bluuebunny/crossref_metadata_2025 huggingface dataset:
Root, Root Hair, and Symbiotic Mutants of the Model Legume Lotus japonicus
Authors: Kawaguchi Masayoshi , Imaizumi-Anraku Haruko , Koiwa Hiroyuki , Niwa Sinobu , Ikuta Akira , Syono Kunihiko , Akao Shoichiro
Published: May 2007

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod¯ (Ljsym70 to Ljsym73), Hist¯ (alb1-1, alb1-2, and Ljsym79), and Fix¯ (fen1, Ljsym75, and Ljsym81).
Differential Effectiveness of Salicylate-Dependent and Jasmonate/Ethylene-Dependent Induced Resistance in Arabidopsis
Authors: Ton Jurriaan , Van Pelt Johan A. , Van Loon L. C. , Pieterse Corné M. J.
Published: May 2007

Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens. These three signals play important roles in induced resistance as well. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR). Both types of induced resistance are effective against a broad spectrum of pathogens. In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen. In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus [TCV]), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv. armoraciae). Activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. Induction of SAR and ISR was equally effective against X. campestris pv. armoraciae. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses. This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively.
Characterization of the salA, syrF, and syrG Regulatory Genes Located at the Right Border of the Syringomycin Gene Cluster of Pseudomonas syringae pv. syringae
Authors: Lu Shi-En , Scholz-Schroeder Brenda K. , Gross Dennis C.
Published: May 2007

Sequence analysis of the right border of the syr gene cluster of Pseudomonas syringae pv. syringae strain B301D revealed the presence of the salA gene 8,113 bp downstream of syrE. The predicted SalA protein of strain B301D differs by one amino acid from that of strain B728a. Two homologs of salA, designated syrF and syrG, were identified between syrE and salA. All three proteins contain helix-turn-helix DNA-binding motifs at their C termini and exhibit homology to regulatory proteins of the LuxR family. A salA mutant failed to produce syringomycin, whereas syrF and syrG mutants produced 12 and 50%, respectively, of syringomycin relative to the wild-type strain. The salA, syrF, and syrG mutants were significantly reduced in virulence, forming small, nonspreading lesions in immature cherry fruits. Translational fusions to the uidA gene were constructed to evaluate expression of syrB1 in regulatory mutant backgrounds and to determine the relationship among the three regulatory loci. Expression of a syrB1::uidA fusion required functional salA and syrF genes and, in series, the expression of a syrF::uidA fusion required a functional salA gene. These results demonstrate that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production and virulence in P. syringae pv. syringae.
Competitive Nodulation Blocking of cv. Afghanistan Pea Is Related to High Levels of Nodulation Factors Made by Some Strains of Rhizobium leguminosarum bv. viciae
Authors: Hogg Bridget , Davies Andrea E. , Wilson Karen E. , Bisseling Ton , Downie J. Allan
Published: May 2007

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this “competitive nodulation-blocking” (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.
Apoptotic Cell Death is a Common Response to Pathogen Attack in Oats
Authors: Yao Nan , Imai Satoshi , Tada Yasuomi , Nakayashiki Hitoshi , Tosa Yukio , Park Pyoyun , Mayama Shigeyuki
Published: May 2007

We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-bterminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.
Attenuation of Cf-Mediated Defense Responses at Elevated Temperatures Correlates With a Decrease in Elicitor-Binding Sites
Authors: de Jong Camiel F. , Takken Frank L. W. , Cai Xinzhong , de Wit Pierre J. G. M. , Joosten Matthieu H. A. J.
Published: May 2007

