bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and Expression Analysis of Uricase mRNA from Lotus japonicus
Authors: Takane Ken-ichi , Tajima Shigeyuki , Kouchi Hiroshi
Published: May 2007

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.
Simultaneous Imaging of Pseudomonas fluorescens WCS365 Populations Expressing Three Different Autofluorescent Proteins in the Rhizosphere: New Perspectives for Studying Microbial Communities
Authors: Bloemberg Guido V. , Wijfjes André H. M. , Lamers Gerda E. M. , Stuurman Nico , Lugtenberg Ben J. J.
Published: May 2007

To visualize simultaneously different populations of pseu-domonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Micro-colonies consisting of mixed populations were frequently observed at the base of the root system, whereas micro-colonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.
The sss Colonization Gene of the Tomato-Fusarium oxysporum f. sp. radicis-lycopersici Biocontrol Strain Pseudomonas fluorescens WCS365 Can Improve Root Colonization of Other Wild-type Pseudomonas spp. Bacteria
Authors: Dekkers Linda C. , Mulders Ine H. M. , Phoelich Claartje C. , Chin-A-Woeng Thomas F. C. , Wijfjes André H. M. , Lugtenberg Ben J. J.
Published: May 2007

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a bio-control strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.
The dnaJ (hsp40) Locus in Rhizobium leguminosarum bv. phaseoli Is Required for the Establishment of an Effective Symbiosis with Phaseolus vulgaris
Authors: Labidi Mohamed , Laberge Serge , Vézina Louis P. , Antoun Hani
Published: May 2007

P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene.
Root Colonization by Phenazine-1-Carboxamide-Producing Bacterium Pseudomonas chlororaphis PCL1391 Is Essential for Biocontrol of Tomato Foot and Root Rot
Authors: Chin-A-Woeng Thomas F. C. , Bloemberg Guido V. , Mulders Ine H. M. , Dekkers Linda C. , Lugtenberg Ben J. J.
Published: May 2007

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.
Avirulence in the Wheat Septoria tritici Leaf Blotch Fungus Mycosphaerella graminicola Is Controlled by a Single Locus
Authors: Kema Gert H. J. , Verstappen Els C. P. , Waalwijk Cees
Published: May 2007

Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici leaf blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.
Fungal Development and Induction of Defense Response Genes During Early Infection of Wheat Spikes by Fusarium graminearum
Authors: Pritsch Clara , Muehlbauer Gary J. , Bushnell William R. , Somers David A. , Vance Carroll P.
Published: May 2007

Fusarium head blight (FHB) of wheat is a crippling disease that causes severe economic losses in many of the wheat-growing regions of the world. Temporal patterns of fungus development and transcript accumulation of defense response genes were studied in Fusarium graminearum-inoculated wheat spikes within the first 48 to 76 h after inoculation (hai). Microscopy of inoculated glumes revealed that the fungus appeared to penetrate through stomata, exhibited subcuticular growth along stomatal rows, colonized glume parenchyma cells, and sporulated within 48 to 76 hai. No major differences in the timing of these events were found between Sumai 3 (resistant) and Wheaton (susceptible) genotypes. In complementary experiments, RNA was extracted from spikes at several time intervals up to 48 hai and temporal expression patterns were determined for defense response genes encoding peroxidase, PR-1, PR-2 (β-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (thaumatin-like protein). In both genotypes, transcripts for the six defense response genes accumulated as early as 6 to 12 hai during F. graminearum infection and peaked at 36 to 48 hai. Greater and earlier PR-4 and PR-5 transcript accumulation was observed in Sumai 3, compared with Wheaton. Our results show that the timing of defense response gene induction is correlated with F. graminearum infection.
Harpin-Induced Hypersensitive Cell Death Is Associated with Altered Mitochondrial Functions in Tobacco Cells
Authors: Xie Zhixin , Chen Zhixiang
Published: May 2007

