bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Defense Reactions in Leaf Tissues of Barley in Response to Infection by Rhynchosporium secalis and to Treatment with a Fungal Avirulence Gene Product
Authors: Steiner-Lange Sabine , Fischer Achim , Boettcher Annette , Rouhara Ila , Liedgens Hiltrud , Schmelzer Elmon , Knogge Wolfgang
Published: May 2007

Expression of defense-associated genes was analyzed in leaf tissues of near-isogenic resistant and susceptible barley cultivars upon infection by Rhynchosporium secalis. The genes encoding pathogenesis-related (PR) proteins PR-1, PR-5, and PR-9 are specifically expressed in the mesophyll of resistant plants, whereas a germin-like protein (OxOLP) is synthesized in the epidermis irrespective of the resistance genotype. Restriction-mediated differential display was employed to identify additional epidermis-specific genes. This resulted in the detection of another PR gene, PR-10, along with a lipoxygenase gene, LoxA, and a gene of unknown function, pI2-4, which are specifically induced in the epidermis of resistant plants. The gene encoding a putative protease inhibitor, SD10, is preferentially but not exclusively expressed in the epidermis. The fungal avirulence gene product NIP1 triggers the induction of the four PR genes only. At least two additional elicitors, therefore, must be postulated, one for the unspecific induction of OxOLP and one for the resistance-specific induction of LoxA, pI2-4, and SD10. PR-10 expression can be assumed to be the consequence of NIP1 perception by epidermis cells. In contrast, gene expression in the mesophyll is likely to be triggered by an as yet unknown signal that appears to originate in the epidermis and that is strongly amplified in the mesophyll.
Isolation of a Polygalacturonase-Inhibiting Protein (PGIP) from Wheat
Authors: Kemp Gabré , Bergmann Carl W. , Clay Ron , Van der Westhuizen Amie J. , Pretorius Zacharias A.
Published: May 2007

Evidence for the presence of a polygalacturonase-inhibiting protein (PGIP) from a monocotyledonous cereal is presented. A 40.3-kDa PGIP that was closely associated with the cell wall was acetone-extracted and purified from wheat (Triticum aestivum L.) leaves and stems. Wheat PGIP exhibited a highly selective inhibitory activity against endopolygalacturonase (EPG) from various fungi. Of nine EPG tested, wheat PGIP only inhibited EPG from Cochliobolus sativus, a pathogen of the tribe Poaceae. A short N-terminal amino acid sequence of wheat PGIP shows no similarity to any other characterized PGIP.
Nitric oxide Does Not Trigger Early Programmed Cell Death Events but May Contribute to Cell-to-Cell Signaling Governing Progression of the Arabidopsis Hypersensitive Response
Authors: Zhang Chu , Czymmek Kirk J. , Shapiro Allan D.
Published: May 2007

Nitric oxide (NO) has been suggested to play a role in the hypersensitive response (HR). Single- and double-label fluorescence microscopy experiments were conducted using Arabidopsis leaves infected with Pseudomonas syringae pv. tomato DC3000 carrying either avrB or avrRpt2. Kinetics of NO production were followed by measurement of green 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) triazole fluorescence in leaves coinfiltrated with DAF-FM diacetate. Kinetics of hypersensitive cell death were followed by measurement of cytoplasmic red fluorescence following internalization of coinfiltrated propidium iodide through compromised plasma membranes. Neither NO accumulation nor cell death was seen until approximately 3 h postinoculation of Columbia leaves with DC3000·avrB or approximately 5.5 h post-inoculation with DC3000·avrRpt2. Subsequent NO accumulation kinetics closely paralleled HR progression in both Columbia and ndr1-1 mutant plants. These data established that NO accumulation does not happen sufficiently early for NO to be a signaling component controlling HR triggering. NO accumulation did contribute to the HR, as proven by an approximately 1-h delay in cell death kinetics caused by an NO scavenger or an NO synthase inhibitor. NO was first seen as punctate foci at the cell surface. Subsequent NO accumulation patterns were consistent with NO being an intercellular signal that functions in cell-to-cell spread of the HR.
Induction of the Hahsp17.7G4 Promoter by Root-Knot Nematodes: Involvement of Heat-Shock Elements in Promoter Activity in Giant Cells
Authors: Escobar Carolina , Barcala Marta , Portillo Mary , Almoguera Concepción , Jordano Juan , Fenoll Carmen
Published: May 2007

