bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted Cell Wall Degradation at the Penetration Site of Cowpea Rust Basidiosporelings
Authors: Xu Haixin , Mendgen Kurt
Published: May 2007

Basidiospore germlings of the cowpea rust fungus (Uromyces vignae) penetrate the epidermal cell wall of the nonhost plant Vicia faba. In order to characterize the wall structure of the penetration site, leaves were high pressure frozen, freeze substituted, and embedded in appropriate resins. With antibodies against epitopes present in pectin, polygalacturonic acid, xyloglucan, and callose, we studied the modification of these wall components during infection. The density of epitopes was determined at the penetration site and compared with noninfected areas of the epidermal wall. Along the fungal penetration hypha, a zone of the plant wall, 0.2 μm wide, exhibited a reduced density of pectin and xyloglucan epitopes. A similar reduction of epitope density was also found for xyloglucan after treatment of sections from noninoculated plants with cellulase and xylanase and for pectin after treatment with pectinase. The density of polygalacturonic acid epitopes remained unchanged in the outer layer of the epidermal wall, but increased over the inner layer. A high density of polygalacturonic acid epitopes was found over a collarlike wall apposition produced by the plant cell along the penetration hypha. These results indicate that the fungus degrades the plant cell wall at the penetration site and that the plant cell secretes new wall material into this area to form the wall apposition.
Expression of the Potato Leafroll Virus ORF0 Induces Viral-Disease-like Symptoms in Transgenic Potato Plants
Authors: van der Wilk Frank , Houterman Petra , Molthoff Jos , Hans Fabienne , Dekker Ben , van den Heuvel Johannes , Huttinga Harm , Goldbach Rob
Published: May 2007

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.
Delayed Resistance to Plum Pox Potyvirus Mediated by a Mutated RNA Replicase Gene: Involvement of a Gene-Silencing Mechanism
Authors: Guo Hui Shan , García Juan Antonio
Published: May 2007

Nicotiana benthamiana plants were transformed with intact and mutated nuclear inclusion b (NIb) gene sequences of plum pox potyvirus, Rankovic isolate (PPV-R). The constructs included additional in-frame initiation and termination codons. All of the eight independently transformed lines showed some kind of protection against PPV; however, this protection was largely overcome at high inoculum doses. Interestingly, a phenotype of total recovery from the initial infection was observed in a high percentage of plants of two lines transformed with an NIb coding sequence that carried a Gly to Val mutation at the GDD motif typical of viral RNA replicases. Recovery frequently started with the emergence of dark green patches in the infected leaves that accumulated much less virus than the surrounding tissue. Newly developing leaves were symptomless and virus free, and showed highly effective, and very specific, resistance to virus reinoculation. Both in the dark green patches and in the symptomless leaves, virus decline was accompanied by a drastic reduction in the accumulation of transgene transcripts. Normal transgene mRNA levels and initial susceptibility to infection were recovered in the progeny plants coming from the autofecundation of a recovered plant, indicating that the recovery phenotype is not meiotically stable. The results are discussed in terms of a previously proposed model that links gene silencing and RNA-mediated virus resistance (J. J. English, E. Mueller, and D. C. Baulcombe, Plant Cell 8:179–188, 1996), adapted to explain the recovery phenomenon.
The Expression Level of the 3a Movement Protein Determines Differences in Severity of Symptoms Between Two Strains of Tomato Aspermy Cucumovirus
Authors: Moreno Ignacio M. , Bernal Juan José , de Blas Blanca García , Rodriguez-Cerezo Emilio , García-Arenal Fernando
Published: May 2007

Two strains of tomato aspermy cucumovirus, 1-TAV and V-TAV, differ in the severity of the symptoms induced in Nicotiana tabacum: 1-TAV induces a severe chlorotic mottle that appears 5 days post inoculation (d.p.i.) in the second systemic leaf, while V-TAV-infected plants show a mild chlorotic mottle, unevenly distributed in the leaf lamina, that appears 7 d.p.i. in the third or fourth systemic leaf. The manipulation of full-length cDNA clones giving infectious transcripts of V-TAV RNAs 1, 2, and 3 and 1-TAV RNA 3 revealed that the slow, mild phenotype of V-TAV maps to the movement protein (MP) gene. By site-directed mutagenesis it was further shown that this phenotype co-segregates with a single nucleotide substitution that introduces an in-frame UAA stop codon at the fourth position of the MP open reading frame of V-TAV. The presence of this stop codon results in a diminished expression of the MP in both tobacco protoplasts and leaves. Analyses of the progress of infection and of the time course of MP and coat protein accumulation show that the low level of MP in V-TAV-infected leaves limits the rate of cell-to-cell movement and leads to the mild phenotype. Data from the infectivity of RNA 3 transcripts with or without this stop codon, plus data from in vitro translation of virion or transcript RNA 3, suggest that the small amount of MP observed in V-TAV-infected leaves is expressed from a minor RNA 3 subpopulation lacking the stop codon.
Nod Factor-Induced Expression of Leghemoglobin to Study the Mechanism of NH4NO3 Inhibition on Root Hair Deformation
Authors: Heidstra Renze , Nilsen Gerd , Martinez-Abarca Francisco , van Kammen Ab , Bisseling Ton
Published: May 2007

