bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid, Cf-9- and Avr9-Dependent Production of Active Oxygen Species in Tobacco Suspension Cultures
Authors: Piedras Pedro , Hammond-Kosack Kim E. , Harrison Kate , Jones Jonathan D. G.
Published: May 2007

The tomato Cf-9 gene confers resistance to races of Cla-dosporium fulvum expressing the corresponding avirulence gene Avr9. The availability of transgenic tobacco lines carrying Cf-9, and the well-characterized 28 amino acid Avr9 elicitor, make this an excellent system to study resistance gene function. In this paper we establish tobacco suspension cultures derived from transgenic tobacco plants containing Cf-9, as well as from plants without Cf-9. Cultures derived from Cf9 tobacco produce active oxygen species (AOS) within 5 min of treatment with pure or synthetic Avr9. This enabled us to perform biochemical and pharmacological analysis in cell culture of the very earliest events in resistance gene function. In addition to AOS production, an increase in oxygen uptake was detected in the Avr9-treated Cf9 cells. Both phenomena were inhibited by low concentrations of diphenyleneiodonium (DPI). Additional pharmacological inhibitor studies suggest that uptake of calcium, activation of protein kinases, and probably phospholipase A2 (PLA2) activity are intermediates in the Cf-9- and Avr9-dependent signaling pathway that leads to AOS production. Interestingly, those defense responses did not result in plant cell death.
Plasmid-Encoded Catabolic Genes in Rhizobium leguminosarum bv. trifolii: Evidence for a Plant-Inducible Rhamnose Locus Involved in Competition for Nodulation
Authors: Oresnik Ivan J. , Pacarynuk Laurie A. , O'Brien Shelley A. P. , Yost Christopher K. , Hynes Michael F.
Published: May 2007

Cosmids carrying genes involved in utilization of rhamnose, sorbitol, and adonitol were isolated from a genomic library of Rhizobium leguminosarum by complementation of plasmid-cured derivatives of strain Rlt100 that were unable to grow on these carbon sources. Transposon mutagenesis was used to identify regions of each cosmid necessary for catabolism of the respective carbon source; partial DNA sequencing, as well as analysis of gene fusions created with transposon Tn5-B20, helped to determine the orientation and possible function of genes required for growth on the three substrates. Representative Tn5 insertions in the cosmids were recombined into the wild-type strain Rlt100 by gene replacement to generate isogenic strains unable to use either rhamnose, sorbitol, or adonitol. These strains were tested for their nodulation competitiveness compared with Rlt100 in co-inoculation experiments on clover plants. While sorbitol and adonitol catabolic mutants were unaltered in their competitive behavior, the nodulation competitiveness of three different rhamnose utilization mutants was significantly impaired. This result, coupled with the fact that the rhamnose catabolic genes were inducible by clover root extracts, suggests an important role for rhamnose catabolism in the early stages of the interaction of R. leguminosarum with clover plants. Hybridization studies with probes derived from the rhamnose, sorbitol, and adonitol catabolic loci demonstrated that these genes are plasmid encoded in virtually all R. leguminosarum strains, including representatives from all three biovars from a variety of different geographic locations.
Analysis of Resistance Gene-Mediated Defense Responses in Arabidopsis thaliana Plants Carrying a Mutation in CPR5
Authors: Boch Jens , Verbsky Michelle L. , Robertson Tara L. , Larkin John C. , Kunkel Barbara N.
Published: May 2007

In resistant plants, pathogen attack often leads to rapid activation of defense responses that limit multiplication and spread of the pathogen. To investigate the signaling mechanisms underlying this process, we carried out a screen for mutants in the signaling pathway governing resistance in Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae. This involved screening for suppressor mutations that restored resistance to a susceptible line carrying a mutation in the RPS2 resistance gene. A mutant that conferred resistance by activating defense responses in the absence of pathogens was isolated. This mutant, which carries a mutation at the CPR5 locus and was thus designated cpr5-2, exhibited resistance to P. syringae, spontaneous development of necrotic lesions, elevated PR gene expression in the absence of pathogens, and abnormal trichomes. Resistance gene-mediated defenses, including the hypersensitive response, restriction of pathogen growth, and induction of defense-related gene expression, were functional in cpr5-2 mutant plants. Additionally, in cpr5-2 plants RPS2-mediated induction of PR-1 expression was enhanced, whereas RPM1-mediated induction of ELI3 was not. These findings suggest that CPR5 encodes a negative regulator of the RPS2 signal transduc-tion pathway.
Tobacco Mosaic Virus Helicase Domain Induces Necrosis in N Gene-Carrying Tobacco in the Absence of Virus Replication
Authors: Abbink Truus E. M. , Tjernberg Pernilla A. , Bol John F. , Linthorst Huub J. M.
Published: May 2007

