bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Uptake of Putrescine in the Rhizosphere Inhibits Competitive Root Colonization by Pseudomonas fluorescens Strain WCS365
Authors: Kuiper Irene , Bloemberg Guido V. , Noreen Sadaf , Thomas-Oates Jane E. , Lugtenberg Ben J. J.
Published: May 2007

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.
Gene C2 of the Monopartite Geminivirus Tomato yellow leaf curl virus-China Encodes a Pathogenicity Determinant That Is Localized in the Nucleus
Authors: van Wezel Rene , Liu Huangting , Tien Po , Stanley John , Hong Yiguo
Published: May 2007

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.
The Endosymbiosis-Induced Genes ENOD40 and CCS52a Are Involved in Endoparasitic-Nematode Interactions in Medicago truncatula
Authors: Favery Bruno , Complainville Arnaud , Vinardell Jose Maria , Lecomte Philippe , Vaubert Daniàle , Mergaert Peter , Kondorosi Adam , Kondorosi Eva , Crespi Martin , Abad Pierre
Published: May 2007

Plants associate with a wide range of mutualistic and parasitic biotrophic organisms. Here, we investigated whether beneficial plant symbionts and biotrophic pathogens induce distinct or overlapping regulatory pathways in Medicago truncatula. The symbiosis between Sinorhizobium meliloti and this plant results in the formation of nitrogen-fixing root nodules requiring the activation of specific genes in the host plant. We studied expression patterns of nodule-expressed genes after infection with the root-knot nematode Meloidogyne incognita. Two regulators induced during nodule organogenesis, the early nodulin gene ENOD40 involved in primordium formation and the cell cycle gene CCS52a required for cell differentiation and en-doreduplication, are expressed in galls of the host plant. Expression analysis of promoter-uidA fusions indicates an accumulation of CCS52a transcripts in giant cells undergoing endoreduplication, while ENOD40 expression is localized in surrounding cell layers. Transgenic plants overexpressing ENOD40 show a significantly higher number of galls. In addition, out of the 192 nodule-expressed genes tested, 38 genes were upregulated in nodules at least threefold compared with control roots, but only two genes, nodulin 26 and cyclin D3, were found to be induced in galls. Taken together, these results suggest that certain events, such as endoreduplication, cell-to-cell communication with vascular tissues, or water transport, might be common between giant cell formation and nodule development.
Isolation of Fungal Cell Wall Degrading Proteins from Barley (Hordeum vulgareL.) Leaves Infected withRhynchosporium secalis
Authors: Zareie Reza , Melanson Dara L. , Murphy Peter J.
Published: May 2007

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa β-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.
Functional Conservation of Wheat and Rice Mlo Orthologs in Defense Modulation to the Powdery Mildew Fungus
Authors: Elliott Candace , Zhou Fasong , Spielmeyer Wolfgang , Panstruga Ralph , Schulze-Lefert Paul
Published: May 2007

Homologs of barley Mlo are found in syntenic positions in all three genomes of hexaploid bread wheat, Triticum aestivum, and in rice, Oryza sativa. Candidate wheat orthologs, designated TaMlo-A1, TaMlo-B1, and TaMlo-D1, encode three distinct but highly related proteins that are 88% identical to barley MLO and appear to originate from the three diploid ancestral genomes of wheat. TaMlo-B1 and the rice ortholog, OsMlo2, are able to complement powdery mildew-resistant barley mlo mutants at the single-cell level. Overexpression of TaMlo-B1 or barley Mlo leads to super-susceptibility to the appropriate powdery mildew formae speciales in both wild-type barley and wheat. Surprisingly, overexpression of either Mlo or TaMlo-B1 also mediates enhanced fungal development to tested inappropriate formae speciales. These results underline a regulatory role for MLO and its wheat and rice orthologs in a basal defense mechanism that can interfere with forma specialis resistance to powdery mildews.
Induction of Systemic Resistance toBotrytis cinereain Tomato byPseudomonas aeruginosa7NSK2: Role of Salicylic Acid, Pyochelin, and Pyocyanin
Authors: Audenaert Kris , Pattery Theresa , Cornelis Pierre , Höfte Monica
Published: May 2007

