bluuebunny/crossref_metadata_2025 huggingface dataset:
Seed Lipoxygenase Products Modulate Aspergillus Mycotoxin Biosynthesis
Authors: Burow G. B. , Nesbitt T. C. , Dunlap J. , Keller N. P.
Published: May 2007

Oilseed crops are frequently subject to contamination by mycotoxins produced by Aspergillus spp., particularly aflatoxin (AF) and to a lesser extent sterigmatocystin (ST). Several studies have suggested that metabolites generated from the plant lipoxygenase (LOX) pathway may either decrease or increase mycotoxin production by Aspergillus spp. We tested the possibility that the occurrence of seed LOX isozymes that produce distinct hydroperoxy fatty acids may account for these different effects on AF biosynthesis. For example, soybean LOX1 catalyzes the addition of O2 to the C13 position of linoleic and linolenic acids while maize embryo LOX catalyzes the addition of O2 to the C9 position. In vitro experiments showed that 13S-hydroperoxy fatty acids at concentrations of 10 and 100 μM repressed AF and ST pathway gene expression and significantly (P = 0.05) reduced AF and ST production in both A. parasiticus (AF producer) and A. nidulans (ST producer). Treatment with 1 μM 13S-hydroperoxy linoleic acid also significantly decreased AF production when introduced into growth media at continuous 24-h intervals. In contrast, the same concentrations of 9S-hydroperoxy linoleic acid did not reduce AF or ST production but extended the length of time AF and ST transcripts were detectable. These results show that 13S-hydroperoxy fatty acids directly or indirectly repress AF and ST biosynthesis and provide in vitro evidence that specific seed lipoxygenase activity could provide resistance to mycotoxin contamination by Aspergillus spp.
Induction of Chalcone Synthase Expression by Rhizobia and Nod factors in Root Hairs and Roots
Authors: Krause Andrea , Lan Vo T. T. , Broughton William J.
Published: May 2007

Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family that is abundantly transcribed in leaves and nodules. Inoculation with Rhizobium sp. NGR234, which nodulates V. unguiculata, or with NGRΔnodABC, a mutant deficient in Nod factor production, induced rapid accumulation of CHS mRNAs in roots and root hairs. As both Nod+ and Nod- bacteria provoke responses, induction of CHS gene expression may involve symbiotic or defense responses. Four days after inoculation with the wild-type Rhizobium sp., the transcript levels increased in roots but decreased in root hairs. Use of a region unique to the 5′ end of a specific CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24 h after inoculation with both rhizobia were specific to this gene. Transcripts of this gene in roots were only detectable 4 days after treatment with NGR234. It is possible therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia entering legume roots. Purified Nod factors induced accumulation of transcripts, showing that they might be part of the signal transduction pathway leading to CHS expression.
Continual Green-Fluorescent Protein Monitoring of Cauliflower Mosaic Virus 35S Promoter Activity in Nematode-Induced Feeding Cells in Arabidopsis thaliana
Authors: Urwin Peter E. , Møller Simon G. , Lilley Catherine J. , McPherson Michael J. , Atkinson Howard J.
Published: May 2007

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressively from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.
Characterization of In Planta—Induced Rust Genes Isolated from a Haustorium-Specific cDNA Library
Authors: Hahn Matthias , Mendgen Kurt
Published: May 2007

Rust fungi are plant parasites that depend on living host tissue for growth. For invasion of leaves, dikaryotic urediospores differentiate germ tubes and infection structures that penetrate through stomata. Biotrophic growth occurs by intercellular mycelia that form haustoria within host cells. A cDNA library was constructed from haustoria isolated from broad bean leaves infected by Uromyces fabae. Differential screening revealed that a high proportion (19%) of the haustorial cDNAs are specifically expressed in planta but are not expressed, or are much weaker, in germlings or infection structures produced in vitro. A total of 31 different in planta-induced genes (PIGs) were identified. Some of the PIGs are highly expressed in haustoria. The PIGs are single or low copy number genes in the rust genome. A variety of developmentally regulated expression patterns of PIG mRNAs were observed. Sequence analysis of PIG cDNAs revealed similarities to genes encoding proteins involved in amino acid transport, thiamine biosynthesis, short-chain dehy-drogenases, metallothioneins, cytochrome P-450 monooxy-genases, and peptidyl-prolyl isomerases.
Molecular Characterization and Expression of the Erwinia carotovora hrpNEcc Gene, Which Encodes an Elicitor of the Hypersensitive Reaction
Authors: Mukherjee Asita , Cui Yaya , Liu Yang , Chatterjee Arun K.
Published: May 2007