Tobacco mosaic virus (TMV) elicits a hypersensitive response (HR) in tobacco plants that carry the N gene. To identify the elicitor of this HR, Agrobacteriumn tumefaciens was used as a vector for the transient expression of TMV replicase proteins, movement protein, and coat protein in NN and nn tobacco. Transient expression of the 126K protein and fragments thereof containing the helicase motifs induced necrosis and systemic expression of the pathogenesis-related PR-1a gene in NN plants but not in nnplants. The results confirm previous evidence that the TMV helicase sequence is the elicitor of the HR (H. S. Padgett, Y. Watanabe, and R. N. Beachy, Mol. Plant-Microbe Interact. 10:709-715, 1997) and demonstrate that this helicase sequence acts as an elicitor of HR in the absence of other viral proteins or RNA replication.
Sequences Related to Transposable Elements and Bacteriophages Flank Avirulence Genes of Pseudomonas syringae
Authors: Kim Jihyun F. , Charkowski Amy O. , Alfano James R. , Collmer Alan , Beer Steven V.
Published: May 2007

Examination of the reported nucleotide sequences containing avirulence genes of Pseudomonas syringae pathovars suggested that avrA, avrB, avrC, avrPphC, avrRpm1, and avrPpiA1 are bordered by sequences similar to those of transposable elements of gram-negative bacteria. Repeat sequences and fragments of at least two different insertion sequence elements were identified at the ends of avrA and avrB, implying multiple transposition events for these areas. A DNA region homologous to a bacteriophage sequence was found upstream of avrPto. The linkage of various virulence/avirulence genes of animal- and plant-pathogenic bacteria with transposable elements and bacteriophage sequences, together with the presence of several of these genes on plasmids, supports the idea of horizontal transfer and frequent exchange of virulence/avirulence genes among bacterial pathogens.
Differential Display Analysis of the Early Compatible Interaction Between Soybean and the Soybean Cyst Nematode
Authors: Hermsmeier Dieter , Mazarei Mitra , Baum Thomas J.
Published: May 2007

The marked cellular changes during feeding site formation of the soybean cyst nematode (Heterodera glycines) indicate major changes in soybean gene expression. We used differential display of mRNA to detect host gene expression changes during the early compatible interaction between soybean and H. glycines. Fifteen cDNA clones corresponding to mRNAs with different abundances in H. glycines-infected versus uninfected roots were identified. Differential display results indicated that abundances of five mRNAs increased in infected roots, whereas abundances of 10 mRNAs decreased. Transcripts for nine of these 15 cDNAs could be detected on RNA blots, and their hybridization signals confirmed the differential display results for eight of these nine cDNAs. Sequence analyses identified five cDNAs with decreased mRNA levels in infected roots as corresponding to two putative aldolase genes, a transcription-factor TFIIA homologue, the soybean small GTP-binding protein gene sra1, and the soybean auxin down-regulated gene ADR12. RNA blot analyses of other auxin down-regulated genes revealed a decrease in their mRNA abundances in H. glycines-infected roots as well.
NifA-Dependent Expression of Glutamate Dehydrogenase in Rhizobium etli Modifies Nitrogen Partitioning During Symbiosis
Authors: Mendoza Alberto , Valderrama Brenda , Leija Alfonso , Mora Jaime
Published: May 2007

Constitutive expression of foreign glutamate dehydrogenase in Rhizobium etli inhibits bean plant nodulation (A. Mendoza, A. Leija, E. Martínez-Romero, G. Hernández, and J. Mora. Mol. Plant-Microbe Interact. 8:584-592, 1995). Here we report that this inhibition is overcome when controlling gdhA expression by NifA, thus delaying the GDH activity onset after nodule establishment. Expression of gdhA modifies the nitrogen partitioning inside the bacteroid, where newly synthesized ammonia is preferentially incorporated into the amino acid pool instead of being exported to the infected cells. As a consequence, the fixed nitrogen transport to the leaves, measured as the ureides content in xylem sap, is significantly reduced. Nitrogenase activity, although not nifHDK expression, is significantly reduced in bacteroids expressing gdhA, probably due to the utilization of energy and reducing power for nitrogen assimilation. Here we show that ammonia assimilation inside R. etli bacteroids is active, albeit at low levels, and when enhanced is deleterious to the symbiotic performance. This leads us to believe that further reduction of the basal nitrogen metabolism in the bacteroid might stimulate the nitrogenase activity and increase the nitrogen supply to the plant.
TheRhizobium etli amtBGene Coding for an NH4+Transporter Is Down-Regulated Early During Bacteroid Differentiation
Authors: Taté Rosarita , Riccio Anna , Merrick Mike , Patriarca Eduardo J.
Published: May 2007

During development of root nodules, Rhizobium bacteria differentiate inside the invaded plant cells into N2-fixing bacteroids. Terminally differentiated bacteroids are unable to grow using the ammonia (NH3) produced therein by the nitrogenase complex. Therefore, the nitrogen assimilation activities of bacteroids, including the ammonium (NH4+) uptake activity, are expected to be repressed during symbiosis. By sequence homology the R. etli amtB (ammonium transport) gene was cloned and sequenced. As previously shown for its counterpart in other organisms, the R. etli amtB gene product mediates the transport of NH4+. The amtB gene is cotranscribed with the glnK gene (coding for a PII-like protein) from a nitrogen-regulated σ54-dependent promoter, which requires the transcriptional activator NtrC. Expression of the glnKamtB operon was found to be activated under nitrogen-limiting, free-living conditions, but down-regulated just when bacteria are released from the infection threads and before transcription of the nitrogenase genes. Our data suggest that the uncoupling between N2-fixation and NH3assimilation observed in symbiosomes is generated by a transcriptional regulatory mechanism(s) beginning with the inactivation of NtrC in younger bacteroids.
Temporal and Spatial Appearance of Recombinant Viruses Formed Between Cauliflower Mosaic Virus (CaMV) and CaMV Sequences Present in Transgenic Nicotiana bigelovii
Authors: Király Lorant , Bourque June E. , Schoelz James E.
Published: May 2007