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (±2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3 Nod factor perception and transduction leading to gene expression are unaffected.
Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean
Authors: Puri Nakul , Jenner Carol , Bennett Mark , Stewart Ruth , Mansfield John , Lyons Nigel , Taylor John
Published: May 2007

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY - strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.
Cloning and Characterization of Four Genes of Rhizobium leguminosarum bv. trifolii Involved in Exopolysaccharide Production and Nodulation
Authors: van Workum Wilbert A. T. , Cremers Hayo C. J. Canter , Wijfjes André H. M. , van der Kolk Christa , Wijffelman Carel A. , Kijne Jan W.
Published: May 2007

Four different genes of Rhizobium leguminosarum bv. tri-folii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an op-eron closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5′ and 3′ parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.
Expression of Cell Cycle Genes During Sesbania rostrata Stem Nodule Development
Authors: Goormachtig Sofie , Alves-Ferreira Marcio , Van Montagu Marc , Engler Gilbert , Holsters Marcelle
Published: May 2007

Upon infection of Sesbania rostrata with Azorhizobium caulinodans, nodules are formed on roots and stems. Stem nodules develop from abundantly distributed dormant root primordia. To acquire more insight into the meristem organization during stem nodule development, the expression patterns of a mitotic B1-type cyclin gene (Sesro; CycB1;1), a cyclin-dependent kinase gene (Cdc2-1Sr), and a histone H4 gene (H4-1Sr) of S. rostrata were followed by in situ hybridization. Cdc2-1Sr transcripts were found in all cells of uninfected and infected root primordia. In uninfected root primordia, Sesro;CycB1;1 transcripts were detected in a few cells of the apical root meristem whereas H4-1Sr transcripts were abundant in this region. Interestingly, after inoculation with A. caulinodans, H4-1Sr transcripts disappeared in the root meristem and a patchy pattern of Sesro;CycB1;1 and H4-1Sr expression appeared in the cortex of the root primordium, reflecting the formation of globular nodule primordia. When bacterial invasion started, a distal nodule meristem was delimited wherein Sesro;CycB1;1 and H4-1Sr expression was concentrated. Approximately 1 week after inoculation, meristem activity ceased, indicated by the loss of Sesro;CycB1;1 and H4-1Sr expression.
Sequence and Mutational Analysis of the 6.7-kb Region Containing nodAFEG Genes of Rhizobium sp. Strain N33: Evidence of DNA Rearrangements
Authors: Cloutier Jean , Laberge Serge , Antoun Hani
Published: May 2007

A 6.7-kb region upstream of nodBC genes in Rhizobium sp. strain N33 was shown to contain the nodAFEG genes and an open reading frame designated orfZ. The open reading frames for these genes contain 591, 282, 1209, 738, and 1,338 nucleotides respectively. Homologues of these genes were found in other rhizobia with the exception of orfZ, for which there was no counterpart found in the GenBank/EMBL database. Tn5 mutagenesis in nodEG and in the intergenic nodG-B region has shown a Nod+ phenotype on their temperate hosts Onobrychis viciifolia and Astragalus cicer. The nodules formed on O. viciifolia plants by these mutants were altered in shape and size. However, on A. cicer there was only a reduction in the number of nodules formed, compared with the wild-type strain. Sequence analysis of the orfZ-nodA and nodG-B intergenic regions indicates the presence of truncated nodD genes.
Comparison of avrD alleles from Pseudomonas syringae pv. glycinea
Authors: Keith Lisa Wolfson , Boyd Carol , Keen Noel T. , Partridge J. E.
Published: May 2007