The interaction between the fungal pathogen Cladosporium fulvum and its only host, tomato, is a well-described gene-for-gene system and several resistance (Cf) genes of tomato and matching fungal avirulence (Avr) genes have been characterized. Transgenic tobacco suspension cells expressing Cf genes respond to matching elicitors with typical defense responses, such as medium alkalization and an oxidative burst. We found that this response is attenuated at elevated ambient temperatures. Tomato seedlings expressing both a Cf and the matching Avr gene rapidly die as a result of systemic necrosis at normal temperatures, but are rescued at 33°C. We demonstrate that, at 33°C, the Cf/Avr-mediated induction of defense-related genes is reversibly suppressed. Furthermore, in cell suspensions, the AVR-induced medium alkalization response is slowly suppressed upon incubation at 33°C, but is quickly restored after transfer to lower temperatures. A high-affinity binding site (HABS) for AVR9 is present on plasma membranes isolated from solanaceous plants and has been suggested to act as a co-receptor for AVR9. The amount of AVR9-HABS is 80% reduced in tobacco cell suspensions incubated at 33°C, as compared with cell suspensions incubated at 20°C. Our data suggest that the temperature sensitivity of Cf-mediated defense responses resides at the level of perception of the fungal avirulence factors.
PopP1, a New Member of the YopJ/AvrRxv Family of Type III Effector Proteins, Acts as a Host-Specificity Factor and Modulates Aggressiveness of Ralstonia solanacearum
Authors: Lavie Muriel , Shillington Edwin , Eguiluz Cédric , Grimsley Nigel , Boucher Christian
Published: May 2007

A functional analysis of an 11-kb-long region of the genome of the plant-pathogenic bacterium Ralstonia solanacearum, previously identified as an alternative codon usage region (ACUR), reveals that it was probably acquired through horizontal gene transfer. This ACUR encodes an insertion sequence and eight potential proteins, one of which is partially homologous with a host-specificity factor from a plant-pathogenic Erwinia sp., and another, PopP1, which is homologous to members of the YopJ/AvrRxv family of type III-secreted bacterial effectors controlling interaction between bacteria and their hosts. The analysis of mutants affecting all except one of the genes identified in the ACUR showed that only the popP1-deficient strain had an altered phenotype in plant infection tests. This mutant strain became pathogenic to a Petunia line that is resistant to the wild-type strain. Therefore, popP1 behaves as a typical avirulence gene. We demonstrate that PopP1 protein is secreted and that secretion of this protein requires a functional type III-secretion pathway. In contrast to the structural genes for other type III-secreted proteins identified in R. solanacearum, transcription of the popP1 gene is not coregulated with transcription of hrp genes but is constitutive.
Ectopic Expression ofTsi1in Transgenic Hot Pepper Plants Enhances Host Resistance to Viral, Bacterial, and Oomycete Pathogens
Authors: Shin Ryoung , Park Jeong Mee , An Jong-Min , Paek Kyung-Hee
Published: May 2007

In many plants, including hot pepper plants, productivity is greatly affected by pathogen attack. We reported previously that tobacco stress-induced gene 1 (Tsi1) may play an important role in regulating stress responsive genes and pathogenesis-related (PR) genes. In this study, we demonstrated that overexpression of Tsi1 gene in transgenic hot pepper plants induced constitutive expression of several PR genes in the absence of stress or pathogen treatment. The transgenic hot pepper plants expressing Tsi1 exhibited resistance to Pepper mild mottle virus (PMMV) and Cucumber mosaic virus (CMV). Furthermore, these transgenic plants showed increased resistance to a bacterial pathogen, Xanthomonas campestris pv. vesicatoria and also an oomycete pathogen, Phytophthora capsici. These results suggested that ectopic expression of Tsi1 in transgenic hot pepper plants enhanced the resistance of the plants to various pathogens, including viruses, bacteria, and oomycete. These results suggest that using transcriptional regulatory protein genes may contribute to developing broad-spectrum resistance in crop plants.
Developmental Control of Promoter Activity Is Not Responsible for Mature Onset of Cf-9B-Mediated Resistance to Leaf Mold in Tomato
Authors: Panter S. N. , Hammond-Kosack K. E. , Harrison K. , Jones J. D. G. , Jones D. A.
Published: May 2007