Cauliflower mosaic virus (CaMV) strain CM1841 is able to recombine with a CaMV transgene sequence present in Nicotiana bigelovii. In the present study we have characterized the temporal and spatial appearance of recombinant viruses formed between CM1841 and the transgene within individual transgenic plants. CM1841 infections were initiated by mechanical inoculation and by agro-inoculation to nontransformed N. bigelovii and transgenic N. bigelovii that expressed the gene VI product of CaMV strain D4. In agroinoculated transgenic plants, inoculated leaf tissue turned necrotic around the point of agroinocu-lation, while chlorotic lesions appeared in the leaves inoculated with CM1841 virions. The first systemic symptoms in both agroinoculated and mechanically inoculated transgenic N. bigelovii consisted of necrotic patches. The predominant type of virus recovered from the inoculated and first systemically infected leaves was the wild-type CM1841 rather than a recombinant. As the infection progressed in the transgenic plants, symptoms changed from necrosis in the lower leaves to a chlorotic mosaic in the upper leaves. This shift in symptom type was associated with the recovery of recombinant viruses, indicating that the recombinants predominated only in later stages of pathogenesis.
A T-DNA from the Agrobacterium tumefaciens Limited-Host-Range Strain AB2/73 Contains a Single Oncogene
Authors: Otten Léon , Schmidt Julien
Published: May 2007

Agrobacterium tumefaciens strain AB2/73 isolated from Lippia canescens has been described as a limited-host-range strain. Its tumor-inducing (Ti) plasmid has been found to lack DNA homology to known T-DNAs (L. Unger, S. F. Ziegler, G. A. Huffman, V. C. Knauf, R. Peet, L. W. Moore, M. P. Gordon, and E. W. Nester. J. Bacteriol. 164:723–730, 1985). We have isolated a T-DNA from AB2/73 by using a heterologous border sequence as a probe. The AB2/73 T-DNA sequence (3,504 bp) is flanked by canonical border sequences, has no detectable DNA homology with other T-DNAs, and contains only two genes: lsn ( Lippia strain nopaline synthaselike gene) and lso ( Lippia strain oncogene). The lso gene induces nondif-ferentiating tumors on a limited number of hosts when transferred by a Ti plasmid from a wide-host-range strain. Part of the predicted Lso protein is weakly homologous to other Agrobacterium oncoproteins encoded by rolB, rolBTR, orf13, gene e, gene 5, and gene 3′. A 28-kb fragment corresponding to the virA to virE region was cloned by using a heterologous vir fragment as probe. The AB2/73 vir region is homologous to most of the C58 virulence region; however, the virA gene is most related to the virA gene of the Agrobacterium vitis limited-host-range strain Ag162.
SyrM1 of Rhizobium sp. NGR234 Activates Transcription of Symbiotic Loci and Controls the Level of Sulfated Nod Factors
Authors: Hanin Moez , Jabbouri Saïd , Broughton William J. , Fellay Rémy
Published: May 2007

One or more transcriptional regulators of the LysR class control transcription of rhizobial nodulation genes. In Rhizobium sp. NGR234, two copies of nodD (nodD1 and nodD2) are present on the symbiotic plasmid pNGR234a. The complete sequence of pNGR234a revealed two additional nodD homologues, syrM1 and syrM2. Competitive RNA hybridization analyses involving a mutant of syrM1 (NGRΔsyrM1) showed that a number of symbiotic genes (e.g., nolXBTUVW) are expressed in an syrM1-dependent manner. Assays in which regions upstream of nolB and nolW were fused to promotorless lacZ confirmed that SyrM1 is required for their late induction. Mutation of syrM1 also drastically reduced production of sulfated Nod factors as shown by reverse phase-thin layer chromatography (RP-TLC). SyrM1 controls sulfation of Nod factors via one of the two chromosomal nodPQ loci. It thus seems likely that syrM1 of NGR234 encodes a transcriptional activator that regulates the expression of genes involved in both the early and late stages of infection.
Identification of Novel Putative Regulatory Genes Induced During Alfalfa Nodule Development with a Cold-Plaque Screening Procedure
Authors: Frugier Florian , Kondorosi Adam , Crespi Martin
Published: May 2007