Avirulence gene D alleles resided on indigenous plasmids in races 0, 2, 3, 4, 5, and 6 of Pseudomonas syringae pv. glycinea (Psg), but the allele in race 1 appeared to be chromosomal. These were all nonfunctional avirulence genes because they neither induced the avirulence phenotype on Rpg4 soybean cultivars nor directed the production of syringolide elicitors when expressed in Escherichia coli cells. The predicted proteins encoded by the seven Psg avrD genes were very similar to that of a functional class II allele from P. syringae pv. phaseolicola G50 race 2, but contained mutations collectively affecting only nine amino acid positions. Despite these relatively small amino acid differences and the location of avrD from each isolate on a 5.6-kb HindIII restriction fragment, the flanking regions varied considerably among the Psg isolates. The presence of avrD alleles with few alterations but different locational contexts in all tested Psg races argues that they provide an important selected function in the bacteria but have been modified to escape defense surveillance in Rpg4 soybean plants.
Mutagenesis by Insertion of Tn4001 into the Genome of Spiroplasma citri: Characterization of Mutants Affected in Plant Pathogenicity and Transmission to the Plant by the Leafhopper Vector Circulifer haematoceps
Authors: Foissac X. , Danet J. L. , Saillard C. , Gaurivaud P. , Laigret F. , Paré C. , Bové J. M.
Published: May 2007

Two hundred and fifty-seven transposon Tn4001 mutants of Spiroplasma citri strain GII3 were used for transmission assays by the leafhopper vector Circulifer haematoceps into periwinkle (Catharanthus roseus) plants. Multiplication of the mutants in the two hosts, the leafhopper and the plant, as well as the symptom expression in the plant were studied. Two mutants, GMT 470 and GMT 553, caused no symptoms on plants. Tn4001 is inserted as a single copy in the genome of these mutants. Mutant GMT 470 did not multiply, or multiplied only poorly, in the leaf-hopper and was not transmitted by the insect to the plant, nor to culture medium through Parafilm membrane. The growth rate of GMT 470 in SP4 medium was twice as slow as that of wild-type strain GII3. Mutant GMT 553 multiplied in the leafhopper as well as the wild-type spiro-plasma, and was transmitted by the leafhoppers into the plants, where it reached the same titers as the wild-type strain but in approximately twice as much time. The plants containing high titers of mutant GMT 553 remained symptomless for several weeks. However, symptoms began to develop at a time when revertants that had lost the transposon were detected.
Two Separate Regions in the Genome of the Tobacco Etch Virus Contain Determinants of the Wilting Response of Tabasco Pepper
Authors: Chu Meihua , Lopez-Moya J. J. , Llave-Correas Cesar , Pirone T. P.
Published: May 2007

Infection of Tabasco pepper by the tobacco etch virus (TEV) typically causes wilting associated with root necrosis. However, a strain of TEV, designated TEV nonwilting (TEV NW), is able to infect Tabasco pepper plants but does not cause wilting. In order to locate the genetic determinants responsible for the wilting response, a full-length cDNA clone of TEV NW from which infectious transcripts can be derived was made. A number of chimeric constructs were prepared by substituting cDNA fragments between TEV HAT (which causes wilting) and TEV NW clones. This approach was used to identify two wilting determinants in TEV HAT: one encompasses the 3′ one-third of the P3 coding region; the other spans the 3′ end of the CI, the 6-kDa protein, and the 5′ end of the VPg-NIa coding regions. Substitution of both these TEV NW fragments into TEV HAT resulted in infection but not wilting of Tabasco pepper, while the replacement of either of the fragments alone did not alter the wilting response. This indicates that both TEV NW regions contain determinants necessary but not sufficient to alter the wilting response and that both must be present in order to avoid the wilting response. There was no difference between the in vitro transcription-translation products derived from constructs containing these regions from TEV HAT and TEV NW.
Environmental Signals Modulate the Expression of an Indole-3-Acetic Acid Biosynthetic Gene in Erwinia herbicola
Authors: Brandl Maria T. , Lindow Steven E.
Published: May 2007