The nucleotide sequence of hrpNEcc DNA, cloned from Erwinia carotovora subsp. carotovora strain Ecc71, reveals a coding region of 1,068 bp which matches the size of hrpNEcc transcripts. hrpNEcc is predicted to encode a glycine-rich protein of approximately 36 kDa. Like the elicitors of the hypersensitive reaction (HR) produced by E. chrysanthemi (HarpinEch) and E. amylovora (HarpinEa), the deduced 36-kDa protein does not possess a typical signal sequence, but it contains a putative membrane-spanning domain. In Escherichia coli strains overexpressing hrpNEcc , the 36-kDa protein has been identified as the hrpNEcc product by Western blot analysis using anti-HarpinEch antibodies. The 36-kDa protein fractionated from E. coli elicits the HR in tobacco leaves. Moreover, a HrpN- and RsmA- double mutant (RsmA = regulator of secondary metabolites) does not produce this 36-kDa protein or elicit the HR, although this strain, like the RsmA- and HrpN+ bacteria, overproduces extracellular enzymes and macerates celery petioles. These observations demonstrate that hrpNEcc encodes the elicitor of the HR, designated HarpinEcc. The levels of hrpNEcc transcripts are affected in both RsmA+ and RsmA- strains by media composition and carbon sources, although the mRNA levels are substantially higher in the RsmA- strains. The expression of hrpNEcc in Ecc71 is cell density dependent and is activated by the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). By contrast, hrpNEcc expression in an RsmA- strain is independent of cell density, and substantial expression occurs in the absence of OHL. The effects of cultural conditions and the occurrence of putative cis-acting sequences, such as consensus σ54 promoters and an hrp promoter upstream of the transcriptional start site, indicate that the production of HarpinEcc in wild-type RsmA+ E. carotovora subsp. carotovora is tightly regulated. These observations, taken along with the finding that the HR is caused by RsmA- mutants but not by RsmA+ strains (Cui et al., 1996, Mol. Plant-Microbe Interact. 9:565–573), strongly support the idea that the inability of the wild-type pectolytic E. carotovora subsp. carotovora to elicit the HR is due to the lack of a significant level of HarpinEcc production.
Structural and Functional Conservation of the Tobamovirus Coat Protein Elicitor Active Site
Authors: Taraporewala Zenobia F. , Culver James N.
Published: May 2007

Elicitation of the N′ gene hypersensitive response (HR) in Nicotiana sylvestris is dependent upon specific structural elements of an elicitor active site located along the right face of the tobacco mosaic tobamovirus (TMV) coat protein (CP) (Z. Taraporewala and J. N. Culver, Plant Cell 8:169-178, 1996). In this study, the functional and structural conservation of this site was examined in three different tobamovirus CPs that vary widely in sequence homology but share a common three-dimensional fold. Results demonstrate that the CPs of TMV-U2, Odontoglossum ringspot, and cucumber green mottle mosaic tobamoviruses elicit the N′ gene HR with strength and temperature stability comparable to the TMV elicitor CP P20L. Within each CP, amino acid substitutions designed to disrupt tertiary structure interfered with N′ gene elicitation. Substitutions of selected surface residues within the previously identified elicitor active site of each CP inhibited HR elicitation, while substitutions outside of this region did not alter elicitor activity. Comparisons of surface characteristics within the elicitor active sites of the different CPs revealed the presence of a conserved central hydrophobic cavity. Surface features at the periphery of this cavity showed less conservation, which may explain the unequal ability of some substitutions in this region to alter the elicitor activity of the different CPs. These findings suggest N′ gene specificity is dependent upon the three dimensional fold of CP as well as upon specific surface features within the elicitor active site.
Two PR-1 Genes from Tomato Are Differentially Regulated and Reveal a Novel Mode of Expression for a Pathogenesis-Related Gene During the Hypersensitive Response and Development
Authors: Tornero Pablo , Gadea José , Conejero Vicente , Vera Pablo
Published: May 2007

