bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of nodD1 and nodD2 in Sinorhizobium fredii, a Nitrogen-Fixing Symbiont of Soybean and Other Legumes
Authors: Machado Daphne , Pueppke Steven G. , Vinardel José Maria , Ruiz-Sainz José E. , Krishnan Hari B.
Published: May 2007

The ability of Sinorhizobium fredii strains USDA191 and USDA257 to form nitrogen-fixing root nodules on legume plants is regulated by nodD1 and nodD2, which sense flavonoid signals from host roots and then activate the expression of inducible nodulation genes. We assessed the interactions between these two loci with nodD1-negative and nodD2-negative mutants and with strains containing extra copies of these genes. Although both nodD1 and nodD2 are expressed constitutively, levels of nodD1 are much higher, as measured by RNA dot blots. Extra plasmid-borne copies of nodD2 reduced transcription of nodD1 to below the level of detection. We employed gel mobility shift assays to demonstrate that cellular proteins from flavonoid-treated cultures of strain USDA191 bind to DNA sequences that lie upstream from nodD1, but not to the corresponding region upstream from nodD2. Extra plasmid-borne copies of nodD2 enhanced protein binding to the nodD1-associated region and rendered the process flavonoid independent. DNase I footprinting analysis and gel retardation experiments with an oligonucleotide DNA probe localized the protein-binding site to a nod box-like sequence that lies just upstream from the nodD1 coding region. Antibodies raised against a NodD2 fusion protein and capable of reacting both with NodD1 and NodD2 blocked formation of the retarded protein-DNA complex.
The Rhizobium sp. BR816 nodO Gene Can Function as a Determinant for Nodulation of Leucaena leucocephala, Phaseolus vulgaris, and Trifolium repens by a Diversity of Rhizobium spp.
Authors: Vlassak K. M. , Luyten E. , Verreth C. , van Rhijn P. , Bisseling T. , Vanderleyden J.
Published: May 2007

Introduction of the subcloned nodO gene of Rhizobium sp. BR816 into R. meliloti 1021, R. leguminosarum bv. trifolii ANU843, and Azorhizobium caulinodans ORS571 significantly stimulated nodulation and infection of Leucaena leucocephala by these transconjugants. The BR816 nodO gene was also shown to suppress the nodulation defect of the nodU mutants of Rhizobium sp. NGR234 and R. tropici CIAT899 on L. leucocephala and of the nodE mutant of R. leguminosarum bv. trifolii ANU843 on white clover. Overproduction of NodO in BR816 extended its host range to Trifolium repens. While a Rhizobium sp. BR816 strain, mutated in nodO, had reduced nodulation and competitiveness on P. vulgaris, no altered phenotype was observed for this mutant on L. leucocephala. Analysis of the transcriptional activation of nodO in BR816 revealed enhanced expression in the presence of apigenin, under both aerobic and microaerobic conditions. Moreover, nodO was also expressed in the bacteroids of L. leucocephala nodules.
The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae: Molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast
Authors: Struck Christine , Siebels Claudia , Rommel Oliver , Wernitz Marcus , Hahn Matthias
Published: May 2007

To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30–35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H+-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (Δ76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H+-ATPases. Compared to the wild-type, the Δ76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.
The Bradyrhizobium japonicum noeD Gene: A Negatively Acting, Genotype-Specific Nodulation Gene for Soybean
Authors: Lohrke S. M. , Day B. , Kolli V. S. Kumar , Hancock R. , Yuen J. P.-Y. , de Souza M. L. , Stacey G. , Carlson R. , Tong Z. , Hur H.-G. , Orf J. H. , Sadowsky M. J.
Published: May 2007

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2–5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378–2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2–5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.
Tm-22 Resistance in Tomato Requires Recognition of the Carboxy Terminus of the Movement Protein of Tomato Mosaic Virus
Authors: Weber Hans , Pfitzner Artur J. P.
Published: May 2007