Root-knot nematodes feed from specialized giant cells induced in the plants that they parasitize. We found that the promoter of the Hahsp17.7G4 gene, which encodes a small heat-shock protein involved in embryogenesis and stress responses, directed GUS expression in tobacco galls induced by the root-knot nematode Meloidogyne incognita. In roots containing a GUS reporter fusion to the Hahsp17.7G4 promoter, 10% of the galls stained for GUS expression 1 to 3 days after infection and the fraction stained increased to 60 to 80% 17 to 20 days after infection. A DNA fragment from −83 to +163, which contains heat-shock element (HSE) core sequences, is sufficient to support a promoter activity largely restricted to giant cells within the galls. Two-point mutations in HSE cores, previously reported to abolish the heat-shock response and to strongly reduce the embryogenesis response of the same promoter, did not reduce expression in giant cells. This suggests a distinct regulation of the promoter by nematodes. However, additional point mutations located at positions crucial for binding of heat-shock transcription factors (HSFs) caused a severe decrease in the nematode response. These results demonstrate that HSEs are involved in the promoter activation in giant cells and suggest that HSFs may mediate this response.
Nitric Oxide-Mediated Transcriptional Changes in Arabidopsis thaliana
Authors: Polverari Annalisa , Molesini Barbara , Pezzotti Mario , Buonaurio Roberto , Marte Mario , Delledonne Massimo
Published: May 2007

Nitric oxide (NO) is an essential regulatory molecule in several developmental processes and in the stress response in both animal and plant systems. Furthermore, key features of plant resistance to pathogens have been shown to depend on NO production, e.g., defense gene expression and the activation of a hypersensitive reaction (HR) in synergy with reactive oxygen species (ROS). Due to the many possible mechanisms of NO action, a clear picture of its involvement in plant resistance to pathogens is far from being achieved. Transcriptional changes related to NO action are likely to play a significant role in resistance and cell death. We investigated the changes in the expression profiles of Arabi-dopsis thaliana following infiltration with the NO donor sodium nitroprusside, by cDNA-amplification fragment length polymorphism (AFLP) transcript profiling. Altered expression patterns were detected for 120 of the approximately 2,500 cDNAs examined. Sequence analysis revealed homologies with genes involved in signal transduction, disease resistance and stress response, photosynthesis, cellular transport, and basic metabolism or with sequences coding for unknown proteins. Comparison of the expression profiles with data from public microarray sources revealed that many of the identified genes modulated by NO were previously reported to be modulated in disease-related experiments.
Oxidative Burst Elicited by Bacillus mycoides Isolate Bac J, a Biological Control Agent, Occurs Independently of Hypersensitive Cell Death in Sugar Beet
Authors: Bargabus Rebecca L. , Zidack Nina K. , Sherwood John E. , Jacobsen Barry J.
Published: May 2007

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and β-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 × 108 cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 × 106 cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and β-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by aviru-lent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.
Acquired Resistance Functions in mlo Barley, Which Is Hypersusceptible to Magnaporthe grisea
Authors: Jarosch Birgit , Jansen Marcus , Schaffrath Ulrich
Published: May 2007

