bluuebunny/crossref_metadata_2025 huggingface dataset:
Arabidopsis thaliana EDS4 Contributes to Salicylic Acid (SA)-Dependent Expression of Defense Responses: Evidence for Inhibition of Jasmonic Acid Signaling by SA
Authors: Gupta Vaijayanti , Willits Michael G. , Glazebrook Jane
Published: May 2007

The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. Maculicola ES4326 (Psm ES4326). Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4. Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326. These phenotypes indicate that EDS4 plays a role in SA-dependent signaling. SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA). Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling. Two possible working models of the role of EDS4 in governing activation of defense responses are presented.
Characterization of SNP1, a Cell Wall-Degrading Trypsin, Produced During Infection by Stagonospora nodorum
Authors: Carlile A. J. , Bindschedler L. V. , Bailey A. M. , Bowyer P. , Clarkson J. M. , Cooper R. M.
Published: May 2007

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depoly-merases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors aprotinin and leupeptin and weakly by phenylmethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clone from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.
Evidence for Agrobacterium-Induced Apoptosis in Maize Cells
Authors: Hansen Geneviève
Published: May 2007

Agrobacterium spp. can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation. One major obstacle is that co-cultivation of Agrobacterium spp. with plant tissues often results in cell death. Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes. Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp. in maize tissues. p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas iap may act upstream to prevent their activation. This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells.
Cloning and Characterization of a Cyclic Peptide Synthetase Gene from Alternaria alternata Apple Pathotype Whose Product Is Involved in AM-Toxin Synthesis and Pathogenicity
Authors: Johnson R. D. , Johnson L. , Itoh Y. , Kodama M. , Otani H. , Kohmoto K.
Published: May 2007

Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. alternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.
Characterizing Rice Lesion Mimic Mutants and Identifying a Mutant with Broad-Spectrum Resistance to Rice Blast and Bacterial Blight
Authors: Yin Zhongchao , Chen Jun , Zeng Lirong , Goh Meiling , Leung Hei , Khush Gurdev S. , Wang Guo-Liang
Published: May 2007

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.
The Amino Terminus of the Coat Protein of Turnip crinkle virus Is the AVR Factor Recognized by Resistant Arabidopsis
Authors: Zhao Yajuan , DelGrosso Leanna , Yigit Erbay , Dempsey D'Maris Amick , Klessig Daniel F. , Wobbe Kristin K.
Published: May 2007

We have isolated three naturally occurring strains of Turnip crinkle virus (TCV) that break resistance in Di-17 Arabidopsis. Two mutations in the N terminus of the TCV coat protein, D4N and P5S, were shown to confer this phenotype. Thus, this region of the coat protein is involved in eliciting resistance responses in Arabidopsis.
Transgenic Expression of Pear PGIP in Tomato Limits Fungal Colonization
Authors: Powell Ann L. T. , van Kan Jan , ten Have Arjen , Visser Jaap , Greve L. Carl , Bennett Alan B. , Labavitch John M.
Published: May 2007

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit. In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009–1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.
Genetic Identification of Multiple Biological Roles Associated with the Capsid Protein of Satellite Panicum Mosaic Virus
Authors: Qiu Wenping , Scholthof Karen-Beth G.
Published: May 2007

Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants.
Cell-to-Cell Movement of Potato virus X: The Role of p12 and p8 Encoded by the Second and Third Open Reading Frames of the Triple Gene Block
Authors: Tamai Atsushi , Meshi Tetsuo
Published: May 2007

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.
Susceptibility to the Sugar Beet Cyst Nematode Is Modulated by Ethylene Signal Transduction in Arabidopsis thaliana
Authors: Wubben Martin J. E. , Su Hong , Rodermel Steven R. , Baum Thomas J.
Published: May 2007

