Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

Deletions of the SACPD-C locus elevate seed stearic acid levels but also result in fatty acid and morphological alterations in nitrogen fixing nodules.

BACKGROUND: Soybean (Glycine max) seeds are the primary source of edible oil in the United States. Despite its widespread utility, soybean oil is oxidatively unstable. Until recently, the majority of soybean oil underwent chemical hydrogenation, a process which also generates trans fats. An alternative to chemical hydrogenation is genetic modification of seed oil through identification and introgression of mutant alleles. One target for improvement is the elevation of a saturated fat with no negative cardiovascular impacts, stearic acid, which typically constitutes a minute portion of seed oil (~3%). RESULTS: We examined radiation induced soybean mutants with moderately increased stearic acid (10-15% of seed oil, ~3-5 X the levels in wild-type soybean seeds) via comparative whole genome hybridization and genetic analysis. The deletion of one SACPD isoform encoding gene (SACPD-C) was perfectly correlated with moderate elevation of seed stearic acid content. However, SACPD-C deletion lines were also found to have altered nodule fatty acid composition and grossly altered morphology. Despite these defects, overall nodule accumulation and nitrogen fixation were unaffected, at least under laboratory conditions. CONCLUSIONS: Although no yield penalty has been reported for moderate elevated seed stearic acid content in soybean seeds, our results demonstrate that genetic alteration of seed traits can have unforeseen pleiotropic consequences. We have identified a role for fatty acid biosynthesis, and SACPD activity in particular, in the establishment and maintenance of symbiotic nitrogen fixation.

Tnt1 retrotransposon mutagenesis: a tool for soybean functional genomics.

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean.

The critical impacts of pyrochar during 2,4,6-trichlorophenol photochemical remediation process: Cooperation between persistent free radicals and oxygenated functional groups.

The widespread use of polychlorophenols poses enormous environmental challenges. Biochar has the potential to accelerate the transformation of polychlorophenols. But the biochar-triggered photochemical decomposition mechanism of polychlorophenols still remains unclear. Herein, the photochemical behavior of pyrochar was comprehensively investigated in 2,4,6-trichlorophenol (TCP) remediation. Researches revealed that persistent free radicals (PFRs) and oxygenated functional groups (OFGs) on the surface of pyrochar cooperatively promoted ROS generation for TCP degradation. PFRs performed a key role of electron-donating and energy transfer in ROS conversion, especially in the activation of H2O2 into •OH. The hydroxyl groups of photosensitive components of pyrochar were photo-excited and provided electrons for enhanced ROS formation as well. With photogenerated ROS involved, more TCP was decomposed through dechlorination under light irradiation than that in the dark, in which 1O2, •OH, and •O2- were the dominant active species. During this process, stronger light intensities (3 W/m2) and shorter light wavelengths (400 nm) can provide more energy for the activation of PFRs and OFGs, promoting the decomposition of TCP. This work casts a new light on the environmental roles of pyrochar in the photochemical removal of polychlorophenol pollutants.

High-Resolution Translatome Analysis Reveals Cortical Cell Programs During Early Soybean Nodulation.

Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development.

Variation in Gene Expression between Two Sorghum bicolor Lines Differing in Innate Immunity Response.

Microbe associated molecular pattern (MAMPs) triggered immunity (MTI) is a key component of the plant innate immunity response to microbial recognition. However, most of our current knowledge of MTI comes from model plants (i.e., Arabidopsis thaliana) with comparatively less work done using crop plants. In this work, we studied the MAMP triggered oxidative burst (ROS) and the transcriptional response in two Sorghum bicolor genotypes, BTx623 and SC155-14E. SC155-14E is a line that shows high anthracnose resistance and the line BTx623 is susceptible to anthracnose. Our results revealed a clear variation in gene expression and ROS in response to either flagellin (flg22) or chitin elicitation between the two lines. While the transcriptional response to each MAMP and in each line was unique there was a considerable degree of overlap, and we were able to define a core set of genes associated with the sorghum MAMP transcriptional response. The GO term and KEGG pathway enrichment analysis discovered more immunity and pathogen resistance related DEGs in MAMP treated SC155-14E samples than in BTx623 with the same treatment. The results provide a baseline for future studies to investigate innate immunity pathways in sorghum, including efforts to enhance disease resistance.