Until now very few plant genes with possible regulatory functions during nodule development have been isolated. We have used a modified cold-plaque screening method to identify new transcripts expressed at low levels that are induced during nodulation. Several clones were isolated and characterized by their mRNA expression patterns during nodule development and in spontaneous nodules. Sequence homology with known genes of other organisms indicated that transcripts corresponded to (i) “basic” genes probably required during the growth of the nodule organ (e.g., structural proteins), (ii) genes related to the metabolic adaptations taking place during nodule morphogenesis and function (e.g., carbonic anhydrase), and (iii) genes containing regulatory motifs and/or homologies (three clones out of the 20 identified). The latter genes encode a zinc-finger-containing protein, a putative protein kinase, and a Wilm's tumor (WT) suppressor homologue, respectively. Expression of the kinase and WT suppressor homologues was induced early in nodulation, although the latter was activated transiently. Accumulation of the Zn-finger gene transcripts was detected at a later stage of development and seems to be regulated in a complex manner. Hence, using a cold-plaque screening procedure, we could identify genes that may play regulatory roles in the signal transduction pathways activated during nodule development.
Magnaporthe grisea Pathogenicity Genes Obtained Through Insertional Mutagenesis
Authors: Sweigard James A. , Carroll Anne M. , Farrall Leonard , Chumley Forrest G. , Valent Barbara
Published: May 2007

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.
Xanthomonas campestris pv. campestris Requires a Functional pigB for Epiphytic Survival and Host Infection
Authors: Poplawsky A. R. , Chun W.
Published: May 2007

When cauliflower plants (Brassica oleraceae) were misted with bacterial suspensions of Xanthomonas campestris pv. campestris (causal agent of black rot of cruciferous plants), two separate populations of the pathogen were associated with the leaves. Initially, bacteria removable by sonication and sensitive to sodium hypochlorite treatment predominated (easily removable epiphytic bacteria, EREB). However, after 2 weeks, bacteria not removable by sonication and insensitive to sodium hypochlorite treatment were dominant. Although the exact location of this second population of the pathogen was not determined, evidence is presented to support its location in protected sites on the leaf surface. pigB of this pathogen is required for production of extracellular polysaccharide (EPS), xanthomonadin pigments, and the diffusible signal molecule, DF (diffusible factor). DF can extracellularly restore EPS and xanthomonadin production to pigB mutant strains. Parent strain B-24 and pigB mutant strain B24-B2 were identical for in planta growth and symptomatology after artificial infection by injection in leaf mid-veins. Subsequently, X. campestris pv. campestris parent strain B-24, Tn3HoHo1 pigB insertion mutation strain B24-B2, chromosomally restored pigB mutation strain B24-B2R, and strain B24-79 with a Tn3HoHo1 insertion in an unrelated part of the genome were compared for epiphytic survival on, and natural infection of, cauliflower. After application, strains B-24, B24-B2R, and B24-79 all maintained leaf EREB populations of between approximately 3 and 6 (log [1 + CFU per g of fresh weight]) over a 3-week period, whereas B24-B2 populations fell to nearly undetectable levels. Plants sprayed with strains B-24, B24-B2R, and B24-79 averaged between 1.0 and 1.2 lesions, whereas those sprayed with B24-B2 averaged only 0.03 lesions per plant after 3 weeks. Differences in EREB population levels did not explain the observed differences in host infection frequencies, and the results indicated that strain B24-B2 was reduced in its ability to infect the host via the hydathodes, but unaffected in infection via wounds. When strains B-24 and B24-B2 were mixed in equal numbers and sprayed on plants together, B24-B2 epiphytic populations were intermediate between those of B-24 applied alone and B24-B2 applied alone. These results indicate that a functional pigB is required for epiphytic survival and natural host infection under the experimental conditions tested, and suggest that DF, xanthomonadins, and EPS could all be important for survival of this pathogen on the leaf surface, and/or for host infection.
The Cryptic Plasmid of Agrobacterium tumefaciens Cointegrates with the Ti Plasmid and Cooperates for Opine Degradation
Authors: Vaudequin-Dransart Valérie , Petit Annik , Chilton W. Scott , Dessaux Yves
Published: May 2007

We crossed the Agrobacterium tumefaciens chrysanthemum strain ANT4, which harbors four plasmids, with the plasmid-free recipient C58.00RS. Transconjugants degrading the Amadori-opines chrysopine and deoxy-fructo-syl-oxo-proline (dfop) harbored the Ti plasmid of ANT4, termed pAtANT4b. Upon transfer to the recipient strain C58.00RS, pAtANT4b (pTiANT4) and pANT4a (the largest of the four plasmids of ANT4) could cointegrate. The cointegration of the two plasmids occurs at various places of the pTiANT4, a feature that may affect several functions of the Ti plasmid (e.g., opine degradation). Transcon-jugants utilizing the opine deoxy-fructosyl-glutamine (dfg) always harbored the large pAtANT4a. Other Agrobacterium strains, including nonpathogenic strains such as C58C1, naturally degraded dfg. Remarkably, strain C58C1 carries a large cryptic plasmid termed pAtC58 that also encodes dfg degradation. A screening of physiological traits additionally revealed that this plasmid allows utilization of octopine as sole nitrogen source after mutation. All these results demonstrate that the larger plasmid of A. tumefaciens is a catabolic plasmid and that both the “cryptic” plasmid and Ti plasmid cooperate for opine degradation.
Can We Explain RNA-Mediated Virus Resistance by Homology-Dependent Gene Silencing?
Authors: van den Boogaart Tom , Lomonossoff George P. , Davies Jeffrey W.
Published: May 2007

