bluuebunny/crossref_metadata_2025 huggingface dataset:
Syringolin-Mediated Activation of the Pir7b Esterase Gene in Rice Cells Is Suppressed by Phosphatase Inhibitors
Authors: Hassa Paul , Granado José , Freydl Ernst , Wäspi Urs , Dudler Robert
Published: May 2007

Inoculation of rice plants (Oryza sativa) with the nonhost pathogen Pseudomonas syringae pv. syringae leads to the activation of defense-related genes and ultimately to induced resistance against the rice blast fungus Pyricularia oryzae. One of the molecular determinants of P. syringae pv. syringae that is recognized by the plant cells and evokes these defense responses is syringolin A, an elicitor that is secreted by the bacteria under appropriate conditions. In order to investigate signal transduction events elicited by syringolin A, the response of cultured rice cells to syringolin A application was analyzed. Cultured rice cells were able to sense syringolin A at concentrations in the nanomolar range as observed by the transient accumulation of Pir7b esterase transcripts. Syringolin A-mediated Pir7b transcript accumulation was inhibited by cycloheximide, indicating that de novo protein synthesis was required. Calyculin and okadaic acid, two protein phosphatase inhibitors, blocked Pir7b gene induction, whereas the serine/threonine protein kinase inhibitors staurosporine and K-252a had no effect on Pir7b transcript levels. Actin transcript levels were essentially not affected by inhibitor treatments over the experimental time span. These results imply that dephosphorylation of a phosphoprotein is an important step in the syringolin A-triggered signal transduction pathway.
Heterologous Expression of Functional Ptr ToxA
Authors: Tuori Robert P. , Wolpert Thomas J. , Ciuffetti Lynda M.
Published: May 2007

Ptr ToxA, a proteinaceous host-selective toxin (HST) produced by the fungus Pyrenophora tritici-repentis, was expressed in Escherichia coli and purified as a polyhistidine-tagged, fusion protein (NC-FP). NC-FP, consisting of both the N and C domains of the ToxA open reading frame (ORF), is produced as an insoluble protein in E. coli at approximately 10 to 16 mg per liter of culture. Following in vitro refolding, NC-FP elicits cultivar-specific necrosis in wheat, with a specific activity similar to that of native Ptr ToxA. A fusion protein consisting of only the C domain has approximately 10 to 20% of the activity of native Ptr ToxA. These data suggest that (i) the N domain is important for maximal activity of Ptr ToxA, (ii) the N domain does not function to eliminate activity of the protoxin, and (iii) post-translational modifications of Ptr ToxA are not essential for activity. A C domain construct with a cysteine residue mutated to glycine is inactive. This, plus the observation that toxin activity is sensitive to reducing agents, provides evidence that the two cysteine residues in Ptr ToxA are involved in a disulfide bond that is essential for activity. The heterologous expression of Ptr ToxA provides a valuable tool for addressing a number of issues such as receptor binding studies, structure/function studies, and screening wheat cultivars for disease resistance.
Identification of Rice Blast Fungal Elicitor-Responsive Genes by Differential Display Analysis
Authors: Kim Cha Young , Lee Sung-Ho , Park Hyeong Cheol , Bae Chang Gyu , Cheong Yong Hwa , Choi Young Ju , Han Chang-deok , Lee Sang Yeol , Lim Chae Oh , Cho Moo Je
Published: May 2007