The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin. Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato. SA-negative mutants of both strains lost the capacity to induce resistance. On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2. In 7NSK2, SA is probably very efficiently converted to Pch. However, Pch alone appeared not to be sufficient to induce resistance. In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage. Reactive oxygen species are known to play an important role in plant defense. To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated. PHZ1 is mutated in the phzM gene encoding an O-methyltransferase. PHZ1 was unable to induce resistance to B. cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance. These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P. aeruginosa 7NSK2.
The AVR4 Elicitor Protein ofCladosporium fulvumBinds to Fungal Components with High Affinity
Authors: Westerink Nienke , Roth Ronelle , Van den Burg Harrold A. , De Wit Pierre J. G. M. , Joosten Matthieu H. A. J.
Published: May 2007

The interaction between tomato and the fungal pathogen Cladosporium fulvum complies with the gene-for-gene system. Strains of C. fulvum that produce race-specific elicitor AVR4 induce a hypersensitive response, leading to resistance, in tomato plants that carry the Cf-4 resistance gene. The mechanism of AVR4 perception was examined by performing binding studies with125I-AVR4 on microsomal membranes of tomato plants. We identified an AVR4 high-affinity binding site (KD= 0.05 nM) which exhibited all the characteristics expected for ligand-receptor interactions, such as saturability, reversibility, and specificity. Surprisingly, the AVR4 high-affinity binding site appeared to originate from fungi present on infected tomato plants rather than from the tomato plants themselves. Detailed analysis showed that this fungus-derived, AVR4-specific binding site is heat- and proteinase K-resistant. Affinity crosslinking demonstrated that AVR4 specifically binds to a component of approximately 75 kDa that is of fungal origin. Our data suggest that binding of AVR4 to a fungal component or components is related to the intrinsic virulence function of AVR4 for C. fulvum.
Analysis of Differences Between Sinorhizobium meliloti 1021 and 2011 Strains Using the Host Calcium Spiking Response
Authors: Wais Rebecca J. , Wells Derek H. , Long Sharon R.
Published: May 2007

In the Rhizobium-legume symbiosis, compatible partners recognize each other through an exchange of signals. Plant inducers act together with bacterial transcriptional activators, the NodD proteins, to regulate the expression of bacterial biosynthetic nodulation (nod) genes. These genes direct the synthesis of a lipochito-oligosaccharide signal called Nod factor (NF). NFs elicit an early host response, root hair calcium spiking, that is initiated in root hair cells within 15 min of NF or live Rhizobium inoculation. We used calcium spiking as an assay to compare two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011, derived from the same field isolate. We found that the two strains show a kinetic difference in the calcium spiking assay: Rm1021 elicits calcium spiking in host root hairs as rapidly as purified NF, whereas Rm2011 shows a significant delay. This difference can be overcome by raising expression levels of either the NodD transcriptional activators or GroEL, a molecular chaperone that affects expression of the biosynthetic nod genes. We further demonstrate that the delay in triggering calcium spiking exhibited by Rm2011 is correlated with a reduced amount of nod gene expression compared with Rm1021. Therefore, calcium spiking is a useful tool in detecting subtle differences in bacterial gene expression that affect the early stages of the Rhizobium-legume symbiosis.
Regulation of the MPG1 Hydrophobin Gene in the Rice Blast Fungus Magnaporthe grisea
Authors: Soanes Darren M. , Kershaw Michael J. , Cooley R. Neil , Talbot Nicholas J.
Published: May 2007

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.
Mutation of Three Cysteine Residues inTomato yellow leaf curl virus-ChinaC2 Protein Causes Dysfunction in Pathogenesis and Posttranscriptional Gene—Silencing Suppression
Authors: van Wezel Rene , Dong Xiangli , Liu Huanting , Tien Po , Stanley John , Hong Yiguo
Published: May 2007

The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses re-vealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59and four potential phosphorylation sites(T52, S61, Y68, and S74) are highly conserved. When ex-pressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana ben-thamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonstrate that TYLCV-C C2, like its counterpart in the bipartite begomovirus African cassava mosaic virus, mediates suppression of posttranscriptional gene silencing (PTGS). Moreover, the individual mutations C36R, C38N, and C46I abolished the ability of C2 protein to suppress PTGS. These results suggest that the three cysteine residues within the putative zinc-finger motif are essential for C2 protein to induce necrosis and to act as a suppressor of PTGS.
Dm3Is One Member of a Large Constitutively Expressed Family of Nucleotide Binding Site—Leucine-Rich Repeat Encoding Genes
Authors: Shen Katherine A. , Chin Doris B. , Arroyo-Garcia Rosa , Ochoa Oswaldo E. , Lavelle Dean O. , Wroblewski Tadeusz , Meyers Blake C. , Michelmore Richard W.
Published: May 2007