Cf-9 confers resistance to tomato seedlings and mature plants against Cladosporium fulvum races expressing the Avr9 elicitor. It is the central member of a cluster of five paralogous genes in an introgressed segment of chromosome 1 derived from Lycopersicon pimpinellifolium. The other four genes have been named Hcr9-9A, Hcr9-9B, Hcr9-9D, and Hcr9-9E. Hcr9-9B, here designated Cf-9B, encodes weaker resistance than Cf-9, recognizes a different elicitor, and protects only mature plants from infection. The onset of Cf-9B-mediated resistance and the molecular basis for its developmental control were investigated in this study. Fungal inoculation of tomato plants containing reciprocal Cf-9/Cf-9B promoter-coding region swaps, analysis of tomato plants containing promoter-gusA fusions, and a reverse transcriptase-polymerase chain reaction study of Cf-9 and Cf-9B transcripts in tomato plants suggested that transcriptional control of Cf-9B did not account for the late onset of Cf-9B-mediated resistance. Alternative explanations for the onset of Cf-9B-mediated resistance in mature plants are discussed.
A Mitogen-Activated Protein Kinase Gene (MGV1) in Fusarium graminearum Is Required for Female Fertility, Heterokaryon Formation, and Plant Infection
Authors: Hou Zhanming , Xue Chaoyang , Peng Youliang , Katan Talma , Kistler H. Corby , Xu Jin-Rong
Published: May 2007

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.
Tissue Specific Localization of Root Infection by Fungal Pathogens: Role of Root Border Cells
Authors: Gunawardena Uvini , Hawes Martha C.
Published: May 2007

When roots of pea seedlings were inoculated uniformly with spores of Nectria haematocca or other pea pathogenic fungi, more than 90% developed lesions in the region of elongation within 3 days. More mature regions of most roots as well as the tip showed no visible signs of infection. Yet, microscopic observation revealed that ‘mantles,’ comprised of fungal hyphae intermeshed with populations of border cells, covered the tips of most roots. After physical detachment of the mantle, the underlying tip of most roots was found to be free of infection. Mantle-covered root tips did not respond to invasion of their border cells by activation of known defense genes unless there was invasion of the tip itself, as revealed by the presence of a lesion. Concomitant with the activation of defense genes was the induction of a cell-wall degrading enzyme whose expression is a marker for renewed production of border cells. Mantle formation did not occur in response to nonpathogens. The data are consistent with the hypothesis that border cells serve as a host-specific ‘decoy’ that protects root meristems by inhibiting fungal infection of the root tip.
Flagella-Driven Chemotaxis Towards Exudate Components Is an Important Trait for Tomato Root Colonization by Pseudomonas fluorescens
Authors: de Weert Sandra , Vermeiren Hans , Mulders Ine H. M. , Kuiper Irene , Hendrickx Nico , Bloemberg Guido V. , Vanderleyden Jos , De Mot René , Lugtenberg Ben J. J.
Published: May 2007

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in non-sterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.
Coupling of Iron Assimilation and Pectinolysis in Erwinia chrysanthemi 3937
Authors: Franza Thierry , Michaud-Soret Isabelle , Piquerel Pierrette , Expert Dominique
Published: May 2007

Two major virulence determinants of the plant-pathogenic enterobacterium Erwinia chrysanthemi strain 3937 are the production of pectate lyase enzymes that degrade plant cell walls and expression of two high-affinity iron uptake systems mediated by two structurally unrelated siderophores, chrysobactin and achromobactin. Low iron availability is a signal that triggers transcription of the genes encoding pectate lyases PelD and PelE as well as that of genes involved in iron transport. This metalloregulation is mediated by the transcriptional repressor Fur. In this study, we analyzed the molecular mechanisms of this control. We purified the Erwinia chrysanthemi Fur protein. Band shift assays showed that Fur specifically binds in vitro to the regulatory regions of the genes encoding the ferrichrysobactin outer membrane receptor Fct and the pectate lyases PelD and PelE. We identified the Fur-binding sites of these promoter regions by performing DNase I footprinting experiments. From these data, we propose that Fur could inhibit the activation of the pelD and pelE genes by the cAMP receptor protein CRP according to an anti-activation mechanism. To identify other possible effectors involved in this control, we screened a bank of insertion mutants for an increase in transcriptional activity of pelD and fct genes in response to iron limitation. We isolated a mutant affected in the kdgK gene encoding the 2-keto-3-deoxygluconate (KDG) kinase, an enzyme involved in pectin catabolism. The growth of this mutant in the presence of pectic compounds led to a constitutive expression of iron transport genes as well as complete derepression of the pectinolysis genes. This effect was caused by intracellular accumulation of KDG. However, the derepression of iron transport genes by KDG does not involve the KdgR regulator of pectinolysis genes, which uses KDG as inducer. Thus, in Erwinia chrysanthemi, iron depletion or presence of KDG induces transcription of the genes involved in iron assimilation and pectinolysis. These important pathogenicity functions are coregulated by responding to common signals encountered in planta.
The P34 Syringolide Elicitor Receptor Interacts with a Soybean Photorespiration Enzyme, NADH-Dependent Hydroxypyruvate Reductase
Authors: Okinaka Yasushi , Yang Ching-Hong , Herman Eliot , Kinney Anthony , Keen Noel T.
Published: May 2007