Mitochondria play important roles in animal apoptosis and are implicated in salicylic acid (SA)-induced plant resistance to viral pathogens. In a previous study, we demonstrated that SA induces rapid inhibition of mitochondrial electron transport and oxidative phosphorylation in tobacco cells. In the present study, we report that plant programmed cell death induced during pathogen elicitor-induced hypersensitive response (HR) is also associated with altered mitochondrial functions. Harpin, an HR elicitor produced by Erwinia amylovora, induced inhibition of ATP synthesis in tobacco cell cultures. Inhibition of ATP synthesis occurred almost immediately after incubation with harpin and preceded hypersensitive cell death induced by the elicitor. Diphenylene iodonium, an inhibitor of the oxidative burst, did not block harpin-induced inhibition of ATP synthesis or cell death, suggesting that oxidative burst was not the direct cause for these two harpin-induced processes. Unlike SA, harpin had no significant effect on total respiratory O2 uptake of treated cells. However, respiration of harpin-treated tobacco cells became very sensitive to the alternative oxidase inhibitors salicyl-hydroxamic acid and n-propyl gallate. Thus, harpin treatment resulted in reduced capacity of mitochondrial cytochrome pathway electron transport, which could lead to the observed inhibition of ATP synthesis. Given the recently demonstrated roles of mitochondria in apoptosis, this rapid inhibition of mitochondrial functions may play a role in harpin-induced hypersensitive cell death.
Identification of Arabidopsis Mutants Exhibiting an Altered Hypersensitive Response in Gene-for-Gene Disease Resistance
Authors: Yu I-ching , Fengler Kevin A. , Clough Steven J. , Bent Andrew F.
Published: May 2007

A mutational study was carried out to isolate Arabidopsis thaliana plants that exhibit full or partial disruption of the RPS2-mediated hypersensitive response (HR) to Pseudomonas syringae that express avrRpt2. Five classes of mutants were identified including mutations at RPS2, dnd mutations causing a “defense, no death” loss-of-HR phenotype, a lesion-mimic mutant that also exhibited an HR¯phenotype, and a number of intermediate or partial-loss-of-HR mutants. Surprisingly, many of these mutants displayed elevated resistance to virulent P. syringae and, in some cases, to Peronospora parasitica. Constitutively elevated levels of pathogenesis-related (PR) gene expression and salicylic acid were also observed. In the lesion-mimic mutant, appearance of elevated resistance was temporally correlated with appearance of lesions. For one of the intermediate lines, resistance was shown to be dependent on elevated levels of salicylic acid. A new locus was identified and named IHR1, after the mutant phenotype of “intermediate HR.” Genetic analysis of the intermediate-HR plant lines was difficult due to uncertainties in distinguishing the partial/intermediate mutant phenotypes from wild type. Despite this difficulty, the intermediate-HR mutants remain of interest because they reveal potential new defense-related loci and because many of these lines exhibit partially elevated disease resistance without dwarfing or other apparent growth defects.
Changes in mRNA Abundance within Heterodera schachtii-Infected Roots of Arabidopsis thaliana
Authors: Hermsmeier Dieter , Hart Jennifer K. , Byzova Marina , Rodermel Steven R. , Baum Thomas J.
Published: May 2007