The use of viral sequences to produce virus-resistant plants is now almost a standard technique. A variety of sequences from a large number of viruses have been used but the mechanisms remain largely unknown. There are probably at least two distinct types of mechanisms operating: one requiring the expression of the viral protein(s) and the other dependent only on the presence of transgene-derived RNA. In this review, we will discuss this RNA-mediated resistance and its similarities with cosuppression, a recently described phenomenon leading to suppression of transgene expression and homologous sequences. We present a critical view of the current models available to explain this type of resistance.
Determining the Relationship Between Salicylic Acid Levels and Systemic Acquired Resistance Induction in Tobacco
Authors: Willits Michael G. , Ryals John A.
Published: May 2007

Salicylic acid (SA) has been proposed as the systemic signal for the induction of systemic acquired resistance (SAR). It has been suggested that SA is synthesized at the site of pathogen-induced necrosis and is translocated to induce SAR in uninfected leaves. Grafting studies between wild-type tobacco plants and plants that are unable to accumulate significant amounts of SA have shown that the large increase in SA accumulation seen in inoculated leaves is not necessary for SAR induction, suggesting that SA is not the primary systemic signal. However, these studies have not addressed whether decreased levels of SA accumulation in inoculated leaves are sufficient to fully induce SAR. In this study, we have determined the relationship between free SA levels in the inoculated leaf and SAR induction in tobacco. These results support our previous conclusion that SA is not likely to be the systemic signal.
Tartrate Utilization Genes Promote Growth ofAgrobacteriumspp. on Grapevine
Authors: Salomone J.-Y. , Szegedi E. , Cobanov P. , Otten L.
Published: May 2007

Crown gall on grapevine is mainly caused by Agrobacterium vitis, which metabolizes tartrate. Competition experiments between a tartrate-utilizing strain and its non-utilizing derivative showed that tartrate utilization confers a selective advantage on grapevine.
Requirements for syrM and nodD Genes in the Nodulation of Medicago truncatula by Rhizobium meliloti 1021
Authors: Smith Lucinda S. , Long Sharon R.
Published: May 2007

Medicago truncatula, a relative of alfalfa (Medicago sativa), has been proposed as a model plant system for study of its interaction with the symbiont Rhizobium meliloti. Differences in M. truncatula and alfalfa may result in distinct symbiosis behaviors for the two hosts when in association with wild-type or mutant bacterial strains. We have found that M. truncatula has a more stringent requirement for syrM and nodD3 than is seen in alfalfa. In particular, the lack of syrM is associated with arrested nodule development.
Targeted Disruption of a Melanin Biosynthesis Gene Affects Conidial Development and UV Tolerance in the Japanese Pear Pathotype of Alternaria alternata
Authors: Kawamura Chie , Tsujimoto Takashi , Tsuge Takashi
Published: May 2007

Structural analysis of the BRM2 gene involved in melanin biosynthesis of the Japanese pear pathotype of Alternaria alternata suggested that this gene encodes 1,3,8-trihydroxynaphthalene reductase. Targeted disruption of the BRM2 gene did not affect pathogenicity, vegetative growth, or the number of conidia produced. Targeted disruption, however, did reduce conidial size and septal number, suggesting that melanin is associated with conidial development. The conidia of brm2 mutant transformants were more sensitive to UV light than those of the wild type, demonstrating that melanin confers UV tolerance.
Carbamoylation of Azorhizobial Nod Factors Is Mediated by NodU
Authors: D'Haeze Wim , Van Montagu Marc , Promé Jean-Claude , Holsters Marcelle
Published: May 2007

Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group. LCO biosynthesis in A. caulinodans is dependent on the nodABCSUIJZnoeC operon. Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear. Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5α expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step.
Lipochitin Oligosaccharides from Rhizobium leguminosarum bv. viciae Reduce Auxin Transport Capacity in Vicia sativa subsp. nigra Roots
Authors: Boot Kees J. M. , van Brussel Anton A. N. , Tak Teun , Spaink Herman P. , Kijne Jan W.
Published: May 2007