Barley plants carrying a mutation in the Mlo (barley [Hordeum vulgare L.] cultivar Ingrid) locus conferring a durable resistance against powdery mildew are hypersusceptible to the rice blast fungus Magnaporthe grisea. It has been speculated that a functional Mlo gene is required for the expression of basic pathogen resistance and that the loss of Mlo function mediating powdery mildew resistance is an exception for this particular disease. Here, we report that the onset of acquired resistance (AR) after chemical as well as biological treatments is sufficient to overcome the hypersusceptible phenotype of backcross line BCIngridmlo5 (mlo) barley plants against M. grisea. Moreover, even barley plants bearing a functional Mlo gene and thus showing a moderate infection phenotype against rice blast exhibit a further enhanced resistance after induction of AR. Cytological investigations reveal that acquired resistance in mlo genotypes is manifested by the restoration of the ability to form an effective papilla at sites of attempted penetration, similarly to wild-type Mlo plants. In addition, the rate of effective papillae formation in Mlo plants was further enhanced after the onset of AR. These results demonstrate that treatments leading to the AR state in barley function independently of the Mlo/mlo phenotype and suggest that the Mlo protein is not a component of the AR signaling network. Moreover, it seems that only concomitant action of Mlo together with AR permits high level resistance in barley against blast. Higher steady state levels of PR1 and barley chemically induced mRNA correlate with higher disease severity rather than with the degree of resistance observed in this particular interaction.
Sulfated Fucan Oligosaccharides Elicit Defense Responses in Tobacco and Local and Systemic Resistance Against Tobacco Mosaic Virus
Authors: Klarzynski Olivier , Descamps Valérie , Plesse Bertrand , Yvin Jean-Claude , Kloareg Bernard , Fritig Bernard
Published: May 2007

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by α-1,4 and α-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 μg ml−1 rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.
Partial Resistance of Tomato to Phytophthora infestans Is Not Dependent upon Ethylene, Jasmonic Acid, or Salicylic Acid Signaling Pathways
Authors: Smart C. D. , Myers K. L. , Restrepo S. , Martin G. B. , Fry W. E.
Published: May 2007

We compared tomato defense responses to Phytophthora infestans in highly compatible and partially compatible interactions. The highly compatible phenotype was achieved with a tomato-specialized isolate of P. infestans, whereas the partially compatible phenotype was achieved with a nonspecialized isolate. As expected, there was induction of the hypersensitive response (HR) earlier during the partially compatible interaction. However, contrary to our expectation, pathogenesis-related (PR) gene expression was not stimulated sooner in the partially compatible interaction. While the level of PR gene expression was quite similar in the two interactions, the LeDES gene (which encodes an enzyme necessary for the production of divinyl ethers) was expressed at a much higher level in the partially compatible interaction at 48 h after inoculation. Host reaction to the different pathogen genotypes was not altered (compared with wild type) in mutant tomatoes that were ethylene-insensitive (Never-ripe) or those with reduced ability to accumulate jasmonic acid (def-1). Similarly, host reaction was not altered in NahG transgenic tomatoes unable to accumulate salicylic acid. These combined data indicate that partial resistance in tomato to P. infestans is independent of ethylene, jasmonic acid, and salicylic acid signaling pathways.
Genetic Analysis of a pH-Regulated Operon from Rhizobium tropici CIAT899 Involved in Acid Tolerance and Nodulation Competitiveness
Authors: Vinuesa Pablo , Neumann-Silkow Frauke , Pacios-Bras Cristina , Spaink Herman P. , Martínez-Romero Esperanza , Werner Dietrich
Published: May 2007

Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5′ end of atvA, encoding a product with high sequence identity to the agrobacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix+ but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several α, β, and γ Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.
Erwinia carotovora subsp. carotovora and Erwinia-Derived Elicitors HrpN and PehA Trigger Distinct but Interacting Defense Responses and Cell Death in Arabidopsis
Authors: Kariola Tarja , Palomäki Tiina A. , Brader Günter , Palva E. Tapio
Published: May 2007