The ipdC gene of Erwinia herbicola strain 299R encodes an indolepyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA). Transcriptional fusions of ipdC to an ice nucleation reporter gene (inaZ) were used to study the expression of ipdC in vitro and in situ on plants. ipdC was expressed only at low levels in liquid media and independently of factors such as richness of the medium, pH, nitrogen availability, the presence of l-tryptophan or oxygen, and growth phase of the culture. However, the transcriptional activity of ipdC increased approximately 18-fold under low solute and matric potentials in culture. ipdC was also induced 32-fold on leaves of bean and tobacco and 1,000-fold on pear flowers. This is the first report of the plant-inducible transcription of a bacterial IAA biosynthetic gene. It strongly supports the role of ipdC, and thus that of the indole-3-pyruvic acid pathway, in IAA biosynthesis by strain 299R in situ. The plant induction and apparent regulation of ipdC by low water availability indicate that this gene, and presumably IAA synthesis, are involved in a response to conditions encountered by E. herbicola in its natural habitat on leaves.
New Rhizobium leguminosarum Flavonoid-Induced Proteins Revealed by Proteome Analysis of Differentially Displayed Proteins
Authors: Guerreiro Nelson , Redmond John W. , Rolfe Barry G. , Djordjevic Michael A.
Published: May 2007

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4′-dihydroxy-flavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N terminus. N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.
Specific Flavonoids Promote Intercellular Root Colonization ofArabidopsis thalianabyAzorhizobium caulinodansORS571
Authors: Gough Clare , Galera Christine , Vasse Jacques , Webster Gordon , Cocking Edward C. , Dénarié Jean
Published: May 2007

The ability of Azorhizobium caulinodans ORS571 and other diazotrophic bacteria to internally colonize roots of Arabidopsis thaliana has been studied. Strains tagged with lacZ or gusA reporter genes were used, and the principal colonization sites were found to be the points of emergence of lateral roots, lateral root cracks (LRCs). High frequencies of colonization were found; 63 to 100% of plants were colonized by ORS571, and 100% of plants were colonized by Herbaspirillum seropedicae. After LRCs were colonized, bacteria moved into intercellular spaces between the cortical and endodermal cell layers. Specific flavonoids, naringenin and daidzein, at 5 × 10-5M, significantly promoted colonization by ORS571. By using a nodC and a nodD mutant of ORS571, it was shown that neither Nod factors nor NodD are involved in colonization or flavonoid stimulation of colonization. Flavonoids did not appear to be stimulating LRC colonization by their activity as nutritional factors. LRC and intercellular colonization by H. seropedicae was stimulated by naringenin and daidzein at the same concentration that stimulated colonization by ORS571.
Evidence That the Pseudomonas syringae pv. syringae hrp-Linked hrmA Gene Encodes an Avr-Like Protein That Acts in an hrp-Dependent Manner Within Tobacco Cells
Authors: Alfano James R. , Kim Han-Suk , Delaney Terrence P. , Collmer Alan
Published: May 2007

A 25-kb DNA region, previously cloned from Pseudomonas syringae pv. syringae 61 in cosmid pHIR11, enables non-pathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum). hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR. DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein. DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P. syringae pv. syringae B728a, P. syringae pv. tabaci 11528, and P. syringae pv. glycinea race 4 U1, but present in P. syringae pv. tomato DC3000. A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P. syringae pv. syringae B728a and P. syringae pv. tabaci 11528. The presence of the hrmA locus had no apparent effect on the ability of P. syringae pv. syringae B728a to cause brown spot of bean, but it caused P. syringae pv. tabaci 11528 to elicit the defense-associated HR rather than disease in N. tabacum cvs. Xanthi N and Xanthi NC and N. clevelandii. Furthermore, N. debeyii, N. glutinosa, N. rustica, and N. tabacum cvs. Petit Havana and Samsun responded with the HR to P. fluorescens(pHIR11). In contrast, N. benthamiana-P. syringae pv. tabaci interactions were unaffected by the presence of HrmA, and P. fluorescens(pHIR11) did not elicit the HR in N. benthamiana. The hrmA ORF was sub-cloned into pFLAG-CTC, which expressed HrmA with a C-terminal FLAG synthetic epitope fusion. Escherichia coli MC4100 cells carrying the functional hrp cluster and the hrmA-FLAG derivative secreted the HrpZ harpin, but not HrmA-FLAG, to the medium, as indicated by immunoblot analysis with M2 anti-FLAG and polyclonal anti-HrpZ antibodies. The hrmA ORF was also subcloned into plant expression vector pFF19 and then biolistically delivered, along with pFF19G (expressing β-glucuroni-dase), into suspension-cultured tobacco cells. Histochemical staining 24 h later revealed substantial β-glucuroni-dase activity in cells receiving pFF19G and pFF19 but not in those receiving pFF19G and pFF19-HrmA. Thus, internal production of HrmA was deleterious to tobacco cells.
Functional and Regulatory Analysis of the Two Copies of the fixNOQP Operon of Rhizobium leguminosarum Strain VF39
Authors: Schlüter Andreas , Patschkowski Thomas , Quandt Jürgen , Selinger L. Brent , Weidner Stephan , Krämer Maria , Zhou Luming , Hynes Michael F. , Priefer Ursula B.
Published: May 2007