Induction of the formation of root nodule primordia in legume roots by symbiotic rhizobia is probably preceded by a change in plant hormone physiology. We used a Vicia sativa (vetch) split root system to study the effect of inoculation with rhizobia or purified Nod factors (lipochitin oligosaccharides, LCOs) on polar auxin transport in roots. Addition of R. leguminosarum bv. viciae, the infective symbiote of vetch, to roots of its host plant reduced polar auxin transport capacity of these roots within 24 h, in contrast to addition of non-nodulating, Sym plasmid-cured rhizobia. Addition of purified vetch-specific LCOs (NodRlv-IV/V[18:4,Ac]) caused a transient reduction in as little as 4 h after application, while after 16 h a second, stronger, and prolonged inhibition was observed that lasted at least 48 h. This reduction of auxin transport capacity was in the same order of magnitude as inhibition by N-(1-naphthyl)phthalamic acid (NPA). Purified LCOs (NodRm-IV[16:2,Ac,S]) from Sinorhizobium meliloti, the symbiote of alfalfa, and chitopentaose were inactive, which indicates a specific effect of LCOs produced by R. leguminosarum bv. viciae. Auxin transport inhibition was restricted to the apical nodulation-susceptible part of the roots, whereas the upper parts of the roots showed no difference in auxin transport after treatment. The effect could be observed with as low as 10-9 M NodRlv-IV/V[18:4,Ac] LCOs. Reduction of auxin transport by LCOs could not be inhibited by nitrate. Since inhibition of auxin transport capacity preceded the first root cortical cell divisions that result in root primordium formation, our results suggest a direct relationship between LCOs, polar auxin transport, and root nodule initiation, consistent with the hypothesis of U. Mathesius, H. R. M. Schlaman, H. P. Spaink, C. Sautter, B. G. Rolfe, and M. A. Djordjevic (Plant J. 14:23–34, 1998). However, nonmitogenic NodRlv-IV/V[18:1,Ac] showed a similar effect, which suggests that mitogenicity results from additional effects, in concert with auxin transport inhibition.
Expression of Erwinia chrysanthemi Pectinase Genes pelI, pelL, and pelZ During Infection of Potato Tubers
Authors: Jafra Sylwia , Figura Izabela , Hugouvieux-Cotte-Pattat Nicole , Lojkowska Ewa
Published: May 2007

Erwinia chrysanthemi mutants, containing transcriptional fusions of one of the minor pectate lyase genes (pelI, pelL, pelZ) with the reporter gene encoding β-glucuronidase activity, were studied for their ability to cause disease symptoms and to synthesize pectinases after inoculation of potato tubers. The strains affected in pelI and pelL genes displayed reduced virulence on potato tubers, demonstrating the important role of these isoenzymes in soft rot disease. Inactivation of the pelZ gene slightly influences the ability to macerate. Analysis of the bacterial population showed rapid multiplication of bacteria during infection. Similar kinetics of growth were observed for all mutants and for the wild-type strain. Comparison of the mutants and the wild-type strain showed that the pelI, pelL, and pelZ mutants synthesized reduced levels of Pels. The expression of pelZ is fivefold higher in planta than in bacterial cultures. In contrast, both pelI and pelL are highly (10-fold factor) induced in planta, which is characteristic of the plant-inducible pectate lyases.
A Novel Class of Ectomycorrhiza-Regulated Cell Wall Polypeptides in Pisolithus tinctorius
Authors: Laurent Pascal , Voiblet Catherine , Tagu Denis , de Carvalho Dulcinéia , Nehls Uwe , De Bellis Roberta , Balestrini Raffaella , Bauw Guy , Bonfante Paola , Martin Francis
Published: May 2007

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.
Molecular Characterization of GmFOX2, an Evolutionarily Highly Conserved Gene from the Mycorrhizal Fungus Glomus mosseae, Down-Regulated During Interaction with Rhizobacteria
Authors: Requena Natalia , Füller Petra , Franken Philipp
Published: May 2007

Arbuscular mycorrhizal (AM) fungi form the most widespread symbiosis of the plant kingdom. More than 80% of vascular plants are susceptible to colonization by the zygomycetous fungi from the order Glomales, and profit significantly by the nutrient exchange between plant and fungus. However, knowledge of the biology of these fungi still remains elusive because of their obligate biotrophism and, up to now, unculturability. The molecular mechanisms underlying the presymbiotic stages and the cell-to-cell communication between AM fungi and other soil microorganisms are, particularly, unknown. Here, we study these aspects by means of a molecular approach to monitor changes in the gene expression of the fungus Glomus mosseae (BEG12) in response to the rhizobacterium Bacillus subtilis NR1. The bacterium was found to induce specific increases in mycelial growth as well as changes in expression of GmFOX2, a highly conserved gene encoding a multifunctional protein of the peroxisomal β-oxidation. We determined the gene structure and studied its expression in response to rhizobacteria at two time points. The results show that the fungus is able to change its gene expression in response to stimuli other than the plant.
Mycorrhiza-Induced Changes in Disease Severity and PR Protein Expression in Tobacco Leaves
Authors: Shaul Orna , Galili Shmuel , Volpin Hanne , Ginzberg Idit , Elad Yigal , Chet Ilan , Kapulnik Yoram
Published: May 2007