Gene expression changes in plant roots infected by plant-parasitic cyst nematodes are involved in the formation of nematode feeding sites. We analyzed mRNA abundance changes within roots of Arabidopsis thaliana during the early compatible interaction with Heterodera schachtii, the sugarbeet cyst nematode. Approximately 1,600 root sections, each containing a single parasitic nematode and its feeding site, and 1,600 adjacent, nematode-free root sections were excised from aseptic A. thaliana cultures 3 to 4 days after inoculation with H. schachtii. These tissue samples were termed infected and uninfected, respectively. Preparasitic nematodes were added to the uninfected tissue sample to maintain the nematode to plant tissue proportion. mRNA extracted from these two tissue samples was subjected to differential display analysis. Thirty-six cDNA clones corresponding to mRNA species with different abundance between both tissue samples were isolated. Of these clones, 24 were of A. thaliana origin and 12 were from H. schachtii. Differential display data predicted that the A. thaliana cDNA clones corresponded to 13 transcripts that were more abundant in the infected root sections and 11 transcripts that were more abundant in the uninfected root sections. H. schachtii cDNA clones were predicted to correspond to four transcripts that were more abundant in parasitic nematodes and to eight transcripts that were more abundant in preparasitic nematodes. In situ hybridization experiments confirmed the mRNA abundance changes in A. thaliana roots predicted by the differential display analyses for two A. thaliana clones.
Resistance Gene Analogs within an Introgressed Chromosomal Segment Derived from Triticum ventricosum That Confers Resistance to Nematode and Rust Pathogens in Wheat
Authors: Seah S. , Spielmeyer W. , Jahier J. , Sivasithamparam K. , Lagudah E. S.
Published: May 2007

A resistance (R) gene-rich 2S chromosomal segment from Triticum ventricosum contains a cereal cyst nematode (CCN; Heterodera avenae) R gene locus CreX and a closely linked group of genes (Sr38, Yr17, and Lr37) that confer resistance to stem rust (Puccinia graminis f. sp. tritici), stripe rust (P. striiformis f. sp. tritici), and leaf rust (P. recondita f. sp. tritici) when introgressed into wheat. The 2S chromosomal segment from T. ventricosum is further delineated in translocations onto chromosome 2A of bread wheat, where the rust genes are retained but not the CreX gene. Using these critical genetic stocks, we have isolated family members of R gene analogs that are associated with either the 2S segment from T. ventricosum carrying the CreX locus or the rust genes. Derivatives of the Cre3 candidate R gene sequence and a rice (Oryza sativa) R gene analog that mapped to the 2S homologous chromosome groups in wheat were used to isolate related gene sequences from T. ventricosum that contain a nucleotide binding site-leucine rich repeat domain. The potential of these gene sequences as entry points for isolating candidate genes or gene family members of the CreX or rust genes and their further applications to plant breeding are discussed.
Differential Induction of Tobacco MAP Kinases by the Defense Signals Nitric Oxide, Salicylic Acid, Ethylene, and Jasmonic Acid
Authors: Kumar Dhirendra , Klessig Daniel F.
Published: May 2007

In tobacco, two mitogen-activated protein (MAP) kinases, designated salicylic acid (SA)-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment. To investigate whether nitric oxide (NO), SA, ethylene, or jasmonic acid (JA) are involved in this phenomenon, the ability of these defense signals to activate these kinases was assessed. Both NO and SA activated SIPK; however, they did not activate WIPK. Additional analyses with transgenic NahG tobacco revealed that SA is required for the NO-mediated induction of SIPK. Neither JA nor ethylene activated SIPK or WIPK. Thus, SIPK may function downstream of SA in the NO signaling pathway for defense responses, while the signals responsible for resistance-associated WIPK activation have yet to be determined.
Cloning and Disruption of pgx4 Encoding an In Planta Expressed Exopolygalacturonase from Fusarium oxysporum
Authors: García-Maceira F. I. , Di Pietro Antonio , Roncero M. Isabel G.
Published: May 2007

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-α1,4-polygalac-turonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxy-sporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.
Transgenic Resistance to PVYO Associated with Post-Transcriptional Silencing of P1 Transgene Is Overcome by PVYN Strains That Carry Highly Homologous P1 Sequences and Recover Transgene Expression at Infection
Authors: Mäki-Valkama Tuula , Valkonen Jari P. T. , Kreuze Jan F. , Pehu Eija
Published: May 2007