In order to study molecular interactions that occur between rice and rice blast fungus upon infection, we isolated fungal elicitor-responsive genes from rice (Oryza sa-tiva cv. Milyang 117) suspension-cultured cells treated with fungal elicitor prepared from the rice blast fungus (Magnaporthe grisea) employing a method that combined mRNA differential display and cDNA library screening. Data base searches with the isolated cDNA clones revealed that the OsERG1 and OsERG2 cDNAs share significant similarities with the mammalian Ca2+-dependent lipid binding (C2) domains. The OsCPX1 cDNA is highly homologous to peroxidases. The OsHin1 cDNA exhibits homology to the tobacco hin1 gene, whose expression is induced by avirulent pathogens. The OsLPL1 and OsMEK1 cDNAs share homologies with lysophospholipases and serine/threonine mitogen-activated protein (MAP) kinase kinases, respectively. The OsWRKY1 and OsEREBP1 cDNAs are homologous to transcription factors, such as the WRKY protein family and the AP2/EREBP family, respectively. Transcripts of the OsERG1, OsHin1, and OsMEK1 genes were specifically elevated only in response to the avirulent race KJ301 of the rice blast fungus. Our study yielded a number of elicitor-responsive genes that will not only provide molecular probes, but also contribute to our understanding of host defense mechanisms against the rice blast fungus.
Temporal and Spatial Order of Events During the Induction of Cortical Cell Divisions in White Clover byRhizobium leguminosarumbv.trifoliiInoculation or Localized Cytokinin Addition
Authors: Mathesius Ulrike , Charon Celine , Rolfe Barry G. , Kondorosi Adam , Crespi Martin
Published: May 2007

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.
Plants Secrete Substances That Mimic Bacterial N-Acyl Homoserine Lactone Signal Activities and Affect Population Density-Dependent Behaviors in Associated Bacteria
Authors: Teplitski Max , Robinson Jayne B. , Bauer Wolfgang D.
Published: May 2007

In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a popula tion density-dependent fashion by N-acyl homoserine lac tone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated be haviors in some strains while inhibiting such behaviors in others. The chemical nature of the active mimic com pounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs. Various species of higher plants in addition to pea were found to secrete AHL mimic activities. The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diver sity of pathogenic, symbiotic, and saprophytic bacteria.
The Dual Function in Virulence and Host Range Restriction of a Gene Isolated from the pPATHEhg Plasmid of Erwinia herbicola pv. gypsophilae
Authors: Ezra David , Barash Isaac , Valinsky Lea , Manulis Shulamit
Published: May 2007

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to gypsophila whereas Erwinia herbicola pv. betae (Ehb) attacks beet as well as gypsophila. Both pathovars contain an indigenous plasmid (pPATHEhg or pPATHEhb) that harbors pathogenicity genes, including the hrp gene cluster. A cosmid library of Ehg824-1 plasmid DNA was mobilized into Ehb4188 and the transconjugants were screened for pathogenicity on beet. One Ehb transconjugant harboring the cosmid pLA173 of pPATHEhg induced a hypersensitive-like response and abolished pathogenicity on beet. Transposon mutagenesis of an open reading frame (ORF) located on this cosmid eliminated its affect on pathogenicity. Marker exchange of this mutation into Ehg824-1 caused a substantial reduction in gall size on gypsophila and caused Ehg824-1 to extend its host range and incite galls on beet. The ORF (1.5 kb) was designated as pthG (pathogenicity gene on gypsophila). DNA sequence analysis of pthG revealed no significant homology to known genes in the data bank. Only remnants of the pthG sequences were identified on the pPATH of Ehb4188. The deduced protein lacked an N-terminal signal peptide but contained a short trans-membrane helix in its C terminus. The gene product, as determined by expression in Escherichia coli and Western blots (immunoblots), was a 56-kDa protein.
The BMP1 Gene Is Essential for Pathogenicity in the Gray Mold Fungus Botrytis cinerea
Authors: Zheng Li , Campbell Mathew , Murphy Jennifer , Lam Stephen , Xu Jin-Rong
Published: May 2007

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.
Nodule Invasion and Symbiosome Differentiation During Rhizobium etli-Phaseolus vulgaris Symbiosis
Authors: Cermola Michele , Fedorova Elena , Taté Rosarita , Riccio Anna , Favre Renee , Patriarca Eduardo J.
Published: May 2007