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the hemecopper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixNgusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation.
Geminivirus Resistance in Transgenic Tobacco Expressing Mutated BC1 Protein
Authors: Duan Y.-P. , Powell C. A. , Webb S. E. , Purcifull D. E. , Hiebert E.
Published: May 2007

Tobacco explants were transformed by Agrobacterium-mediated transformation with sense and antisense constructs of the movement protein genes (BC1 and BV1) of tomato mottle geminivirus (TMoV). Transgenic plants were tested for virus resistance either by exposure to viruliferous whiteflies carrying TMoV or cabbage leaf curl geminivirus (CabLCV) for a 72-h inoculation period or by continuous exposure to TMoV during the life of the plants. The transgenic lines were scored for disease symptoms, and virus replication and distribution were determined by enzyme-linked immunosorbent assay and dot blot hybridizations. Transgenic plants which expressed a mutated form (identified in a previous study) of the BC1 gene showed TMoV and CabLCV resistance. Three resistant phenotypes were observed: a delay in symptom development, a recovery from early symptoms, and an absence of virus symptoms at all stages. Geminivirus was detected in inoculated leaves but was not readily detected in leaves beyond the inoculation sites in the highly resistant plants. The geminivirus resistance conferred by expression of the spontaneously mutated BC1 gene (detected after transformation) in transgenic tobacco may involve transdominant negative interference.
Characterization of Acquired Resistance in Lesion-Mimic Transgenic Potato Expressing Bacterio-Opsin
Authors: Abad Mark S. , Hakimi Salim M. , Kaniewski Wojciech K. , Rommens Caius M. T. , Shulaev Vladimir , Lam Eric , Shah Dilip M.
Published: May 2007

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this “engineered” resistance is likely to be limited to certain pathogens and particular cultivars.
Spatial and Temporal Induction of Cell Death, Defense Genes, and Accumulation of Salicylic Acid in Tobacco Leaves Reacting Hypersensitively to a Fungal Glycoprotein Elicitor
Authors: Dorey Stephan , Baillieul Fabienne , Pierrel Marie-Agnès , Saindrenan Patrick , Fritig Bernard , Kauffmann Serge
Published: May 2007

We have analyzed the spatial and temporal expression of defense responses induced in attached tobacco leaves treated with a Phytophthora megasperma glycoprotein that was previously shown to be an efficient elicitor of the hypersensitive reaction. The infiltrated tissue (zone 1), the surrounding tissue (zone 2), which is 5 mm in width, and tissue at a distance >2 cm from zone 2 (zone 3) were analyzed separately. Cell death occurred only in zone 1 and was completed by 14 h. Defense gene expression was induced in zones 1 and 2 with striking differential patterns, but not in zone 3. There was a rapid (1 to 4 h) induction of genes of the phenylpropanoid, salicylic acid, and ses-quiterpenoid pathways in zones 1 and 2. However, it was strong and transient in the former and of lesser extent but sustained in the latter. High amounts of scopoletin, a phenylpropanoid metabolite, were found synthesized in zone 2. Pathogenesis-related (PR) transcripts and the corresponding PR proteins accumulated in high amounts in zone 2 rather late after the treatment (24 h). Salicylic acid was synthesized in both zone 1 and zone 2. Using a radioactively labeled elicitor, we demonstrated that the elicitor remained strictly localized to zone 1. The results provide strong evidence that an endogenous cell-to-cell signaling mechanism was triggered and occurred between the elici-tor-treated cells (zone 1) and the surrounding cells (zone 2), where it induces strong defense responses without cell death.
The In Planta-Produced Extracellular Proteins ECP1 and ECP2 of Cladosporium fulvum Are Virulence Factors
Authors: Laugé Richard , Joosten Matthieu H. A. J. , Van den Ackerveken Guido F. J. M. , Van den Broek Henk W. J. , De Wit Pierre J. G. M.
Published: May 2007