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site—leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length ge-nomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Trans-genics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.
Functional Analyses of the Pto Resistance Gene Family in Tomato and the Identification of a Minor Resistance Determinant in a Susceptible Haplotype
Authors: Chang Jeff H. , Tai Yin-Shan , Bernal Adriana J. , Lavelle Daniel T. , Staskawicz Brian J. , Michelmore Richard W.
Published: May 2007

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4,) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophos-phorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a ‘susceptible’ haplotype encoding a minor recognition determinant.
Molecular Determinants Required for the Avirulence Function of AvrPphB in Bean and Other Plants
Authors: Tampakaki Anastasia P. , Bastaki Marina , Mansfield John W. , Panopoulos Nickolas J.
Published: May 2007

The avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants. The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa. We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoy-lation motif, which lies adjacent to the proteolytic cleavage site. The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X. The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression. Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains. Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.
An Arabidopsis thaliana Pectin Acetylesterase Gene Is Upregulated in Nematode Feeding Sites Induced by Root-knot and Cyst Nematodes
Authors: Vercauteren Isabel , de Almeida Engler Janice , De Groodt Ruth , Gheysen Godelieve
Published: May 2007

By using differential display, gene expression was investigated in Arabidopsis thaliana roots shortly after nematode infection, and a putative pectin acetylesterase (PAE) homolog (DiDi 9C-12) was found to be upregulated. PAEs catalyze the deacetylation of pectin, a major compound of primary cell walls. mRNA in situ hybridization experiments showed that the expression of DiDi 9C-12 was enhanced very early after infection in initiating giant-cells and in cells surrounding the nematodes. Later on, the level of DiDi 9C-12 mRNA was lower in giant-cells and transcripts were mainly found in parenchyma, endodermis, and pericycle cells of the root gall. Twenty days after infection, DiDi 9C-12 transcripts could no longer be detected. DiDi 9C-12 transcripts were also found in young syncytia and in the cells surrounding the expanding syncytium. Our results suggest that plant parasitic nematodes can modulate the rapid growth of the feeding cells and the expansion of the root gall by triggering the expression of DiDi 9C-12. PAEs, which probably act together with a range of other pectin-degrading enzymes, could be involved in softening and loosening the primary cell wall in nematode-infected plant roots.
Roles for Riboflavin in the Sinorhizobium-Alfalfa Association
Authors: Yang Guoping , Bhuvaneswari T. V. , Joseph Cecillia M. , King Maria D. , Phillips Donald A.
Published: May 2007

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.
rpfF Mutants of Xanthomonas oryzae pv. oryzae Are Deficient for Virulence and Growth Under Low Iron Conditions
Authors: Chatterjee Subhadeep , Sonti Ramesh V.
Published: May 2007

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.
hrp Genes of Erwinia chrysanthemi 3937 Are Important Virulence Factors
Authors: Yang Ching-Hong , Gavilanes-Ruiz Marina , Okinaka Yasushi , Vedel Regine , Berthuy Isabelle , Boccara Martine , Chen Jason Wei-Ta , Perna Nicole T. , Keen Noel T.
Published: May 2007

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN Δpel (Δpel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.
Auxotrophic Mutant Strains of Rhizobium etli Reveal New Nodule Development Phenotypes
Authors: Ferraioli Simona , Tatè Rosarita , Cermola Michele , Favre Renée , Iaccarino Maurizio , Patriarca Eduardo J.
Published: May 2007

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix¯ mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and citology before considerations of metabolic exchange are made.
Enhanced Resistance to Seed-Transmitted Bacterial Diseases in Transgenic Rice Plants Overproducing an Oat Cell-Wall-Bound Thionin
Authors: Iwai Takayoshi , Kaku H. , Honkura R. , Nakamura S. , Ochiai H. , Sasaki T. , Ohashi Y.
Published: May 2007

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.
Phylogeny and Genomic Organization of the TIR and Non-TIR NBS-LRR Resistance Gene Family in Medicago truncatul
Authors: Zhu Hongyan , Cannon Steven B. , Young Nevin D. , Cook Douglas R.
Published: May 2007