By means of a detailed ultrastructural analysis of nodules induced by Rhizobium etli on the roots of Phaseolus vulgaris, we observe that the development of host-invaded cells is not synchronous. An accumulation of mitochondria was found in freshly invaded host cells, containing only a few symbiosomes (SBs) that are released from highly branched intracellular ramification of the infection threads. Moreover, besides the fusion between the SB membrane with host secretory vesicles, we observe also a great number of fusions between the outer leaflets of adjoining SB membranes, thus resulting in structures that resemble the tight junction network (zona occludens with a five-layered structure) of epithelian cells. This process was found to be induced strongly and earlier both in the invaded host cells of ineffective nodules (elicited by Fix-mutant strains of R. etli) and in the older (senescence) invaded cells of effective nodules, whereas bacteroid division is seldom if ever observed. Our observations strongly suggest that multiple-occupancy SBs also arise by fusion of single-occupancy SBs and the physiological consequence of this process is discussed.
The Ustilago maydis ubc4 and ubc5 Genes Encode Members of a MAP Kinase Cascade Required for Filamentous Growth
Authors: Andrews David L. , Egan John D. , Mayorga María E. , Gold Scott E.
Published: May 2007

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.
Sinorhizobium fredii USDA257, a Cultivar-Specific Soybean Symbiont, Carries Two Copies of y4yA and y4yB, Two Open Reading Frames That Are Located in a Region That Encodes the Type III Protein Secretion System
Authors: Jiang Guoqiao , Krishnan Hari B.
Published: May 2007

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.
Resistance of Tomato and Pepper to T3 Strains of Xanthomonas campestris pv. Vesicatoria Is Specified by a Plant-Inducible Avirulence Gene
Authors: Astua-Monge Gustavo , Minsavage Gerald V. , Stall Robert E. , Davis Michael J. , Bonas Ulla , Jones Jeffrey B.
Published: May 2007

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.
Interactions of Pseudomonas syringae pv. glycinea with Host and Nonhost Plants in Relation to Temperature and Phytotoxin Synthesis
Authors: Budde Ina P. , Ullrich Matthias S.
Published: May 2007

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coro-natine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18°C whereas no detectable synthesis occurs at 28°C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18°C prior to inoculation but not when derived from cultures grown at 28°C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18°C, compared with parallels incubated at 28°C. CFA biosynthesis by PG4180.N9 was optimal at 18°C and negligible at 28°C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.
Cell-to-Cell Movement of Potexviruses: Evidence for a Ribonucleoprotein Complex Involving the Coat Protein and First Triple Gene Block Protein
Authors: Lough Tony J. , Netzler Natalie E. , Emerson Sarah J. , Sutherland Paul , Carr Fiona , Beck David L. , Lucas William J. , Forster Richard L. S.
Published: May 2007

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for inter-cellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potex-viruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.
Structural and Functional Complexity of the Genomic Region Controlling AK-Toxin Biosynthesis and Pathogenicity in the Japanese Pear Pathotype of Alternaria alternata
Authors: Tanaka Aiko , Tsuge Takashi
Published: May 2007

The Japanese pear pathotype of Alternaria alternata produces host-specific AK-toxin and causes black spot of Japanese pear. Previously, a cosmid clone, pcAKT-1, was isolated that contains two genes, AKT1 and AKT2, within a 5.0-kb region required for AK-toxin biosynthesis. The wild-type strain has multiple, nonfunctional copies of these genes. In the present study, two additional genes, AKTR-1 and AKT3-1, downstream of AKT2 were identified. Transformation of the wild type with AKTR-1- and AKT3-1-targeting vectors produced toxin-deficient (Tox¯), nonpathogenic mutants. DNA gel blot analysis, however, demonstrated that the fragments targeted in Tox¯ mutants were different from those containing AKTR-1 and AKT3-1 on the transforming vectors. A cosmid clone, pcAKT-2, containing the targeted DNA was isolated and shown to carry two genes, AKTR-2 and AKT3-2, with high similarity to AKTR-1 and AKT3-1, respectively. Transcripts from not only AKTR-2 and AKT3-2 but also AKTR-1 and AKT3-1 were found in the wild type. DNA gel blot analysis with pulsed-field gel electrophoresis showed that AKT1, AKT2, AKT3, and AKTR and their homologues are on a single chromosome. These results indicate the structural and functional complexity of the genomic region controlling AK-toxin biosynthesis.
Proteome Analysis of Differentially Displayed Proteins As a Tool for the Investigation of Symbiosis
Authors: Natera Siria H. A. , Guerreiro Nelson , Djordjevic Michael A.
Published: May 2007