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.
The 50-kDa Protein of Apple chlorotic leaf spot virus Interferes with Intracellular and Intercellular Targeting and Tubule-Inducing Activity of the 39-kDa Protein of Grapevine berry inner necrosis virus
Authors: Isogai M. , Saitou Y. , Takahashi N. , Itabashi T. , Terada M. , Satoh H. , Yoshikawa N.
Published: May 2007

To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.
Comparative Study of Regulatory Mechanisms for Pectinase Production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi
Authors: Matsumoto Hiroyuki , Jitareerat Pongphen , Baba Yasuhiro , Tsuyumu Shinji
Published: May 2007

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalac-turonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.
The HrpX/HrpY Two-Component System Activates hrpS Expression, the First Step in the Regulatory Cascade Controlling the Hrp Regulon in Pantoea stewartii subsp. stewartii
Authors: Merighi Massimo , Majerczak Doris R. , Stover Elizabeth H. , Coplin David L.
Published: May 2007

A regulatory cascade activating hrp/hrc type III secretion and effector genes was delineated in Pantoea stewartii subsp. stewartii, a bacterial pathogen of corn. Four hrp regulatory genes were characterized: hrpX and hrpY encode the sensor kinase and response regulator, respectively, of a two-component signal transduction system; hrpS encodes an NtrC-like transcriptional enhancer; and hrpL encodes an alternative sigma factor. Epistasis analysis, expression studies using gene fusions, and genetic reconstruction of each step in Escherichia coli were used to delineate the following pathway: HrpY activates hrpS and also positively auto-regulates the hrpXY operon. In turn, HrpS is required for full activation of the σ54-dependent hrpL promoter. Finally, HrpL controls expression of all known hrp and wts genes. In vitro, hrpS and all downstream hrp genes were regulated by pH and salt concentration. Mutants with in-frame deletions in hrpX were still partially virulent on corn but were unable to sense the chemical or metabolic signals that induce hrp genes in vitro. Site-directed mutagenesis of HrpY indicated that aspartate 57 is the probable phosphorylation site and that it is needed for activity. These findings suggest that both HrpX and an alternate mechanism are involved in the activation of HrpY in planta.
Transcriptional Changes in Response to Arbuscular Mycorrhiza Development in the Model Plant Medicago truncatula
Authors: Wulf Anne , Manthey Katja , Doll Jasmin , Perlick Andreas M. , Linke Burkhard , Bekel Thomas , Meyer Folker , Franken Philipp , Küster Helge , Krajinski Franziska
Published: May 2007

Significant changes in root morphology and physiology during arbuscular mycorrhiza (AM) development are likely to be controlled by specific gene expression pattern in the host plant. Until now, little was known about transcriptional changes which occur AM-exclusively; that is, they do not occur during other root-microbe associations, nor are they induced by improved phosphate nutrition. In order to identify such AM-exclusive gene inductions of Medicago truncatula, we used a pool of different RNA samples as subtractor population in a suppressive subtractive hybridization (SSH) experiment. This approach resulted in the identification of a number of new AM-regulated genes. None of these genes were expressed in nonmycorrhiza roots or leaves. Electronic data obtained by comparison of the cDNA sequences to expressed sequence tag (EST) sequences from a wide range of cDNA libraries in the M. truncatula EST database (Gene Index, MtGI) support the mycorrhiza specificity of the corresponding genes, because sequences in the MtGI that were found to match the identified SSH-cDNA sequences originated exclusively from AM cDNA libraries. The promoter of one of those genes, MtGst1, showing similarities to plant glutathione-S-transferase (GST) encoding genes, was cloned and used in reporter gene studies. In contrast to studies with the potato GST gene PRP, MtGst 1 promoter activity was detected in all zones of the root cortex colonized by Glomus intraradices, but nowhere else.
A Chorismate Mutase from the Soybean Cyst Nematode Heterodera glycines Shows Polymorphisms that Correlate with Virulence
Authors: Bekal Sadia , Niblack Terry L. , Lambert Kris N.
Published: May 2007

