bluuebunny/crossref_metadata_2025 huggingface dataset:
Five Avirulence Genes from Xanthomonas campestris pv. malvacearum Cause Genotype-Specific Cell Death When Expressed Transiently in Cotton
Authors: de Feyter Rob , McFadden Helen , Dennis Liz
Published: May 2007

The coding regions from five avirulence (avr) genes from Xanthomonas campestris pv. malvacearum (Xcm), the causal agent of bacterial blight of cotton, were joined to the cauliflower mosaic virus 35S promoter and expressed transiently in cotton leaves after Agrobacterium tumefaciens-mediated gene transfer. A genotype-specific necrosis was observed 3 to 6 days post inoculation for constructs derived from avrB4, avrb7, and avrBIn on cotton lines containing the resistance (R) genes B4, b7, and BIn, respectively, but not on susceptible cotton plants. No necrosis was obtained with plasmids that direct expression of avr genes within A. tumefaciens, showing that the cotton response required transfer of the genes into the plant cells. Addition of a signal peptide sequence into the avr constructs to target expressed Avr protein to the apoplast significantly reduced the responses. The results indicate that intracellular expression of Xcm Avr proteins in cotton having the corresponding R gene causes specific host cell death.
Cloning, Characterization, and Targeted Disruption of cpcat1, Coding for an in Planta Secreted Catalase of Claviceps purpurea
Authors: Garre Victoriano , Müller Ulrike , Tudzynski Paul
Published: May 2007

Claviceps purpurea has been shown to secrete catalases in axenic and parasitic culture. In order to determine the importance of these enzymes in the host-parasite interaction, especially their role in overcoming oxidative stress imposed on the pathogen by the plant's defense system, the catR gene from A. niger was used to isolate a putative catalase gene from a genomic library of C. purpurea. cpcat1 consists of an open reading frame of 2,148 bp that is interrupted by five introns. Its derived gene product shows significant homology to fungal catalases and contains a putative signal peptide of 19 amino acids and three putative N-glycosylation sites, which indicates that CPCAT1 is a secreted catalase. Disruption of the gene by a gene replacement approach resulted in the loss of two catalase isoforms, CATC and CATD, strongly suggesting that they are both encoded by cpcat1. CATD is the major secreted catalase of C. purpurea and is furthermore the only catalase present in the honeydew of infected rye ears. Deletion mutants of cpcat1 were inoculated on rye plants and showed no significant reduction in virulence. Ovarian tissue and honeydew of plants inoculated with the mutants lacked CATD, confirming that this catalase is not essential for colonization of the host tissue by C. purpurea.
Modified Development in Transgenic Tobacco Plants Expressing a rolA∷GUS Translational Fusion and Subcellular Localization of the Fusion Protein
Authors: Vilaine Françoise , Rembur Jacques , Chriqui Dominique , Tepfer Mark
Published: May 2007

The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling. The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases. We have introduced into the tobacco genome a gene encoding a rolA∷GUS fusion protein. Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene. The distribution of β-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA∷gus gene or a control gus construct. As expected, in the control plants, GUS activity was essentially cytosolic. In contrast, in plants expressing the rolA∷gus gene the highest specific activity was associated with the plasmalemma fraction.
Mycorrhiza Mutants of Lotus japonicus Define Genetically Independent Steps During Symbiotic Infection
Authors: Wegel Eva , Schauser Leif , Sandal Niels , Stougaard Jens , Parniske Martin
Published: May 2007

Four nodulation mutants of Lotus japonicus affected in three different genetic loci blocked infection by Glomus intraradices within rhizodermal cells where abnormal deformations of hyphae occurred. Rare successful infections led to the formation of arbuscules. The affected plant genes are therefore essential during the early infection process, but are dispensable for later developmental stages.
The Rhizobium etli metZ Gene Is Essential for Methionine Biosynthesis and Nodulation of Phaseolus vulgaris
Authors: Taté Rosarita , Riccio Anna , Caputo Emilia , laccarino Maurizio , Patriarca Eduardo J.
Published: May 2007