The two extracellular proteins ECP1 and ECP2 are abundantly secreted by the plant-pathogenic fungus Cladosporium fulvum during colonization of the intercellular space of tomato leaves. We examined the involvement of both proteins in pathogenicity and virulence of this fungus. ECP1-deficient, ECP2-deficient, and ECP1/ECP2- deficient isogenic C. fulvum strains were created by targeted gene replacement. Upon inoculation onto susceptible 6-week-old tomato plants, all three mutants showed reduced virulence. Deficiency in ECP2 resulted in a strain that poorly colonized the leaf tissue and secreted lower amounts of the in planta-produced ECP3, AVR4, and AVR9 proteins than the wild-type strain. The ECP2-deficient strain produced little emerging mycelium and few conidia. Deficiency in ECP1 did not significantly modify colonization of the leaf tissue, but reduced secretion of in planta-produced proteins. The ECP1-deficient strain emerged from stomata of the lower epidermis, but failed to sporulate as abundantly as the wild-type strain. A strain deficient in both ECP1 and ECP2 proteins had a phenotype similar to that of the ECP2-deficient strain. Accumulation of pathogenesis-related proteins and induction of late responses, such as leaf desiccation and abscission, occurred more quickly and more severely in tomato after inoculation with the ECP1-, ECP2-, and ECP1/ECP2-deficient strains than after inoculation with the wild-type strain. Moreover, partial collapse of stomatal guard cells occurred at emergence of the ECP2-deficient strain. These results indicate that the ECP1 and ECP2 proteins play a role in virulence of C. fulvum on tomato and suggest that both are involved in suppression of host defense responses.
Tissue Dependence and Differential Cordycepin Sensitivity of Race-Specific Resistance Responses in the Barley—Powdery Mildew Interaction
Authors: Schiffer Ruth , Görg Regina , Jarosch Birgit , Beckhove Uli , Bahrenberg Gregor , Kogel Karl-Heinz , Schulze-Lefert Paul
Published: May 2007

Epidermal cell monolayers prepared from partially dissected barley (Hordeum vulgare) coleoptiles were used for in vivo analysis of race-specific resistance to powdery mildew (Erysiphe graminis f. sp. hordei) specified by host genes Mla-1, Mla-12, and Mlg. Complete resistance governed by each of these genes is closely associated with hypersensitive cell death (hypersensitive response, HR) in primary leaf tissue. In contrast, Mla-12 coleoptile tissue reveals a fully compatible, Mla-1 coleoptile tissue a partially compatible, and Mlg coleoptile tissue an incompatible interaction upon challenge with pathogen races carrying corresponding avirulence functions. Quantitative recording of single plant-fungus interaction sites showed arrest of fungal development in papillae on Mlg coleoptiles. On Mla-1 and Mla-12 coleoptiles, attacked cells become predominantly penetrated by the fungus. Approximately one third of penetrated cells on Mla-1 coleoptiles subsequently undergo an HR. These sites reveal no further fungal development. Both Mlg and Mla-12 coleoptiles fail to mount an HR. The effect of cordycepin (3′-deoxyadenosine), an inhibitor of mRNA synthesis, was studied in planta on primary leaf tissue of Mla-12 and Mlg genotypes. Host cell death triggered by either gene is reduced to background levels observed in the near-isogenic compatible interaction and exhibits the same dose-dependent cordycepin sensitivity. Inhibition of Mlg-triggered, single-cell HR is not accompanied by release of fungal growth arrest, indicating cordycepin insensitivity of a papillae-associated resistance component. The data suggest that host cell death is a requisite component for expression of Mla-type but not Mlg-type resistance.
Characterization of Two Plasmid-borne lpsβ Loci of Rhizobium etli Required for Lipopolysaccharide Synthesis and for Optimal Interaction with Plants
Authors: García-de los Santos Alejandro , Brom Susana
Published: May 2007

In Rhizobium etli CFN42, both the symbiotic plasmid (pd) and plasmid b (pb) are required for effective bean nodulation. This is due to the presence on pb of a region (lpsβ) involved in lipopolysaccharide (LPS) biosynthesis. We report here the genetic array and functional features of this plasmid-borne region. The sequence analysis of a 3,595-bp fragment revealed the presence of a transcriptional unit integrated by two open reading frames (lpsβ1 and lpsβ2) essential for LPS biosynthesis and symbiosis. The lpsβ1 encodes a putative 193 amino acid polypeptide that shows strong homology with glucosyl-1P and galactosyl-1P transferases. The deduced amino acid sequence of the protein encoded by lpsβ2 was very similar to that of proteins involved in surface polysaccharide biosynthesis, such as Pseudomonas aeruginosa WpbM, Bordetella pertussis BplL, and Yersinia enterocolitica TrsG. DNA sequences homologous to lpsβ1 and lpsβ2 of R. etli CFN42 were consistently found in functionally equivalent plasmids of R. etli, R. leguminosarum bv. viciae, and R. leguminosarum bv. trifolii strains, but not in R. meliloti, R. loti, R. tropici, R. fredii, Bradyrhizobium, Azorhizobium, and Agrobacterium tumefaciens. Even though Rhizobium and Agrobacterium do not share lpsβ sequences, their presence is required for crown-gall tumor induction by R. etli transconjugants carrying the Ti plasmid.
Compounds Exuded byPhaseolus vulgarisThat Induce a Modification ofRhizobium etliLipopolysaccharide
Authors: Duelli Dominik M. , Noel K. Dale
Published: May 2007

