bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and Comparative Bioinformatic Analysis of Expressed Genes from Wheat Spikes Infected with Fusarium graminearum
Authors: Kruger Warren M. , Pritsch Clara , Chao Shiaoman , Muehlbauer Gary J.
Published: May 2007

Fusarium head blight, caused by the fungus Fusarium graminearum, is a major disease on wheat (Triticum aestivum L.). Expressed sequence tags (ESTs) were used to identify genes expressed during the wheat-F. graminearum interaction. We generated 4,838 ESTs from a cDNA library prepared from spikes of the partially resistant cultivar Sumai 3 infected with F. graminearum. These ESTs were composed of 2,831 singlet (single-copy transcripts) and 715 contigs (multiple-copy transcripts) for a total of 3,546 non-redundant sequences. Four sets of nonredundant sequences were identified. One set contains numerous, common biotic and abiotic stress-related genes. Many of these stress-related genes were represented by multiple ESTs, indicating that they are abundantly expressed. A second set comprised 16 nonredundant sequences from F. graminearum that may be required for pathogenicity. A subset of these fungal genes encodes proteins associated with plant cell wall degradation. A third set of 326 nonredundant sequences had no DNA or amino acid sequence similarity to almost 1 million plant and over 7 million animal sequences in dbEST (as of 22 June 2001). Thus, these 326 nonredundant sequences have only been found in our F. graminearum-infected ‘Sumai 3’ cDNA library. A fourth set of 29 nonredundant sequences was found in our F. graminearum-infected wheat and another plant-pathogen interaction cDNA library. Some of these sequences encode proteins that may act in establishing various plant-fungal interactions.
A Molecular Genetic Map and Electrophoretic Karyotype of the Plant Pathogenic Fungus Cochliobolus sativus
Authors: Zhong Shaobin , Steffenson Brian J. , Martinez J. Patrick , Ciuffetti Lynda M.
Published: May 2007

A molecular genetic map was constructed and an electrophoretic karyotype was resolved for Cochliobolus sativus, the causal agent of spot blotch of barley and wheat. The genetic map consists of 27 linkage groups with 97 amplified fragment length polymorphism (AFLP) markers, 31 restriction fragment length polymorphism (RFLP) markers, two polymerase chain reaction amplified markers, the mating type locus (CsMAT), and a gene (VHv1) conditioning high virulence on barley cv. Bowman. These linkage groups covered a map distance of 849 cM. The virulence gene VHv1 cosegregated with six AFLP markers and was mapped on one of the major linkage groups. Fifteen chromosome-sized DNAs were resolved in C. sativus isolates ND93-1 and ND90Pr with contour-clamped homogeneous electric field (CHEF) electrophoresis combined with telo-mere probe analysis of comigrating chromosome-sized DNAs. The chromosome sizes ranged from 1.25 to 3.80 Mbp, and the genome size of the fungus was estimated to be approximately 33 Mbp. By hybridizing genetically mapped RFLP and AFLP markers to CHEF blots, 25 of the 27 linkage groups were assigned to specific chromosomes. The barley-specific virulence locus VHv1 was localized on a chromosome of 2.80 Mbp from isolate ND90Pr in the CHEF gel. The total map length of the fungus was estimated to be at least 1,329 cM based on the map distance covered by the linked markers and the estimated gaps. Therefore, the physical to genetic distance ratio is approximately 25 kb/cM. Construction of a high-resolution map around target loci will facilitate the cloning of the genes conferring virulence and other characters in C. sativus by a map-based cloning strategy.
Cloning and Characterization of an Esophageal-Gland-Specific Pectate Lyase from the Root-Knot NematodeMeloidogyne javanica
Authors: Doyle Elizabeth A. , Lambert Kris N.
Published: May 2007

Root-knot nematodes (Meloidogyne javanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.
Deleterious Impact of a Virulent Bacteriophage on Survival and Biocontrol Activity of Pseudomonas fluorescens Strain CHA0 in Natural Soil
Authors: Keel Christoph , Ucurum Zöhre , Michaux Patrick , Adrian Marc , Haas Dieter
Published: May 2007