Regulation of motility in Erwinia carotovora subsp. carotovora: quorum-sensing signal controls FlhDC, the global regulator of flagellar and exoprotein genes, by modulating the production of RsmA, an RNA-binding protein.

Erwinia carotovora subsp. carotovora causes soft-rotting (tissue-macerating) disease in many plants and plant organs. Although pectinases are the primary determinants of virulence, several ancillary factors that augment bacterial virulence have also been identified. One such factor is bacterial motility. Flagellum formation and bacterial movement are regulated in many enterobacteria, including E. carotovora subsp. carotovora, by FlhDC, the master regulator of flagellar genes and FliA, a flagellum-specific σ factor. We document here that motility of E. carotovora subsp. carotovora is positively regulated by the quorum-sensing signal, N-acylhomoserine lactone (AHL), and negatively regulated by RsmA, a post-transcriptional regulator. RsmA, an RNA-binding protein, causes translational repression and promotes RNA decay. Our data show that RsmA negatively regulates flhDC and fliA expression. Moreover, the chemical stabilities of transcripts of these genes are greater in an RsmA- mutant than in RsmA+ bacteria. These observations contrast with positive regulation of flhDC and motility by CsrA (=RsmA) in Escherichia coli. In the absence of AHL, the AHL receptors ExpR1/ExpR2 (=AhlR) in Erwinia carotovora subsp. carotovora negatively regulate motility and expression of flhDC and fliA by activating RsmA production. In the presence of AHL, regulatory effects of ExpR1/ExpR2 are neutralized, resulting in reduced levels of rsmA expression and enhanced motility.

RsmC of Erwinia carotovora subsp. carotovora negatively controls motility, extracellular protein production, and virulence by binding FlhD and modulating transcriptional activity of the master regulator, FlhDC.

RsmC and FlhDC are global regulators controlling extracellular proteins/enzymes, rsmB RNA, motility, and virulence of Erwinia carotovora subsp. carotovora. FlhDC, the master regulator of flagellar genes, controls these traits by positively regulating gacA, fliA, and rsmC and negatively regulating hexA. RsmC, on the other hand, is a negative regulator of extracellular proteins/enzymes, motility, and virulence since the deficiency of RsmC in FlhDC(+) strain results in overproduction of extracellular proteins/enzymes, hypermotility, and hypervirulence. These phenotypes are abolished in an RsmC(-) FlhDC(-) double mutant. We show that RsmC interferes with FlhDC action. Indeed, the expression of all three targets (i.e., gacA, rsmC, and fliA) positively regulated in E. carotovora subsp. carotovora by FlhDC is inhibited by RsmC. RsmC also partly relieves the inhibition of hexA expression by FlhDC. The results of yeast two-hybrid analysis revealed that RsmC binds FlhD and FlhDC, but not FlhC. We propose that binding of RsmC with FlhD/FlhDC interferes with its regulatory functions and that RsmC acts as an anti-FlhD(4)FlhC(2) factor. We document here for the first time that RsmC interferes with activation of fliA and motility in several members of the Enterobacteriaceae family. The extent of E. carotovora subsp. carotovora RsmC-mediated inhibition of FlhDC-dependent expression of fliA and motility varies depending upon enterobacterial species. The data presented here support the idea that differences in structural features in enterobacterial FlhD are responsible for differential susceptibility to E. carotovora subsp. carotovora RsmC action.

Identification of QTL for Target Leaf Spot resistance in Sorghum bicolor and investigation of relationships between disease resistance and variation in the MAMP response.

Target leaf spot (TLS) of sorghum, a foliar disease caused by the necrotrophic fungus Bipolaris cookei (also known as Bipolaris sorghicola), can affect grain yield in sorghum by causing premature drying of leaves and defoliation. Two sorghum recombinant inbred line (RIL) populations, BTx623/BTx642 and BTx623/SC155-14E, were assessed for TLS resistance in replicated trials. Using least square mean trait data, four TLS resistance QTL were identified, two in each population. Of these, three were previously unidentified while a major QTL on chromosome 5 in the BTx623/BTx642 RIL population corresponded to the previously identified TLS resistance gene ds1. A set of sorghum lines were assessed for production of reactive oxygen species induced by treatment with the microbe-associated molecular pattern (MAMP) flg22 (a derivative of flagellin). Flg22-induced ROS production varied between lines in a consistent fashion. One QTL associated with variation in the flg22 response was detected in the RIL populations. No evidence was found to link variation in the MAMP response to variation in TLS resistance.

Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA(-) FlhDC(-) double mutant and multiple copies of rsmB(+) DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora.

Discovery and characterization of a novel tryptophan hydroxylase 1 inhibitor as a prodrug.

Serotonin (5-HT) is an important neurotransmitter and paracrine signaling molecule in the gastrointestinal tract. Two distinct tryptophan hydroxylases (TPH), TPH1 and TPH2, are the rate-limiting enzymes in the 5-HT biosynthesis process. TPH1 expression is mainly limited in the enterochromaffin cells and distributed in peripheries such as the skin and gut, while TPH2 is the predominant isoform in the CNS. In this study, mol002291 was screened as a drug-like compound from the TCM database for the inhibitor of TPH. After the enzymological analysis of mol002291, the analgesic effect of mol002291 was also further investigated in a PI-IBS visceral hyperalgesia rat model. Results from kinetic analysis showed that mol002291 specifically inhibited the TPH1 but did not act on TPH2, and the inhibitory action displayed characteristics of competitive inhibition. In addition, the results from abdominal withdrawal reflex (AWR) tests and electromyography (EMG) recordings showed that mol002291 significantly (p < .05) alleviated the visceral hyperalgesia. This result is entirely consistent with the fact that mol002291 significantly decreased the 5-HT content. These data demonstrated that mol002291 can attenuate visceral hyperalgesia mediated via reducing colonic 5-HT content. More important is that mol002291 could be developed as a novel prodrug and offer therapeutic avenues for the diseases where there is dysregulation of peripheral serotonergic pathways.

Analgesic activity of cynaropicrinon on post-inflammatory irritable bowel syndrome visceral hypersensitivity in a rat model.

Visceral hypersensitivity is one of the most common symptoms in patients with post-inflammatory-irritable bowel syndrome (PI-IBS). Enterochromaffin (EC) cells and 5-hydroxytryptamine (5-HT) are important in the development of visceral hyperalgesia, and EC cells are influenced by helper T-cell subtype 1 or 2 cytokine predominant environments. In the present study, the analgesic effect of cynaropicrin and its underlying mechanism on the treatment of trinitrobenzene sulfonic (TNBS)-induced PI-IBS visceral hyperalgesia rats was investigated. The results from the abdominal withdrawal reflex tests and electromyography recordings indicated that treatment with cynaropicrin significantly and dose-dependently alleviated the visceral hyperalgesia of PI-IBS rats (P<0.05). In addition, the increased colonic 5-HT content, colonic tryptophan hydroxylase expression, EC cell number and the cytokine levels, including tumor necrosis factor-α and interleukin-6 in PI-IBS rats were significantly alleviated by cynaropicrin (P<0.05). These data demonstrate that the analgesic activity of cynaropicrin on TNBS-induced PI-IBS visceral hypersensitive rats was mediated via reduction of cytokines levels. Thus, cynaropicrin as a bioactive natural product may offer promising therapeutic avenues for visceral hypersensitivity in IBS.

Diurnal cycling of rhizosphere bacterial communities is associated with shifts in carbon metabolism.