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.
Role of the Nucleoid-Associated Protein H-NS in the Synthesis of Virulence Factors in the Phytopathogenic Bacterium Erwinia chrysanthemi
Authors: Nasser William , Faelen Michel , Hugouvieux-Cotte-Pattat Nicole , Reverchon Sylvie
Published: May 2007

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25°C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.
Overlapping Plant Signal Transduction Pathways Induced by a Parasitic Nematode and a Rhizobial Endosymbiont
Authors: Koltai Hinanit , Dhandaydham Murali , Opperman Charles , Thomas Judith , Bird David
Published: May 2007

Root-knot nematodes and rhizobia establish interactions with roots characterized by the de novo induction of host structures, termed giant cells and nodules, respectively. Two transcription regulators, PHAN and KNOX, required for the establishment of meristems were previously shown to be expressed in tomato giant cells. We isolated the orthologues of PHAN and KNOX (Mt-phan and Mt-knox-1) from the model legume Medicago truncatula, and established the spatial distribution of their expression in situ. We confirmed that Mt-phan and Mt-knox-1 are expressed in lateral root initials and in nematode-induced giant cells and showed that they are expressed in nodules induced by Sinorhizobium meliloti. Expression of both genes becomes spatially restricted as the nodules develop. We further examined nematode feeding sites for the expression of two genes involved in nodule formation, ccs52 (encodes a mitotic inhibitor) and ENOD40 (encodes an early, nodulation mitogen), and found transcripts of both genes to be present in and around giant cells induced in Medicago. Collectively, these results reveal common elements of host responses to mutualistic and parasitic plant endosymbionts and imply that overlapping regulatory pathways lead to giant cells and nodules. We discuss these pathways in the context of phytohormones and parallels between beneficial symbiosis and disease.
Expression Studies of Superoxide Dismutases in Nodules and Leaves of Transgenic Alfalfa Reveal Abundance of Iron-Containing Isozymes, Posttranslational Regulation, and Compensation of Isozyme Activities
Authors: Rubio Maria C. , Ramos Javier , Webb K. Judith , Minchin Frank R. , González Esther , Arrese-Igor Cesar , Becana Manuel
Published: May 2007

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1–10), MnSOD in the chloroplasts (line 4–6), and FeSOD in the chloroplasts (line 10–7). In nodules of line 10–7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.
Biological Activity of Harpin Produced by Pantoea stewartii subsp. stewartii
Authors: Ahmad Musharaf , Majerczak Doris R. , Pike Sharon , Hoyos Mary Elizabeth , Novacky Anton , Coplin David L.
Published: May 2007

Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpNPnss. Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpNPnss to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpNPnss in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpNPnss was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.
Actin Monoubiquitylation Is Induced in Plants in Response to Pathogens and Symbionts
Authors: Dantán-González Edgar , Rosenstein Yvonne , Quinto Carmen , Sánchez Federico
Published: May 2007

Most dramatic examples of actin reorganization have been described during host-microbe interactions. Plasticity of actin is, in part, due to posttranslational modifications such as phosphorylation or ubiquitylation. Here, we show for the first time that actins found in root nodules of Phaseolus vulgaris are modified transiently during nodule development by monoubiquitylation. This finding was extended to root nodules of other legumes and to other plants infected with mycorrhiza or plant pathogens such as members of the genera Pseudomonas and Phytophthora. However, neither viral infections nor diverse stressful conditions (heat shock, wounding, or osmotic stress) induced this response. Additionally, this phenomenon was mimicked by the addition of a yeast elicitor or H2O2 to Phaseolus vulgaris suspension culture cells. This modification seems to provide increased stability of the microfilaments to proteolytic degradation and seems to be found in fractions in which the actin cytoskeleton is associated with membranes. All together, these data suggest that actin monoubiquitylation may be considered an effector mechanism of a general plant response against microbes.
Transgenic Tobacco Plants Expressing thePotato virus XOpen Reading Frame 3 Gene Develop Specific Resistance and Necrotic Ring Symptoms After Infection with the Homologous Virus
Authors: Kobayashi Ken , Cabral Silvia , Calamante Gabriela , Maldonado Sara , Mentaberry Alejandro
Published: May 2007