The Tm-22 resistance gene is used in most commercial tomato cultivars for protection against infection with tomato mosaic virus (ToMV). It has been suggested that Tm-22 resistance interferes with viral cell-to-cell movement in plants; ToMV strain ToMV-22 requires two amino acid (aa) exchanges in the carboxy-terminal region of the viral 30-kDa movement protein (at positions 238 and 244) to overcome Tm-22 resistance. For further analysis of this region of the 30-kDa protein, two stop codons were introduced into ToMV movement proteins at aa positions 235 and 237, leading to deletion of the terminal 30 aa. The mutant virus strains were able to infect wild-type tomato plants systemically, suggesting the carboxy-terminal portion of the ToMV 30-kDa protein is dispensable for virus transport in tomato. Even more important, the deletion mutants overcame the Tm-22 resistance gene. These data indicate the carboxy-terminal domain of the ToMV movement protein serves as a recognition target in the context of the Tm-22 resistance gene. Furthermore, expression of the 30-kDa movement protein from wild-type ToMV, but not from ToMV-22, in transgenic tomato plants with the Tm-22 resistance gene led to elicitation of a necrotic reaction in tomato seedlings, showing that the 30-kDa protein on its own is able to induce the plant's defense reaction.
Differential Induction of the Arabidopsis thaliana Thi2.1 Gene by Fusarium oxysporum f. sp. matthiolae
Authors: Epple Petra , Vignutelli Alberto , Apel Klaus , Bohlmann Holger
Published: May 2007

The Arabidopsis thaliana Thi2.1 gene is inducible by necrotrophic fungi through a signal transduction pathway different from that for pathogenesis-related (PR) proteins. We have identified three ecotypes that are susceptible (Col-2, Ler, and Ws) and two ecotypes that are resistant (Mt-0 and Uk-4) to spray inoculation with Fusarium oxysporum f. sp. matthiolae. The Thi2.1 transcript level after infection correlates with resistance, being 5 to 10 times higher in the resistant than in the susceptible ecotypes. The β-glucuronidase (GUS) expression of a Thi2.1-promoter-uidA fusion (with a promoter derived from Col-2) is on the average almost 10 times higher in the Uk-4 background than in the Col-2 background. This confirms the results obtained by Northern (RNA) blots and indicates that Uk-4, and probably other resistant ecotypes too, might have a more sensitive recognition system for F. oxysporum f. sp. matthiolae or might have a signal transduction system that gives a higher amplification of the original recognition signal. Our results suggest a role of the Thi2.1 gene in resistance against F. oxysporum f. sp. matthiolae and perhaps other necrotrophic fungi.
Expression of a Defense-Related 3-Hydroxy-3-Methylglutaryl CoA Reductase Gene in Response to Parasitization by Orobanche spp.
Authors: Westwood James H. , Yu Xueshu , Foy Chester L. , Cramer Carole L.
Published: May 2007

Orobanche spp. are angiosperms that live parasitically on the roots of other plants, and are capable of significantly reducing the yield and quality of their crop hosts. We have demonstrated that parasitization by Orobanche induces expression of hmg2, a defense-related isogene of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in tobacco. Transgenic tobacco plants expressing a construct containing 2.3 kb of the tomato hmg2 gene promoter fused to the β-glucuronidase (GUS) reporter gene were parasitized by O. aegyptiaca. Expression of the hmg2:GUS construct was detected within 1 day following penetration of the host root by the O. aegyptiaca radicle and was localized to the region immediately around the site of parasite invasion. This expression continued and intensified over the course of O. aegyptiaca development. In addition, the hmg2:GUS expression was induced by secondary parasitization, where secondary roots of O. aegyptiaca contacted the host root at a distance from the primary attachment site. This GUS expression was specific to plants containing the hmg2:GUS construct, and was not observed in control plants transformed with a construct of the cauliflower mosaic virus 35S promoter fused to the GUS gene. These results indicate that Orobanche parasitization initiates rapid and sustained induction of a defense-related gene in the host root.
Entry of Xanthomonas campestris pv. campestris into Hydathodes of Arabidopsis thaliana Leaves: A System for Studying Early Infection Events in Bacterial Pathogenesis
Authors: Hugouvieux Véronique , Barber Christine E. , Daniels Michael J.
Published: May 2007

