bluuebunny/crossref_metadata_2025 huggingface dataset:
The CTB1 Gene Encoding a Fungal Polyketide Synthase Is Required for Cercosporin Biosynthesis and Fungal Virulence of Cercospora nicotianae
Authors: Choquer Mathias , Dekkers Katherine L. , Chen Hui-Qin , Cao Lihua , Ueng Peter P. , Daub Margaret E. , Chung Kuang-Ren
Published: May 2007

Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.
NopA Is Associated with Cell Surface Appendages Produced by the Type III Secretion System of Rhizobium sp. Strain NGR234
Authors: Deakin William J. , Marie Corinne , Saad Maged M. , Krishnan Hari B. , Broughton William J.
Published: May 2007

Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretionminus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.
Identification of Arabidopsis Loci Required for Susceptibility to the Downy Mildew Pathogen Hyaloperonospora parasitica
Authors: Van Damme Mireille , Andel Annemiek , Huibers Robin P. , Panstruga Ralph , Weisbeek Peter J. , Van den Ackerveken Guido
Published: May 2007

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line L er eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.
Selection of Heterodera glycines Chorismate Mutase-1 Alleles on Nematode-Resistant Soybean
Authors: Lambert Kris N. , Bekal Sadia , Domier Leslie L. , Niblack Terry L. , Noel Gregory R. , Smyth Charles A.
Published: May 2007

The soybean cyst nematode Heterodera glycines is the most destructive pathogen of soybean in the Unites States. Diversity in the parasitic ability of the nematode allows it to reproduce on nematode-resistant soybean. H. glycines chorismate mutase-1 (Hg-CM-1) is a nematode enzyme with the potential to suppress host plant defense compounds; therefore, it has the potential to enhance the parasitic ability of nematodes expressing the gene. Hg-cm-1 is a member of a gene family where two alleles, Hg-cm-1A and Hg-cm-1B, have been identified. Analysis of the Hg-cm-1 gene copy number revealed that there are multiple copies of Hg-cm-1 alleles in the H. glycines genome. H. glycines inbred lines were crossed to ultimately generate three F2 populations of second-stage juveniles (J2s) segregating for Hg-cm-1A and Hg-cm-1B. Segregation of Hg-cm-1A and 1B approximated a 1:2:1 ratio, which suggested that Hg-cm-1 is organized in a cluster of genes that segregate roughly as a single locus. The F2H. glycines J2 populations were used to infect nematode- resistant (Hartwig, PI88788, and PI90763) and susceptible (Lee 74) soybean plants. H. glycines grown on Hartwig, Lee 74, and PI90763 showed allelic frequencies similar to Hg-cm-1A/B, but nematodes grown on PI88788 contained predominately Hg-cm-1A allele as a result of a statistically significant drop of Hg-cm-1B in the population. This result suggests that specific Hg-cm-1 alleles, or a closely linked gene, may aid H. glycines in adapting to particular soybean hosts.
The Zinc Uptake Regulator Zur Is Essential for the Full Virulence of Xanthomonas campestris pv. campestris
Authors: Tang Dong-Jie , Li Xiang-Jiang , He Yong-Qiang , Feng Jia-Xun , Chen Baoshan , Tang Ji-Liang
Published: May 2007

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 μM and in nonrich medium supplemented with Zn2+ at a concentration of 110 μM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 μM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.
Profiling of Wheat Class III Peroxidase Genes Derived from Powdery Mildew-Attacked Epidermis Reveals Distinct Sequence-Associated Expression Patterns
Authors: Liu Guosheng , Sheng Xiaoyan , Greenshields David L. , Ogieglo Adam , Kaminskyj Susan , Selvaraj Gopalan , Wei Yangdou
Published: May 2007

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.
β-Aminobutyric Acid-Induced Resistance Against Downy Mildew in Grapevine Acts Through the Potentiation of Callose Formation and Jasmonic Acid Signaling
Authors: Hamiduzzaman Mollah Md. , Jakab Gabor , Barnavon Laurent , Neuhaus Jean-Marc , Mauch-Mani Brigitte
Published: May 2007