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.
Regulation of Expression of Avirulence Gene avrRxv and Identification of a Family of Host Interaction Factors by Sequence Analysis of avrBsT
Authors: Ciesiolka L. D. , Hwin T. , Gearlds J. D. , Minsavage G. V. , Saenz R. , Bravo M. , Handley V. , Conover S. M. , Zhang H. , Caporgno J. , Phengrasamy N. B. , Toms A. O. , Stall R. E. , Whalen M. C.
Published: May 2007

Resistance in tomato line Hawaii 7998 as well as in several nonhost plants to Xanthomonas campestris pv. vesicatoria tomato strain (XcvT) is mediated in part by the avirulence gene avrRxv. Analysis of growth of wild-type and avrRxv deletion strains indicates that avrRxv plays a crucial role in the ability of XcvT 92–14 to induce resistance on Hawaii 7998. We used avrRxv reporter gene fusions and Northern (RNA) blot analysis to test several growth environments for inductive potential. We found that avrRxv is constitutively expressed at high levels and that growth in planta, in tobacco conditioned medium, and in hrp-inductive medium XVM2 did not affect the high levels of expression. In addition, hrp structural and regulatory mutant backgrounds had no effect. We mutated the bipartite plant inducible promoter (PIP)-box sequence and found that avrRxv activity appears to be independent of an intact PIP-box element. We present the sequence of the avrRxv homologue called avrBsT and align the six AvrRxv host interaction factor family members including mammalian pathogen virulence factors YopJ and YopP from Yersinia spp. and AvrA from Salmonella typhimurium, and open reading frame Y4LO with unknown function from the symbiont Rhizobium sp.
Localization of a Pathogenicity Gene in Ophiostoma novo-ulmi and Evidence That It May Be Introgressed from O. ulmi
Authors: Et-Touil Abdelali , Brasier Clive M. , Bernier Louis
Published: May 2007

Ophiostoma novo-ulmi, the principal agent of Dutch elm disease, has recently replaced another species of Dutch elm disease pathogen, O. ulmi, across much of the Northern Hemisphere. Field inoculations of the moderately resistant elms Ulmus procera and Ulmus × Commelin were carried out with progeny of a genetic cross between AST27, a Eurasian (EAN) O. novo-ulmi isolate with an unusually low level of pathogenicity, and H327, a highly aggressive EAN isolate. These confirmed the results of a previous study that indicated that the difference in phenotype was controlled by a single nuclear gene. This pathogenicity gene, designated here Pat1, is the first putative pathogenicity gene to be identified in O. novo-ulmi. In a bulked segregant analysis, involving 80 random primers, 10 RAPD (random amplified polymorphic DNA) markers were identified linked to Pat1. Linkage distances between these markers and Pat1 were confirmed by genetic analysis of all individual progeny. Five RAPD amplicons identified in AST27 were O. ulmi and not O. novo-ulmi specific amplicons, and of these two were linked to Pat1. This suggests that the Pat1 allele conferring unusually low aggressiveness in AST27 may have been acquired from O. ulmi via introgression. When RAPD marker OPK31050, linked to Pat1, was used as a probe for Southern hybridization with electrophoretically separated chromosomes of O. novo-ulmi and O. ulmi, the results indicated that Pat1 is located on a 3.5 Mb chromosome previously designated chDNA II.
Naturally Induced Secretions of the Potato Cyst Nematode Co-stimulate the Proliferation of Both Tobacco Leaf Protoplasts and Human Peripheral Blood Mononuclear Cells
Authors: Goverse Aska , van der Voort Jeroen Rouppe , van der Voort Charlotte Rouppe , Kavelaars Annemieke , Smant Geert , Schots Arjen , Bakker Jaap , Helder Johannes
Published: May 2007

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones α-naphtha-leneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (<3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue—these nematodes normally invade the roots of potato plants—suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.
The Alfalfa (Medicago sativa) TDY1 Gene Encodes a Mitogen-Activated Protein Kinase Homolog
Authors: Schoenbeck Mark A. , Samac Deborah A. , Fedorova Maria , Gregerson Robert G. , Gantt J. Stephen , Vance Carroll P.
Published: May 2007

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5′ flanking sequence of the TDY1 gene fused to the β-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.
Identification of a Locus in Arabidopsis Controlling Both the Expression of Rhizobacteria-Mediated Induced Systemic Resistance (ISR) and Basal Resistance Against Pseudomonas syringae pv. tomato
Authors: Ton Jurriaan , Pieterse Corné M. J. , Van Loon Leendert C.
Published: May 2007