Parasitism genes from phytoparasitic nematodes are thought to be essential for nematode invasion of the host plant, to help the nematode establish feeding sites, and to aid nematodes in the suppression of host plant defenses. One gene that may play several roles in nematode parasitism is chorismate mutase (CM). This secreted enzyme is produced in the nematode's esophageal glands and appears to function within the plant cell to manipulate the plant's shikimate pathway, which controls plant cell growth, development, structure, and pathogen defense. Using degenerate polymerase chain reaction primers, we amplified and cloned a chorismate mutase (Hg-cm-1) from Heterodera glycines, the soybean cyst nematode (SCN), and showed it had CM activity. RNA in situ hybridization of Hg-cm-1 cDNA to SCN sections confirms that it is specifically expressed in the nematodes' esophageal glands. DNA gel blots of genomic DNA isolated from SCN inbred lines that have differing virulence on SCN resistant soybean show Hg-cm-1 is a member of a polymorphic gene family. Some Hg-cm family members predominate in SCN inbred lines that are virulent on certain SCN resistant soybean cultivars. The same polymorphisms and correlation with virulence are seen in the Hg-cm-1 expressed in the SCN second-stage juveniles. Based on the enzymatic activity of Hg-cm-1 and the observation that different forms of the mutase are expressed in virulent nematodes, we hypothesize that the Hg-cm-1 is a virulence gene, some forms of which allow SCN to parasitize certain resistant soybean plants.
RNA Silencing in the Phytopathogenic Fungus Magnaporthe oryzae
Authors: Kadotani Naoki , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki
Published: May 2007

Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.
Distinct Patterns of Symbiosis-Related Gene Expression in Actinorhizal Nodules from Different Plant Families
Authors: Pawlowski Katharina , Swensen Susan , Guan Changhui , Hadri Az-Eddine , Berry Alison M. , Bisseling Ton
Published: May 2007

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).
Apoplastic pH Signaling in Barley Leaves Attacked by the Powdery Mildew Fungus Blumeria graminis f. sp. hordei
Authors: Felle Hubert H. , Herrmann Almut , Hanstein Stefan , Hückelhoven Ralph , Kogel Karl-Heinz
Published: May 2007

To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H+- and Ca2+-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H+- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.
NPR1-Independent Activation of Immediate Early Salicylic Acid-Responsive Genes in Arabidopsis
Authors: Uquillas Carolina , Letelier Ingrid , Blanco Francisca , Jordana Xavier , Holuigue Loreto
Published: May 2007

Salicylic acid (SA) is a key signal for the activation of defense genes in response to stress. The activation of late defense genes by SA, such as PR-1, involves the participation of the NPR1 protein. This protein acts as coactivator of the TGA factors that recognize as-1-like elements in the PR-1 promoter. Considering that functional as-1-like elements are also found in the promoter of SA- and auxin-responsive immediate early genes, we tested the hypothesis that NPR1 is also required for activation of these genes. The expression of the immediate early genes glutathione S-transferase (GST6) and glucosyltransferase (EIGT) was studied in npr1 mutant and wild-type Arabidopsis plants. In the npr1 mutant background, SA and 2,4-dichlorophen-oxyacetic acid were unable to promote transcription of PR-1 but effectively stimulated the expression of GST6 and EIGT. Furthermore, increased binding of proteins to the GST6 as-1-like promoter element was detected in nuclear extracts from npr1 and wild-type plants after treatment with SA. In summary, these results indicate that activation of immediate early genes by SA proceeds through an NPR1-independent pathway. Therefore, we propose that activation by SA of immediate early and late genes occur by different mechanisms.
Patterns of Gene Expression Upon Infection of Soybean Plants byPhytophthora sojae
Authors: Moy Pat , Qutob Dinah , Chapman B. Patrick , Atkinson Ian , Gijzen Mark
Published: May 2007