The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean. However, the mechanism underlying P34 signal transmission after syringolide binding is unknown. In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column. A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing. HPR is an important enzyme involved in the plant photorespiration system. Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2). Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34. Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2. Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR. These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean.
Effect of Leaf Surface Waxes on Leaf Colonization by Pantoea agglomerans and Clavibacter michiganensis
Authors: Marcell Lise M. , Beattie Gwyn A.
Published: May 2007

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.
The Mitogen-Activated Protein Kinase Gene MAF1 Is Essential for the Early Differentiation Phase of Appressorium Formation in Colletotrichum lagenarium
Authors: Kojima Kaihei , Kikuchi Taisei , Takano Yoshitaka , Oshiro Eriko , Okuno Tetsuro
Published: May 2007

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.
CPR1: A Gene Encoding a Putative Signal Peptidase That Functions in Pathogenicity of Colletotrichum graminicola to Maize
Authors: Thon M. R. , Nuckles E. M. , Takach J. E. , Vaillancourt L. J.
Published: May 2007

Colletotrichum graminicola causes anthracnose leaf blight and stalk rot of maize. We used restriction-enzyme mediated insertional (REMI) mutagenesis to identify a gene in this fungus that is required for pathogenicity to both stalks and leaves. The predicted polypeptide encoded by this gene, which we have named CPR1, is similar to a family of proteins that comprise one subunit of the eukaryotic micro-somal signal peptidase. The nonpathogenic CPR1 REMI mutant contains a plasmid integration in the 3′ untranslated region of the gene, 19 bp downstream from the stop codon. The result is a significant reduction in transcript levels in comparison to the wild type, perhaps as a result of increased transcript instability. We were unable to knock out the CPR1 gene, and it may be essential for viability. Microscopic examination of the REMI mutant on maize leaves revealed that it is fully capable of penetrating and colonizing host cells during the initial, biotrophic phases of the disease interaction but, unlike the wild type, it appears to be unable to switch to a necrotrophic mode of growth. We suggest that the CPR1 REMI mutant may be unable to secrete sufficient quantities of degradative enzymes to support that transition. The CPR1 REMI mutant provides us with a useful tool for future studies of the role of fungal protein transport in this important stalk rot disease of maize.
Expression of Genes Encoding Late Nodulins Characterized by a Putative Signal Peptide and Conserved Cysteine Residues Is Reduced in Ineffective Pea Nodules
Authors: Kato Takashi , Kawashima Kazuya , Miwa Masami , Mimura Yoshifumi , Tamaoki Masanori , Kouchi Hiroshi , Suganuma Norio
Published: May 2007

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II–III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.
Sinorhizobium fredii HH103 Has a Truncated nolO Gene Due to a -1 Frameshift Mutation That Is Conserved Among Other Geographically Distant S. fredii Strains
Authors: Madinabeitia Nuria , Bellogín Ramón A. , Buendía-Clavería Ana M. , Camacho María , Cubo Teresa , Espuny M. Rosario , Gil-Serrano Antonio M. , Lyra María C. C. P. , Moussaid Ahmed , Ollero F. Javier , Soria-Díaz M. Eugenia , Vinardell José M. , Zeng Jing , Ruiz-Sainz José E.
Published: May 2007

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103, suggesting a polar effect of the transposon insertion into nolO over the noeI gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.
Fdb1 and Fdb2, Fusarium verticillioides Loci Necessary for Detoxification of Preformed Antimicrobials from Corn
Authors: Glenn A. E. , Gold S. E. , Bacon C. W.
Published: May 2007