Selected nonpathogenic rhizobacteria with biological disease control activity are able to elicit an induced systemic resistance (ISR) response that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Ten ecotypes of Arabidopsis thaliana were screened for their potential to express rhizobacteria-mediated ISR and pathogen-induced SAR against the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). All ecotypes expressed SAR. However, of the 10 ecotypes tested, ecotypes RLD and Wassilewskija (Ws) did not develop ISR after treatment of the roots with nonpathogenic Pseudomonas fluorescens WCS417r bacteria. This nonresponsive phenotype was associated with relatively high susceptibility to Pst infection. The F1 progeny of crosses between the non-responsive ecotypes RLD and Ws on the one hand, and the responsive ecotypes Columbia (Col) and Landsberg erecta (Ler) on the other hand, were fully capable of expressing ISR and exhibited a relatively high level of basal resistance, similar to that of their WCS417r-responsive parent. This indicates that the potential to express ISR and the relatively high level of basal resistance against Pst are both inherited as dominant traits. Analysis of the F2 and F3 progeny of a Col × RLD cross revealed that inducibility of ISR and relatively high basal resistance against Pst cosegregate in a 3 : 1 fashion, suggesting that both resistance mechanisms are monogenically determined and genetically linked. Neither the responsiveness to WCS417r nor the relatively high level of basal resistance against Pst were complemented in the F1 progeny of crosses between RLD and Ws, indicating that RLD and Ws are both affected in the same locus, necessary for the expression of ISR and basal resistance against Pst. The corresponding locus, designated ISR1, was mapped between markers B4 and GL1 on chromosome 3. The observed association between ISR and basal resistance against Pst suggests that rhizo-bacteria-mediated ISR against Pst in Arabidopsis requires the presence of a single dominant gene that functions in the basal resistance response against Pst infection.
Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis
Authors: Ramírez Mario , Valderrama Brenda , Arredondo-Peter Raúl , Soberón Mario , Mora Jaime , Hernández Georgina
Published: May 2007

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.
LSD1 Regulates Salicylic Acid Induction of Copper Zinc Superoxide Dismutase in Arabidopsis thaliana
Authors: Kliebenstein Daniel J. , Dietrich Robert A. , Martin Adam C. , Last Robert L. , Dangl Jeffery L.
Published: May 2007

We characterized the accumulation patterns of Arabidopsis thaliana proteins, two CuZnSODs, FeSOD, MnSOD, PR1, PR5, and GST1, in response to various pathogen-associated treatments. These treatments included inoculation with virulent and avirulent Pseudomonas syringae strains, spontaneous lesion formation in the lsd1 mutant, and treatment with the salicylic acid (SA) analogs INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadia-zole). The PR1, PR5, and GST1 proteins were inducible by all treatments tested, as expected from previous mRNA blot analysis. The two CuZnSOD proteins were induced by SA analogs and in conjunction with lsd1-mediated spreading cell death. Additionally, LSD1 is a part of a signaling pathway for the induction of the CuZnSOD proteins in response to SA but not in lsd1-mediated cell death. We suggest that the spreading lesion phenotype of lsd1 results from a lack of up-regulation of a CuZnSOD responsible for detoxification of accumulating superoxide before the reactive oxygen species can trigger a cell death cascade.
Involvement of cAMP and Protein Kinase A in Conidial Differentiation by Erysiphe graminis f. sp. hordei
Authors: Hall Alison A. , Bindslev Lene , Rouster Jacques , Rasmussen Søren W. , Oliver Richard P. , Gurr Sarah J.
Published: May 2007

Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus.
Expression Pattern of Genes Encoding Farnesyl Diphosphate Synthase and Sesquiterpene Cyclase in Cotton Suspension-Cultured Cells Treated with Fungal Elicitors
Authors: Liu Chang-Jun , Heinstein Peter , Chen Xiao-Ya
Published: May 2007