The development of leaf disease symptoms and the accumulation of pathogenesis-related (PR) proteins were monitored in leaves of tobacco (Nicotiana tabacum cv. Xanthinc) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices. Leaves of mycorrhizal plants infected with the leaf pathogens Botrytis cinerea or tobacco mosaic virus showed a higher incidence and severity of necrotic lesions than those of nonmycorrhizal controls. Similar plant responses were obtained at both low (0.1 mM) and high (1.0 mM) nutritional P levels and with mutant plants (NahG) that are unable to accumulate salicylic acid. Application of PR-protein activators induced PR-1 and PR-3 expression in leaves of both nonmycorrhizal and mycorrhizal plants; however, accumulation and mRNA steady-state levels of these proteins were lower, and their appearance delayed, in leaves of the mycorrhizal plants. Application of 0.3 mM phosphate to the plants did not mimic the delay in PR expression observed in the mycorrhizal tobacco. Together, these data strongly support the existence of regulatory processes, initiated in the roots of mycorrhizal plants, that modify disease-symptom development and gene expression in their leaves.
FISH Chromosome Mapping Allowing Karyotype Analysis in Medicago truncatula Lines Jemalong J5 and R-108-1
Authors: Cerbah Malika , Kevei Zoltan , Siljak-Yakovlev Sonja , Kondorosi Eva , Kondorosi Adam , Trinh Toan Hanh
Published: May 2007

Two lines of Medicago truncatula, R-108-1 and Jemalong J5, exhibit differences in their symbiotic properties, morphological markers, and capacity for regeneration and transformation. Using cytogenetic techniques, we detected differences in chromomycin A3 staining on the two sides of the secondary constriction of the nucleolar chromosome pair of J5 and R-108-1, while DAPI (4′, 6-diamidino-2-phenylindole) staining revealed no differences between the two lines. FISH (fluorescent in situ hybridization) analysis showed a difference between J5 and R-108-1 for the same 5S rRNA genes; three loci were found in J5 but only two loci in R-108-1. However, for the 18S-5.8S-26S rDNA genes only a single locus was found in both lines. Based on these analyses, a karyotype for the two lines was established.
A Putative ECF σ Factor Gene, rpoI, Regulates Siderophore Production in Rhizobium leguminosarum
Authors: Yeoman Kay H. , May Alex G. , deLuca Nicola G. , Stuckey Daniel B. , Johnston Andrew W. B.
Published: May 2007

A cloned Rhizobium leguminosarum gene, termed rpoI, when transferred to wild-type strains, caused overproduction of the siderophore vicibactin. An rpoI mutant was defective in Fe uptake but was unaffected in symbiotic N2 fixation. The RpoI gene product was similar in sequence to extra-cytoplasmic σ factors of RNA polymerase. Transcription of rpoI was reduced in cells grown in medium that was replete with Fe.
A Positive Role for Rhizobitoxine in Rhizobium-legume Symbiosis
Authors: Duodu Samuel , Bhuvaneswari T. V. , Stokkermans Thomas J. W. , Peters N. Kent
Published: May 2007

Although Bradyrhizobium elkanii is a mutualistic symbiont of legumes, it synthesizes a phytotoxin, rhizobitoxine, that causes chlorosis on a variety of legume hosts, giving a pathogenic character to these interactions. No positive role for rhizobitoxine has been previously demonstrated. Interestingly, rhizobitoxine inhibits the rate-limiting step for ethylene biosynthesis, a plant hormone known to inhibit or down-regulate nodule development. We hypothesized that rhizobitoxine plays a positive role in nodule development through its inhibition of ethylene biosynthesis. To test this hypothesis, host plants of B. elkanii were screened for a differential nodulation response to the wild-type and rhizobitoxine mutant strains. In Vigna radiata (mungbean), the rhizobitoxine mutant strains induced many aborted nodules arrested at all stages of pre-emergent and post-emergent development and formed significantly fewer mature nodules than the wild type. Experiments revealed that nodulation of mungbean plants is sensitive to exogenous ethylene, and that the ethylene inhibitors aminoethoxyvinylglycine and Co2+ were able to partially restore a wild-type nodulation pattern to the rhizobitoxine mutants. This is the first demonstration of a nodulation phenotype of the rhizobitoxine mutants and suggests that rhizobitoxine plays a positive and necessary role in Rhizobium-legume symbiosis through its inhibition of ethylene biosynthesis.
Mitotic Stability of Infection-Induced Resistance to Plum Pox Potyvirus Associated with Transgene Silencing and DNA Methylation
Authors: Guo Hui Shan , López-Moya Juan José , García Juan Antonio
Published: May 2007

Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated with resetting of transgene DNA methylation. On the basis of these and other published results, we present a general model for post-transcriptional gene silencing in which RNA signals, generated either by a silenced nuclear gene or by virus replication, both activate a specific cytoplasmic RNA degradation pathway and induce changes (in particular, DNA methylation) in homologous nuclear genes that switch them from an active to a silenced status.
A Synergism Induced by Satellite Panicum Mosaic Virus
Authors: Scholthof Karen-Beth G.
Published: May 2007

Panicum mosaic virus (PMV) induces a mild mottle on millet plants, but the addition of its satellite virus (SPMV) causes a severe chlorosis and stunting. Immunoblots, RNA blots, and sucrose density gradient analyses revealed increases of PMV RNA and its p8 and capsid proteins when plants were co-infected with SPMV. A unique feature associated with this satellite virus interaction was an increased rate of systemic infection of PMV.
Comparison of Tobamovirus Coat Protein Structural Features That Affect Elicitor Activity in Pepper, Eggplant, and Tobacco
Authors: Dardick Christopher D. , Taraporewala Zenobia , Lu Bin , Culver James N.
Published: May 2007