Previously, we identified Arabidopsis thaliana mutant rhd1-4 as hypersusceptible to the sugar beet cyst nematode Heterodera schachtii. We assessed rhd1-4 as well as two other rhd1 alleles and found that each exhibited, in addition to H. schachtii hypersusceptibility, decreased root length, increased root hair length and density, and deformation of the root epidermal cells compared with wild-type A. thaliana ecotype Columbia (Col-0). Treatment of rhd1-4 and Col-0 with the ethylene inhibitors 2-aminoeth-oxyvinylglycine and silver nitrate and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid suggests that the rhd1-4 hypersusceptibility and root morphology phenotypes are the result of an increased ethylene response. Assessment of known ethylene mutants further support the finding that ethylene plays a role in mediating A. thaliana susceptibility to H. schachtii because mutants that overproduce ethylene (eto1-1, eto2, and eto3) are hypersusceptible to H. schachtii and mutants that are ethylene-insensitive (etr1-1, ein2-1, ein3-1, eir1-1, and axr2) are less susceptible to H. schachtii. Because the ethylene mutants tested show altered susceptibility and altered root hair density and length, a discrimination between the effects of altered ethylene signal transduction and root hair density on susceptibility was accomplished by analyzing the ttg and gl2 mutants, which produce ectopic root hairs that result in greatly increased root hair densities while maintaining normal ethylene signal transduction. The observed normal susceptibilities to H. schachtii of ttg and gl2 indicate that increased root hair density, per se, does not cause hypersusceptibility. Furthermore, the results of nematode attraction assays suggest that the hypersusceptibility of rhd1-4 and the ethylene-overproducing mutant eto3 may be the result of increased attraction of H. schachtii-infective juveniles to root exudates of these plants. Our findings indicate that rhd1 is altered in its ethylene response and that ethylene signal transduction positively influences plant susceptibility to cyst nematodes.
Salicylic Acid and NIM1/NPR1-Independent Gene Induction by Incompatible Peronospora parasitica in Arabidopsis
Authors: Rairdan Gregory J. , Donofrio Nicole M. , Delaney Terrence P.
Published: May 2007

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.
A Protein from the Mold Aspergillus giganteus Is a Potent Inhibitor of Fungal Plant Pathogens
Authors: Vila L. , Lacadena V. , Fontanet P. , del Pozo A. Martinez , Segundo B. San
Published: May 2007

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.
Bradyrhizobium japonicum Mutants with Enhanced Sensitivity to Genistein Resulting in Altered nod Gene Regulation
Authors: Ip H. , D'Aoust F. , Begum A. A. , Zhang H. , Smith D. L. , Driscoll B. T. , Charles T. C.
Published: May 2007

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY∷lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5-bromo-4chloro-3indolyl-β-d-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY∷lacZ reporter gene, were isolated. β-Galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY∷lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY∷lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.
Vascular Defense Responses in Rice: Peroxidase Accumulation in Xylem Parenchyma Cells and Xylem Wall Thickening
Authors: Hilaire Emmanuel , Young Scott A. , Willard Lloyd H. , McGee J. David , Sweat Teresa , Chittoor J. M. , Guikema James A. , Leach Jan E.
Published: May 2007

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced perox-idases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.
Chromosomal Deletion in Isolates of Phytophthora infestans Correlates with Virulence on R3, R10, and R11 Potato Lines
Authors: van der Lee Theo , Testa Antonino , van 't Klooster John , van den Berg-Velthuis Grardy , Govers Francine
Published: May 2007

In Phytophthora infestans, a cluster of three dominant avirulence genes is located on the distal part of linkage group VIII. In a mapping population from a cross between two Dutch field isolates, probe M5.1, derived from an amplified fragment length polymorphism (AFLP) marker linked to the Avr3-Avr10-Avr11 cluster, hybridized only to DNA from the parent and F1 progeny that is avirulent on potato lines carrying the R3, R10, and R11 resistance gene. In the virulent parent and the virulent progeny, no M5.1 homologue was detected, demonstrating a deletion on that part of linkage group VIII. P. infestans is diploid, so the avirulent strains must be hemizygous for the region concerned. A similar situation was found in another mapping population from two Mexican strains. The deletion was also found to occur in many field isolates. In a large set of unique isolates collected in The Netherlands from 1980 to 1991, 37% had no M5.1 homologue and the deletion correlated strongly with gain of virulence on potato lines carrying R3, R10, and R11. Also, in some old isolates that belong to a single clonal lineage (US-1) and are thus highly homogenous, deletions at the M5.1 locus were detected, indicating that this region is unstable.
Maize Ribosome-Inactivating Protein Inhibits Normal Development of Aspergillus nidulans and Aspergillus flavus
Authors: Nielsen Kirsten , Payne Gary A. , Boston Rebecca S.
Published: May 2007