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen of cruciferous plants that normally gains entry to plants via hydathodes. In order to study the basis of the preference for this portal of entry we have developed an Arabidopsis thaliana model with attached or detached leaves partially immersed in a bacterial suspension. Entry of bacteria into leaves, assessed by resistance to surface sterilization, could be detected after 1 h. Dissection of leaves and histochemical staining for β-glucuronidase produced by the bacteria indicated that they were located in hydathodes. In contrast, similar experiments with the leaf-spotting pathogen X. campestris pv. armoraciae gave patterns of localized staining dispersed over the leaf area, indicative of entry through stomata. A survey of 41 A. thaliana accessions showed that they fell into three classes distinguishable by total numbers of Xcc that entered under standard conditions and by preference for hydathode colonization. Previously isolated Xcc mutants affected in pathogenicity were tested for hydathode colonization: an hrp mutant behaved indistinguishably from the wild type, and rpf regulatory mutants gave 10-fold reduced colonization, whereas with rfaX mutants with altered lipopolysaccharide, few if any viable bacteria were recoverable from hydathodes. This fact, together with the rapid induction of superoxide dismutase in the bacteria located in hydathodes, suggests that an early defense reaction is mounted in the hydathode.
Activation of hsr203, a Plant Gene Expressed During Incompatible Plant-Pathogen Interactions, Is Correlated with Programmed Cell Death
Authors: Pontier Dominique , Tronchet Maurice , Rogowsky Peter , Lam Eric , Roby Dominique
Published: May 2007

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.
The hrpC and hrpN Operons of Erwinia chrysanthemi EC16 Are Flanked by plcA and Homologs of Hemolysin/Adhesin Genes and Accompanying Activator/Transporter Genes
Authors: Kim Jihyun F. , Ham Jong Hyun , Bauer David W. , Collmer Alan , Beer Steven V.
Published: May 2007

The hrpC operon of Erwinia chrysanthemi EC16 encodes five genes conserved in Erwinia amylovora and Pseudomonas syringae. Mutagenesis indicated that hrcC is required for elicitation of the hypersensitive reaction in tobacco leaves. The unexpected presence of plcA and homologs of hemolysin/activator genes in the regions flanking the hrcC and hrpN operons is reported.
The Myristylation Motif of Pto Is Not Required for Disease Resistance
Authors: Loh Ying-Tsu , Zhou Jianmin , Martin Gregory B.
Published: May 2007

The tomato Pto kinase confers resistance to bacterial speck disease caused by strains of Pseudomonas syringae pv. tomato that express the avirulence gene avrPto. Pto contains a putative myristylation site at its amino terminus that was hypothesized to play a role in localizing Pto in the plant cell. Site-directed mutagenesis was used to change the invariant glycine residue in the myristylation motif to an alanine. Transgenes encoding the mutant Pto(G2A) and wild-type Pto were placed behind the cauliflower mosaic virus 35S promoter and transformed into tomato plants that are susceptible to bacterial speck disease. Both the mutant and wild-type forms of Pto conferred resistance to a strain of P. syringae pv. tomato expressing avrPto. These results indicate that the myristylation motif of Pto is not required for bacterial speck disease resistance.
A Virulence-Associated, 6.4-kb, Double-Stranded RNA from Rhizoctonia solani Is Phylogenetically Related to Plant Bromoviruses and Electron Transport Enzymes
Authors: Jian JianHua , Lakshman Dilip K. , Tavantzis Stellos M.
Published: May 2007

We have recently shown that acquisition of a 6.4-kb, double-stranded (ds) RNA (M1) by hyphal anastomosis is associated with enhanced vigor and virulence, whereas its removal by hyphal tipping correlates with diminished virulence in the plant-pathogenic basidiomycete Rhizoctonia solani. The M1 dsRNA is not encapsidated by a typical nucleocapsid, has a circular and/or concatemeric form, and is associated with the mitochondrial and cytosolic fractions. M1 possesses six open reading frames (ORFs) the longest of which (ORF 2) is located on the (+) strand, and encodes a putative polypeptide consisting of 1,747 amino acids or 199.4 kDa. This polypeptide has a significant amino acid sequence similarity, including six conserved helicase domains and an ATP/GTP binding motif, with the 1A protein of broad bean mottle virus (BBMV) and other bromoviruses. ORF 5, which is located on the (-) strand of M1 and is complementary to a region of ORF 2, codes for a putative polypeptide that has a significant amino acid sequence similarity with the cytochrome c oxidase assembly factor. This complementarity provides direct evidence suggesting that the long-standing hypothesis of viruses evolving from cellular genes may be valid.
Cell-to-Cell Movement of Beet Necrotic Yellow Vein Virus: I. Heterologous Complementation Experiments Provide Evidence for Specific Interactions Among the Triple Gene Block Proteins
Authors: Lauber Emmanuelle , Bleykasten-Grosshans Claudine , Erhardt M. , Bouzoubaa S. , Jonard G. , Richards K. E. , Guilley H.
Published: May 2007