β-Aminobutyric acid (BABA) was used to induce resistance in grapevine, (Vitis vinifera) against downy mildew (Plasmopara viticola). This led to a strong reduction of mycelial growth and sporulation in the susceptible cv. Chasselas. Comparing different inducers, the best protection was achieved with BABA followed by jasmonic acid (JA), whereas benzo (1,2,3)-thiadiazole-7-carbothionic acid-Smethyl ester (a salicylic acid [SA] analog) and abscisic acid (ABA) treatment did not increase the resistance significantly. Marker genes for the SA and JA pathways showed potentiated expression patterns in BABA-treated plants following infection. The callose synthesis inhibitor 2-deoxy- D-glucose partially suppressed BABA- and JA-induced resistance against P. viticola in Chasselas. Application of the phenylalanine ammonia lyase inhibitor 2-aminoindan-2- phosphonic acid and the lipoxygenase (LOX) inhibitor 5, 8, 11, 14-eicosatetraynoic acid (ETYA) also led to a reduction of BABA-induced resistance (BABA-IR), suggesting that callose deposition as well as defense mechanisms depending on phenylpropanoids and the JA pathways all contribute to BABA-IR. The similar phenotype of BABA- and JA-induced resistance, the potentiated expression pattern of JA-regulated genes (LOX-9 and PR-4) following BABA treatment, and the suppression of BABA-IR with ETYA suggest an involvement of the JA pathway in BABA-IR of grapevine leading to a primed deposition of callose and lignin around the infection sites.
Pathogen-Induced Production of the Antifungal AFP Protein from Aspergillus giganteus Confers Resistance to the Blast Fungus Magnaporthe grisea in Transgenic Rice
Authors: Moreno Ana Beatriz , Peñas Gisela , Rufat Mar , Bravo Juan Manuel , Estopà Montserrat , Messeguer Joaquima , San Segundo Blanca
Published: May 2007

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the β-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus.
Phosphorus-Free Membrane Lipids of Sinorhizobium meliloti Are Not Required for the Symbiosis with Alfalfa but Contribute to Increased Cell Yields Under Phosphorus-Limiting Conditions of Growth
Authors: López-Lara Isabel M. , Gao Jun-Lian , Soto María José , Solares-Pérez Alhondra , Weissenmayer Barbara , Sohlenkamp Christian , Verroios George P. , Thomas-Oates Jane , Geiger Otto
Published: May 2007

The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaAand btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.
The Effect of Cellulose Overproduction on Binding and Biofilm Formation on Roots by Agrobacterium tumefaciens
Authors: Matthysse Ann G. , Marry Mazz , Krall Leonard , Kaye Mitchell , Ramey Bronwyn E. , Fuqua Clay , White Alan R.
Published: May 2007

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic β-(1,2)-D-glucans, respectively. We have identified two genes (celG and celI) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.
The Cf-4 and Cf-9 Resistance Genes Against Cladosporium fulvum are Conserved in Wild Tomato Species
Authors: Kruijt Marco , Kip Diana J. , Joosten Matthieu H. A. J. , Brandwagt Bas F. , de Wit Pierre J. G. M.
Published: May 2007

The Cf-4 and Cf-9 genes originate from the wild tomato species Lycopersicon hirsutum and L. pimpinellifolium and confer resistance to strains of the leaf mold fungus Cladosporium fulvum that secrete the Avr4 and Avr9 elicitor proteins, respectively. Homologs of Cf-4 and Cf-9 (Hcr9s) are located in several clusters and evolve mainly through sequence exchange between homologs. To study the evolution of Cf genes, we set out to identify functional Hcr9s that mediate recognition of Avr4 and Avr9 (designated Hcr9-Avr4s and Hcr9-Avr9s) in all wild tomato species. Plants responsive to the Avr4 and Avr9 elicitor proteins were identified throughout the genus Lycopersicon. Open reading frames of Hcr9s from Avr4- and Avr9-responsive tomato plants were polymerase chain reaction-amplified. Several Hcr9s that mediate Avr4 or Avr9 recognition were identified in diverged tomato species by agroinfiltration assays. These Hcr9-Avr4s and Hcr9-Avr9s are highly identical to Cf-4 and Cf-9, respectively. Therefore, we conclude that both Cf-4 and Cf-9 predate Lycopersicon speciation. These results further suggest that C. fulvum is an ancient pathogen of the genus Lycopersicon, in which Cf-4 and Cf-9 have been maintained by selection pressure imposed by C. fulvum.
Natural Variation in the Arabidopsis Response to the Avirulence Gene hopPsyA Uncouples the Hypersensitive Response from Disease Resistance
Authors: Gassmann Walter
Published: May 2007