To investigate patterns of gene expression in soybean (Glycine max) and Phytophthora sojae during an infection time course, we constructed a 4,896-gene microarray of host and pathogen cDNA transcripts. Analysis of rRNA from soybean and P. sojae was used to estimate the ratio of host and pathogen RNA present in mixed samples. Large changes in this ratio occurred between 12 and 24 h after infection, reflecting the rapid growth and proliferation of the pathogen within host tissues. From the microarray analysis, soybean genes that were identified as strongly upregulated during infection included those encoding enzymes of phytoalexin biosynthesis and defense and pathogenesis-related proteins. Expression of these genes generally peaked at 24 h after infection. Selected lipoxygenases and peroxidases were among the most strongly downregulated soybean genes during the course of infection. The number of pathogen genes expressed during infection reached a maximum at 24 h. The results show that it is possible to use a single microarray to simultaneously probe gene expression in two interacting organisms. The patterns of gene expression we observed in soybean and P. sojae support the hypothesis that the pathogen transits from biotrophy to necrotrophy between 12 and 24 h after infection.
Transcriptome Profiling in Root Nodules and Arbuscular Mycorrhiza Identifies a Collection of Novel Genes Induced During Medicago truncatula Root Endosymbioses
Authors: Manthey Katja , Krajinski Franziska , Hohnjec Natalija , Firnhaber Christian , Pühler Alfred , Perlick Andreas M. , Küster Helge
Published: May 2007

Transcriptome profiling based on cDNA array hybridizations and in silico screening was used to identify Medicago truncatula genes induced in both root nodules and arbuscular mycorrhiza (AM). By array hybridizations, we detected several hundred genes that were upregulated in the root nodule and the AM symbiosis, respectively, with a total of 75 genes being induced during both interactions. The second approach based on in silico data mining yielded several hundred additional candidate genes with a predicted symbiosis-enhanced expression. A subset of the genes identified by either expression profiling tool was subjected to quantitative real-time reverse-transcription polymerase chain reaction for a verification of their symbiosis-induced expression. That way, induction in root nodules and AM was confirmed for 26 genes, most of them being reported as symbiosis-induced for the first time. In addition to delivering a number of novel symbiosis-induced genes, our approach identified several genes that were induced in only one of the two root endo-symbioses. The spatial expression patterns of two symbiosis-induced genes encoding an annexin and a β-tubulin were characterized in transgenic roots using promoter-reporter gene fusions.
Enhancer Trapping IdentifiesTRI, anArabidopsisGene Up-Regulated by Pathogen Infection
Authors: Fridborg Ingela , Williams Alan , Yang Aidong , MacFarlane Stuart , Coutts Katherine , Angell Susan
Published: May 2007

Enhancer trap Arabidopsis thaliana plants were screened for genes up-regulated by virus infection. The plants carried T-DNA insertions comprising a minimal -60-bp Cauliflower mosaic virus 35S promoter fused to the β-glucuronidase (GUS) reporter gene. Approximately 12,000 plants were assayed for GUS activity before and after rub-inoculation with Tobacco rattle virus (TRV) tagged with the green fluorescent protein (GFP). One plant and its progeny consistently showed upregulation of GUS activity in response to TRV-GFP infection, indicating that a virus-responsive enhancer element was “tagged” by the T-DNA in this line. Other viruses, bacteria, and oomycetes, but not wounding, up-regulated GUS activity in the enhancer trap line, indicating that the response was not specific to TRV-GFP infection. A pathogen-inducible, alternatively spliced gene was identified, which we have termed TRI for TRV-induced gene. A pathogen-responsive element was localized to a 1.1-kb region upstream of the T-DNA insertion, and two different cis-acting elements, both implicated in defense responses, were found in the sequence upstream of TRI. Sequence analyses revealed that TRI is similar to ACRE169, a gene that is up-regulated in Cf-9-expressing tobacco when treated with Avr-9, the Cladosporium fulvum elicitor of the Cf-9 resistance response.
Candidate Defense Genes as Predictors of Quantitative Blast Resistance in Rice
Authors: Liu Bin , Zhang Shaohong , Zhu Xiaoyuan , Yang Qiyun , Wu Shangzhong , Mei Mantong , Mauleon Ramil , Leach Jan , Mew Tom , Leung Hei
Published: May 2007