Cell-to-cell movement of beet necrotic yellow vein virus (BNYVV) requires three proteins encoded by a triple gene block (TGB) on viral RNA 2. A BNYVV RNA 3-derived replicon was used to express movement proteins of other viruses and the ability of these proteins to functionally substitute for the BNYVV TGB proteins was tested by coinoculation of TGB-defective BNYVV with the various replicons to Chenopodium quinoa. Trans-heterocomplementation was successful with the movement protein (P30) of tobacco mosaic virus but not with the tubule-forming movement proteins of alfalfa mosaic virus and grapevine fanleaf virus. Trans-complementation of BNYVV movement was also observed when all three TGB proteins of the distantly related peanut clump virus were supplied together but not when they were substituted for their BNYVV counterparts one by one. When P30 was used to drive BNYVV movement in trans, accumulation of the first TGB protein of BNYVV was adversely affected by null mutations in the second and third TGB proteins. Taken together, these results suggest that highly specific interactions among cognate TGB proteins are important for their function and/or stability in planta.
Differential Involvement of Indole-3-Acetic Acid Biosynthetic Pathways in Pathogenicity and Epiphytic Fitness of Erwinia herbicola pv. gypsophilae
Authors: Manulis Shulamit , Haviv-Chesner Anat , Brandl Maria T. , Lindow Steve E. , Barash Isaac
Published: May 2007

Erwinia herbicola pv. gypsophilae (Ehg), which induces galls on Gypsophila paniculata, harbors two major pathways for indole-3-acetic acid (IAA) synthesis, the indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) routes, as well as cytokinin biosynthetic genes. Mutants were generated in which the various biosynthetic routes were disrupted separately or jointly in order to assess the contribution of IAA of various origins and cytokinins to pathogenicity and epiphytic fitness. Inactivation of the IAM pathway or cytokinin biosynthesis caused the largest reduction in gall size. Inactivation of the IPyA pathway caused a minor, nonsignificant decrease in pathogenicity. No further reduction in gall size was observed by the simultaneous inactivation of both IAA pathways only or in combination with that of cytokinin production. However, inactivation of the IPyA pathway caused a 14-fold reduction in the population of Ehg on bean plants. Inactivation of the IAM pathway or cytokinin production did not affect epiphytic fitness. While the apparent transcriptional activity of iaaM-inaZ fusion increased slightly in cells of Ehg on bean and gypsophila leaves, compared with that in culture, very high levels of induction were observed in cells injected into gypsophila stems. In contrast, moderate levels of induction of ipdC-inaZ in Ehg were observed on leaves of these plants and in gypsophila stems, when compared with that in culture. These results suggest that the IAM pathway is involved primarily in gall formation and support the main contribution of the IpyA pathway to the epiphytic fitness of this bacterial species.
Role of the Agrobacterium tumefaciens VirD2 Protein in T-DNA Transfer and Integration
Authors: Mysore Kirankumar S. , Bassuner Burgund , Deng Xiao-bing , Darbinian Nune S. , Motchoulski Andrei , Ream Walt , Gelvin Stanton B.
Published: May 2007

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called ω that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the ω region are not essential for nuclear uptake of T-DNA, and further suggested that the ω domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the ω domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of β-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid (X-gluc). However, using an ω-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 ω deletion/substitution. Taken together, these data indicate that the VirD2 ω domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the ω mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an ω mutation. In addition, a mutant VirD2 protein lacking the ω domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the ω and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the ω domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.
Use of Monoclonal Antibodies to Determine Biomass of Cladosporium fulvum in Infected Tomato Leaves
Authors: Karpovich-Tate Natalia , Spanu Pietro , Dewey Frances M.
Published: May 2007