The plant hypersensitive response (HR) is tightly associated with gene-for-gene resistance and has been proposed to function in containing pathogens at the invasion site. This tight association has made it difficult to unequivocally evaluate the importance of HR for plant disease resistance. Here, hopPsyA from Pseudomonas syringae pv. syringae 61 is identified as a new avirulence gene for Arabidopsis that triggers resistance in the absence of macroscopic HR. Resistance to P. syringae pv. tomato DC3000 expressing hopPsyA was EDS1-dependent and NDR1-independent. Intriguingly, several Arabidopsis accessions were resistant to DC3000(hopPsyA) in the absence of HR. This is comparable to the Arabidopsis response to avrRps4, but it is shown that hopPsyA does not signal through RPS4. In a cross between two hopPsyA-resistant accessions that differ in their HR response, the HR segregated as a recessive phenotype regulated by a single locus. This locus, HED1 (HR regulator in EDS1 pathway), is proposed to encode a protein whose activity can cause suppression of the EDS1-dependent HR signaling pathway. HED1-regulated symptomless gene-for-gene resistance responses may explain some cases of Arabidopsis resistance to bacteria that are classified as nonhost resistance.
PecS and PecT Coregulate the Synthesis of HrpN and Pectate Lyases, Two Virulence Determinants in Erwinia chrysanthemi 3937
Authors: Nasser William , Reverchon Sylvie , Vedel Regine , Boccara Martine
Published: May 2007

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of ectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.
Membrane Release and Destabilization of Arabidopsis RIN4 Following Cleavage by Pseudomonas syringae AvrRpt2
Authors: Takemoto Daigo , Jones David A.
Published: May 2007

The Arabidopsis RIN4 protein mediates interaction between the Pseudomonas syringae type III effector proteins AvrB, AvrRpm1, and AvrRpt2 and the Arabidopsis disease-resistance proteins RPM1 and RPS2. Confocal laser-scanning fluorescence microscopy following particle bombardment of tobacco leaf epidermal cells was used to examine the subcellular localization of fusions between GFP and RIN4 or several of its homologs and to examine the effects of co-bombardment with AvrRpt2 or AvrRpm1. This study showed that RIN4 was attached to the plasma membrane at its carboxyl terminus and that a carboxyl-terminal CCCFxFxxx prenylation or acylation (typically palmitoylation) motif, or both, was essential for this attachment. RIN4 was cleaved by AvrRpt2 at two PxFGxW motifs, one releasing a large portion of RIN4 from the plasma membrane and both exposing amino-terminal residues that destabilized the carboxyl-terminal cleavage products by targeting them for N-end ubiquitylation and proteasomal degradation. Plasma-membrane localization of RIN4 was not affected by AvrRpm1. RIN4 was found to be part of a protein family comprising two full-length homologs and at least 11 short carboxyl-terminal homologs. Representatives of this family, comprising a full-length RIN4 homolog and two short carboxyl-terminal RIN4 homologs, were also attached to the plasma membrane and cleaved near their amino termini by AvrRpt2, but in contrast to RIN4, the major portions of these proteins remained on the plasma membrane. N-end degradation may play a minor role in RIN4 degradation but probably plays a major role in the degradation of RIN4 homologs and is, therefore, a major pathogenic consequence of AvrRpt2 cleavage.
In Planta Secretion of a Calreticulin by Migratory and Sedentary Stages of Root-Knot Nematode
Authors: Jaubert Stéphanie , Milac Adina L. , Petrescu Andrei J. , de Almeida-Engler Janice , Abad Pierre , Rosso Marie-Noëlle
Published: May 2007

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.
Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence
Authors: Liu Huanli , Zhang Shuping , Schell Mark A. , Denny Timothy P.
Published: May 2007