Cotton plants accumulate sesquiterpene aldehydes in pigment glands. The two enzymes farnesyl diphosphate synthase (FPS) and (+)-δ-cadinene synthase (CAD), a sesquiterpene cyclase, are involved in the biosynthesis of these secondary metabolites. A full-length cDNA (garfps) encoding FPS was isolated from Gossypium arboreum and identified by in vitro enzymatic assay of the garfps protein heterologously expressed in Escherichia coli. Treatment of G. arboreum suspension-cultured cells with an elicitor preparation obtained from the phytopathogenic fungus Verticillium dahliae dramatically induced transcription of both FPS and CAD, paralleling the accumulation of the sesquiterpene aldehydes in these cells. For G. australe, a wild species from Australia, the V. dahliae elicitor preparation also caused an induction of FPS but only a low rate of induction of CAD, apparently because of a constitutive expression of the sesquiterpene cyclase gene in suspension-cultured cells. Two transcripts and proteins of FPS were detected in the elicited G. australe cells; the smaller FPS seemed to be de novo synthesized after elicitation. Furthermore, G. australe-cultured cells accumulated the cadinene, instead of sesquiterpene aldehydes, indicating that the biosynthetic pathway leading to sesquiterpene aldehydes was absent or blocked after FPP cyclization.
Stunting Induced by Cucumber Mosaic Cucumovirus-Infected Nicotiana glutinosa Is Determined by a Single Amino Acid Residue in the Coat Protein
Authors: Szilassy Dénes , Salánki Katalin , Balázs Ervin
Published: May 2007

R-CMV, a subgroup II strain of cucumber mosaic cucumovirus (CMV) induces a very strong stunting response in Nicotiana glutinosa plants, while Trk7-CMV causes green mosaic in this host. The genetic determinant of this phenotype was mapped to a 534-nucleotide region at the 3′ end of RNA3 with biologically active, full-length cDNA clones of R-CMV and Trk7-CMV and RNA3 chimeras of the two strains. Within this region, R-CMV differs from Trk7-CMV by a single amino acid at position 193 in the coat protein. Changing the codon for Lys at this position to Asn or Ser, by site-directed mutagenesis, also changed the phenotype of the viruses from green mosaic to induction of stunting. Profound differences in both the spread and the accumulation of the viruses causing stunting and green mosaic were observed, although these did not correlate with the host specificity of stunting. Since expression of R-CMV coat protein with the PVX vector did not cause stunting, the data suggest that the presence of other CMV components is necessary for the induction of this symptom.
Virus Particles of Cucumber Green Mottle Mosaic Tobamovirus Move Systemically in the Phloem of Infected Cucumber Plants
Authors: Simón-Buela Laureano , García-Arenal Fernando
Published: May 2007

Systemic movement through the phloem of infected host plants is a key process in the life cycle of plant viruses, knowledge of which is scant. A main point to be elucidated is the structural form in which virus infection moves within the phloem. Indirect evidence suggests that virions might be the viral structure that moves in the phloem, but data from direct analysis in phloem sap have not been reported. We have done such analysis in the system cucumber (from which phloem exudate can be collected) / cucumber green mottle mosaic tobamovirus (CGMMV). CGMMV has structurally well-characterized particles. Both CGMMV coat protein and RNA were found in phloem exudate from infected cucumbers. Analysis of the accessibility of CGMMV RNA in phloem exudate to RNase A indicates that it is protected within a ribonucleoprotein structure. The accessibility to RNase A of the RNA in these structures was as in virus particles. Centrifugation analyses showed that the ribonucleoprotein structures in the phloem exudate have the same mass and isopycnic density as virions. Virus particles indistinguishable from purified virions were detected by electron microscopy in phloem exudate. No evidence of free RNA or other CGMMV-related structure was found in phloem exudate of infected plants. These results indicate that CGMMV movement in the phloem occurs mainly, if not exclusively, in the form of virus particles.
Iron Regulation and Pathogenicity in Erwinia chrysanthemi 3937: Role of the Fur Repressor Protein
Authors: Franza Thierry , Sauvage Christele , Expert Dominique
Published: May 2007

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.
Induction of the Maize GapC4 Promoter in Transgenic Potato under Anaerobiosis and in Erwinia carotovora-Inoculated Tuber Tissue
Authors: Bülow Lorenz , Köhler Uwe , Cerff Rüdiger , Hehl Reinhard , Düring Klaus
Published: May 2007