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.
De novo Cortical Cell Division Triggered by the Phytopathogen Rhodococcus fascians in Tobacco
Authors: de O. Manes Carmem-Lara , Van Montagu Marc , Prinsen Els , Goethals Koen , Holsters Marcelle
Published: May 2007

Plant growth, development, and morphology can be affected by several environmental stimuli and by specific interactions with phytopathogens. In many cases, plants respond to pathogenic stimuli by adapting their hormone levels. Here, the interaction between the phytopathogen Rhodococcus fascians and one of its host plants, tobacco, was analyzed phenotypically and molecularly. To elucidate the basis of the cell division modulation and shoot primordia initiation caused by R. fascians, tobacco plants were infected at leaf axils and shoot apices. Adventitious meristems that gave rise to multiple-shoot primordia (leafy galls) were formed. The use of a transgenic line carrying the mitotic CycB1 promoter fused to the reporter gene coding for β-glucuronidase from Escherichia coli (uidA), revealed that stem cortical cells were stimulated to divide in an initial phase of the leafy gall ontogenesis. Local cytokinin and auxin levels throughout the infection process as well as modulation of expression of the cell cycle regulator gene Nicta;CycD3;2 are discussed.
Catharanthus roseus Genes Regulated Differentially by Mollicute Infections
Authors: Jagoueix-Eveillard Sandrine , Tarendeau Frank , Guolter Karine , Danet Jean-Luc , Bové Joseph M. , Garnier Monique
Published: May 2007

A differential display of mRNAs was used to isolate periwinkle cDNAs differentially expressed following infection with one of three mollicutes: Spiroplasma citri, Candidatus Phytoplasma aurantifolia, and stolbur phytoplasma. Twenty-four differentially expressed cDNAs were characterized by Northern blots and sequence analysis. Eight of them had homologies with genes in databanks coding for proteins involved in photosynthesis, sugar transport, response to stress, or pathways of phytosterol synthesis. The regulation of these genes in periwinkle plants infected by additional phloem-restricted bacteria showed that they were not specific to a given mollicute, but correlations with particular symptoms could be established. Expression of transketolase was down regulated following infection with a pathogenic strain of S. citri. No down regulation was observed for the nonphytopathogenic mutant GMT553, which is deficient for fructose utilization.
Definition of Tissue-Specific and General Requirements for Plant Infection in a Phytopathogenic Fungus
Authors: Dufresne Marie , Osbourn Anne E.
Published: May 2007

Although plant diseases are usually characterized by the part of the plant that is affected (e.g., leaf spots, root rots, wilts), surprisingly little is known about the factors that condition the ability of pathogens to colonize different plant tissues. Here we demonstrate that the leaf blast pathogen Magnaporthe grisea also can infect plant roots, and we exploit this finding to distinguish tissue-specific and general requirements for plant infection. Tests of a M. grisea mutant collection identified some mutants that were defective specifically in infection of either leaves or roots, and others such as the map kinase mutant pmk1 that were generally defective in pathogenicity. Conservation of a functional PMK1-related MAP kinase in the root pathogen Gaeumannomyces graminis was also demonstrated. Exploitation of the ability of M. grisea to infect distinct plant tissues thus represents a powerful tool for the comprehensive dissection of genetic determinants of tissue specificity and global requirements for plant infection.
Biological Activity of the rolB-like 5′ End of the A4-orf8 Gene from the Agrobacterium rhizogenes TL-DNA
Authors: Otten Léon , Helfer Anne
Published: May 2007