Pathogenesis-related (PR) proteins form a heterogeneous family of plant proteins that are likely to be involved in defense and are inducible by pathogen attacks. One group of PRs, represented by the subfamily PR-1, are low-molecular-weight proteins of unknown biochemical function. Here we describe the cloning and characterization of two closely related genes encoding a basic and an acidic PR-1 protein (PR1b1 and PR1a2) from tomato (Lycopersicon esculentum). We present a comparative study of the mode of transcriptional regulation of these two genes in transgenic tobacco plants using a series of promoter-GUS fusions. Unexpectedly, the chimeric PR1a2/GUS gene is not induced by pathogenic signals but instead shows constitutive expression with a reproducible developmental expression pattern. It is expressed in shoot meristems, trichomes, and cortical cells as well as in vascular and nearby tissues of the mature stem. This constitutive expression pattern may represent preemption of plant defenses against potential pathogens. Conversely, the chimeric PR1b1/GUS gene does not show any constitutive expression in the plant, but it is transcriptionally activated following pathogen attack. Upon infection by tobacco mosaic virus, the PR1b1 gene is strongly activated locally in tissues undergoing the hypersensitive response but not systemically in uninoculated tissues. Furthermore, its expression is induced by both salicylic acid and ethylene precursors, two signals that coexist and apparently mediate the activation of local defenses during the hypersensitive response. We speculate that the different mode of expression of the two genes presented here, together with that reported previously for the induction of other PR-1 genes in systemic, uninoculated tissues, may all be complementary and necessary for the plant to acquire an efficient refractory state to resist pathogen attacks.
A Nodule-Specific Gene Family from Alnus glutinosa Encodes Glycine- and Histidine-Rich Proteins Expressed in the Early Stages of Actinorhizal Nodule Development
Authors: Pawlowski Katharina , Twigg Paul , Dobritsa Svetlana , Guan Changhui , Mullin Beth C.
Published: May 2007

Two cDNAs representing different members (agNt84 and ag164) of a gene family encoding glycine- and histidinerich proteins have been isolated from cDNA libraries from Alnus glutinosa root nodules. Expression of the corresponding genes could only be detected in nodules. With in situ hybridization, the expression in nodules was found to occur in young, infected cells of the prefixation zone (zone 2). The encoded proteins contain putative signal peptides for targeting to the endomembrane system, sharing sequence similarity with signal peptides from plant glycinerich proteins, among them nodulin 24, a nodule-specific protein from soybean. This similarity suggests that, analogous to nodulin-24, proteins encoded by agNt84/ag164 may be located at the interface between the host plant membrane and the matrix surrounding the endosymbiont. The 3′untranslated regions of the cDNAs contain unusual poly(AT)n stretches that may play a role in the regulation of RNA stability. The protein encoded by agNt84 cDNA was expressed in Escherichia coli as a fusion with maltosebinding protein, and was shown to have the ability to bind to a nickel-chelating resin, indicating that it may function as a metal-binding protein.
Mapping the Elicitor and Necrotic Sites of Phytophthora Elicitins with Synthetic Peptides and Reporter Genes Controlled by Tobacco Defense Gene Promoters
Authors: Perez Valérie , Huet Jean-Claude , Nespoulous Claude , Pernollet Jean-Claude
Published: May 2007

Elicitins are 10-kDa proteins secreted by Phytophthora and Pythium fungi that elicit a hypersensitive-like necrotic reaction, leading to resistance against fungal and bacterial plant pathogens. Induction of necrosis and resistance were previously shown to be borne by different sites of the molecule. Furthermore, sequence comparison indicated several potential residues necessary for necrosis. The role of one of these residues was previously evidenced with site-directed mutagenesis. In order to locate other necrosis-determining sites and reveal the defense-eliciting sites, we synthesized a series of synthetic peptides. Tests were performed on two types of transgenic tobacco plants, both transformed with a construction containing the β-glucuronidase reporter gene, in one case controlled by the promoter of the multiple stimulus response gene str 246C and in the other by the promoter of the pathogenesis-related gene PR1a. We report that only certain peptides were found to be active. Whereas PR1a induction was consistently correlated with induction of necrosis, four peptides were observed to induce only str 246C expression without necrosis, which led to differentiate the defense-eliciting sites from the necrotic sites. From the structure-function relationships thus obtained, two different defense pathways were inferred to be independently induced by elic-itins.
Polygalacturonase-Inhibiting Proteins (PGIPs) with Different Specificities Are Expressed inPhaseolus vulgaris
Authors: Desiderio A. , Aracri B. , Leckie F. , Mattei B. , Salvi G. , Tigelaar H. , Van Roekel J. S. C. , Baulcombe D. C. , Melchers L. S. , De Lorenzo G. , Cervone F.
Published: May 2007

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367–373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.
Role for Salicylic Acid in the Activation of Defense Responses in Catalase-Deficient Transgenic Tobacco
Authors: Du He , Klessig Daniel F.
Published: May 2007

Transgenic tobacco plants with severely reduced catalase activity were crossed with NahG plants that do not accumulate salicylic acid. Some of the progeny from the cross spontaneously developed necrosis when grown under strong light, similar to that observed on the parental severely catalase-deficient plants. However, in contrast to the parental catalase-deficient plants, these progeny plants did not constitutively express PR-1 genes or develop enhanced resistance. Thus, salicylic acid appears to be required for the induction of these defense responses in catalase-deficient tobacco plants.
A Rhizobium leguminosarum Biovar trifolii Locus Not Localized on the Sym Plasmid Hinders Effective Nodulation on Plants of the Pea Cross-Inoculation Group
Authors: Roest Henk P. , Mulders Ine H. M. , Spaink Herman P. , Wijffelman Carel A. , Lugtenberg Ben J. J.
Published: May 2007