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (δpehA, δpehB, δpehC, δpme, and δegl) and one inactivated allele (cbhA::aphA-3) resulted in 15 mutants missing one to six CWDE. In soil-drench inoculation assays, virulence of mutants lacking only pectic enzymes (PehA, PehB, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.
The Coexistence of Symbiosis and Pathogenicity-Determining Genes in Rhizobium rhizogenes Strains Enables Them to Induce Nodules and Tumors or Hairy Roots in Plants
Authors: Velázquez Encarna , Peix Alvaro , Zurdo-Piñiro José Luis , Palomo José Luis , Mateos Pedro F. , Rivas Raúl , Muñoz-Adelantado Estefanía , Toro Nicolás , García-Benavides Pablo , Martínez-Molina Eustoquio
Published: May 2007

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.
The Endo-β-1,4-Xylanase Xyn11A Is Required for Virulence in Botrytis cinerea
Authors: Brito Nélida , Espino José Juan , González Celedonio
Published: May 2007

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-β-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A coding for an endo-β-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-β-1-4-xy-lanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.
Genetically Engineered Resistance to Fusarium Head Blight in Wheat by Expression of Arabidopsis NPR1
Authors: Makandar Ragiba , Essig Juliane S. , Schapaugh Melissa A. , Trick Harold N. , Shah Jyoti
Published: May 2007

Fusarium head blight (FHB) is a devastating disease of wheat and barley which causes extensive losses worldwide. Monogenic, gene-for-gene resistance to FHB has not been reported. The best source of resistance to FHB is a complex, quantitative trait derived from the wheat cv. Sumai 3. Here, we show that the Arabidopsis thaliana NPR1 gene (AtNPR1), which regulates the activation of systemic acquired resistance, when expressed in the FHB-susceptible wheat cv. Bobwhite, confers a heritable, type II resistance to FHB caused by Fusarium graminearum. The heightened FHB resistance in the transgenic AtNPR1-expressing wheat is associated with the faster activation of defense response when challenged by the fungus. PR1 expression is induced rapidly to a high level in the fungus-challenged spikes of the AtNPR1-expressing wheat. Furthermore, benzothiadiazole, a functional analog of salicylic acid, induced PR1 expression faster and to a higher level in the AtNPR1-expressing wheat than in the nontransgenic plants. We suggest that FHB resistance in the AtNPR1-expressing wheat is a result of these plants being more responsive to an endogenous activator of plant defense. Our results demonstrate that NPR1 is an effective candidate for controlling FHB.
Two Polyketide Synthase-Encoding Genes Are Required for Biosynthesis of the Polyketide Virulence Factor, T-toxin, by Cochliobolus heterostrophus
Authors: Baker Scott E. , Kroken Scott , Inderbitzin Patrik , Asvarak Thipa , Li Bi-Yu , Shi Liang , Yoder O. C. , Turgeon B. Gillian
Published: May 2007

Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A, and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for Ttoxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, ligation specificity-based expression analysis display (LEAD), a comparative amplified fragment length polymorphism/gel fractionation/capillary sequencing procedure, was applied to cDNAs from a near-isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both Ttoxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member likely is assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.
Novel Extracellular Chitinases Rapidly and Specifically Induced by General Bacterial Elicitors and Suppressed by Virulent Bacteria as a Marker of Early Basal Resistance in Tobacco
Authors: Ott Péter G. , Varga Gabriella J. , Szatmári Ágnes , Bozsó Zoltán , Klement Éva , Medzihradszky Katalin F. , Besenyei Eszter , Czelleng Arnold , Klement Zoltán
Published: May 2007

Early basal resistance (EBR, formerly known as early induced resistance) is triggered by general bacterial elicitors. EBR has been suggested to inhibit or retard expression of the type III secretion system of pathogenic bacteria and may also prevent nonpathogenic bacteria from colonizing the plant tissue. The quickness of EBR here plays a crucial role, compensating for a low bactericidal efficacy. This inhibitory activity should take place in the cell wall, as bacteria do not enter living plant cells. We found several soluble proteins in the intercellular fluid of tobacco leaf parenchyma that coincided with EBR under different environmental (light and temperature) conditions known to affect EBR. The two most prominent proteins proved to be novel chitinases (EC 3.2.1.14) that were transcriptionally induced before and during EBR development. Their expression in the apoplast was fast and not stress-regulated as opposed to many pathogenesis-related proteins. Nonpathogenic, saprophytic, and avirulent bacteria all induced EBR and the chitinases. Studies using these chitinases as EBR markers revealed that the virulent Pseudomonas syringae pv. tabaci, being sensitive to EBR, must suppress it while suppressing the chitinases. EBR, the chitinases, as well as their suppression are quantitatively related, implying a delicate balance determining the outcome of an infection.
Ethylene Signaling Mediates a Maize Defense Response to Insect Herbivory
Authors: Harfouche Antoine L. , Shivaji Renuka , Stocker Russell , Williams Paul W. , Luthe Dawn S.
Published: May 2007