The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA). Unlike the IaaM proteins from other bacteria, Agrobacterium spp. T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family. This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined. Deletion of the 5′rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants. When expressed in tobacco, the rolB-like fragment did not affect growth or morphology. An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated. Neither the full-size A4-orf8 gene nor the 5′-truncated form induced detectable IAM synthesis. Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch.
Reduced Expression of the Tomato Ethylene Receptor Gene LeETR4 Enhances the Hypersensitive Response to Xanthomonas campestris pv. vesicatoria
Authors: Ciardi Joseph A. , Tieman Denise M. , Jones Jeffrey B. , Klee Harry J.
Published: May 2007

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 105 to 107 CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.
Isolation and Characterization of the Gene Coding for the Amidinotransferase Involved in the Biosynthesis of Phaseolotoxin in Pseudomonas syringae pv. phaseolicola
Authors: Hernández-Guzmán Gustavo , Alvarez-Morales Ariel
Published: May 2007

Pseudomonas syringae pv. phaseolicola is the causal agent of the “halo blight” disease of beans. A key component in the development of the disease is a nonhost-specific toxin, Nδ-(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine, known as phaseolotoxin. The homoarginine residue in this molecule has been suggested to be the product of Larginine:lysine amidinotransferase activity, previously detected in extracts of P. syringae pv. phaseolicola grown under conditions of phaseolotoxin production. We report the isolation and characterization of an amidinotransferase gene (amtA) from P. syringae pv. phaseolicola coding for a polypeptide of 362 residues (41.36 kDa) and showing approximately 40% sequence similarity to Larginine:inosamine-phosphate amidinotransferase from three species of Streptomyces spp. and 50.4% with an Larginine:glycine amidinotransferase from human mitochondria. The cysteine, histidine, and aspartic acid residues involved in substrate binding are conserved. Furthermore, expression of the amtA and argK genes and phaseolotoxin production occurs at 18°C but not at 28°C. An amidinotransferase insertion mutant was obtained that lost the capacity to synthesize homoarginine and phaseolotoxin. These results show that the amtA gene isolated is responsible for the amidinotransferase activity detected previously and that phaseolotoxin production depends upon the activity of this gene.
Purification of the Major Outer Membrane Protein ofAzospirillum brasilense, Its Affinity to Plant Roots, and Its Involvement in Cell Aggregation
Authors: Burdman Saul , Dulguerova Gabriella , Okon Yaacov , Jurkevitch Edouard
Published: May 2007

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.
Cell-Cycle, Phase-Specific Activation of Maize streak virus Promoters
Authors: Nikovics Krisztina , Simidjieva Julietta , Peres Adrian , Ayaydin Ferhan , Pasternak Taras , Davies Jeffrey W. , Boulton Margaret I. , Dudits Dénes , Horváth Gábor V.
Published: May 2007

It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize streak virus (MSV) in transgenic maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing “long and short” C-sense promoter sequences upstream of the GUS (β-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas the C-sense promoter sequences produced two peaks of activity in the S and G2 cell-cycle phases.
Isolation and Characterization of a Symbiosis-Regulated ras from the Ectomycorrhizal Fungus Laccaria bicolor
Authors: Sundaram S. , Kim S. J. , Suzuki H. , Mcquattie C. J. , Hiremath S. T. , Podila G. K.
Published: May 2007

Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants. Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation. The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells. Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots. Northern analysis showed that expression was detectable in L. bicolor 48 h after interaction as well as in the established mycorrhizal tissue. Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans.
Lectin-Like Glycoprotein PsNLEC-1 Is Not Correctly Glycosylated and Targeted in Boron-Deficient Pea Nodules
Authors: Bolaños Luis , Cebrián Arancha , Redondo-Nieto Miguel , Rivilla Rafael , Bonilla Ildefonso
Published: May 2007