Tobacco plants were transformed with the open reading frame 3 gene from Potato virus X (PVX) coding for the p12 protein. Although the transgenic plants exhibited a normal morphological aspect, microscopic examination revealed extensive alterations in leaf tissue structure. After being challenged with PVX, the transgenic plants showed resistance to PVX infection and formation of specific leaf symptoms consisting of concentric rings encircled by necrotic borders. These novel symptoms were accompanied by biochemical changes normally associated with the hypersensitive response (HR) and were absent in noninfected transgenic plants or in PVX-infected nontransgenic plants. No equivalent virus resistance was observed after inoculation with Tobacco mosaic virus or Potato virus Y, suggesting the presence of a specific resistance mechanism. Despite development of HR-like symptoms, systemic acquired resistance was not induced in PVX-infected p12 transgenic plants. No evidence of an RNA-mediated resistance mechanism was found.
Ornithine Decarboxylase Knockout in Tapesia yallundae Abolishes Infection Plaque Formation In Vitro but Does Not Reduce Virulence Toward Wheat
Authors: Mueller E. , Bailey A. , Corran A. , Michael A. J. , Bowyer P.
Published: May 2007

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.
A Highly Efficient Transposon Mutagenesis System for the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis
Authors: Kirchner Oliver , Gartemann Karl-Heinz , Zellermann Eva-Maria , Eichenlaub Rudolf , Burger Annette
Published: May 2007

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.
Potato spindle tuber viroid as Inducer of RNA Silencing in Infected Tomato
Authors: Itaya Asuka , Folimonov Alexey , Matsuda Yoshie , Nelson Richard S. , Ding Biao
Published: May 2007

Potato spindle tuber viroid (PSTVd), an RNA plant pathogen encoding no known proteins, induces systemic symptoms on tomato plants. We report detection of small RNAs of approximately 25 nucleotides with sequence specificity to PSTVd in infected plants: an indication of the presence of RNA silencing. RNA silencing, however, did not appear to be responsible for the differing symptoms induced by a mild and a severe strain of PSTVd. The unique structural and biological features of viroids make them attractive experimental tools to investigate mechanisms of RNA silencing and pathogen counterdefense.
Analysis of Genes Expressed During Rice-Magnaporthe griseaInteractions
Authors: Kim Soonok , Ahn II-Pyung , Lee Yong-Hwan
Published: May 2007

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.
Regulatory Roles of the GacS/GacA Two-Component System in Plant-Associated and Other Gram-Negative Bacteria
Authors: Heeb Stephan , Haas Dieter
Published: May 2007

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of Gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.
A Major Quantitative Trait Locus for Resistance to Potato leafroll virus Is Located in a Resistance Hotspot on Potato Chromosome XI and Is Tightly Linked to N-Gene-Like Markers
Authors: Marczewski Waldemar , Flis Bogdan , Syller Jerzy , Schäfer-Pregl Ralf , Gebhardt Christiane
Published: May 2007

