published: May 2007

Recently, the helicase domain of the Tobacco mosaic virus (TMV)-U1 replicase proteins (designated MOREHEL:U1) was identified as the elicitor of the N gene-mediated hypersensitive response (HR) in tobacco. In this study, we used agroinfiltration to express the equivalent MOREHEL domain of the non-HR-inducing tobamovirus strain TMV-Ob. It appeared that this MOREHEL:Ob sequence did not elicit a HR in N gene-carrying tobacco. Both MOREHEL sequences were divided into eight subdomains, and chimeras of MOREHEL sequences from U1 and Ob were constructed. Expression of these chimeric MOREHEL sequences revealed that, in the TMV-U1 MOREHEL sequence, at least four domains involved in full HR induction were present. The presence of at least three of these four domains seems a minimal requirement for HR induction. Two additional domains may play a minor role in HR induction. To study the elicitor function of the chimeras during the TMV life cycle, chimeric MOREHEL domains were introduced into full-length TMV cDNA clones. These constructs, however, were unable to establish an infection in Nicotiana benthamiana or Nicotiana tabacum plants.
Local Modulation of Host pH byColletotrichumSpecies as a Mechanism to Increase Virulence
Authors: Prusky Dov , McEvoy James L. , Leverentz Britta , Conway William S.
Published: May 2007

The phytopathogenic fungus Colletotrichum gloeosporioides produces one pectate lyase (PL) that is a key virulence factor in disease development. During growth of C. gloeosporioides, Colletotrichum acutatum, and Colletotrichum coccodes in acidified yeast extract medium, the fungus secreted ammonia and increased the medium pH. Ammonia accumulation and the consequent pH change increased as a function of initial pH and buffer capacity of the medium. PL secretion by C. gloeosporioides correspondingly increased as the pH of the medium increased. The C. gloeosporioides pelB gene-disrupted mutant was able to increase ammonia accumulation and pH of the media similarly to the wild-type isolate. C. gloeosporioides in avocado, C. coccodes in tomato, and C. acutatum in apple showed ammonia accumulation in the infected area where pH increased to 7.5 to 8 and PL activity is optima. In nonhost interactions where C. gloeosporioides was inoculated in apples, the addition of ammonia-releasing compounds significantly enhanced pathogenicity to levels similar to those caused by the compatible C. acutatum-apple interaction. The results therefore suggest the importance of ammonia secretion as a virulence factor, enhancing environmental pH and pathogenicity of the Colletotrichum species.