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.
Identification of Defense-Related Rice Genes by Suppression Subtractive Hybridization and Differential Screening
Authors: Xiong Lizhong , Lee Min-Woo , Qi Min , Yang Yinong
Published: May 2007

Identification of host genes involved in defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in plants. In this study, two different cloning strategies were employed to identify defense-related genes from a tropical japonica rice cultivar (Oryza sativa cv. Drew). With the use of bacterial colony arrays, differential screening of a blast fungus (Pyricularia grisea)-induced rice cDNA library led to the isolation of 22 distinct rice genes that are expressed differentially in response to blast infection. Sequence analysis indicates that most of them are full-length cDNAs encoding pathogenesis-related proteins or other relatively abundant proteins. In combination with treatments of cycloheximide plus jasmonic acid (JA) or benzothiadiazole (BTH) in rice seedlings, the polymerase chain reaction-based suppression subtractive hybridization also was conducted to search for immediate early (IE) defense-related genes whose transcription is independent of de novo protein synthesis. The initial screening of only 768 subtracted clones resulted in the identification of 34 distinct IE genes that are induced by JA, BTH, and/or blast infection. Database searches revealed that these IE genes encode putative mitogen-activated protein kinase, diacylglycerol kinase, zinc finger protein, RelA-SpoT protein, ankyrin-containing protein, ABC transporter, β-ketoacyl-CoA synthase, and other potential defense-signaling components. Further characterization of these novel IE genes will likely facilitate the elucidation of defense signal transduction in rice plants.
Induction of Plant gp91 phox Homolog by Fungal Cell Wall, Arachidonic Acid, and Salicylic Acid in Potato
Authors: Yoshioka Hirofumi , Sugie Kenichi , Park Hae-Jun , Maeda Hirotaka , Tsuda Naoki , Kawakita Kazuhito , Doke Noriyuki
Published: May 2007

The oxidative burst has been suggested to be a primary event responsible for triggering the cascade of defense responses in various plant species against infection with avirulent pathogens or pathogen-derived elicitors. The molecular mechanisms of rapid production of active oxygen species (AOS), however, are not well known. We isolated homologs of gp91 phox, a plasma membrane protein of the neutrophil NADPH oxidase, from a potato cDNA library. Molecular cloning of the cDNA showed that there are two isogenes, designated StrbohA and StrbohB, respectively. The RNA gel blot analyses showed that StrbohA was constitutively expressed at a low level, whereas StrbohB was induced by hyphal wall components (HWC elicitor) from Phytophthora infestans in potato tubers. Treatment of potato tubers with HWC elicitor caused a rapid but weak transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6 to 9 h after treatment (phase II). Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocked both bursts, whereas pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolished only the second burst. These results suggest that the expression of StrbohA and StrbohB contributes to phase I and II bursts, respectively. The same is true for arachidonic acid, a lipid component of P. infestans-stimulated biphasic oxidative burst, whereas an endogenous signaling molecule, salicylic acid, only induced a weak phase II burst. Both molecules induced the StrbohB expression, which is in agreement with the second burst. To characterize the signal transduction pathway leading to the oxidative burst, we examined the role of protein phosphorylation in HWC-stimulated StrbohB gene expression. K252a and staurosporine, two protein kinase inhibitors, blocked the transcript accumulation. Two inhibitors of extracellular Ca2+ movement, however, did not abolish the transcript accumulation of StrbohB, suggesting that certain calcium-independent protein kinases are involved in the process of StrbohB gene expression. Additionally, we examined a causal relationship between the oxidative burst and expression of defense genes induced by the HWC elicitor. The transcript accumulation of genes related to sesquiterpenoid phytoalexin synthesis (lubimin and rishitin) and phenylpropanoid pathway was inhibited slightly by the DPI treatment, suggesting that the oxidative burst is not essential to activate these genes. Interestingly, the concomitant presence of DPI with the elicitor resulted in an increase in lubimin accumulation and a decrease in rishitin accumulation. Because it is known that lubimin is metabolized into rishitin via oxylubimin, we propose that AOS mediates the synthesis of rishitin from lubimin.
Root Mucilage from Pea and Its Utilization by Rhizosphere Bacteria as a Sole Carbon Source
Authors: Knee Emma M. , Gong Fang-Chen , Gao Mensheng , Teplitski Max , Jones Angela R. , Foxworthy Angel , Mort Andrew J. , Bauer Wolfgang D.
Published: May 2007