Fusarium verticillioides is a fungus of significant economic importance because of its deleterious effects on plant and animal health and on the quality of their products. Corn (Zea mays) is the primary host for F. verticillioides, and we have investigated the impact of the plant's antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) on fungal virulence and systemic colonization. F. verticillioides is able to metabolize these antimicrobials, and genetic analyses indicated two loci, Fdb1 and Fdb2, were involved in detoxification. Mutation at either locus caused sensitivity and no detoxification. In vitro physiological complementation assays resulted in detoxification of BOA and suggested that an unknown intermediate compound was produced. Production of the intermediate compound involved Fdb1, and a lesion in fdb2 preventing complete metabolism of BOA resulted in transformation of the intermediate into an unidentified metabolite. Based on genetic and physiological data, a branched detoxification pathway is proposed. Use of genetically characterized detoxifying and nondetoxifying strains indicated that detoxification of the corn antimicrobials was not a major virulence factor, since detoxification was not necessary for development of severe seedling blight or for infection and endophytic colonization of seedlings. Production of the antimicrobials does not appear to be a highly effective resistance mechanism against F. verticillioides.
MST12 Regulates Infectious Growth But Not Appressorium Formation in the Rice Blast Fungus Magnaporthe grisea
Authors: Park Gyungsoon , Xue Chaoyang , Zheng Li , Lam Stephen , Xu Jin-Rong
Published: May 2007

In the rice blast fungus Magnaporthe grisea, a mitogen-activated protein kinase gene, PMK1, is known to regulate ap-pressorium formation and infectious hyphae growth. Since PMK1 is homologous to the FUS3 and KSS1 genes that regulate the transcription factor STE12 in yeast, we functionally characterized the STE12 homologue in M. grisea (MST12). A polymerase chain reaction-based approach was used to isolate the MST12 gene that is homologous to yeast STE12. Four mst12 deletion mutants were isolated by gene replacement. No obvious defect in vegetative growth, conidiation, or conidia germination was observed in mst12 mutants. However, mst12 mutants were nonpathogenic on rice and barley leaves. In contrast to pmk1 mutants that did not form appressoria, mst12 mutants produced typical dome-shaped and melanized appressoria. However, the ap-pressoria formed by mst12 mutants failed to penetrate onion epidermal cells. When inoculated through wound sites, mst12 mutants failed to cause spreading lesions and appeared to be defective in infectious growth. These data indicate that MST12 may function downstream of PMK1 to regulate genes involved in infectious hyphae growth. A transcription factor or factors other than MST12 must exist in M. grisea and function downstream from PMK1 for ap-pressorium formation.
Efficient Infection of Nicotiana benthamiana by Tomato bushy stunt virus Is Facilitated by the Coat Protein and Maintained by p19 Through Suppression of Gene Silencing
Authors: Qu Feng , Morris T. Jack
Published: May 2007

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana ben-thamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. ben-thamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.
Transformation and Transposon Mutagenesis of Leifsonia xyli subsp. xyli, Causal Organism of Ratoon Stunting Disease of Sugarcane
Authors: Brumbley Stevens M. , Petrasovits Lars A. , Birch Robert G. , Taylor Paul W. J.
Published: May 2007

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/μg of plas-mid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/μg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/μg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam¯/dcm¯ E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-μm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetra-cycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.
Lotus japonicus Gene Ljsbp Is Highly Conserved Among Plants and Animals and Encodes a Homologue to the Mammalian Selenium-Binding Proteins
Authors: Flemetakis Emmanouil , Agalou Adamantia , Kavroulakis Nektarios , Dimou Maria , Martsikovskaya Anna , Slater Andrian , Spaink Herman P. , Roussis Andreas , Katinakis Panagiotis
Published: May 2007

We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L. japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L. japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a poly-peptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.
Identification of a Family of Extensin-Like Glycoproteins in the Lumen of Rhizobium-Induced Infection Threads in Pea Root Nodules
Authors: Rathbun Elizabeth A. , Naldrett Michael J. , Brewin Nicholas J.
Published: May 2007