Resistance to Potato virus Y (PVY) has been obtained in our previous studies through expression of the PVY P1 gene in sense or antisense orientation in potato cv. Pito. In the present study, the mechanism and strain specificity of the resistance were analyzed. Several features including low steady-state P1 mRNA expression in the resistant P1 plants indicated that resistance was based on post-transcriptional gene silencing (PTGS). Resistance was specific to PVYO isolates, the PVY strain group from which the P1 transgene was derived. However, according to group analyses, there was no distinguishing characteristic between the PVYO and PVYN strains P1 gene sequences. Therefore, the ability of the PVYN strains to overcome resistance could not be explained solely based on their P1 gene sequences. Infection with PVYN of the PVYO-resistant transgenic lines led to a recovery of expression of the P1 transgene. These data suggested that factors other than sequence homology are required in determination of the resistance specificity.
Interacting Signal Pathways Control Defense Gene Expression in Arabidopsis in Response to Cell Wall-Degrading Enzymes from Erwinia carotovora
Authors: Norman-Setterblad Cecilia , Vidal Sabina , Palva E. Tapio
Published: May 2007

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.
A Lotus japonicus Nodulation System Based on Heterologous Expression of the Fucosyl Transferase NodZ and the Acetyl Transferase NolL in Rhizobium leguminosarum
Authors: Bras Cristina Pacios , Jordá Meritxell Alberich , Wijfjes André H. M. , Harteveld Marga , Stuurman Nico , Thomas-Oates Jane E. , Spaink Herman P.
Published: May 2007

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizo-bium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-α-(2-aminoethoxyvinyl) glycine (AVG).
Host Suppressors in Arabidopsis thaliana of Mutations in the Movement Protein Gene of Cauliflower mosaic virus
Authors: Callaway Anton S. , Huang Zhong , Howell Stephen H.
Published: May 2007

A novel genetic screen was used to identify host factors in Arabidopsis thaliana that suppress mutations in the Cauliflower mosaic virus (CaMV) movement protein gene (gene I). A series of small mutations was made in gene I and the mutations were tested for their suitability in a suppressor screen. The first round of screening yielded only revertants or second-site mutations in gene I. A derivative of one of the second-site mutant viruses (N7) that was delayed in symptom production was used in a second round of screening for suppressor plants that accelerated symptom production. Two candidate suppressor plants were found that accelerated by 1 to 4 days the first appearance of symptoms caused by the mutant viruses. One of the suppressors (5-2), called asc1 (acceleration of symptoms by CaMV N7), was mapped to chromosome 1. Two additional loci that differentially affect N7 virus susceptibility in the parental Columbia and Ler ecotypes were mapped to chromosomes 3 and 4 by quantitative trait locus (QTL) analysis.
Regulation of Tomato Leaf Curl Viral Gene Expression in Host Tissues
Authors: Dry Ian , Krake Les , Mullineaux Phil , Rezaian Ali
Published: May 2007

The regulation of expression of the two virion-sense (V1 and V2) and four complementary-sense (C1, C2, C3, and C4) open reading frames (ORFs) of Tomato leaf curl virus (TLCV) was studied in both stably and transiently transformed Nicotiana tabacum tissues with fusions with the β-glucuronidase (GUS) reporter gene. GUS-expressing transgenic lines were obtained with each of the four complementary-sense gene-GUS fusion constructs and with truncated versions of the virion-sense gene-GUS fusion constructs (V1GUSΔC and V2GUSΔC) lacking complementary-sense sequences encoding the C1, C2, and C3 ORFs. However, little or no GUS expression was observed in kanamycin-resistant plants transformed with full-length, virion-sense gene constructs (V1GUS and V2GUS) constituting the complete viral genome. In contrast, V1GUS and V2GUS were found to direct high-level GUS expression in transient assays with tobacco protoplasts, suggesting that integration of viral constructs containing functional, complementary-sense genes may lead to repression or deletion of the introduced constructs in trans-genic tissues. V2GUS expression in the transient proto-plast assay was found to be severely curtailed by specific mutation of the C2 ORF, supporting a role for the C2 protein in transactivation of TLCV virion-sense gene expression. TLCV ORF-GUS constructs displayed distinctive tissue expression patterns in transgenic tobacco plants that could be divided into constitutive (C1, C4, and V2GUSΔC), predominantly vascular (C2, C3), or reduced expression in cells associated with the vascular bundles (V1GUSΔC). The significance of these results is discussed in terms of current models of gene function and regulation in geminiviruses.
Constitutive Accumulation of a Resveratrol-Glucoside in Transgenic Alfalfa Increases Resistance to Phoma medicaginis
Authors: Hipskind John D. , Paiva Nancy L.
Published: May 2007