A monoclonal antibody, OX-CH1, was raised against surface washings of Cladosporium herbarum. This antibody recognizes an epitope that is found in various fungi belonging to the genus Cladosporium, including C. fulvum, the causal agent of tomato leaf mold. The epitope is present at comparable levels in two different races of C. fulvum and in transgenic isolates derived from them. The epitope is heat- and protease-resistant but sensitive to oxidation with periodate and it is constitutively expressed in C. fulvum grown in pure culture and on the plant. C. fulvum can be detected in infected tissues at levels starting from around 1 mg fresh weight of fungus per g fresh weight of leaf tissue. Noninfected tomato leaves do not cross-react with OX-CH1. We have developed an enzyme-linked immunosorbent assay (ELISA) for fungal biomass in tomato leaves and compared it with the assay based on measurements of β-glucuronidase (GUS) activity in tissues infected with a transgenic isolate of C. fulvum race 4 carrying a uidA gene; the two assays give similar results.
Two-Component Regulators Involved in the Global Control of Virulence in Erwinia carotovora subsp. carotovora
Authors: Eriksson Anders R. B. , Andersson Robert A. , Pirhonen Minna , Palva E. Tapio
Published: May 2007

Production of extracellular, plant cell wall degrading enzymes, the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, is coordinately controlled by a complex regulatory network. Insertion mutants in the exp (extracellular enzyme production) loci exhibit pleiotropic defects in virulence and the growth-phase-dependent transcriptional activation of genes encoding extracellular enzymes. Two new exp mutations, designated expA and expS, were characterized. Introduction of the corresponding wild-type alleles to the mutants complemented both the lack of virulence and the impaired production of plant cell wall degrading enzymes. The expA gene was shown to encode a 24-kDa polypeptide that is structurally and functionally related to the uvrY gene product of Escherichia coli and the GacA response regulator of Pseudomonas fluorescens. Functional similarity of expA and uvrY was demonstrated by genetic complementation. The expA gene is organized in an operon together with a uvrC-like gene, identical to the organization of uvrY and uvrC in E. coli. The unlinked expS gene encodes a putative sensor kinase that shows 92% identity to the recently described rpfA gene product from another E. carotovora subsp. carotovora strain. Our data suggest that ExpS and ExpA are members of two-component sensor kinase and response regulator families, respectively. These two proteins might interact in controlling virulence gene expression in E. carotovora subsp. carotovora.
A Pathogenicity Gene Isolated from the pPATH Plasmid of Erwinia herbicola pv. gypsophilae Determines Host Specificity
Authors: Valinsky Lea , Manulis Shulamit , Nizan Roni , Ezra David , Barash Isaac
Published: May 2007

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to the gypsophila plant whereas E. herbicola pv. betae (Ehb) incites galls on beet as well as gypsophila. The pathogenicity of Ehg and Ehb was previously shown to be dependent on a plasmid (pPATH). Transposon mutagenesis was used to generate mutants on the cosmid pLA150 of the pPATH from Ehg824-1. A cluster of nonpathogenic mutations flanked by two IS1327 elements was identified on a 3.2-kb NdeI DNA fragment. All mutants were restored to pathogenicity by complementation in trans with the wild-type Ehg DNA. DNA sequence analysis of the 3.2-kb NdeI fragment revealed a single open reading frame (ORF) of 2 kb as well as a potential ribosome binding site and a putative hrp box upstream to the ORF. The ORF had no significant homology to known genes. Southern analysis also revealed the presence of DNA sequences that hybridized to the ORF in the beet pathovar Ehb4188. This gene was isolated and sequenced. Marker exchange mutants generated in the ORF of Ehb eliminated the pathogenicity of Ehb on gypsophila but fully retained its pathogenicity on beet. Since the putative gene appeared to encode a host-specific virulence factor for gypsophila it was designated as hsvG.
Role of the O-Antigen of Lipopolysaccharide, and Possible Roles of Growth Rate and of NADH:ubiquinone Oxidoreductase (nuo) in Competitive Tomato Root-Tip Colonization by Pseudomonas fluorescens WCS365
Authors: Dekkers Linda C. , van der Bij Arjan J. , Mulders Ine H. M. , Phoelich Claartje C. , Wentwoord Rino A. R. , Glandorf Deborah C. M. , Wijffelman Carel A. , Lugtenberg Ben J. J.
Published: May 2007