Exudates released from germinating seeds and roots of a black-seeded bean (Phaseolus vulgaris cv. Midnight Black Turtle Soup) induce an antigenic change in the lipopolysaccharide (LPS) of Rhizobium etli CE3. By spectroscopic analyses and chromatographic comparisons with derived standards, the chemical structures of the aglycone portions of the major inducing molecules from seed exudate were deduced, and they were identified as delphinidin, cyanidin, petunidin, and malvidin. These anthocyanidins were present in seed exudate mainly as glycosides, the chief inducer being delphinidin 3-glucoside. Also present were 3-glucosides of petunidin and malvidin and glycosides of cyanidin and delphinidin. Seed exudate from a bean variety deficient in anthocyanins did not induce the LPS conversion. The ability of root exudate to induce an antigenic change in the LPS was due to compounds other than anthocyanins.
The Activity of Lipid A and Core Components of Bacterial Lipopolysaccharides in the Prevention of the Hypersensitive Response in Pepper
Authors: Newman Mari-Anne , Daniels Michael J. , Dow J. Maxwell
Published: May 2007

Pre-treatment of leaves of pepper (Capsicum annuum) with lipopolysaccharide (LPS) preparations from enteric bacteria and Xanthomonas campestris could prevent the hypersensitive response caused by an avirulent X. campestris strain. By use of a range of deep-rough mutants, the minimal structure in Salmonella LPS responsible for the elicitation of this effect was determined to be lipid A attached to a disaccharide of 2-keto-3-deoxyoctulosonate; lipid A alone and the free core oligosaccharide from a Salmonella Ra mutant were not effective. For Xanthomonas, the core oligosaccharide alone had activity although lipid A was not effective. The results suggest that pepper cells can recognize different structures within bacterial LPS to trigger alterations in plant response to avirulent pathogens.
The Coat Protein Is Dispensable for the Establishment of Systemic Infections by Pea Enation Mosaic Enamovirus
Authors: Skaf Jihad S. , Rucker Deborah G. , Demler Steven A. , Wobus Christiane E. , de Zoeten Gustaaf A.
Published: May 2007

Deletions in the coat protein (CP) open reading frame (ORF)-3 of pea enation mosaic enamovirus (PEMV) did not inhibit the systemic movement of PEMV. As early as 48 h postinoculation, truncated viral RNAs were detected in the roots and uninoculated leaves of infected plants. The detected concentrations of truncated RNAs varied significantly between experiments but were similar to those extracted from plants infected with wild-type (WT)-PEMV. Pea plants infected with CP-less mutants exhibited wilting and necrosis that appeared faster and were more severe than observed in infections with WT-PEMV. Moreover, the absence of the CP-ORF and its protein product did not affect the formation of either double-membrane bound vesicles or replication complexes characteristic of infections by PEMV. The potential role of these structures as the movement vehicles of the infectious entity of PEMV in plants is discussed.
Association of Distinct Double-Stranded RNAs with Enhanced or Diminished Virulence in Rhizoctonia solani Infecting Potato
Authors: Jian JianHua , Lakshman Dilip K. , Tavantzis Stellos M.
Published: May 2007