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-β-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 μg per g fresh weight) and oldest stem internode segments (>10 μg per g fresh weight) while roots contained only trace amounts (<0.2 μg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malo-nyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.
Decreased Inositol 1,4,5-Trisphosphate Content in Pathogen-Challenged Soybean Cells
Authors: Shigaki Toshiro , Bhattacharyya Madan K.
Published: May 2007

Phosphoinositide-specific phospholipase C (PI-PLC) has been shown to be transiently activated when plant cells were treated with elicitors. We thus investigated the activity of PI-PLC when soybean cells were infected with the bacterial pathogen Pseudomonas syringae pv. glycinea, by measuring cellular cytosolic inositol 1,4,5-trisphosphate (IP3) levels. We observed that IP3 content decreased in both compatible and incompatible interactions. In vitro phosphatase activities were similar in both water control and infected cells with slightly lower IP3 degradation observed for infected cells, indicating that the reduced IP3 content in infected cells most likely results from reduced PI-PLC activity. We hypothesize that reduced IP3 content following infection may lead to suppression of various housekeeping activities of the cells, thus diverting the cellular resources either to the synthesis of defense-related compounds against pathogens, and/or to the growth of pathogens.
Multiresistance Genes of Rhizobium etli CFN42
Authors: González-Pasayo Ramón , Martínez-Romero Esperanza
Published: May 2007

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.
Cell-to-Cell Movement of the 25K Protein of Potato virus X Is Regulated by Three Other Viral Proteins
Authors: Yang Yang , Ding Biao , Baulcombe David C. , Verchot Jeanmarie
Published: May 2007

The 25K, 12K, and 8K proteins and coat protein (CP) of Potato virus X (PVX) are required for virus cell-to-cell movement. In this study, experiments were conducted to determine whether the PVX 25K protein moves cell to cell and to explore potential interactions between the PVX 25K, 12K, and 8K proteins and CP. The PVX 25K gene was fused to the green fluorescent protein (GFP) gene and inserted into plasmids adjacent to the cauliflower mosaic virus 35S promoter. These plasmids were introduced by biolistic bombardment to transgenic tobacco expressing the PVX 12K, 8K, and CP genes. The GFP:25K fused proteins moved cell to cell on nontransgenic tobacco and tobacco expressing either the 12K or 8K proteins. However, the GFP:25K proteins did not move on transgenic tobacco expressing the combined 12K/8K genes or the CP gene. Thus, movement of the PVX 25K protein through plas-modesmata may be regulated by interactions with other PVX proteins.
High Level Activation of Vitamin B1 Biosynthesis Genes in Haustoria of the Rust Fungus Uromyces fabae
Authors: Sohn Jürgen , Voegele Ralf T. , Mendgen Kurt , Hahn Matthias
Published: May 2007