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (ΦGP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. ΦGP100 was found to be a doublestranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of ΦGP100 was negatively affected at temperatures higher than 25°C. ΦGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of ΦGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a ΦGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.
The FixM Flavoprotein Modulates Inhibition by AICAR or 5′AMP of Respiratory and Nitrogen Fixation Gene Expression in Sinorhizobium meliloti
Authors: Cosseau Céline , Garnerone Anne Marie , Batut Jacques
Published: May 2007

AICAR, a purine-related metabolite, was recently shown to inhibit respiratory and nifA gene expression in Sinorhizobium meliloti. Here, we demonstrate that AICAR has essentially no or little effect in a wild-type S. meliloti strain and inhibits respiratory and nitrogen fixation gene expression only in specific mutant backgrounds. We have analyzed in detail a mutant in which addition of AICAR inhibited fixK, fixN, fixT, and nifA expression. The corresponding gene, fixM, is located just downstream of fixK1 on pSymA megaplasmid and encodes a flavoprotein oxidore-ductase. 5′AMP, a structural analogue of AICAR, mimicked AICAR effect as well as the nucleoside precursors AICAriboside and adenosine. The mode of action of AICAR and 5′AMP in vivo was investigated. We demonstrate that AICAR does not affect FixK transcriptional activity and instead regulates fixK and nifA gene expression. We hypothesize that AICAR and 5′AMP may modulate, possibly indirectly, the activity of the FixLJ two-component regulatory system. The possible physiological roles of AICAR, 5′AMP, and fixM in the context of symbiosis are discussed.
Virulence and Differential Local and Systemic Spread of Cucumber mosaic virus in Tobacco are Affected by the CMV 2b Protein
Authors: Soards Avril J. , Murphy Alex M. , Palukaitis Peter , Carr John P.
Published: May 2007

A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVΔ2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVΔ2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVΔ2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVΔ2b results in the absence of disease symptoms in tobacco.
Analysis of Molecular Markers Genetically Linked to the Leptosphaeria maculans Avirulence Gene AvrLm1 in Field Populations Indicates a Highly Conserved Event Leading to Virulence on Rlm1 Genotypes
Authors: Attard Agnès , Gout Lilian , Gourgues Mathieu , Kühn Marie-Line , Schmit Jacques , Laroche Sandrine , Ansan-Melayah Delphine , Billault Alain , Cattolico Laurence , Balesdent Marie-Hélène , Rouxel Thierry
Published: May 2007

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5′ end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race “AvrLm1” at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.
Mapping of Viral Genomic Regions Important in Cross-Protection Between Strains of a Potyvirus
Authors: Valkonen Jari P. T. , Rajamäki Minna-Liisa , Kekarainen Tuija
Published: May 2007

Cross-protection was tested between potato and tobacco strains of Potato virus A, a member of the genus Potyvirus (PVA), in tobacco plants. Cross-protection was effective only at the initiation of infection. The potato strains provided only weak cross-protection against the tobacco strain, whereas the tobacco strain provided strong cross-protection against potato strains. The tamarillo strain (TamMV) showed cross-protection phenotypes mostly resembling those of the potato strains. Chimera of the PVA strains were utilized to map viral genomic regions important for cross-protection. The coat protein (CP) encoding region and the helper component proteinase (HCpro) affected cross-protection and virus accumulation. An amino acid substitution at the CP N-terminus reduced virus accumulation and the ability to overcome cross-protection, whereas amino acid substitutions introduced to the HCpro increased virus accumulation and the ability to overcome cross-protection. Closer sequence relatedness between the protector and challenger isolate, as determined by the CP-encoding sequence, was correlated with an increased cross-protection ability. Cross-protection was not overcome by inoculation with nonencapsidated viral RNA. Thus, the differences in cross-protection abilities between PVA strains and chimera were not explained with the “re-encapsidation model” described for strains of Tobacco mosaic tobamovirus but may be associated with a virus infection-induced RNA silencing mechanism.
Rhizobium etli Mutant Modulates Carbon and Nitrogen Metabolism in Phaseolus vulgaris Nodules
Authors: Silvente Sonia , Blanco Lourdes , Camas Alberto , Ortega José-Luis , Ramírez Mario , Lara-Flores Miguel
Published: May 2007