Sequences homologous to the nucleotide binding site (NBS) domain of NBS-leucine-rich repeat (LRR) resistance genes were retrieved from the model legume M. truncatula through several methods. Phylogenetic analysis classified these sequences into TIR (toll and interleukin-1 receptor) and non-TIR NBS subfamilies and further subclassified them into several well-defined clades within each subfamily. Comparison of M. truncatula NBS sequences with those from several closely related legumes, including members of the tribes Trifoleae, Viceae, and Phaseoleae, reveals that most clades contain sequences from multiple legume species. Moreover, sequences from species within the closely related Trifoleae and Viceae tribes (e.g., Medicago and Pisum spp.) tended to be cophyletic and distinct from sequences of Phaseoleae species (e.g., soybean and bean). These results suggest that the origin of major clades within the NBS-LRR family predate radiation of these Papilionoid legumes, while continued diversification of these sequences mirrors speciation within this legume subfamily. Detailed genetic and physical mapping of both TIR and non-TIR NBS sequences in M. truncatula reveals that most NBS sequences are organized into clusters, and few, if any, clusters contain both TIR and non-TIR sequences. Examples were found, however, of physical clusters that contain sequences from distinct phylogenetic clades within the TIR or non-TIR subfamilies. Comparative mapping reveals several blocks of resistance gene loci that are syntenic between M. truncatula and soybean and between M. truncatula and pea.
PPI1: A Novel Pathogen-Induced Basic Region-Leucine Zipper (bZIP) Transcription Factor from Pepper
Authors: Lee Sang Jik , Lee Mi Yeon , Yi So Young , Oh Sang Keun , Choi Soon Ho , Her Nam Han , Choi Doil , Min Byung Whan , Yang Seung Gyun , Harn Chee Hark
Published: May 2007

We have isolated a full-length cDNA, PPI1 (pepper-PMMV interaction 1), encoding a novel basic region-leucine zipper (bZIP) DNA-binding protein, from expressed sequence tags differentially expressed in Capsicum chinense PI257284 infected with Pepper mild mottle virus (PMMV). PPI1 encodes a predicted protein of 170 amino acids and contains a putative DNA-binding domain that shares significant amino acid identity with ACGT-binding domains of members of the bZIP DNA-binding protein family. PPI1 was localized in the nucleus and had transcriptional activation activity in yeast. Transcripts of the PPI1 gene were preferentially induced during an incompatible interaction by inoculation with PMMV, Pseudomonas syringae pv. syringae 61, and Xanthomonas campestris pv. vesicatoria race 3. However, the PPI1 gene was not induced by abiotic stressors that activate the plant defense-signaling pathway. Our data provide the first evidence that a bZIP transcription factor is preferentially induced by pathogen attack, suggesting that PPI1 may play a specific functional role in the regulation of expression of plant defense-related genes.
Identification and Characterization of a Soybean Ethylene-Responsive Element-Binding Protein Gene Whose mRNA Expression Changes During Soybean Cyst Nematode Infection
Authors: Mazarei Mitra , Puthoff David P. , Hart Jennifer K. , Rodermel Steven R. , Baum Thomas J.
Published: May 2007

Ethylene-responsive element-binding proteins (EREBPs) are members of a family of plant transcription factors. Conserved EREBP domains of these proteins bind to the GCC box, an ethylene-responsive promoter element found in many pathogenesis-related (PR) genes. Using degenerate primers to the EREBP domain from diverse plant species, an EREBP homolog was isolated from a soybean cDNA library. Gel mobility-shift assays revealed that the translation product of this cDNA bound specifically to GCC box sequences. We, therefore, named this gene Glycine max ethylene-responsive element-binding protein 1 (GmEREBP1), i.e., a gene coding for the first confirmed GCC box-binding protein of soybean. GmEREBP1 mRNA abundance was analyzed by RNA blot hybridizations in soybean roots and shoots of cultivars Corsoy 79 and Hartwig, which are susceptible and resistant, respectively, to the soybean cyst nematode (Heterodera glycines). These analyses revealed that GmEREBP1 is expressed in a root-preferential manner and that GmEREBP1 mRNA abundance is changed after H. glycines infection. GmEREBP1 mRNA abundance decreased in infected (susceptible) ‘Corsoy 79’ roots, whereas it increased in abundance in infected (resistant) ‘Hartwig’ roots. Furthermore, ethephon treatment repressed GmEREBP1 mRNA accumulation in both cultivars, whereas wounding increased expression in both cultivars. These changes in mRNA steady-state levels suggest that GmEREBP1 plays a role in soybean-H. glycines interactions.
Plant Defense Genes Associated with Quantitative Resistance to Potato Late Blight in Solanum phureja × Dihaploid S. tuberosum Hybrids
Authors: Trognitz Friederike , Manosalva Patricia , Gysin Rene , Niño-Liu David , Simon Reinhard , del Rosario Herrera Ma. , Trognitz Bodo , Ghislain Marc , Nelson Rebecca
Published: May 2007