The signaling pathways that enable plants to mount defenses against insect herbivores are known to be complex. It was previously demonstrated that the insect-resistant maize (Zea mays L.) genotype Mp708 accumulates a unique defense cysteine proteinase, Mir1-CP, in response to caterpillar feeding. In this study, the role of ethylene in insect defense in Mp708 and an insect-susceptible line Tx601 was explored. Ethylene synthesis was blocked with either cobalt chloride or aminoethoxyvinylglycine. Alternatively, ethylene perception was inhibited with 1-methylcyclopropene. Blocking ethylene synthesis and perception resulted in Mp708 plants that were more susceptible to caterpillar feeding. In addition, fall armyworm (Spodoptera frugiperda) larvae that fed on inhibitor-treated Mp708 plants had signifycantly higher growth rates than those reared on untreated plants. In contrast, these responses were not significantly altered in Tx601. The ethylene synthesis and perception inhibitors also reduced the accumulation of Mir1-CP and its transcript mir1 in response to herbivory. These results indicate that ethylene is a component of the signal transduction pathway leading to defense against insect herbivory in the resistant genotype Mp708.
Inhibition of Resistance Gene-Mediated Defense in Rice by Xanthomonas oryzae pv. oryzicola
Authors: Makino Seiko , Sugio Akiko , White Frank , Bogdanove Adam J.
Published: May 2007

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.
Pseudomonas syringae Effector avrB Confers Soybean Cultivar-Specific Avirulence on Soybean mosaic virus Adapted for Transgene Expression but Effector avrPto Does Not
Authors: Wang Li , Eggenberger Alan , Hill John , Bogdanove Adam J.
Published: May 2007

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P. syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein 1a, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.
Insights into Symbiotic Nitrogen Fixation in Medicago truncatula
Authors: Tesfaye Mesfin , Samac Deborah A. , Vance Carroll P.
Published: May 2007

In silico analysis of the Medicago truncatula gene index release 8.0 at The Institute for Genomic Research identified approximately 530 tentative consensus sequences (TC) clustered from 2,700 expressed sequence tags (EST) derived solely from Sinorhizobium meliloti-inoculated root and nodule tissues. A great majority (76%) of these TC were derived exclusively from nitrogen-fixing and senescent nodules. A cDNA filter array was constructed using approximately 58% of the in silico-identified TC as well as cDNAs representing selected carbon and nitrogen metabolic pathways. The purpose of the array was to analyze transcript abundance in M. truncatula roots and nodules following inoculation by a wild-type S. meliloti strain, a mutant strain that forms ineffective nodules, an uninoculated root control, and roots following nitrate or ammonium treatments. In all, 81 cDNAs were upregulated in both effective and ineffective nodules, and 78% of these cDNAs represent in silico-identified TC. One group of in silico-identified TC encodes genes with similarity to putative plant disease resistance (R) genes of the nucleotide binding site-leucine-rich repeat type. Expression of R genes was enhanced in effective nodules, and transcripts also were detected in ineffective nodules at 14 days postinoculation (dpi). Homologous R gene sequences also have been identified in the Medicago genome. However, their functional importance in nodules remains to be established. Genes for enzymes involved in organic acid synthesis along with genes involved in nitrogen metabolism were shown to be coexpressed in nitrate-fed roots and effective nodules of M. truncatula.
Suppression of Defense Response in Plants by the avrBs3/pthA Gene Family of Xanthomonas spp.
Authors: Fujikawa Takashi , Ishihara Hiromichi , Leach Jan E. , Tsuyumu Shinji
Published: May 2007