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared with CE3. However, plants inoculated with strain CE3-vhb, which express the Vitreoscilla sp. hemoglobin and also displays increased respiratory capacity, did not have altered ureide transport in N2-fixing plants. The data suggest that inoculation with special selected mutant strains of R. etli can modulate nodule N assimilation and N transport compounds.
Pseudomonas putida Strain PCL1444, Selected for Efficient Root Colonization and Naphthalene Degradation, Effectively Utilizes Root Exudate Components
Authors: Kuiper Irene , Kravchenko Lev V. , Bloemberg Guido V. , Lugtenberg Ben J. J.
Published: May 2007

Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies. After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv. Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots. Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components. High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids. Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P. stutzeri. Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase. Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid. Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase. When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid. These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P. putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components. Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.
pH Regulates Endoglucanase Expression and Virulence of Alternaria alternata in Persimmon Fruit
Authors: Eshel Dani , Miyara Itay , Ailing Tong , Dinoor Amos , Prusky Dov
Published: May 2007

The phytopathogenic fungus Alternaria alternata produces one endo-1,4-β-glucanase, AaK1, which is an important factor in disease development in persimmon fruit. During growth of A. alternata in media containing acidified yeast extract or cell walls from persimmon fruit, the fungus secreted ammonia and raised the medium pH. A rise in media pH from 3.8 to 6.0 in the presence of cell walls induced the expression of AaK1, whereas a glucose-induced decline in pH to 2.5 repressed transcription and enzymatic production. Treatments with buffered solutions at pH 6.0 during growth of A. alternata in the presence of glucose derepressed AaK1 expression and endo-1,4-β-glucanase production and enhanced decay development on the fruit. The results suggest that conditions affecting environmental pH modulate gene expression of AaK1 and virulence of A. alternata in persimmon fruit.
Extending Chestnut Blight Hypovirus Host Range Within Diaporthales by Biolistic Delivery of Viral cDNA
Authors: Sasaki Atsuko , Onoue Mari , Kanematsu Satoko , Suzaki Kouich , Miyanishi Masaki , Suzuki Nobuhiro , Nuss Donald L. , Yoshida Kouji
Published: May 2007

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C. parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V. ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.
Oomycete Plant Pathogens Use Electric Fields to Target Roots
Authors: van West P. , Morris B. M. , Reid B. , Appiah A. A. , Osborne M. C. , Campbell T. A. , Shepherd S. J. , Gow N. A. R.
Published: May 2007

Plant roots generate electrical currents and associated electrical fields as a consequence of electrogenic ion transport at the root surface. Here we demonstrate that the attraction of swimming zoospores of oomycete plant pathogens to plant roots is mediated in part by electrotaxis in natural root-generated electric fields. The zones of accumulation of anode- or cathode-seeking zoospores adjacent to intact and wounded root surfaces correlated with their in vitro electrotactic behavior. Manipulation of the root electrical field was reflected in changes in the pattern of zoospore accumulation and imposed focal electrical fields were capable of overriding endogenous signals at the root surface. The overall pattern of zoospore accumulation around roots was not affected by the presence of amino acids at concentrations expected within the rhizosphere, although higher concentrations induced encystment and reduced root targeting. The data suggest that electrical signals can augment or override chemical ones in mediating short-range tactic responses of oomycete zoospores at root surfaces.
Engineering Bacterial Competitiveness and Persistence in the Phytosphere
Authors: Savka Michael A. , Dessaux Yves , Oger Phil , Rossbach Silvia
Published: May 2007

Several tactics exist to improve the survival of an introduced microorganism of interest in the plant environment. One, derived from studies on the Agrobacterium-plant interaction and the role of opines in this interaction, proposes to promote growth of the inoculant in the plant environment via the establishment of a bias in the rhizosphere. It is supported by the occurrence of natural biases, such as those generated by opine-like molecules, by calestegins, or by mimosine. Opine-mediated biases have allowed several investigators to favor the growth of opine-degrading bacteria or communities under sterile or axenic environments or in microcosms mimicking near field conditions. Another way to favor a given microbe consists in impeding growth of competing microorganisms. Experiments performed using detergent or bacteriostatic agents as amendments under field or near field conditions yielded promising results. Research perspectives for engineering plant-microbe interactions also include specific engineering of predation and strategies designed to interfere with some of the signals perceived by the microbes, provided these signals control the expression of functions central to microbial fitness. In this respect, quorum-sensing signal molecules, such as N-acyl-homoserine lactones, may be valuable targets for the development of biocontrol agents and procedures.
Characterization and Evolutionary Analysis of a Large Polygalacturonase Gene Family in the Oomycete Plant PathogenPhytophthora cinnamomi
Authors: Götesson Arvid , Marshall Jerry S. , Jones David A. , Hardham Adrienne R.
Published: May 2007

Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration. In this study, PG activity was demonstrated in culture filtrates of the oo-mycete plant pathogen, Phytophthora cinnamomi. A P. cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P. cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes. Gel blotting of genomic DNA indicated that there may be even more pg genes in the P. cinnamomi genome. P. cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity. The predicted mature P. cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure. Evidence for birth-and-death and reticulate evolution in the P. cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P. cinnamomi PGs were shown to have been subject to diversifying selection. Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P. cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants.
Phospholipase D in Phytophthora infestans and Its Role in Zoospore Encystment
Authors: Latijnhouwers Maita , Munnik Teun , Govers Francine
Published: May 2007

We show that differentiation of zoospores of the late blight pathogen Phytophthora infestans into cysts, a process called encystment, was triggered by both phosphatidic acid (PA) and the G-protein activator mastoparan. Mastoparan induced the accumulation of PA, indicating that encystment by mastoparan most likely acts through PA. Likewise, mechanical agitation of zoospores, which often is used to induce synchronized encystment, resulted in increased levels of PA. The levels of diacylglycerolpyrophosphate (DGPP), the phosphorylation product of PA, increased simultaneously. Also in cysts, sporangiospores, and mycelium, mastoparan induced increases in the levels of PA and DGPP. Using an in vivo assay for phospholipase D (PLD) activity, it was shown that the mastoparan-induced increase in PA was due to a stimulation of the activity of this enzyme. Phospholipase C in combination with diacylglycerol (DAG) kinase activity also can generate PA, but activation of these enzymes by mastoparan was not detected under conditions selected to highlight 32P-PA production via DAG kinase. Primary and secondary butanol, which, like mastoparan, have been reported to activate G-proteins, also stimulated PLD activity, whereas the inactive tertiary isomer did not. Similarly, encystment was induced by n- and sec-butanol but not by tert-butanol. Together, these results show that Phytophthora infestans contains a mastoparan- and bu-tanol-inducible PLD pathway and strongly indicate that PLD is involved in zoospore encystment. The role of G-proteins in this process is discussed.
Localization of a Potyvirus and the Viral Genome-Linked Protein in Wild Potato Leaves at an Early Stage of Systemic Infection
Authors: Rajamäki Minna-Liisa , Valkonen Jari P. T.
Published: May 2007

The upper noninoculated ‘sink’ leaves of the wild potato species, Solanum commersonii, were studied for distribution of Potato virus A (PVA) at an early stage of systemic infection. Viral RNA was detected by in situ hybridization, and five viral proteins were localized using immunohistochemical staining in leaf sections. Initial systemic infection foci were found at the vicinity of major and minor veins. In these infection foci, the viral coat protein, cylindrical inclusion protein, and helper component-proteinase colocalized with viral RNA in parenchyma and mesophyll cells, but none of these were detected in companion cells (CC). In contrast, VPg, which is the N-proximal half of the NIa protein (separated from the C-terminal proteinase domain, NIapro, by an autocatalytic cleavage) and acts as a viral genome-linked protein, was detected in CC in the infection foci, but only at an early stage of virus unloading. Outside the infection foci, conspicuous signals for VPg were readily and exclusively detected in CC of many veins in all vein classes in the absence of signals for NIapro, other viral proteins, and viral RNA. Taken together, our data indicate that both major and minor veins may unload PVA in the sink leaves of potato. The data suggest that VPg is translocated from inoculated source leaves to the sink leaves, where it accumulates in CC at an early stage of systemic infection. These findings suggest that VPg may be a ‘phloem protein’ that specifically acts in CC in the sink leaves to facilitate virus unloading.
Differential Induction of Symptoms in Arabidopsis by P6 of Cauliflower mosaic virus
Authors: Yu Weichang , Murfett Jane , Schoelz James E.
Published: May 2007