In the rust fungus Uromyces fabae, the transition from the early stages of host plant invasion toward parasitic growth is accompanied by the activation of many genes (PIGs = in planta induced genes). Two of them, PIG1 = THI1) and PIG4 (= THI2), were found to be highly transcribed in haustoria, and are homologous to genes involved in thiamine (vitamin B1) biosynthesis in yeast. Their functional identity was confirmed by complementation of Schizosac-charomyces pombe thiamine auxotrophic thi3 (nmt1) and thi2 (nmt2) mutants, respectively. In contrast to thiamine biosynthesis genes of other fungi that are completely suppressed by thiamine, THI1 and THI2 expression was not affected by the addition of thiamine to rust hyphae grown either in vitro or in planta. Immunoblot analysis revealed decreasing amounts of THI1p in extracts from spores, germlings, and in vitro-grown infection structures with increasing time after inoculation. Immunofluorescence microscopy of rust-infected leaves detected high concentrations of THI1p in haustoria, and only low amounts in intercellu-lar hyphae. In the sporulating mycelium, THI1p was found in the basal hyphae of the uredia, but not in the pedicels and only at very low levels in uredospores. These data indicate that the haustorium is an essential structure of the biotrophic rust mycelium not only for nutrient uptake but also for the biosynthesis of metabolites such as thiamine.
The Endo-β-1,4-glucanase CelA of Clavibacter michiganensis subsp. michiganensis Is a Pathogenicity Determinant Required for Induction of Bacterial Wilt of Tomato
Authors: Jahr Holger , Dreier Jens , Meletzus Dietmar , Bahro Rainer , Eichenlaub Rudolf
Published: May 2007

The phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D. Meletzus, A. Bermpohl, J. Dreier, and R. Eichenlaub, 1993, J. Bacteriol. 175:2131–2136; J. Dreier, D. Meletzus, and R. Eichenlaub, 1997, Mol. Plant-Microbe Interact. 10:195–206). The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation. RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped. The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-β-1,4-glucanases of family A1 cellulases. CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain. We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA- gene-replacement mutant CMM-H4 with the wild-type celA gene.
Nodule-Expressed Cyp15a Cysteine Protease Genes Map to Syntenic Genome Regions in Pisum and Medicago spp.
Authors: Vincent Jason L. , Knox Maggie R. , Ellis T. H. Noel , Kaló Péter , Kiss György B. , Brewin Nicholas J.
Published: May 2007

PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5′ to the coding sequence of the gene was cloned. Varying lengths of 5′ untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into “hairy roots” of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.
Differential Expression of Eight Chitinase Genes in Medicago truncatula Roots During Mycorrhiza Formation, Nodulation, and Pathogen Infection
Authors: Salzer Peter , Bonanomi Athos , Beyer Katinka , Vögeli-Lange Regina , Aeschbacher Roger A. , Lange Jürg , Wiemken Andres , Kim Dongjin , Cook Douglas R. , Boller Thomas
Published: May 2007

Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.
Growth Temperature Regulation of Host-Specific Modifications of Rhizobial Lipo-Chitin Oligosaccharides: The Function of nodX Is Temperature Regulated
Authors: Olsthoorn Maurien M. A. , Stokvis Ellen , Haverkamp Johan , Spaink Herman P. , Thomas-Oates Jane E.
Published: May 2007

Lipo-chitin oligosaccharides (LCOs) are usually produced and isolated for structural analysis from bacteria cultured under laboratory rather than field conditions. We have studied the influence of bacterial growth temperature on the LCO structures produced by different Rhizobium leguminosarum strains, using thin-layer chromatographic, high-performance liquid chromatographic, and mass spectrometric analyses. Wild-type R. leguminosarum bv. viciae A1 was shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12°C than at 28°C, indicating that the activity of nodX (a gene encoding an LCO O-acetyl transferase) is temperature dependent. Interestingly, symbiotic resistance genes sym1 and sym2 found in primitive pea cultivars are also temperature sensitive, only being active at low temperatures, at which they block nodulation by R. leguminosarum bv. viciae strains lacking nodX. We therefore propose that the gene-for-gene relationship between plant and bacterium has a temperature-sensitive mechanism as an adaptation to environmental conditions. An R. leguminosarum bv. trifolii strain was also shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12°C than at 28°C. The major components synthesized by the two strains are produced at both temperatures but in different relative amounts, while some minor components are only produced at one of the two temperatures.
Phosphoproteins Involved in the Signal Transduction of Cryptogein, an Elicitor of Defense Reactions in Tobacco
Authors: Lecourieux-Ouaked Fatma , Pugin Alain , Lebrun-Garcia Angela
Published: May 2007