Effector genes of some plant-pathogenic bacteria, including some members of the avrBs3/pthA effector gene family from Xanthomonas spp., confer not only genotype-specific disease resistance but also pathogen aggressiveness or virulence. In addition, some effector gene products suppress induction of a nonspecific (or general) hypersensitive response (HR). To determine whether the Xanthomonas avrBs3/pthA gene family members apl1, avrXa7, or avrXa10 also confer suppressor activity, we introduced constructs with each effector gene into Pseudomonas fluorescens 55 that expressed the entire hrp cluster from P. syringae pv. syringae in cosmid pHIR11. When inoculated to tobacco ‘Bright Yellow’, P. fluorescens (pHIR11) induces the HR and expression of four tobacco defense response genes: HIN1, RbohB, PAL, and PR1. When P. fluorescens double transformants that contained pHIR11 and constructs with apl1, avrXa7, or avrXa10 were infiltrated into tobacco, the HR and expression of three defense response genes, RbohB, PAL, and PR1, were suppressed. The suppression of the HR and defense gene expression was more efficient in the transformants with the apl1 and avrXa7 than the transformant with avrXa10. Although expression of other defense genes was suppressed by the double transformants, HIN1 expression was the same level as was observed after infiltration with P. fluorescens (pHIR11), suggesting that HIN1 may not be involved directly in HR. Taken together, our data suggest that avrXa7, avrXa10, and apl1, when delivered to plant cells by the P. syringae pv. syringae hrp secretion system, can suppress nonhost HR and associated phenotypes.
Spontaneous Root-Nodule Formation in the Model LegumeLotus japonicus: A Novel Class of Mutants Nodulates in the Absence of Rhizobia
Authors: Tirichine Leïla , James Euan K. , Sandal Niels , Stougaard Jens
Published: May 2007

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.
A Root-Knot Nematode Secretory Peptide Functions as a Ligand for a Plant Transcription Factor
Authors: Huang Guozhong , Dong Ruihua , Allen Rex , Davis Eric L. , Baum Thomas J. , Hussey Richard S.
Published: May 2007

Parasitism genes expressed in the esophageal gland cells of root-knot nematodes encode proteins that are secreted into host root cells to transform the recipient cells into enlarged multinucleate feeding cells called giant-cells. Expression of a root-knot nematode parasitism gene which encodes a novel 13-amino-acid secretory peptide in plant tissues stimulated root growth. Two SCARECROW-like transcription factors of the GRAS protein family were identified as the putative targets for this bioactive nematode peptide in yeast two-hybrid analyses and confirmed by in vitro and in vivo coimmunoprecipitations. This discovery is the first demonstration of a direct interaction of a nematode-secreted parasitism peptide with a plant-regulatory protein, which may represent an early signaling event in the root-knot nematode-host interaction.
Analysis of Silencing Escape of Tomato leaf curl virus: An Evaluation of the Role of DNA Methylation
Authors: Bian Xue-Yu , Rasheed M. Saif , Seemanpillai Mark J. , Rezaian M. Ali
Published: May 2007

RNA silencing is a sequence-specific mechanism regulating gene expression and has been used successfully for antiviral defense against RNA viruses. Similar strategies to develop resistance against DNA containing Tomato leaf curl virus (TLCV) and some other geminiviruses have been unsuccessful. To analyze this silencing escape, we transformed tomato plants with a hairpin construct from the TLCV C2 open reading frame (ORF). The transgenic plants showed a strong RNA silencing response, and following TLCV inoculation, their infection was delayed. However, the viral infection was not prevented and TLCV DNA accumulated to the levels found in nontransgenic plants. To determine the fate of a transgene carrying homology to the virus, we used transgenic plants carrying the TLCV C4 gene, which induces a distinct phenotype. Upon TLCV infection, the phenotype was abolished and C4 transcript disappeared. Concurrently, TLCV-specific small interfering RNAs were produced. In situ hybridization showed abundant levels of TLCV DNA in phloem cells of TLCV-infected C4 trans-genic plants. However, the C4 transcripts were no longer detectable in nonvascular cells. Analysis of the transgene by methylation sequencing revealed a high level of de novo methylation of asymmetric cytosines in both the C4 ORF and its 35S promoter. A high level of methylation also was found at both symmetric and asymmetric cytosines of the complementary-sense strand of TLCV double-stranded DNA. Given the previous finding that methylated geminivi-ral DNA is not competent for replication, we provide a model whereby TLCV evades host defense through a population of de novo synthesized unmethylated DNA.