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.
Production ofSinorhizobium meliloti nodGene Activator and Repressor Flavonoids fromMedicago sativaRoots
Authors: Zuanazzi José Angelo Silveira , Clergeot Pierre Henri , Quirion Jean-Charles , Husson Henri-Philippe , Kondorosi Adam , Ratet Pascal
Published: May 2007

During symbiosis between leguminous plants and rhizobia, flavonoids exuded by the plants act as chemoattractants and nodulation (nod) gene regulators in the other partner. To better understand the role of these compounds during the early steps of the alfalfa-Sinorhizobium meliloti symbiosis and the regulation of their production we have isolated nod gene inducers from alfalfa roots. All the compounds that we identified in this study as nod gene inducers in the root are flavonoids, indicating that other compounds with nod gene activator capacity may have little contribution, if any, to nod gene activation. Most of the intermediates of the flavonoid pathway were found in Medicago sativa roots and nodules, but only end products of the flavonoid pathway were identified in the root exudate. We have also studied flavonoid production in different parts of the root and found that it is developmentally regulated during root growth. Finally, we have shown that coumestrol and medicarpin, present in the exudates and previously described as phytoalexins, possess nod gene repressing activity, indicating that the in vivo nod gene inducing activity of the root exudate results from positive as well as negative controls of nod gene expression by the flavonoids.
Molecular Dissection of the Mechanism by Which Potexvirus Triple Gene Block Proteins Mediate Cell-to-Cell Transport of Infectious RNA
Authors: Lough Tony J. , Shash Khalid , Xoconostle-Cázares Beatriz , Hofstra Katrina R. , Beck David L. , Balmori Ezequiel , Forster Richard L. S. , Lucas William J.
Published: May 2007

The triple gene block (TGB; consisting of proteins TGB1–3) and coat protein (CP) of white clover mosaic potexvirus (WClMV) are required for cell-to-cell movement of viral RNA. Cell-to-cell spread of WClMV mutants in which the TGB open reading frames had been mutated was rescued in transgenic plants expressing specific TGB proteins (TGBPs). This indicated that there are no requirements for the synthesis in cis of viral TGBPs. These transgenic plants provided an experimental framework to explore the roles performed by the TGBPs and CP in cell-to-cell movement of WClMV RNA. Microinjection experiments established that TGB1 functions as the WClMV cell-to-cell movement protein (MP). Furthermore, combined microinjection and dual-channel confocal laser scanning microscopy provided direct evidence that infectious transcripts of WClMV move cell to cell as a ribonucleoprotein complex, consisting of single-stranded RNA, TGB1, and CP. Movement of this ribonucleoprotein complex displayed an absolute requirement for the presence of both TGB2 and TGB3. A model consistent with these findings is presented.
AvrXa10 Contains an Acidic Transcriptional Activation Domain in the Functionally Conserved C Terminus
Authors: Zhu Weiguang , Yang Bing , Chittoor Jaishree M. , Johnson Lowell B. , White Frank F.
Published: May 2007

The avrXa10 gene of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, is a member of the avrBs3 avirulence gene family and directs the elicitation of resistance in a gene-for-gene manner on rice lines carrying the resistance gene Xa10. The carboxyl (C) terminus of AvrXa10 has a previously undescribed domain that is structurally similar to the acidic activation domain of many eukaryotic transcription factors in addition to three nuclear localization signal (NLS) sequences. Removal of the C-terminal 38 codons containing the putative activation domain, but retaining the NLS sequences, was concomitant with the loss of avirulence activity. The C-terminal coding regions of avrBs3 and avrXa7 can be replaced by the corresponding region of avrXa10, and the genes retained specificity for the resistance genes Bs3 in pepper and Xa7 in rice, respectively. The avrBs3 and avrXa7 avirulence activities of the hybrid genes were also lost upon removal of the terminal 38 codons. When fused to the coding sequence of the Gal4 DNA binding domain, AvrXa10 activated transcription in yeast and Arabidopsis thaliana. Removal of the carboxyl region severely reduced transcriptional activation. AvrXa10 would have to be localized to the host cell nucleus to function autonomously in transcriptional activation. Consistent with this requirement, mutations in all three NLS sequences of avrXa10 caused a loss in avirulence activity. The findings demonstrate the requirement of the C terminus for AvrXa10 function and the potential for the members of this family of avirulence gene products to enter the host nucleus and alter host transcription.
Involvement of Phenazines and Anthranilate in the Antagonism of Pseudomonas aeruginosa PNA1 and Tn5 Derivatives Toward Fusarium spp. and Pythium spp.
Authors: Anjaiah Vanamala , Koedam Nico , Nowak-Thompson Brian , Loper Joyce E. , Höfte Monica , Tambong James Tabi , Cornelis Pierre
Published: May 2007

Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpea in India, suppressed Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris, and Pythium damping-off of bean, caused by Pythium splendens. When grown in culture, PNA1 produced the phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine, and inhibited mycelial growth of F. oxysporum f. sp. ciceris, P. splendens, and certain other phytopathogenic fungi. Two mutants (FM29 and FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1. The transposon in the genome of FM29 was localized to phnA, which is thought to encode a subunit of anthranilate synthase II involved in the phenazine biosynthesis. The FM13 mutation was complemented by trpC, which encodes indole glycerol phosphate synthase in the tryptophan biosynthesis pathway; consequently, FM13 could not grow on a minimal medium in the absence of tryptophan. Neither FM29 nor FM13 suppressed Fusarium wilt of chickpea to the level achieved by the wild-type strain, indicating that phenazine production contributed to the biocontrol of this disease by P. aeruginosa PNA1. FM29 was also less effective than the phenazine-producing parental strain in biological control of Pythium damping-off of bean, but FM13 was as effective as the parental strain in suppressing this disease. Anthranilate, an intermediate in the tryptophan biosynthesis pathway, suppressed mycelial growth of Pythium spp. in culture and Pythium damping-off of bean and lettuce. Anthranilate, excreted by FM13 as a consequence of the trpC mutation, may have contributed to the suppression of Pythium damping-off by the mutant.
Xylem Colonization by an HrcV¯ Mutant of Ralstonia solanacearum Is a Key Factor for the Efficient Biological Control of Tomato Bacterial Wilt
Authors: Etchebar Christophe , Trigalet-Demery Danièle , van Gijsegem Frédérique , Vasse Jacques , Trigalet André
Published: May 2007

Microscopic studies of the colonization of the vascular tissues of tomato by an HrcV¯ (formerly HrpO¯) mutant strain of Ralstonia solanacearum were carried out after either root inoculation of the mutant strain alone or delayed challenge inoculation by a pathogenic strain. The use of two different marker genes, lacZ and uidA, introduced into either mutant or wild-type strains, respectively, permitted histological observation for the presence of both strains simultaneously. In roots, both strains could be found together in infected root tips and in lateral root emergence sites (lateral root cracks), but these bacterial strains subsequently invaded separate xylem vessels in the root system. At the hypocotyl level, a novel staining procedure, in conjunction with bacterial isolation and counting, showed three vascular colonization patterns: exclusive colonization by each of the competitors or simultaneous presence of each strain in separate xylem vessels. The relative frequencies of these patterns depended upon the root inoculation techniques used. The presence of one population always influenced the density of the other challenge-inoculated population. In plants inoculated with both wild-type and mutant strains, the population of the wild-type strain is lower than in plants inoculated with the wild type alone. In contrast, growth of the HrcV¯ mutant strain was significantly increased in the presence of the pathogenic strain. Two agriculturally acceptable techniques for plant inoculation were tested. Inoculation of plants by transplanting them into soil amended with clay micro-granules impregnated with the HrcV¯ mutant strain gave higher and more reproducible colonization of the plants than inoculation by watering a bacterial suspension on the roots. Significant percentages of exclusive colonization by the HrcV¯ mutant strain were only obtained after the clay microgranule inoculation technique. Competition for space in xylem vessels is one of the possible explanations for the protective ability of the HrcV¯ mutant strain against subsequent invasion by a pathogenic strain.
Soybean (lbc3), Parasponia, and Trema Hemoglobin Gene Promoters Retain Symbiotic and Nonsymbiotic Specificity in Transgenic Casuarinaceae: Implications for Hemoglobin Gene Evolution and Root Nodule Symbioses
Authors: Franche Claudine , Diouf Diaga , Laplaze Laurent , Auguy Florence , Frutz Thierry , Rio Maryannick , Duhoux Emile , Bogusz Didier
Published: May 2007