Introduction of the Sym plasmid pRL1JI into the cured Rhizobium leguminosarum bv. trifolii strain RCR5 resulted in a strain, designated RBL5523, that was expected to nodulate plants of the pea cross-inoculation group. However, effective nodulation occurred only on Vicia sativa plants, not on V. hirsuta or Pisum sativum. After random Tn5 mutagenesis, a derivative of RBL5523 was isolated that effectively nodulated and fixed nitrogen on P. sativum and V. hirsuta. Characterization of the mutant, RBL5787, indicated the cell surface components, extracellular polysaccharides, lipopolysaccharides, and outer membrane proteins, as well as the pattern of Nod metabolites, were indistinguishable from those of the parental strain. To obtain an indication of the function of the mutated locus, the flanking regions were sequenced and used to perform searches in protein and nonredundant nucleotide databases. No significant similarity or homology with any known sequence was detected.
Host Range Limitation of a Pseudorecombinant Virus Produced by Two Distinct Bipartite Geminiviruses
Authors: Höfer Petra , Engel Margit , Jeske Holger , Frischmuth Thomas
Published: May 2007

The production of pseudorecombinant viruses following exchange of cloned genomic components of the distinct geminiviruses Sida golden mosaic virus (SiGMV-Co) and Abutilon mosaic virus (AbMV) has been investigated. The pseudorecombinant virus produced by DNA A of AbMV and DNA B of SiGMV-Co was infectious in various host plants, although showing a limited host range compared with the two parental viruses. The reciprocal exchange of genomic components did not result in an infectious pseudorecombinant virus. However, in leaf disks SiGMV-Co DNA A was able to trans-replicate the heterogenomic AbMV DNA B component.
An Immunoreactive Protein to Wheat-Germ Agglutinin Antibody Is Induced in Oat Roots Following Invasion of the Cereal Cyst Nematode Heterodera avenae, and by Jasmonate
Authors: Oka Y. , Chet I. , Spiegel Y.
Published: May 2007

A protein that cross-reacts to a wheat-germ agglutinin antibody was induced in oat roots following the invasion of second-stage juveniles (J2) of the cereal cyst nematode Heterodera avenae. This protein, designated ASP45, was acid soluble, and its molecular mass was about 45 kDa on a sodium dodecyl sulfate-polyacrylamide gel. ASP45 was induced in both compatible and incompatible interactions between the nematode and the plant, and also in roots by exposure to jasmonic acid (JA) or methyl jasmonate. However, ASP45 was not induced by elicitors of pathogenesis-related proteins, abscisic acid, or wounding. Lipoxygenase activity, which is involved in JA synthesis, was higher in nematode-infected and JA-treated roots than in their noninfected, untreated counterparts. Inhibition of lipoxygenase activity in roots abolished ASP45 induction in the nematode-infected roots. Amino acid sequences similar to that of ASP45 were found in chitinases of poplar tree and Arabidopsis, even though ASP45 showed no chitinase activity. Although the biological role of ASP45 in infected roots is not clear, JA is suggested to be involved in signal transduction after pathogen invasion of the plant.
A Host-Controlled, Serogroup-Specific, Ineffective-Nodulation System in the Bradyrhizobium-Soybean (Glycine max) Symbiosis
Authors: Pazdernik David L. , Vance Carroll P. , Sadowsky Michael J. , Graham Peter H. , Orf James H.
Published: May 2007