Although quantitative trait loci (QTL) underpin many desirable agronomic traits, their incorporation into crop plants through marker-assisted selection is limited by the low predictive value of markers on phenotypic performance. Here we used candidate defense response (DR) genes to dissect quantitative resistance in rice using recombinant inbred (RI) and advanced backcross (BC) populations derived from a blast-resistant cultivar, Sanhuangzhan 2 (SHZ-2). Based on DNA profiles of DR genes, RI lines were clustered into two groups corresponding to level of resistance. Five DR genes, encoding putative oxalate oxidase, dehydrin, PR-1, chitinase, and 14-3-3 protein, accounted for 30.0, 23.0, 15.8, 6.7, and 5.5% of diseased leaf area (DLA) variation, respectively. Together, they accounted for 60.3% of the DLA variation and co-localized with resistance QTL identified by interval mapping. Average phenotypic contributions of oxalate oxidase, dehydrin, PR-1, chitinase, and 14-3-3 protein in BC lines were 26.1, 19.0, 18.0, 11.5, and 10.6%, respectively, across environments. Advanced BC lines with four to five effective DR genes showed enhanced resistance under high disease pressure in field tests. Our results demonstrate that the use of natural variation in a few candidate genes can solve a long-standing problem in rice production and has the potential to address other problems involving complex traits.
Role of Chemotaxis Toward Fusaric Acid in Colonization of Hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365
Authors: de Weert Sandra , Kuiper Irene , Lagendijk Ellen L. , Lamers Gerda E. M. , Lugtenberg Ben J. J.
Published: May 2007

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.
Diverse Members of the AvrBs3/PthA Family of Type III Effectors Are Major Virulence Determinants in Bacterial Blight Disease of Rice
Authors: Yang Bing , White Frank F.
Published: May 2007

AvrXa7 is a member of the avBs3/pthA gene family and the only known type III secretion system effector gene from Xanthomonas oryzae pv. oryzae with a major contribution to bacterial growth and lesion formation in bacterial blight disease of rice. We examined the general requirement for effectors of the AvrBs3/PthA family in bacterial blight of rice by identifying effectors from diverse strains of the pathogen. Inactivation of single effector genes in representative strains from Japan, Korea, and the Philippines resulted in severely limited growth in plants. Five strains harbored one gene of the avrBs3/pthA family, while one strain had two genes with the equivalent virulence activity of avrXa7. Sequence analysis revealed three genes with unique repeat arrangements in comparison to avrXa7. Comparison of the repetitive regions revealed a potential motif for the group that was also present in the repetitive region of avrBs3. However, the repetitive region of avrBs3 could not support virulence activity but, in combination with the C-terminal coding region of avrXa7, triggered a Xa7-dependent avirulence reaction. The results revealed diverse members of the avrBs3/pthA gene family with virulence activity in X. oryzae pv. oryzae and supported the hypothesis that bacterial blight disease of rice is highly dependent on a single class of type III effectors. The results also indicated that avrXa7 avirulence specificity is separable from virulence activity.
Cloning, Characterization, and Evolution of the NBS-LRR-Encoding Resistance Gene Analogue Family in Polyploid Cotton (Gossypium hirsutum L.)
Authors: He Limei , Du Chunguang , Covaleda Lina , Xu Zhanyou , Robinson A. Forest , Yu John Z. , Kohel Russell J. , Zhang Hong-Bin
Published: May 2007