The purpose of this study was to compare the control of expression of legume and nonlegume hemoglobin genes. We used the Casuarina glauca and Allocasuarina verticillata transformation system to examine the properties of the soybean (lbc3), Parasponia andersonii, and Trema tomentosa hemoglobin gene promoters in actinorhizal plants. Expression of the hemoglobin promoters gus genes was examined by fluorometric and histochemical assays. The fluorometric assays in various organs showed that the soybean and P. andersonii promoters were most active in nodules whereas the T. tomentosa promoter gave a very high activity in roots. The histochemical study showed that GUS activity directed by the soybean and the P. andersonii gus chimeric genes appeared mainly confined to the infected cells of the C. glauca and A. verticillata nodules. The T. tomentosa hemoglobin promoter was primarily expressed in the root's cortex and vascular tissue. The results indicate that the soybean, P. andersonii, and T. tomentosa hemoglobin promoters retain their cell-specific expression in transgenic members of the Casuarinaceae, suggesting a close relationship between legume, Ulmaceae member, and actinorhizal hemoglobin genes. The conservation of the mechanism for nodule-specific expression of soybean, P. andersonii, and C. glauca and A. verticillata hemoglobin genes is discussed in view of recent molecular phylogenetic data that suggest a single origin for the predisposition to form root nodule symbioses.
In Planta Localization of a β-1,4-Endoglucanase Secreted by Heterodera glycines
Authors: Wang Xiaohong , Meyers Diane , Yan Yitang , Baum Thomas , Smant Geert , Hussey Richard , Davis Eric
Published: May 2007

Polyclonal sera specific to β-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.
(—)-Jasmonic Acid Accumulation in Tobacco Hypersensitive Response Lesions
Authors: Kenton Paul , Mur Luis A. J. , Atzorn Rainer , Wasternack Claus , Draper John
Published: May 2007

Tobacco infected with Pseudomonas syringae pv. phaseolicola undergoes a hypersensitive response (HR). Jasmonic acid (JA) accumulated within the developing lesion 3 to 9 h after infection and this accumulation preceded protein loss, cell death, and malondialdehyde accumulation. Accumulating JA consisted largely of the (—)-JA stereoisomer and was essentially restricted to the HR lesion.
Maize Streak Virus Coat Protein Is Karyophyllic and Facilitates Nuclear Transport of Viral DNA
Authors: Liu H. , Boulton M. I. , Thomas C. L. , Prior D. A. M. , Oparka K. J. , Davies J. W.
Published: May 2007

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.
Systemic Cell Death Is Elicited by the Interaction of a Single Gene in Nicotiana clevelandii and Gene VI of Cauliflower Mosaic Virus
Authors: Király Lóránt , Cole Anthony B. , Bourque June E. , Schoelz James E.
Published: May 2007

Cauliflower mosaic virus (CaMV) strains D4 and W260 can be distinguished by the type of symptoms they induce in Nicotiana clevelandii and N. edwardsonii. W260 induces systemic cell death in addition to a mosaic symptom in N. clevelandii and a hypersensitive response (HR) in N. edwardsonii, whereas D4 induces a systemic mosaic in both hosts. To determine which W260 genes are responsible for systemic cell death, chimeric viruses were constructed between the D4 and W260 strains. It was found that W260 gene VI was responsible for the elicitation of systemic cell death; previous studies had shown that this same gene elicited HR in N. edwardsonii. An immunological analysis of plants infected with W260 or D4 indicated that the systemic cell death symptom was not associated with enhanced levels of either W260 virions or the W260 gene VI product. To investigate the inheritance of systemic cell death, crosses were made between N. clevelandii and N. bigelovii, a host that reacts with a systemic mosaic symptom upon infection with W260. All F1 plants developed a systemic mosaic after inoculation with W260, whereas the F2 generation segregated 3:1 for systemic mosaic versus cell death. The plant gene responsible for cell death was designated ccd1, for CaMV cell death gene. These results demonstrate that the systemic cell death symptom in N. clevelandii is induced by the interaction between a single host gene and gene VI of CaMV.
The Rhizobium etli trpB Gene Is Essential for an Effective Symbiotic Interaction with Phaseolus vulgaris
Authors: Taté Rosarita , Riccio Anna , Caputo Emilia , Cermola Michele , Favre Renee , Patriarca Eduardo J.
Published: May 2007

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix¯) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.