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaïc acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospholabeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.
An Efficient cDNA-AFLP-Based Strategy for the Identification of Putative Pathogenicity Factors from the Potato Cyst Nematode Globodera rostochiensis
Authors: Qin Ling , Overmars Hein , Helder Johannes , Popeijus Herman , van der Voort Jeroen Rouppe , Groenink Wouter , van Koert Paul , Schots Arjen , Bakker Jaap , Smant Geert
Published: May 2007

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.
Induced Expression of Sarcotoxin IA Enhanced Host Resistance Against Both Bacterial and Fungal Pathogens in Transgenic Tobacco
Authors: Mitsuhara Ichiro , Matsufuru Hiroki , Ohshima Masahiro , Kaku Hisatoshi , Nakajima Yuki , Murai Norimoto , Natori Shunji , Ohashi Yuko
Published: May 2007

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.
Expression of Pectate Lyase from Colletotrichum gloeosporioides in C. magna Promotes Pathogenicity
Authors: Yakoby Nir , Freeman Stanley , Dinoor Amos , Keen Noel T. , Prusky Dov
Published: May 2007

To test the contribution of pectate lyase (PL) to promoting fungal pathogenicity, a pectate lyase gene (pel) from the avocado pathogen Colletotrichum gloeosporioides, isolate Cg-14, was expressed in C. magna isolate L-2.5, a pathogen of cucurbits that causes minor symptoms in watermelon seedlings and avocado fruits. Isolate L-2.5 was transformed with pPCPH-1 containing hph-B as a selectable marker and the 4.1-kb genomic pel clone. Southern hybridization, with the 4.1-kb genomic pel clone or 2.13-kb hph-B cassette as probes, detected integration of pel in transformed C. magna isolates Cm-PL-3 and Cm-PL-10. Western blot (immunoblot) analysis with antibodies against Cg-14 PL detected a single PL secreted by L-2.5 at a molecular mass of 41.5 kDa, whereas the PL of C. gloeosporioides had a molecular mass of 39 kDa. When PL activity was measured 4 days after inoculation in pecto-lytic enzyme-inducing media (PEIM), transformed isolates Cm-PL-3 and Cm-PL-10 showed additive PL activity relative to both Cg-14 and L-2.5. Transformed isolates also showed additive maceration capabilities on avocado pericarp relative to the wild-type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, more severe maceration and damping off developed in watermelon seedlings inoculated with the transformed isolates compared with the two wild-type isolates, which showed no symptom development on these seedlings during the same period. Results clearly show the contribution of a single pel to the pathogenic abilities of C. magna and suggest that PL is a pathogenicity factor required for the penetration and colonization of Colletotrichum species.
Construction of Transgenic Pea Lines with Modified Expression of Diamine Oxidase and Modified Nodulation Responses with Exogenous Putrescine
Authors: Wisniewski J.-P. , Brewin N. J.
Published: May 2007

Diamine oxidase (DAO) might influence pea nodule development either by regulating the peroxide-driven cross-linking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity in extracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing co-suppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in the cross-linking of matrix glycoprotein, indicating that putrescine-derived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serve to reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues.
Uncoupling Resistance from Cell Death in the Hypersensitive Response of Nicotiana Species to Cauliflower mosaic virus Infection
Authors: Cole Anthony B. , Király Lóránt , Ross Kathleen , Schoelz James E.
Published: May 2007

Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the resistance of N. edwardsonii to W260 infection was conditioned by a gene derived from N. glutinosa, whereas cell death was conditioned by a gene derived from N. clevelandii. An analysis of pathogenesis-related (PR) protein expression in response to W260 infection revealed that elicitation of PR proteins was associated with resistance rather than with the onset of cell death.