We have previously reported a soybean plant introduction, PI 437153A, which was ineffectively nodulated (definition of G. Vest, D. F. Weber, and C. Sloger, Agronomy 16:353–390, 1973) by Bradyrhizobium japonicum strain UMR (University of Minnesota Rhizobium) 161. In this study, we further characterize this nodulation system by (i) examining the nodulation of 21 B. japonicum strains, representing 15 serogroups, with PI 437153A, (ii) ascertaining the inheritance of the ineffective nodulation, and (iii) characterizing the symbiosis both morphologically and physiologically. Only USDA 126 (serogroup 125) and UMR 161 failed to produce effective nodules with PI 437153A. Segregation among F2 and F3 plants from a cross between cv. Lambert and PI 437153A was consistent with control of this trait by a single, dominant gene. In reciprocal grafts between cv. Lambert and PI 437153A, ineffective nodulation was determined by the root genotype, and was independent of plant growth temperature and inoculant concentration. The nodule dry weight of UMR 161-inoculated plants of PI 437153A plants was approximately 10-fold less than that of comparable plants inoculated with USDA 110; nodule soluble protein levels were also significantly reduced. Nitrogenase activity and leghemoglobin could not be detected in the ineffectively nodulated phenotype, but 1 to 6 days after inoculation (DAI) phenylalanine ammonialyase RNA level was higher in the ineffectively nodulated phenotype than in the effectively nodulated one. Microscopic examination of effective and ineffective nodules 20 and 35 DAI revealed major morphological differences. Ineffective nodules contained large, deteriorating regions near apparently infected plant cells at 35 DAI, and schlerenchyma cells that were stained red, suggestive of lignin deposition. These changes could be the result of incompatibility between host and Bradyrhizobium, though the lack of chalcone synthase expression and the appearance of infected cells within the ineffective nodules suggest that some later steps in the infection process do occur.
Transgenic Arabidopsis Lines Expressing Gene VI from Cauliflower Mosaic Virus Variants Exhibit a Range of Symptom-Like Phenotypes and Accumulate Inclusion Bodies
Authors: Cecchini Edi , Gong Zhenhui , Geri Chiara , Covey Simon N. , Milner Joel J.
Published: May 2007

Gene VI of cauliflower mosaic virus (CaMV) is an important determinant of symptom expression during infection. We have constructed a series of transgenic Arabidopsis lines that express gene VI protein (P6) from two CaMV isolates (Bari-1 and Cabb B-JI) that cause mild and severe symptoms, respectively, in Arabidopsis, and from a recombinant virus (Baji-31) with a hybrid gene VI that causes very severe symptoms. From 41 transgenic lines analyzed, 17 showed symptom-like phenotypes that ranged from mild vein chlorosis to severe chlorosis and stunting. P6 levels in trans-genic lines varied from undetectable in the lowest expressors to levels greater than those in CaMV-infected plants. There was a strong correlation between phenotype severity and the level of P6, and with the gene VI origin in the order, Baji-31 > B-JI > Bari-1. This was similar to symptom severity in Arabidopsis infected with the respective CaMV variant. We also found that transgenic P6 accumulated in inclusion bodies that were similar to those found in infected plants but lacking virions. We conclude that expression of P6, in the absence of virus replication, elicits a subset of the host symptom responses normally observed during infection and that the level, sequence, and possibly the form of P6 are important in potentiating the process.
The Omega Sequence of VirD2 Is Important but Not Essential for Efficient Transfer of T-DNA by Agrobacterium tumefaciens
Authors: Bravo-Angel Ana María , Hohn Barbara , Tinland Bruno
Published: May 2007

The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells. We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far. In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs. However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence. The importance of the C terminus of VirD2 for T-DNA transfer is discussed.
Synthesis of Extracellular Polysaccharide, Extracellular Enzymes, and Xanthomonadin in Xanthomonas campestris: Evidence for the Involvement of Two Intercellular Regulatory Signals
Authors: Poplawsky Alan R. , Chun Wesley , Slater Holly , Daniels Michael J. , Dow J. Maxwell
Published: May 2007

In previous work, the pigB transcriptional unit of Xanthomonas campestris was implicated in the generation of a diffusible factor (DF) that regulates xanthomonadin and extracellular polysaccharide (EPS) production. Similarly, the rpfF and rpfB genes were implicated in the generation of a diffusible factor (DSF) that regulates the production of extracellular enzymes and EPS. In this study, extracel-lular cross-feeding studies with rpfF and pigB mutant strains provided evidence for the existence of two separate intercellular signaling systems with overlapping roles in the regulation of EPS production.
Genetic Mapping of thepvr1Locus inCapsicumspp. and Evidence That Distinct Potyvirus Resistance Loci Control Responses That Differ at the Whole Plant and Cellular Levels
Authors: Murphy John F. , Blauth James R. , Livingstone Kevin D. , Lackney Vincent K. , Jahn Molly Kyle
Published: May 2007

Two unlinked, recessive loci have been characterized in Capsicum spp. that independently confer distinct types of resistance to pepper mottle potyvirus (PepMoV). The first locus, from C. annuum cv. Avelar, affected the spread of PepMoV through the plant and had no discernible effect on tobacco etch potyvirus (HAT isolate; TEV-HAT) infection (J. F. Murphy and M. K. Kyle, Phytopathology 85:561-566, 1995). The second locus, found in C. chinense PI 159236 and PI 152225, interfered with PepMoV and TEV-HAT infection in plants and in isolated protoplasts. The resistant responses displayed by the C. chinense accessions to PepMoV and TEV-HAT appeared indistinguishable; however, these accessions were both susceptible to a second isolate of TEV, TEV-Mex21. We propose the symbols pvr1 for recessive PepMoV and TEV resistance from the C. chinense accessions and pvr3 for recessive PepMoV resistance from Avelar. pvr1 has been genetically mapped to a small linkage group with synteny to the short arm of tomato chromosome 3.
A Secretory Cellulose-Binding Protein cDNA Cloned from the Root-Knot Nematode (Meloidogyne incognita)
Authors: Ding X. , Shields J. , Allen R. , Hussey R. S.
Published: May 2007