BACKGROUND: The circadian clock regulates plant metabolic functions and is an important component in plant health and productivity. Rhizosphere bacteria play critical roles in plant growth, health, and development and are shaped primarily by soil communities. Using Illumina next-generation sequencing and high-resolution mass spectrometry, we characterized bacterial communities of wild-type (Col-0) Arabidopsis thaliana and an acyclic line (OX34) ectopically expressing the circadian clock-associated cca1 transcription factor, relative to a soil control, to determine how cycling dynamics affected the microbial community. Microbial communities associated with Brachypodium distachyon (BD21) were also evaluated. RESULTS: Significantly different bacterial community structures (P = 0.031) were observed in the rhizosphere of wild-type plants between light and dark cycle samples. Furthermore, 13% of the community showed cycling, with abundances of several families, including Burkholderiaceae, Rhodospirillaceae, Planctomycetaceae, and Gaiellaceae, exhibiting fluctuation in abundances relative to the light cycle. However, limited-to-no cycling was observed in the acyclic CCAox34 line or in soil controls. Significant cycling was also observed, to a lesser extent, in Brachypodium. Functional gene inference revealed that genes involved in carbohydrate metabolism were likely more abundant in near-dawn, dark samples. Additionally, the composition of organic matter in the rhizosphere showed a significant variation between dark and light cycles. CONCLUSIONS: The results of this study suggest that the rhizosphere bacterial community is regulated, to some extent, by the circadian clock and is likely influenced by, and exerts influences, on plant metabolism and productivity. The timing of bacterial cycling in relation to that of Arabidopsis further suggests that diurnal dynamics influence plant-microbe carbon metabolism and exchange. Equally important, our results suggest that previous studies done without relevance to time of day may need to be reevaluated with regard to the impact of diurnal cycles on the rhizosphere microbial community.

Purification of Translating Ribosomes and Associated mRNAs from Soybean (Glycine max).

Cell identity and function are largely determined by specific gene expression patterns and ultimately by the proteome. Current high-throughput sequencing technologies offer the possibility of quantifying gene expression at high resolution, with minimum input and without the constraints of array-based systems, such as the need for specific probes. In addition, techniques are now available to capture genes that are actively being translated. These techniques use either density gradients or epitope-based immunoprecipitation to purify translating ribosomes and associated mRNAs (i.e., translatomes). More recently, the combination of tissue-specific promoters driving epitope-tagged ribosomes with high-throughput sequencing has allowed the identification of genes and networks unique to specific cell types. Translatome analyses have the potential to unravel genetic programs and cellular responses to environmental stresses at cell-specific resolution. This unit describes steps for the use of epitope-based immunoprecipitation to purify translating ribosomes from soybean and the recovery of mRNA for downstream applications such as gene expression analysis. © 2016 by John Wiley & Sons, Inc.

Regulation of Erwinia carotovora hrpL(Ecc) (sigma-L(Ecc)), which encodes an extracytoplasmic function subfamily of sigma factor required for expression of the HRP regulon.

In Erwinia carotovora subsp. carotovora (Ecc) strain 71 (Ecc71), HrpL(Ecc), an alternate sigma factor of the extracytoplasmic function subfamily, plays a central role in the expression of the hrp (hypersensitive reaction and pathogenicity) regulon. We document here that sigma-54 (RpoN) is required for full expression of hrpL(Ecc) and that HrpS, in conjunction with sigma-54, activates hrpL(Ecc) transcription. We also made the novel observation that integration host factor is required for the activation of the hrpL(Ecc) promoter. Our findings reveal that the RsmA/rsmB RNA-mediated post-transcriptional system, known to control extracellular enzyme and harpin production, affects hrpL(Ecc) expression as well. For example, hrpL(Ecc) RNA levels are barely detected in an RsmB- strain. Conversely, hrpL(Ecc) mRNA levels are much higher in RsmA- bacteria than in the RsmA+ parent. This effect is due to RsmA-promoted decay of hrpL(Ecc) RNA. Moreover, the following regulators known to control the production of either RsmA, rsmB RNA, or both also affect hrpL(Ecc) expression: GacA (response regulator of a two-component system), KdgR (an IcII type repressor), HexA (a LysR type repressor), RsmC (a putative transcriptional adapter). Based upon the data now available for Ecc and extrapolating from the evidence in other systems, we propose a tentative model that depicts the Hrp regulatory system of Ecc and explains the basis for coregulation of extracellular enzyme production and expression of the Hrp regulon.

GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors.