Rhizobium leguminosarum bv. viciae normally gains access to pea host cells through tubular cell wall ingrowths termed infection threads. Matrix glycoprotein (MGP), a major component of the infection thread lumen, is also secreted from the tips of uninoculated roots and can be released into solution under reducing conditions. Monoclonal antibody MAC265, which recognizes MGP through a carbohydrate epitope, was used for immunoaffinity purification of the glycoprotein from pea roots. Following treatment with chymotrypsin, a peptide fragment was obtained and subjected to N-terminal sequencing. Using PCR primers based on this sequence, cDNA clones were isolated with RNA from inoculated roots and nodules. DNA sequencing of 30 of these clones revealed a family of closely related and repetitive polypeptides with (hydroxy)proline-rich motifs. The cDNA sequences showed over 70% identity with the deduced amino acid sequences of plant extensins, particularly with VfNDS-E from Vicia faba and MtN12 from Medicago truncatula, both of which are strongly upregulated in legume root nodules. Root nodule extensins from pea were of variable length but showed strong sequence conservation of the N-terminus, of the C-terminus, and of a central domain comprising 33 amino acids that were sometimes reiterated. The distribution of tyrosine residues suggested the possible importance of intramolecular and intermolecular cross-linking. There was strong sequence conservation with MtN12 in the 3′-untranslated region, suggesting a possible involvement in posttranscriptional regulation of gene expression.
Expressed Sequence Tags from Roots and Nodule Primordia of Lotus japonicus Infected with Mesorhizobium loti
Authors: Poulsen Carsten , Pødenphant Lone
Published: May 2007

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.
Pathogen Challenge, Salicylic Acid, and Jasmonic Acid Regulate Expression of Chitinase Gene Homologs in Pine
Authors: Davis John M. , Wu Haiguo , Cooke Janice E. K. , Reed Jon M. , Luce K. Scott , Michler Charles H.
Published: May 2007

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable β-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.
Novel Aspects of Symbiotic Nitrogen Fixation Uncovered by Transcript Profiling with cDNA Arrays
Authors: Colebatch Gillian , Kloska Sebastian , Trevaskis Ben , Freund Susanne , Altmann Thomas , Udvardi Michael K.
Published: May 2007

An array of 2,304 cDNA clones derived from nitrogen-fixing nodules of Lotus japonicus was produced and used to detect differences in relative gene transcript abundance between nodules and uninfected roots. Transcripts of 83 different genes were found to be more abundant in nodules than in roots. More than 50 of these have never before been identified as nodule-induced in any species. Expression of 36 genes was detected in nodules but not in roots. Several known nodulin genes were included among the nodule-induced genes. Also included were genes involved in sucrose breakdown and glycolysis, CO2 recycling, and amino acid synthesis, processes that are known to be accelerated in nodules compared with roots. Genes involved in membrane transport, hormone metabolism, cell wall and protein synthesis, and signal transduction and regulation of transcription were also induced in nodules. Genes that may subvert normal plant defense responses, including two encoding enzymes involved in detoxification of active oxygen species and one that may prohibit phytoalexin synthesis, were also identified. The data represent a rich source of information for hypothesis building and future exploration of symbiotic nitrogen fixation.
Expression of Different Calmodulin Genes in Bean (Phaseolus vulgaris L.): Role of Nod Factor on Calmodulin Gene Regulation
Authors: Camas Alberto , Cárdenas Luis , Quinto Carmen , Lara Miguel
Published: May 2007

Three calmodulin (PvCaM-1, PvCaM-2, and PvCaM-3) clones were isolated from a Phaseolus vulgaris nodule cDNA library. All clones contain the complete coding region and are 62 to 74% homologous within this region. Compared to plant CaM consensus sequences, PvCaM-2 has a novel tyrosine118 residue, representing a putative phosphorylation site. Southern analysis suggested that calmodulin is encoded by a gene family. These three CaM clones are expressed mainly in young tissues and meristems. The expression pattern of PvCaM-2 and PvCaM-3 is almost identical but different from that of PvCaM-1, suggesting that PvCaM-1 is a well-defined CaM gene, whereas PvCaM-2 and PvCaM-3 could be alleles. PvCaM clones are expressed early in nodules, and transcript levels increase from nodule primordia to nodule-like structures induced by the Nod factor. Conversely, in roots, Nod factor lowers mRNA levels of all three PvCaM clones, but especially of PvCaM-1. Inhibition of PvCaM-1 expression also is observed when 2,3,5-triiodobenzoic acid is added and is prevented when roots are treated with indole-3-acetic acid, suggesting that PvCaM-1 regulation is related to the Nod factor inhibition of polar auxin transport. These results could suggest that CaM clones do not participate in the early signaling generated by the Nod factor but do participate in early events of nodule formation.