A cDNA encoding a secretory cellulose-binding protein was cloned from the root-knot nematode (Meloidogyne incognita) with RNA fingerprinting. The putative full-length cDNA, named Mi-cbp-1, encoded a 203 amino acid protein containing an N-terminal secretion signal peptide. The C-terminal sequence of the putative MI-CBP-1 was similar to a bacterial-type cellulose-binding domain, whereas the N-terminal sequence did not show significant similarity to any proteins in data bases. Recombinant MI-CBP-1 lacked cellulase activity, but bound to cellulose and plant cell walls. In Southern blot hybridization, Mi-cbp-1 hybridized with genomic DNA from M. incognita, M. arenaria, and M. javanica, but not M. hapla, Heterodera glycines, or Caenorhabditis elegans. Polyclonal antibodies raised against recombinant MI-CBP-1 strongly labeled secretory granules in subventral gland cells of second-stage juveniles in indirect immunofluorescence microscopy. Enzyme-linked immunosorbent assay detection of MI-CBP-1 in stylet secretions of second-stage juveniles with the polyclonal antibodies indicated MI-CBP-1 could be secreted through the nematodes' stylet, suggesting that the cellulose-binding protein may have a role in pathogenesis.
A Plasmid Isolated from Phytopathogenic Onion Yellows Phytoplasma and Its Heterogeneity in the Pathogenic Phytoplasma Mutant
Authors: Kuboyama Tsutomu , Huang Chieh-Chen , Lu Xiaoyun , Sawayanagi Toshimi , Kanazawa Tokiko , Kagami Takashi , Matsuda Izumi , Tsuchizaki Tsuneo , Namba Shigetou
Published: May 2007

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.
Tomato Cell Death Mediated By Complementary Plant Viral Satellite RNA Sequences
Authors: Taliansky Michael E. , Ryabov Eugene V. , Robinson David J. , Palukaitis Peter
Published: May 2007

Cell death (necrosis) and severe yellowing (chlorosis) in tomato are induced by cucumber mosaic virus (CMV) supporting particular satellite RNAs. To determine whether CMV RNA sequences also are needed to induce necrosis or chlorosis, tomato seedlings were infected with the potato virus X vector expressing either a necrogenic or chlorosis-inducing satellite RNA of CMV. The infected plants did not develop chlorosis, although they did develop necrosis, but only when all or part of a 335-nucleotide necrogenic satellite RNA was expressed in the (-) polarity; i.e., the strand not packaged in virus particles. Computer-assisted secondary structure analysis suggests that the necrogenicity domain is an octanucleotide loop and adjacent base-paired stem of a thermodynamically stable hairpin structure.
Induction of Hypersensitive Cell Death by a Fungal Protein in Cultures of Tobacco Cells
Authors: Yano Akira , Suzuki Kaoru , Uchimiya Hirofumi , Shinshi Hideaki
Published: May 2007

Treatment of suspension-cultured tobacco (Nicotiana tabacum cv. Xanthi) cells (line XD6S) with fungal proteinaceous elicitors, namely, xylanase (EC 3.2.1.8) from Trichoderma viride (TvX) and xylanase from T. reesei (TrX), induced shrinkage of the cytoplasm, condensation of the nucleus, and, finally, cell death, which were accompanied by typical defense responses that included an oxidative burst and expression of defense genes. A Ca2+ channel blocker, Gd3+, inhibited the typical response of XD6S cells to TvX, which resembled the hypersensitive reaction (HR). These results suggested that the influx of Ca2+ ions plays an important role as a secondary signal. The HR was not observed in TvX-treated tobacco cells (line BY-2) derived from cv. Bright Yellow 2. This result suggests that key features of cultivar-specific interaction can be observed in cultures of tobacco cells. Xylanase from Bacillus circulans (BcX) and B. subtilis (BsX), which has enzymatic properties similar to those of TvX but an amino acid sequence different from that of TvX, did not induce the HR-like response in XD6S cells. These results suggest that the elicitor action of TvX is not due to its ability to hydrolyze cell walls but requires the TvX-specific recognition factors in plant cells. Thus, TvX-induced cell death was not due to some general toxic effect, but seems to be mediated by the activation of a specific cellular signal-transduction cascade that converges with a pathway that activates the intracellular cell death program.
clk1, a Serine/Threonine Protein Kinase-Encoding Gene, Is Involved in Pathogenicity ofColletotrichum lindemuthianumon Common Bean
Authors: Dufresne Marie , Bailey John A. , Dron Michel , Langin Thierry
Published: May 2007