Markers corresponding to 27 plant defense genes were tested for linkage disequilibrium with quantitative resistance to late blight in a diploid potato population that had been used for mapping quantitative trait loci (QTLs) for late blight resistance. Markers were detected by using (i) hybridization probes for plant defense genes, (ii) primer pairs amplifying conserved domains of resistance (R) genes, (iii) primers for defense genes and genes encoding transcriptional regulatory factors, and (iv) primers allowing amplification of sequences flanking plant defense genes by the ligation-mediated polymerase chain reaction. Markers were initially screened by using the most resistant and susceptible individuals of the population, and those markers showing different allele frequencies between the two groups were mapped. Among the 308 segregating bands detected, 24 loci (8%) corresponding to six defense gene families were associated with resistance at χ2 ≥ 13, the threshold established using the permutation test at P = 0.05. Loci corresponding to genes related to the phenylpropanoid pathway (phenylalanine ammonium lyase [PAL], chalcone isomerase [CHI], and chalcone synthase [CHS]), loci related to WRKY regulatory genes, and other defense genes (osmotin and a Phytophthora infestans-induced cytochrome P450) were significantly associated with quantitative disease resistance. A subset of markers was tested on the mapping population of 94 individuals. Ten defense-related markers were clustered at a QTL on chromosome III, and three defense-related markers were located at a broad QTL on chromosome XII. The association of candidate genes with QTLs is a step toward understanding the molecular basis of quantitative resistance to an important plant disease.
NHL25 and NHL3, Two NDR1/HIN1-Like Genes in Arabidopsis thaliana with Potential Role(s) in Plant Defense
Authors: Varet Anne , Parker Jane , Tornero Pablo , Nass Norbert , Nürnberger Thorsten , Dangl Jeffery L. , Scheel Dierk , Lee Justin
Published: May 2007

The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene. Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation. Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv. tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4. Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation. However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced. Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3. Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected. This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes. Bacteria-induced gene expression was not abolished in ethylene- (etr1-3 and ein2-1) and jasmonate- (coi1-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1). Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv. tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv. tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis. Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance.
The Xanthomonas Type III Effector Protein AvrBs3 Modulates Plant Gene Expression and Induces Cell Hypertrophy in the Susceptible Host
Authors: Marois Eric , Van den Ackerveken Guido , Bonas Ulla
Published: May 2007

Xanthomonas campestris pv. vesicatoria bacteria expressing the type III effector protein AvrBs3 induce a hypersensitive response in pepper plants carrying the resistance gene Bs3. Here, we report that infection of susceptible pepper and tomato plants leads to an AvrBs3-dependent hypertrophy of the mesophyll tissue. Agrobacterium-mediated transient expression of the avrBs3 gene in tobacco and potato plants resulted in a similar phenotype. Induction of hypertrophy was shown to depend on the repeat region, nuclear localization signals, and acidic transcription activation domain (AAD) of AvrBs3, suggesting that the effector modulates the host's transcriptome. To search for host genes regulated by AvrBs3 in an AAD-dependent manner, we performed a cDNA-amplified fragment length polymorphism analysis of pepper mRNA populations. Thirteen AvrBs3-induced transcripts were identified and confirmed by reverse transcriptase-poly-merase chain reaction. Sequence analysis revealed homologies to auxin-induced and expansinlike genes, which play a role in cell enlargement. These results suggest that some of the AvrBs3-induced genes may be involved in hypertrophy development and that xanthomonads possess type III effectors that steer host gene expression.
Ingestion of Double-Stranded RNA by Preparasitic Juvenile Cyst Nematodes Leads to RNA Interference
Authors: Urwin P. E. , Lilley Catherine J. , Atkinson Howard J.
Published: May 2007