The induction pattern of the GapC4 promoter from maize in transgenic potato has been analyzed by fusion to the β-glucuronidase (gus) gene. Under anaerobic conditions this promoter confers high level expression not only in leaves, stems, and roots but also in tubers. After inoculation of potato tuber disks with Erwinia carotovora subsp. atroseptica, β-glucuronidase (GUS) activity could be detected in macerated tissue as well as in surrounding intact tissue. In mock controls no induction was detected, ruling out any induction due to an overall limitation in oxygen in the experimental system. In addition, it could be proven that no diffusion of GUS protein from macerated into intact tissue occurred. The promoter was shown to be aerobically induced even in the absence of live bacteria by incubation with purified Erwinia spp. pectolytic enzymes alone. Therefore, promoter induction seems to be mediated by a mobile factor instead of by limitation in oxygen. These results demonstrate that the maize GapC4 promoter is suitable for directing foreign genes encoding antibacterial proteins in transgenic potato.
Tight Physical Linkage of the Nematode Resistance GeneGpa2and the Virus Resistance GeneRxon a Single Segment Introgressed from the Wild SpeciesSolanum tuberosumsubsp.andigenaCPC 1673 into Cultivated Potato
Authors: van der Voort Jeroen Rouppe , Kanyuka Konstantin , van der Vossen Edwin , Bendahmane Abdelhafid , Mooijman Paul , Klein-Lankhorst René , Stiekema Willem , Baulcombe David , Bakker Jaap
Published: May 2007

Comparative mapping studies within several plant genera have shown that genes for resistance to taxonomically diverse pathogens are often co-localized on the maps of these genotypes. Here we describe the genetic analysis of such a gene cluster on chromosome 12 of potato. This cluster harbors the resistance loci Gpa2 and Rx, which confer resistance to the potato cyst nematode Globodera pallida and potato virus X (PVX), respectively. By screening two mapping populations with 10 CAPS (cleaved amplified polymorphic sequence) markers derived from AFLP (amplified fragment length polymorphism) markers and the ends of overlapping bacterial artificial chromosome (BAC) clones, it is demonstrated that Gpa2 and Rx are tightly linked. From a recombination fraction of 1/3386, found after screening two different mapping populations, a genetic distance of 0.02 cM between Gpa2 and Rx was calculated. The physical length of this genetic interval does not exceed 300 kb of DNA as determined by the total size of overlapping BAC clones in the Rx/Gpa2 contig. Evidence is provided that both genes have been introgressed from a single wild species clone, Solanum tuberosum subsp. andigena CPC 1673. This suggests that these genes underlying the disease resistance specificities to potato virus X and potato cyst nematodes have a common evolutionary origin.
Analysis of Cell-to-Cell and Long-Distance Movement of a Bean Dwarf Mosaic Geminivirus-Green Fluorescent Protein Reporter in Host and Nonhost Species: Identification of Sites of Resistance
Authors: Wang H. L. , Sudarshana M. R. , Gilbertson R. L. , Lucas W. J.
Published: May 2007

A bean dwarf mosaic geminivirus-green fluorescent protein (BDMV-GFP) reporter system was employed to analyze the viral infection process in host and nonhost species. Five classes of BDMV/host interaction were identified: (i) adapted hosts (susceptible Phaseolus vulgaris cultivars) permissive for systemic infection; (ii) adapted hosts (resistant P. vulgaris cv. Othello) displaying the development of a hypersensitive response (HR) associated with resistance to systemic infection; (iii) adapted (resistant P. vulgaris cv. Black Turtle Soup T-39) and nonadapted (Vigna unguiculata) hosts in which cell-to-cell, but not long-distance, movement was permitted; (iv) nonadapted hosts (Glycine max) in which systemic infection was coat protein-dependent; and (v) nonhosts (Cucurbita maxima, Gossypium barbadense, and Zea mays) in which the virus was confined to inoculated cells. Confocal laser scanning microscopy, fluorescence microscopy, and histochemical analyses were used to identify the cellular distribution of BDMV-GFP and the host response to viral infection. With this approach, the HR in P. vulgaris cv. Othello was visualized within cells of the epidermis, cortex, and phloem of inoculated hypocotyls. Infection studies performed with four begomoviruses and infectious BDMV/tomato mottle geminivirus pseudorecombinants revealed that the HR determinant(s) mapped to the BDMV DNA-B component.
Altered Patterns of Gene Expression in Arabidopsis Elicited by Cauliflower Mosaic Virus (CaMV) Infection and by a CaMV Gene VI Transgene
Authors: Geri Chiara , Cecchini Edi , Giannakou Maria E. , Covey Simon N. , Milner Joel J.
Published: May 2007