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.
Interactions Between Spore Morphogenetic Mutations Affect Cell Types, Sporulation, and Pathogenesis in Magnaporthe grisea
Authors: Shi Zhixin , Christian David , Leung Hei
Published: May 2007

We have previously defined four single-gene mutations, con1, con2, con4, and con7, that control various stages of spore morphogenesis in the rice blast fungus. To delineate the developmental pathway of spore morphogenesis, we investigated the interactions among these morphogenetic genes by generating strains with double mutations via transformation-mediated gene disruption. Plasmids containing portions of the inactivated CON4 and CON7 genes were introduced into strains harboring single mutation to produce double mutants. Interaction between con1 and con4 resulted in reduced vegetative growth and suppression of sporulation. Interaction between con1 and con7 suppressed spore production but not vegetative growth. The con2/con4 and con4/con7 double mutants produced hybrid spore types with characteristic features of both parental mutants. The con2/con7 mutant produced con2 type spores, indicating that con2 is epistatic to con7 in spore morphogenesis. The epistatic relationship, however, reversed when traits related to pathogenesis were considered. Double mutants harboring the con7 mutation could not form appressoria or colonize plant tissue, indicating that con7 is epistatic to con2 and con4 in appressorium formation and pathogenesis. Thus, morphogenetic genes interact at multiple levels leading to different epistatic relationships in the pathways of spore morphogenesis, appressorium formation, and pathogenesis.
Cecropin A—Derived Peptides Are Potent Inhibitors of Fungal Plant Pathogens
Authors: Cavallarin Laura , Andreu David , San Segundo Blanca
Published: May 2007

Cecropins are naturally occurring peptides that play an important role in the immune response of insects. Cecropin A-derived and cecropin A-melittin hybrid peptides, all smaller than the natural compound cecropin A, were synthesized and tested for their ability to inhibit growth of several agronomically important fungal pathogens. We found that an 11-amino-acid sequence, corresponding to the N-terminal amphipathic α-helix domain of cecropin A, exhibited antifungal activity. Differences in susceptibility of the various pathogens were observed, Phytophthora infestans being particularly sensitive to the shortened cecropin A peptides (IC50 = 2 × 10−6 M). Biotoxicity of the shortest cecropin A-derived peptide was variously affected by the presence of proteins extracted from leaves of tobacco and tomato plants, either total extracts or intercellular fluids (ICFs). Overall, there was a greater tolerance to tomato protein extracts than to tobacco extracts. These findings suggest that tobacco should not be used as a model for testing the possible protective effects of transgenically expressed, cecropin-based genes. The feasibility of tailoring cecropin A genes to enhance crop protection in particular plant/fungus combinations is discussed.
Additional Resistance Gene(s) Against Cladosporium fulvum Present on the Cf-9 Introgression Segment Are Associated with Strong PR Protein Accumulation
Authors: Laugé Richard , Dmitriev Alexander P. , Joosten Matthieu H. A. J. , De Wit Pierre J. G. M.
Published: May 2007

The existence of a gene or genes conferring weak resistance against the fungal tomato pathogen Cladosporium fulvum, in addition to the Cf-9 resistance gene, present on the Lycopersicon pimpinellifolium Cf-9 segment introgressed into L. esculentum, was demonstrated with strains of C. fulvum lacking a functional Avr9 avirulence gene and tomato genotypes lacking a functional Cf-9 gene, respectively. Two mutant strains, obtained by disruption of Avr9 in race 4 and race 5 of C. fulvum, do not trigger the hypersensitive response-mediated resistance on MM-Cf9 genotypes that is normally induced after recognition of the AVR9 elicitor. However, when these strains are inoculated onto MM-Cf0 and MM-Cf9 genotypes, adult MM-Cf9 plants still show weak resistance. This resistance is not related to the Cf-9 gene, as ethyl methanesulfonate (EMS)-generated Cf-9 mutants retained weak resistance. Growth of the fungus in the leaf mesophyll is strongly inhibited, whereas re-emergence of fungal mycelium and conidiation are poor. Strong accumulation of pathogenesis-related proteins and early leaf chlorosis are associated with this phenotype of weak resistance. A search among natural strains lacking the Avr9 gene revealed that one strain is able to overcome this weak resistance. Possible mechanisms underlying this weak resistance are discussed. The presence of the additional weak resistance gene(s) could explain why the resistance of Cf9 genotypes has not been overcome so far in practice.
Restriction of Host Range by the sym2 Allele of Afghan Pea Is Nonspecific for the Type of Modification at the Reducing Terminus of Nodulation Signals
Authors: Ovtsyna Alexandra O. , Geurts Rene , Bisseling Ton , Lugtenberg Ben J. J. , Tikhonovich Igor A. , Spaink Herman P.
Published: May 2007