The gene VI protein (P6) of Cauliflower mosaic virus (CaMV) functions as a virulence factor in crucifers by eliciting chlorotic symptoms in infected plants. The ability to induce chlorosis has been associated previously with P6 through gene-swapping experiments between strains and through the development of transgenic plants that express P6. The primary role that has been identified for P6 in the CaMV infection cycle is to modify the host translation machinery to facilitate the translation of the polycistronic CaMV 35S RNA. This function for P6 has been designated as the translational transactivator (TAV) function. In the present study, we have characterized an unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana. The level of D4 P6 produced in transgenic Arabidopsis line D4-2 was comparable to the amount found in transgenic plants homozygous for W260 and CM1841 P6, two versions of P6 that induce strong chlorotic symptoms and stunting in Arabidopsis. A complementation assay proved that P6 expressed in the D4-2 line was functional, as it could support the systemic infection of a CM1841 mutant that contained a lethal frame-shift mutation within gene VI. This complementation assay allowed us to separately assess the contribution of CM1841 gene VI to symptom development versus the contribution of other CM1841 genes. Furthermore, a previous study had shown that the TAV activity of D4 P6 was comparable to that of W260 P6. That comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line in the present paper, indicates that the TAV function of P6 may play only a minor role in the development of chlorotic symptoms.
Infection-Blocking Genes of a Symbiotic Rhizobium leguminosarum Strain That Are Involved in Temperature-Dependent Protein Secretion
Authors: Bladergroen M. R. , Badelt K. , Spaink H. P.
Published: May 2007

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.
Salicylic Acid Inhibits Indeterminate-Type Nodulation But Not Determinate-Type Nodulation
Authors: van Spronsen Paulina C. , Tak Teun , Rood Anita M. M. , van Brussel Anton A. N. , Kijne Jan W. , Boot Kees J. M.
Published: May 2007

LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation. In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway). Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10−4 M SA. This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae association with vetch roots. In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants. SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.
A Mating-Induced Protein of Phytophthora infestans Is a Member of a Family of Elicitors with Divergent Structures and Stage-Specific Patterns of Expression
Authors: Fabritius Anna-Liisa , Judelson Howard S.
Published: May 2007

Five members of an elicitor-like gene family from Phytophthora infestans were examined. The family was identified through the analysis of M81, a mating-induced gene. The predicted M81 product resembled a 42-kDa P. sojae glycoprotein known to elicit defense reactions in plants, including a host of P. infestans, potato. M81 was the most structurally and functionally divergent of the P. infestans genes compared with the P. sojae sequence. M81 lacked elicitor activity, had the lowest protein identity (47%), displayed mating-specific transcription, and had a novel C-terminal domain. The latter contained a 30-residue proline- and threonine-rich motif, which, remarkably, was tandemly repeated 24 to 36 times in different alleles. M81C, M81D, and M81E better resembled the P. sojae protein based on amino acid identity (63 to 75%) and conserved elicitor activity. M81C and M81D mRNA accumulated only during zoosporogenesis, while M81E expression was restricted to hyphae. M81B, an apparent pseudogene, was physically linked to M81. The protein products of each gene were predicted to be extracellular transglutaminases ranging in size from 436 to 1,607 amino acids. Genes with an elicitor, proline- and threonine-rich repeat, and both elicitor and repeat domains were widely distributed throughout Phytophthora infestans. These findings help explain the natural functions of elicitors in pathogen biology and plant-microbe interactions.
Nodulation of Mimosa spp. by the β-Proteobacterium Ralstonia taiwanensis
Authors: Chen Wen-Ming , James Euan K. , Prescott Alan R. , Kierans Martin , Sprent Janet I.
Published: May 2007

Several β-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.
The Glucocorticoid-Inducible GVG System Causes Severe Growth Defects in Both Root and Shoot of the Model Legume Lotus japonicus
Authors: Andersen Stig Uggerhøj , Cvitanich Cristina , Hougaard Birgit Kristine , Roussis Andreas , Grønlund Mette , Jensen Dorthe Bødker , Frøkjær Line Askou , Jensen Erik Østergaard
Published: May 2007