A virulent field isolate of Rhizoctonia solani AG 3, Rhs 1AP, has given rise to three sectors in a period of several years. Subculturing of these sectors resulted in three hypovirulent isolates—Rhs 1A1, Rhs 1A2, and Rhs 1A3. We reported previously that five genetically different double-stranded (ds) RNAs occur in these four isolates, with each isolate containing a unique combination of two or more dsRNAs. We report here that all five dsRNA elements occur in the cytoplasm, and none in the nucleus. The mitochondria contains low to moderate concentrations of the four larger dsRNAs. The four isolates were paired in selected combinations in an attempt to transmit specific dsRNAs to cultures lacking these dsRNAs. This approach generated groups of near-isogenic lines possessing the same dsRNA elements. As many as six cultures possessing the same dsRNA genotype were found to have the same degree of pathogenicity. Consistently, acquisition of a 6.4-kb dsRNA brought about increased virulence, whereas the presence or acquisition of a 3.6-kb dsRNA resulted in diminished virulence in the recipient culture. All cultures had the same mitochondrial DNA haplotype as that of the original field isolate, Rhs 1AP. This, in conjunction with the fact that all cultures in this study had a single nuclear DNA origin (Rhs 1AP), indicates a strong correlation between certain phenotypic characters or changes thereof and dsRNA profiles or acquisition of particular dsRNAs, respectively.
Alfalfa Mosaic Virus Movement Protein Induces Tubules in Plant Protoplasts
Authors: Zheng Huanquan , Wang Guiling , Zhang Lee
Published: May 2007

The alfalfa mosaic virus (AlMV) movement protein (MP) was fused with the green fluorescence protein (GFP). The MP-GFP fusion was transiently expressed in protoplasts prepared from Nicotiana tabacum, resulting in the production of long, extending, tubular structures protruding from the protoplast surface. Deletions of MP amino acids 1 to 77, 84 to 142, or 226 to 300 all affected tubule formation. Hence, the AlMV MP is solely required for the induction of the tubular structures, and the results suggest that AlMV may move from cell to cell via tubules.
A Novel Class of Elicitin-like Genes from Phytophthora infestans
Authors: Kamoun Sophien , Lindqvist Hannele , Govers Francine
Published: May 2007

Elicitins are a family of structurally related proteins that induce hypersensitive response in specific plant species. Two Phytophthora infestans cDNAs, inf2A and inf2B, potentially encoding novel elicitin-like proteins, were isolated from a cDNA library made from infected potato tissue. Multiple sequence alignments and phylogenetic analyses of 19 elicitins and elicitin-like proteins from nine Phytophthora spp. and from Pythium vexans suggest that there are at least five distinct classes within the elicitin family.
A Regulatory Locus, pehSR, Controls Polygalacturonase Production and Other Virulence Functions in Ralstonia solanacearum
Authors: Allen Caitilyn , Gay Jaqueline , Simon-Buela Laureano
Published: May 2007

We previously identified a locus that regulates production of polygalacturonase (PG), an extracellular plant cell wall-degrading enzyme important in bacterial wilt of plants caused by Ralstonia (Pseudomonas) solanacearum. The DNA sequence of this locus, called pehSR, was determined and two consecutive open reading frames (ORFs) of 1,905 and 1,680 bp were identified. The amino acid sequences predicted to be encoded by these ORFs are similar to those of regulators of pilin synthesis in Pseudomonas aeruginosa and Myxococcus xanthus and to a regulator of flagellin synthesis and adhesion in P. aeruginosa, as well as to other two-component regulators of the NtrB/C subfamily. pehSR mutants produced negligible levels of endo-PG activity, while exo-PG activity was reduced by 50%. Northern (RNA) blot analysis showed that PehSR regulates endo-PG expression at the transcriptional level. pehSR mutants grew normally in culture and in planta but were dramatically reduced in virulence; this loss of virulence was substantially greater than that observed for endo-PG structural gene mutants, suggesting that pehSR regulates additional factors important in virulence. Although pehSR mutants were essentially nonmotile, like the wild-type strain, multiple copies of pehSR conferred motility on the bacterium. Reporter gene studies indicated that pehSR expression increased when bacteria grew in plant tissue, and that the pehSR locus was itself negatively regulated by the global virulence gene regulator PhcA.
Iron-Dependent Transcription of the Regulatory Gene ros of Agrobacterium radiobacter
Authors: Hussain H. , Johnston A. W. B.
Published: May 2007

Transcription of the regulatory gene ros of Agrobacterium radiobacter requires growth in the presence of Fe although this regulation was not mediated by ros itself. The ros gene repressed its own transcription, independently of the Fe status of the growth media. It was shown that the two cysteine residues in the Ros protein were essential for the complementation of the exopolysaccharide synthesis defect of ros mutant strains. It was found that the mutation in one exo mutant strain that was complemented both by ros and by the “structural” exoY gene was not, in fact, in ros but is in some other, unknown gene. The two cysteines were also essential for the correction of this mutant. This mutant affected the expression of exoY but not of ros.