Rhizobium leguminosarum bv. viciae strains producing lipo-chitin oligosaccharides (LCOs) that are O-acetylated at the reducing terminus are required for nodulation of wild pea cultivars originating from Afghanistan that possess the recessive sym2A allele. The O-acetylation of the reducing sugar of LCOs is mediated by the bacterial nodX gene, which presumably encodes an acetyltransferase. We found that for nodulation on Afghan pea cultivars and sym2A introgression lines the nodX gene can be functionally replaced by the nodZ gene of Bradyrhizobium japonicum, which encodes a fucosyltransferase that fucosylates the reducing terminus of LCOs. The structure of the nodules, which were induced with normal frequency, was typical for effective pea nodules, and they fixed nitrogen with the same efficiency as nodules induced by nodX-carrying strains.
The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids
Authors: Schrammeijer B. , Hemelaar J. , Hooykaas P. J. J.
Published: May 2007

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.
Transformation of Nicotiana benthamiana with the Potato Mop-Top Virus Coat Protein Gene Produces a Novel Resistance Phenotype Mediated by the Coat Protein
Authors: Barker Hugh , Reavy Brian , McGeachy Kara D. , Dawson Sheila
Published: May 2007

Transformed lines of Nicotiana benthamiana expressing a translatable transgene (CPtrans) encoding the coat protein (CP) gene of potato mop-top virus (PMTV) differed considerably in steady-state levels of transgene RNA transcript and CP that were positively correlated. A mean of 88% of transgenic T1 plants from seven transgenic lines were resistant to manual PMTV inoculation. Resistance was identified by failure to develop symptoms in plants and inability to recover infectious virus with a sensitive infectivity assay: many of the 12% of transgenic plants that gave a positive infectivity assay did not produce symptoms and virus was not detected in repeat assays on the same plants. Resistant plants did not express a “recovery” phenotype and resistance was not overcome by use of RNA inoculum. Strong resistance was expressed in all CPtrans lines irrespective of the amount of transcript RNA or CP detected, and there was no evidence that resistance was mediated by a “transgene silencing” mechanism. Lines transformed with PMTV CP gene sequence in a nontranslatable form (CPnontrans) also contained differing steady-state levels of RNA transcript, but were not resistant to PMTV, although in some lines symptoms were slightly milder and were slower to develop than those in nontransgenic control plants. These results suggest that CPtrans is unique because, although it depends on CP translation to be effective, it mediates very strong resistance in 10 independent transformed lines (tested here and in other work).
Genetic Characterization of RRS1, a Recessive Locus in Arabidopsis thaliana that Confers Resistance to the Bacterial Soilborne Pathogen Ralstonia solanacearum
Authors: Deslandes Laurent , Pileur Frédéric , Liaubet Laurence , Camut Sylvie , Can Canan , Williams Kevin , Holub Eric , Beynon Jim , Arlat Matthieu , Marco Yves
Published: May 2007

The soilborne, vascular pathogen Ralstonia solanacearum, the causative agent of bacterial wilt, was shown to infect a range of Arabidopsis thaliana accessions. The pathogen was capable of infecting the Col-5 accession in an hrp-dependent manner, following root inoculation. Elevated bacterial population levels were found in leaves of Col-5, 4 to 5 days after root inoculation by the GMI1000 strain. Bacteria were found predominantly in the xylem vessels and spread systemically throughout the plant. The Nd-1 accession of A. thaliana was resistant to the GMI1000 strain of R. solanacearum. Bacterial concentrations detected in leaves of Nd-1, inoculated with an hrp+ strain of R. solanacearum, were only slightly higher than those detected in the susceptible accession, Col-5, following inoculation with a strain whose hrp gene cluster was deleted. Leaf inoculation of the GMI1000 strain on the resistant accession Nd-1 induced the formation of lesions in the older leaves of the rosette whereas the same strain of R. solanacearum provoked complete wilting of Col-5. Resistance to strain GMI1000 of R. solanacearum segregated as a simply inherited recessive trait in a genetic cross between Col-5 and Nd-1. F9 recombinant inbred lines generated between these two accessions were used to map a locus, RRS1, that was the major determinant of resistance between restriction fragment length polymorphism markers mi83 and mi61 on chromosome V. This region of the A. thaliana genome is known to contain many other pathogen recognition capabilities.