RNA interference is of value in determining gene function in many organisms. Plant parasitic nematodes are refractory to microinjection as a means of introducing RNA and do not show any oral uptake until they are within plants. We have used octopamine to stimulate uptake by prepara-sitic second stage juveniles of two cyst nematodes, Heterodera glycines and Globodera pallida. This new technique was used to facilitate uptake of double stranded RNA (dsRNA) together with fluoroscein isothiocyanate as a visual marker. Targeting cysteine proteinases did not reduce the number of parasites but caused a shift from the normal female/male ratio of 3:1 to 1:1 by 14 days postinfection (dpi). Exposure of H. glycines to dsRNA corresponding to a newly characterized protein with homology to C-type lectins did not affect sexual fate, but 41% fewer parasites were recovered from the plants. As expected, treatment with dsRNA corresponding to the major sperm protein (MSP) had no effect on either parasite development or sexual fate over 14 days. Northern analysis showed lower transcript abundance for the two targeted mRNAs that occur in J2, plus a later inhibition for MSP transcripts when males developed sperm at 15 dpi. These findings establish a procedure for RNAi of plant parasitic nematodes.
Differential Expression of Genes Encoding Arabidopsis Phospholipases After Challenge with Virulent or Avirulent Pseudomonas Isolates
Authors: de Torres Zabela Marta , Fernandez-Delmond Isabelle , Niittyla Totte , Sanchez Pedro , Grant Murray
Published: May 2007

Phospholipase D (PLD; EC 3.1.4.4) has been linked to a number of cellular processes, including Tran membrane signaling and membrane degradation. Four PLD genes (α, β, γ1, and γ2) have been cloned from Arabidopsis thalami. They encode isoforms with distinct regulatory and catalytic properties but little is known about their physiological roles. Using cDNA amplified fragment length polymorphism display and RNA blot analysis, we identified Arabidopsis PLDγ1 and a gene encoding a lysophospholipase (EC 3.1.1.5), lysoPL1, to be differentially expressed during host response to virulent and avirulent pathogen challenge. Examination of the expression pattern of phospholipase genes induced in response to pathogen challenge was undertaken using the lysoPL1 and gene-specific probes corresponding to the PLD isoforms α, β, and γ1. Each mRNA class exhibited different temporal patterns of expression after infiltration of leaves with Pseudomonas syringae pv. tomato with or without avrRpm1. PLDα was rapidly induced and remained constitutively elevated regardless of treatment. PLDβ was transiently induced upon pathogen challenge. However, mRNA for the lysoPL1 and PLDγ1 genes showed enhanced and sustained elevation during an incompatible interaction, in both ndr1 and overexpressing NahG genetic backgrounds. Further evidence for differential engagement of these PLD mRNA during defense responses, other than gene-for-gene interactions, was demonstrated by their response to salicylic acid treatment or wounding. Our results indicate that genes encoding lysoPL1, PLDγ1, and PLDβ are induced during early responses to pathogen challenge and, additionally, PLDγ1 and lysoPL1 are specifically upregulated during gene-for-gene interactions, leading to the hypersensitive response. We discuss the possible role of these genes in plant-pathogen interactions.
Pisatin Demethylase Genes Are on Dispensable Chromosomes While Genes for Pathogenicity on Carrot and Ripe Tomato Are on Other Chromosomes in Nectria haematococca
Authors: Funnell Deanna L. , VanEtten Hans D.
Published: May 2007

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda¯ isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda¯ progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda¯ isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.
Transcript Profiling in the Barley Mildew Pathogen Blumeria graminis by Serial Analysis of Gene Expression (SAGE)
Authors: Thomas Stephen W. , Glaring Mikkel A. , Rasmussen Søren W. , Kinane Julia T. , Oliver Richard P.
Published: May 2007

The fungal pathogen Blumeria graminis f. sp. hordei develops on the barley leaf via distinct, morphologically well-defined stages. After landing on a host plant, the conidia rapidly germinate to form a primary germ tube. Subsequently, an appressorial germ tube emerges from the conidium and differentiates an appressorium from which penetration of the host cell wall is attempted. We have used serial analysis of gene expression to provide a measurement of messenger RNA contents in ungerminated conidia, during conidial germination, and during appressorium formation. The resulting data provide a resource for the characterization of changes in transcript accumulation during early development of B. graminis.
A Decarboxylase Encoded at the Cochliobolus heterostrophus Translocation-Associated Tox1B Locus Is Required for Polyketide (T-toxin) Biosynthesis and High Virulence on T-cytoplasm Maize
Authors: Rose Mark S. , Yun Sung-Hwan , Asvarak Thipa , Lu Shun-Wen , Yoder O. C. , Turgeon B. Gillian
Published: May 2007

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B¯ mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to aceto-acetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydro-genase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.