Concerted investigations of factors affecting host-pathogen interactions are now possible with the model plant Arabidopsis thaliana and its model pathogen Pseudomonas syringae pv. tomato DC3000, as their whole genome sequences have become available. As a prelude to analysis of the regulatory genes and their targets, we have focused on GacA, the response regulator of a two-component system. The DC3000 gene was cloned by testing for the reversal of phenotypes of an Erwinia GacA- mutant. A GacA- mutant of DC3000 constructed by marker exchange produces much-reduced levels of transcripts of three alternate sigma factors: HrpL, required for the production of effector proteins and their translocation via the type III secretion system; RpoS, required for stress responses and secondary metabolite production; and RpoN, required for an assortment of metabolic processes and expression of hrpL. GacA deficiency also reduces the expression of hrpR and hrpS, which specify enhancer-binding proteins of the NtrC family required for hrpL transcription; ahlI and ahlR, the genes for quorum sensing signal; salA, a regulatory gene known to control virulence; CorS, a sensor kinase; CorR, the cognate response regulator that controls coronatine biosynthetic genes; and rsmB and rsmZ, which specify untranslatable regulatory RNA species. gacA expression itself is regulated by environmental conditions in DC3000, since transcript levels are affected by growth phase and media composition. The observations that high levels of gacA RNA occur in the hrp-inducing medium and GacA deficiency reduces the levels of rpoS expression implicate an important role of GacA in stress responses of DC3000. Consistent with the effects on hrpL expression, the GacA- mutant produces lower levels of transcripts of avr, hrp, and hop genes controlled by HrpL. In addition, GacA deficiency results in reduced levels of transcripts of several HrpL-independent genes. As would be expected, these effects on gene expression cause drastic changes in bacterial behavior: virulence towards A. thaliana and tomato; multiplication in planta; efficiency of the induction of the hypersensitive reaction (HR); production of pigment and N-acyl-homoserine lactone (AHL), the presumed quorum-sensing signal; and swarming motility. Our findings establish that GacA, located at the top in a regulatory cascade in DC3000, functions as a central regulator by controlling an assortment of transcriptional and posttranscriptional factors.

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system.

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

Fluorescence in situ hybridization-based karyotyping of soybean translocation lines.

Soybean (Glycine max [L.] Merr.) is a major crop species and, therefore, a major target of genomic and genetic research. However, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty of cytogenetic analysis of its small, morphologically homogeneous chromosomes. The recent development of a fluorescence in situ hybridization -based karyotyping system for soybean has enabled our characterization of most of the chromosomal translocation lines identified to date. Utilizing genetic data from existing translocation studies in soybean, we identified the chromosomes and approximate breakpoints involved in five translocation lines.

PsrA, the Pseudomonas sigma regulator, controls regulators of epiphytic fitness, quorum-sensing signals, and plant interactions in Pseudomonas syringae pv. tomato strain DC3000.

Pseudomonas syringae pv. tomato strain DC3000, a pathogen of tomato and Arabidopsis, occurs as an epiphyte. It produces N-acyl homoserine lactones (AHLs) which apparently function as quorum-sensing signals. A Tn5 insertion mutant of DC3000, designated PsrA(-) (Psr is for Pseudomonas sigma regulator), overexpresses psyR (a LuxR-type regulator of psyI) and psyI (the gene for AHL synthase), and it produces a ca. 8-fold-higher level of AHL than does DC3000. The mutant is impaired in its ability to elicit the hypersensitive reaction and is attenuated in its virulence in tomato. These phenotypes correlate with reduced expression of hrpL, the gene for an alternate sigma factor, as well as several hrp and hop genes during early stages of incubation in a Hrp-inducing medium. PsrA also positively controls rpoS, the gene for an alternate sigma factor known to control various stress responses. By contrast, PsrA negatively regulates rsmA1, an RNA-binding protein gene known to function as negative regulator, and aefR, a tetR-like gene known to control AHL production and epiphytic fitness in P. syringae pv. syringae. Gel mobility shift assays and other lines of evidence demonstrate a direct interaction of PsrA protein with rpoS promoter DNA and aefR operator DNA. In addition, PsrA negatively autoregulates and binds the psrA operator. In an AefR(-) mutant, the expression of psyR and psyI and AHL production are lower than those in DC3000, the AefR(+) parent. In an RpoS(-) mutant, on the other hand, the levels of AHL and transcripts of psyR and psyI are much higher than those in the RpoS(+) parent, DC3000. We present evidence, albeit indirect, that the RpoS effect occurs via psyR. Thus, AefR positively regulates AHL production, whereas RpoS has a strong negative effect. We show that AefR and RpoS do not regulate PsrA and that the PsrA effect on AHL production is exerted via its cumulative, but independent, effects on both AefR and RpoS.