Plant roots secrete a complex polysaccharide mucilage that may provide a significant source of carbon for microbes that colonize the rhizosphere. High molecular weight mucilage was separated by high-pressure liquid chromatography gel filtration from low molecular weight components of pea root exudate. Purified pea root mucilage generally was similar in sugar and glycosidic linkage composition to mucilage from cowpea, wheat, rice, and maize, but appeared to contain an unusually high amount of material that was similar to arabinogalactan protein. Purified pea mucilage was used as the sole carbon source for growth of several pea rhizosphere bacteria, including Rhizobium leguminosarum 8401 and 4292, Burkholderia cepacia AMMD, and Pseudomonas fluorescens PRA25. These species grew on mucilage to cell densities of three- to 25-fold higher than controls with no added carbon source, with cell densities of 1 to 15% of those obtained on an equal weight of glucose. Micromolar concentrations of nod gene-inducing flavonoids specifically stimulated mucilage-dependent growth of R. leguminosarum 8401 to levels almost equaling the glucose controls. R. leguminosarum 8401 was able to hydrolyze p-nitrophenyl glycosides of various sugars and partially utilize a number of purified plant polysaccharides as sole carbon sources, indicating that R. leguminosarum 8401 can make an unexpected variety of carbohydrases, in accordance with its ability to extensively utilize pea root mucilage.
Characterization of a PR-10 Pathogenesis-Related Gene Family Induced in Rice During Infection with Magnaporthe grisea
Authors: McGee J. David , Hamer John E. , Hodges Thomas K.
Published: May 2007

A partial cDNA with homology to the PR-10 class of pathogenesis-related proteins was used to screen a rice genomic library. One 16-kb genomic clone contained three genes with PR-10 similarity. These genes, RPR10a, RPR10b, and RPR10c, were arranged in tandem and separated by approximately 2.5 kb. RPR10a cDNA was obtained by reverse transcription-polymerase chain reaction, and sequence analysis revealed that RPR10a and RPR10b encode predicted proteins of 158 and 160 amino acids, respectively, and share 71% amino acid identity. RPR10c appears to be a nonfunctional pseudogene. Gene-specific probes were used to study transcript accumulations of the three RPR10 genes in rice plants following inoculation with Magnaporthe grisea. RPR10a transcripts were induced from a low basal level within 12 h after inoculation and showed a second higher level induction at 48 h, which continued throughout the 144 h it was examined. In addition, RPR10a was induced strongly by salicylic and jasmonic acid applications to rice plants. Transcripts of RPR10b also were enhanced by M. grisea, but were not strongly visible until 48 h after inoculation. Tissue prints of M. grisea-infected rice leaves when the RPR10a-specific probe was used indicate that RPR10a is expressed most strongly in a localized fashion in response to the pathogen.
Global Regulators ExpA (GacA) and KdgR Modulate Extracellular Enzyme Gene Expression Through the RsmA-rsmB System in Erwinia carotovora subsp. carotovora
Authors: Hyytiäinen Heidi , Montesano Marcos , Palva E. Tapio
Published: May 2007

The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp. carotovora is controlled by a complex regulatory network. One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen. To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant. Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways. This interaction was mediated by the RsmA-rsmB system. Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA. Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR. This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E. carotovora subsp. carotovora.
Intracellular Localization and Movement Phenotypes of Alfalfa mosaic virus Movement Protein Mutants
Authors: Huang Mei , Jongejan Laurian , Zheng Huanquan , Zhang Lee , Bol John F.
Published: May 2007

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.