The nucleotide-binding site-leucine-rich repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. We cloned the NBS-LRR-encoding genes from polyploid cotton by a polymerase chain reaction-based approach. A sample of 150 clones was selected from the NBS-LRR gene sequence library and was sequenced, and 61 resistance gene analogs (RGA) were identified. Sequence analysis revealed that RGA are abundant and highly diverged in the cotton genome and could be categorized into 10 distinct subfamilies based on the similarities of their nucleotide sequences. The numbers of members vary many fold among different subfamilies, and gene index analysis showed that each of the subfamilies is at a different stage of RGA family evolution. Genetic mapping of a selection of RGA indicates that the RGA reside on a limited number of the cotton chromosomes, with those from a single subfamily tending to cluster and two of the RGA loci being colocalized with the cotton bacterial blight resistance genes. The distribution of RGA between the two subgenomes A and D of cotton is uneven, with RGA being more abundant in the A subgenome than in the D subgenome. The data provide new insights into the organization and evolution of the NBS-LRR-encoding RGA family in polyploid plants.
Mutations in Wheat Exhibiting Growth-Stage-Specific Resistance to Biotrophic Fungal Pathogens
Authors: Smith Phil H. , Howie John A. , Worland Anthony J. , Stratford Rebecca , Boyd Lesley A.
Published: May 2007

Two mutants were isolated in wheat that showed enhanced resistance towards Puccinia striiformis f. sp. tritici, the fungal causal agent of yellow rust. The altered phenotype of I3-48 is due to a minimum of two mutation events, each showing a partial, additive effect, with one mutation segregating with a deletion on the long arm of chromosome 4D. In the case of I3-54, the enhanced resistance is due to a single, dominant mutation. In both mutants, the expression of the enhanced resistance is growth-stage specific. With I3-54, the full resistance phenotype is apparent from the third seedling leaf onwards, while with I3-48, a full resistance phenotype is only seen on the tenth and subsequent leaves. In addition to the enhanced resistance towards yellow rust, I3-48 also shows enhanced resistance towards brown rust, and I3-54 shows enhanced resistance to powdery mildew.
Insertion of Transposon Tn5tac1 in the Sinorhizobium meliloti Malate Dehydrogenase (mdh) Gene Results in Conditional Polar Effects on Downstream TCA Cycle Genes
Authors: Dymov Sergiy l. , Meek David J. J. , Steven Blaire , Driscoll Brian T.
Published: May 2007

To isolate Sinorhizobium meliloti mutants deficient in malate dehydrogenase (MDH) activity, random transposon Tn5tac1 insertion mutants were screened for conditional lethal phenotypes on complex medium. Tn5tac1 has an outward-oriented isopropyl-β-D-thiogalactopyranoside (IPTG)- inducible promoter (Ptac). The insertion in strain Rm30049 was mapped to the mdh gene, which was found to lie directly upstream of the genes encoding succinyl-CoA synthetase (sucCD) and 2-oxoglutarate dehydrogenase (sucAB and lpdA). Rm30049 required IPTG for wild-type growth in complex media, and had a complex growth phenotype in minimal media with different carbon sources. The mdh∷ Tn5tac1 insertion eliminated MDH activity under all growth conditions, and activities of succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase were affected by the addition of IPTG. Reverse-transcriptase polymerase chain reaction (RT-PCR) studies confirmed that expression from Ptac was induced by IPTG and leaky in its absence. Alfalfa plants inoculated with Rm30049 were chlorotic and stunted, with small white root nodules, and had shoot dry weight and percent-N content values similar to those of uninoculated plants. Cosmid clone pDS15 restored MDH activity to Rm30049, complemented both the mutant growth and symbiotic phenotypes, and was found to carry six complete (sdhB, mdh, sucCDAB) and two partial (lpdA, sdhA) tricarboxylic acid cycle genes.