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.
Self-regulation of Pir, a Regulatory Protein Responsible for Hyperinduction of Pectate Lyase in Erwinia chrysanthemi EC16
Authors: Nomura Kinya , Nasser William , Tsuyumu Shinji
Published: May 2007

Previously, we have cloned and characterized the pir (plant inducible regulator) gene, which is responsible for hyperinduction of the synthesis of an isozyme of pectate lyase (PLe) in Erwinia chrysanthemi EC16 in the presence of potato extract and sodium polypectate (NaPP). The Pir protein purified from Escherichia coli overexpressing pir is able to bind to the promoter region of pir as a dimer. Self-regulation of pir by its own translational product (Pir) was suggested from the findings that Pir binds at the promoter region of pir and that the hyperinduction of the pir-lux construct in response to plant extract was observed only in pir+ but not in pir mutant EC16. Thus, hyperinduction of PLe was thought to be mainly due to overproduction of Pir. On the other hand, KdgR and PecS, which have been reported to be the major regulatory proteins for the synthesis of pectic enzymes, did not bind to the promoter region of pir. Thus, the regulation of Pir synthesis seems to be independent of KdgR and PecS. Also, its expression was insensitive to catabolite repression as predicted from failure of cyclic AMP (cAMP)-CRP (cAMP recognizing protein) to bind at the pir promoter region.
Disruption of the ech42 (Endochitinase-Encoding) Gene Affects Biocontrol Activity in Trichoderma harzianum P1
Authors: Woo S. L. , Donzelli B. , Scala F. , Mach R. , Harman G. E. , Kubicek C. P. , Del Sorbo G. , Lorito M.
Published: May 2007

The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.
cDNA Cloning of a Sorghum Pathogenesis-Related Protein (PR-10) and Differential Expression of Defense-Related Genes Following Inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum
Authors: Lo Sze-Chung Clive , Hipskind John D. , Nicholson Ralph L.
Published: May 2007

A sorghum cDNA clone was isolated by differential screening of a cDNA library prepared from mesocotyls (cultivar DK18) inoculated with fungal pathogens. The deduced translation product shows sequence similarity to a family of intracellular pathogenesis-related proteins (PR-10) with a potential ribonuclease function. We studied the accumulation of PR-10 and chalcone synthase (CHS) transcripts in mesocotyls following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. CHS is involved in phytoalexin synthesis in sorghum. Coordinate expression of PR-10 and CHS genes was localized in the area of inoculation along with the accumulation of phytoalexins. C. heterostrophus is a nonpathogen of sorghum and cytological studies indicated that cultivar DK18 is resistant to C. sublineolum, a sorghum pathogen. We demonstrated that the two fungi triggered different time courses of plant defense reactions. Inoculation with C. heterostrophus resulted in rapid accumulation of PR-10 and CHS transcripts after appressoria had become mature. Accumulation of these transcripts was delayed in plants inoculated with C. sublineolum until penetration of host tissue had been completed and infection vesicles had formed. Results suggest that different recognition events are involved in the expression of resistance to the two fungi used or that C. sublineolum suppresses the nonspecific induction of defense responses.
Expression of a Pathogen-Induced Gene Can Be Mimicked by Auxin Insensitivity
Authors: Mayda Esther , Marqués Carmen , Conejero Vicente , Vera Pablo
Published: May 2007