Specific mutations designed to affect the tertiary, quaternary, and surface structures of the tobacco mosaic tobamovirus (TMV) coat protein (CP) were compared for their effect on CP elicitor activity in pepper (L1 gene), eggplant, and tobacco (N′ gene). Recognition by all three hosts was dependent upon the maintenance of the CP's α-helical bundle. However, variations in recognition resulted from mutations that affected different CP surface features and/or quaternary configurations. The relationship between CP structure and recognition in these hosts is discussed.
Separate Regions on the Tomato Bushy Stunt Virus p22 Protein Mediate Cell-to-Cell Movement versus Elicitation of Effective Resistance Responses
Authors: Chu Meihua , Park Jong-Won , Scholthof Herman B.
Published: May 2007

The objective of the present study on tomato bushy stunt virus (TBSV) was to determine the relationship between TBSV p22 protein domains that control its viral cell-to-cell movement function versus those that regulate the p22-mediated elicitation of necrotic local lesions on selected resistant plants. For this purpose, eight site-specific p22 mutants were generated that targeted clustered or individual charged amino acids that were presumed likely to be exposed on the protein surface and therefore candidates for interaction with viral RNA or host factors. Three cell-to-cell movement impaired mutants were identified, but expression of two such mutant p22 genes from a potato virus X gene vector showed that their encoded proteins retained the ability to elicit the formation of necrotic local lesions on Nicotiana edwardsonii. Conversely, inoculation of this normally resistant plant with two movement-positive p22 mutants resulted in chlorotic rather than necrotic lesions on inoculated leaves and infectious virus penetrated into the upper parts of young plants. These results suggest that p22 protein residues essential for cell-to-cell movement are separable from those that are crucial for the elicitation of effective resistance responses.
Origin of de Novo Synthesized Proteins in the Different Compartments of Pea-Rhizobium sp. Symbiosomes
Authors: Simonsen Anna Carina Wiborg , Rosendahl Lis
Published: May 2007

The origin of de novo synthesized proteins in the interface between pea and a Rhizobium sp. in root nodules has been determined. The symbiotic interface is defined as the peribacteroid space including proteins associated with either the symbiosome membrane or the bacteroid outer membrane. Two approaches have been used to study the origin of proteins in the symbiotic interface. First, to determine the localization of de novo synthesized plant-produced proteins in the symbiosomes, an in vitro protein translocation assay was established. To produce plant proteins poly A+ RNA was isolated from root nodules followed by in vitro translation in the presence of [35S]methionine. Subsequently, purified symbiosomes were incubated with the [35S]methionine-labeled plant proteins. The symbiosomes were subfractionated and de novo synthesized plant proteins in the different fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These import studies demonstrate the presence of plant proteins in the symbiosome membrane, peribacteroid space, and bacteroid membrane pellet. In contrast, no proteins of plant origin were detected in the bacteroid cytosol. Second, the presence of de novo synthesized bacteroid proteins in the interface was examined by incubation of symbiosomes isolated under micro-aerobic or aerobic conditions with [35S]methionine. Analysis of symbiosome compartments by SDS-PAGE and phosphor image revealed that proteins of bacteroid origin are primarily detected in the bacteroid cytosol and bacteroid membrane pellet. However, a few bacteroid-produced proteins are also observed in the symbiosome membrane. Together these data demonstrate that the majority of de novo synthesized proteins in the pea-Rhizobium sp. symbiotic interface are of plant origin.
Cloning and Characterization of an Esophageal-Gland-Specific Chorismate Mutase from the Phytoparasitic NematodeMeloidogyne javanica
Authors: Lambert Kris N. , Allen Keith D. , Sussex Ian M.
Published: May 2007

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant feeder cells. It is thought that nematodes inject secretions from their esophageal glands into plant cells while feeding, and that these secretions cause giant cell formation. To elucidate the mechanisms underlying the formation of giant cells, a strategy was developed to clone esophageal gland genes from the root-knot nematode Meloidogyne javanica. One clone, shown to be expressed in the nematode's esophageal gland, codes for a potentially secreted chorismate mutase (CM). CM is a key branch-point regulatory enzyme in the shikimate pathway and converts chorismate to prephenate, a precursor of phenylalanine and tyrosine. The shikimate pathway is not found in animals, but in plants, where it produces aromatic amino acids and derivative compounds that play critical roles in growth and defense. Therefore, we hypothesize that this CM is involved in allowing nematodes to parasitize plants.
The 5′ Noncoding Region of Grapevine Chrome Mosaic Nepovirus RNA-2 Triggers a Necrotic Response on ThreeNicotianaspp
Authors: Fernandez Isabelle , Candresse Thierry , Le Gall Olivier , Dunez Jean
Published: May 2007

The 5′ noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5′ NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5′ NCR showed that the three stem-loop structures at the 3′ end of the 5′ NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5′-most 70 nucleotides of the 5′ NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3′ NCR, the GCMV 5′ NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5′ NCR might modify PVX symptoms are discussed.