During the past decade, the legume Lotus japonicus has emerged as an important model system for study of symbiotic nitrogen fixation. Controlled expression of genes involved in symbiosis from an inducible promoter at specific time points would be a valuable tool for investigating gene function in L. japonicus. We have attempted to study the function of the putative transcription factors LjNDX and LjCPP1 by expression from the GVG inducible system. This study showed that the GVG system itself causes growth disturbances in L. japonicus. Shoot internode elongation and root pericycle cell division are affected when the chimeric GVG transcription factor is activated. We suggest that deficient auxin signaling could cause the phenotype observed and conclude that the GVG inducible system is not well suited for use in the model legume L. japonicus.
A Set of Genes Differentially Expressed Between Avirulent and Virulent Meloidogyne incognita Near-Isogenic Lines Encode Secreted Proteins
Authors: Neveu Cédric , Jaubert Stéphanie , Abad Pierre , Castagnone-Sereno Philippe
Published: May 2007

A cDNA-amplification fragment length polymorphism (AFLP)-based strategy has been used to identify genes differentially expressed between two pairs of near-isogenic lines (NIL) of the root-knot nematode Meloidogyne incognita either avirulent or virulent against the tomato Mi resistance gene. Gene expression profiles from infective second-stage juveniles (J2) were compared, and 22 of the 24,025 transcript-derived fragments (TDF) generated proved to be differential, i.e., present in both avirulent NIL and absent in both virulent NIL. Fourteen of the TDF sequences did not show any significant similarity to known proteins, while eight matched reported sequences from nematodes and other invertebrates. The differential expression of nine genes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) experiments. In situ hybridization conducted with five of the sequences showed that two were specifically expressed in the intestinal cells (HM10 and PM1), one in the subventral esophageal glands (HM1), and two in the dorsal esophageal gland of J2 (HM7 and HM12). Analysis of full-length cDNA sequences revealed the presence of a signal peptide for HM1, HM10, and HM12, indicating that the encoded proteins are putatively secreted. Since secreted products in general and esophageal gland secretions in particular are thought to be among the main M. incognita pathogenicity factors, this result suggests a possible dual role for some of the genes encoding such secretions, i.e., they could be involved in both pathogenicity and virulence or avirulence of these biotrophic parasites.
The sypA, sypB, and sypC Synthetase Genes Encode Twenty-Two Modules Involved in the Nonribosomal Peptide Synthesis of Syringopeptin by Pseudomonas syringae pv. syringae B301D
Authors: Scholz-Schroeder Brenda K. , Soule Jonathan D. , Gross Dennis C.
Published: May 2007

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.
Characterization and Expression Analysis of Genes Encoding Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase of Lotus japonicus, a Model Legume
Authors: Nakagawa Tomomi , Izumi Tomoko , Banba Mari , Umehara Yosuke , Kouchi Hiroshi , Izui Katsura , Hata Shingo
Published: May 2007

Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.
Rhizobium-Induced Calcium Spiking in Lotus japonicus
Authors: Harris Jeanne M. , Wais Rebecca , Long Sharon R.
Published: May 2007

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.
Nitrogen Comes Down to Earth: Report from the 5th European Nitrogen Fixation Conference
Authors: De Hoff Peter , Hirsch Ann M.
Published: May 2007

For four days and four nights, with almost 50 presentations and more than 175 posters, the 5th European Nitrogen Fixation Conference continued a tradition of excellence, bringing scientists from diverse fields such as microbiology, biochemistry, computational genomics, and plant physiology together to address the complex problems associated with biological nitrogen fixation (BNF). The conference was hosted by the John Innes Center and the University of East Anglia in Norwich, England and took place from September 6 through 10, 2002. A diverse range of topics was presented, from the evolution of rhizobial genomes to the plant genes involved in bacterial and fungal symbiosis, to the structure of nitrogenase, and to the means by which nitrogen is shuttled between the symbiotic bacteria and the plant. Additionally, sessions involving broader issues, such as nitrogen fertilizer use and work being done in developing countries, brought home the importance of the research being carried out in BNF around the world.
Ectopic Expression of Ralstonia solanacearum Effector Protein PopA Early in Invasion Results in Loss of Virulence
Authors: Kanda Ayami , Yasukohchi Masahiko , Ohnishi Kouhei , Kiba Akinori , Okuno Tetsuro , Hikichi Yasufumi
Published: May 2007

Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (ΔABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.