Following perception of a pathogenic attack, plants are able to develop a strong response with the corresponding activation of a plethora of defense-related genes. In this study we have characterized the mode of expression of the CEVI-1 gene from tomato plants, which encodes an anionic peroxidase. CEVI-1 expression is induced during the course of compatible viral and subviral infections, like many other defense-related genes, but is induced neither in incompatible interactions nor by signal molecules such as salicylic acid, ethylene, or methyl jasmonate. Additionally, CEVI-1 is induced in detached leaf tissues following a pathway distinct from that related to the classical wound response. We also describe the characterization of the structural CEVI-1 gene and compare the mode of expression in different transgenic plant species harboring a CEVI-1::GUS construct. Furthermore, we have isolated mutants in Arabidopsis, called dth mutants, that are deregulated in the control of expression of this gene. From the initial analysis of some of these mutants it seems that activation of CEVI-1 gene expression correlates with a defect in the perception of auxins by the plant. All these results may suggest that, during systemic infections with viruses, auxin homeostasis is one of the components participating in the regulation of the overall defense response.
Expression Profiles of 22 Novel Molecular Markers for Organogenetic Pathways Acting in Alfalfa Nodule Development
Authors: Jiménez-Zurdo José I. , Frugier Florian , Crespi Martín D. , Kondorosi Adam
Published: May 2007

During symbiotic nodule development, a variety of molecular signals of rhizobia and plant origin are likely to be involved in the control of the expression of specific genes in the legume Medicago sativa (alfalfa). Twenty-two new, nodule-associated expressed sequence tags (ESTs, MsNod clones) as well as 16 clones for previously reported alfalfa nodulins were identified by cold-plaque screening. Protein homologs were found for 10 of the 22 MsNod-encoded polypeptides, revealing putative novel functions associated with this symbiosis. Expression of these MsNod genes was investigated in spontaneous nodules (generated in the absence of bacteria), in nodules induced by a Sinorhizobium meliloti wild-type strain and Eps-and Bac-mutant derivatives, as well as in roots inoculated with a Nod-mutant strain. This analysis enabled us to correlate plant gene expression with the different stages of nodule ontogeny and invasion. The effect of phytohormones on MsNod gene expression was analyzed in cytokinin- and auxin-treated alfalfa roots. Cytokinin induced the accumulation of seven MsNod transcripts, four of them were also regulated by the synthetic auxin 2,4-D (2,4-dichlorophenoxyacetic acid). Comparison of MsNod expression profiles in wild-type and transgenic M. truncatula roots overexpressing the early nodulin Enod40 suggested that one clone, the M. sativa L3 ribosomal protein homolog (MsNod377), is a putative component of an Enod40-dependent pathway acting during nodule development. These novel molecular markers may help in the investigation of gene networks and regulatory circuits controlling nodule organogenesis.
Differential Expression of Two Soybean Apyrases, One of Which Is an Early Nodulin
Authors: Day R. Bradley , McAlvin Crystal B. , Loh John T. , Denny Roxanne L. , Wood Todd C. , Young Nevin D. , Stacey Gary
Published: May 2007

Two cDNA clones were isolated from soybean (Glycine soja) by polymerase chain reaction with primers designed to conserved motifs found in apyrases (nucleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e., GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers. Neither gene was expressed in leaves or cotyledons. Inoculation of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybean, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The level of GS50 mRNA expression was not affected by bacterial inoculation. Western blot (immunoblot) analysis of GS50 expression mirrored the results obtained by mRNA analysis. However, in contrast to the mRNA results, GS52 protein was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 antibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum. Fractionation of cellular membranes in sucrose density gradients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymorphism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean. These data suggest that GS50 is likely an endo-apyrase involved in Golgi function, while GS52 is localized on the root surface and appears to play an important role in nodulation.
Susceptibility and Symptom Development in Arabidopsis thaliana to Tobacco mosaic virus Is Influenced by Virus Cell-to-Cell Movement
Authors: Dardick Christopher D. , Golem Sheetal , Culver James N.
Published: May 2007

To identify host factors that regulate susceptibility to Tobacco mosaic virus(TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.
Saprophytic Intracellular Rhizobia in Alfalfa Nodules
Authors: Timmers Antonius C. J. , Soupène Eric , Auriac Marie-Christine , de Billy Françoise , Vasse Jacques , Boistard Pierre , Truchet Georges
Published: May 2007

In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bac-teroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule.
A guaB Mutant Strain of Rhizobium tropici CIAT899 Pleiotropically Defective in Thermal Tolerance and Symbiosis
Authors: Riccillo Pablo M. , Collavino Monica M. , Grasso Daniel H. , England Reg , de Bruijn Frans J. , Aguilar O. Mario
Published: May 2007

Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using Tn5-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.