Potato leafroll virus (PLRV) causes one of the most widespread and important virus diseases in potato. Resistance to PLRV is controlled by genetic factors that limit plant infection by viruliferous aphids or virus multiplication and accumulation. Quantitative trait locus (QTL) analysis of resistance to virus accumulation revealed one major and two minor QTL. The major QTL, PLRV.1, mapped to potato chromosome XI in a resistance hotspot containing several genes for qualitative and quantitative resistance to viruses and other potato pathogens. This QTL explained between 50 and 60% of the phenotypic variance. The two minor QTL mapped to chromosomes V and VI. Genes with sequence similarity to the tobacco N gene for resistance to Tobacco mosaic virus were tightly linked to PLRV.1. The cDNA sequence of an N-like gene was used to develop the sequence characterized amplified region (SCAR) marker N1271164 that can assist in the selection of potatoes with resistance to PLRV.
A Physical Map of the Syringomycin and Syringopeptin Gene Clusters Localized to an Approximately 145-kb DNA Region of Pseudomonas syringae pv. syringae Strain B301D
Authors: Scholz-Schroeder Brenda K. , Soule Jonathan D. , Lu Shi-En , Grgurina Ingeborg , Gross Dennis C.
Published: May 2007

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.
Overexpression of Pti5 in Tomato Potentiates Pathogen-Induced Defense Gene Expression and Enhances Disease Resistance to Pseudomonas syringae pv. tomato
Authors: He Ping , Warren Randall F. , Zhao Tiehan , Shan Libo , Zhu Lihuang , Tang Xiaoyan , Zhou Jian-Min
Published: May 2007

The tomato Pti5 gene encodes a pathogen-inducible ethylene response element-binding protein-like transcription factor that interacts with the disease resistance gene product Pto. Overexpression of Pti5 or Pti5-VP16, a translational fusion with a constitutive transcriptional activation domain, in tomato enhanced resistance to Pseudomonas syringae pv. tomato. Constitutive expression of Pti5 or Pti5-VP16 did not affect the basal level of pathogenesis-related gene expression, but it accelerated pathogen-induced expression of GluB and Catalase. The results demonstrate a positive role of Pti5 in defense gene regulation and disease resistance and suggest that a pathogen-activated posttran-scriptional regulatory step is necessary for the pathogen induction of the defense gene expression.
Functional Analysis of the Type III Effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the Use of a Single-Copy Genomic Integration System
Authors: Guttman David S. , Greenberg Jean T.
Published: May 2007

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpm1 effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.
Lotus japonicus Forms Early Senescent Root Nodules with Rhizobium etli
Authors: Banba Mari , Siddique Abu-Baker M. , Kouchi Hiroshi , Izui Katsura , Hata Shingo
Published: May 2007

Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors. Lotus japonicus efficiently formed root nodules with R. etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene. On a nitrogen-free medium, however, the host plant inoculated with R. etli showed a severe nitrogen deficiency symptom. Initially, the nodules formed with R. etli were pale pink and leghemoglobin mRNA was detectable at significant levels. Nevertheless, the nodules became greenish with time. Acetylene-reduction activity of nodules formed with R. etli was comparable with that formed by M. loti 3 weeks postinoculation, but thereafter it decreased rapidly. The nodules formed with R. etli contained much more starch granules than those formed with M. loti. R. etli developed into bacteroids in the L. japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M. loti. The nodules formed with R. etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules. Thus, L. japonicus started and then ceased a symbiotic relationship with R. etli at the final stage.
Replicase-Derived Resistance AgainstPea early browning virusinNicotiana benthamianaIs an Unstable Resistance Based upon Posttranscriptional Gene Silencing
Authors: van den Boogaart Tom , Wen Fuijang , Davies Jeffrey W. , Lomonossoff George P.
Published: May 2007

Virus resistance in Nicotiana benthamiana plants containing a translatable Pea early browning virus (PEBV) 54K sequence from the 201K replicase gene has been reported previously. Resistant plants contain multiple transgene copies divided between two loci. Analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. The resistance observed in R2 and R3 generations of lines containing both transgene loci in homozygous condition became less consistent in R4 and R5 generations. This inversely correlated with steady-state transgene transcript levels of the segregating populations. The use of recombinant Potato virus X vectors carrying PEBV 54K sequences showed that the resistance is based upon posttranscriptional gene silencing, is non-strand specific, and recognizes 3′ located sequences within the PEBV 54K sequence.