Project description:RpoN (σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include alpha- and beta-rhizobia. Our previous studies showed that a rpoN mutant of the beta-rhizobial strain Paraburkholderia phymatum formed root nodules on Phaseolus vulgaris that were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by the P. phymatum rpoN Fix- mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix+ and Fix- nodules – combined with a search for RpoN binding sequences in promoter regions of regulated genes – confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also identified additional target genes, whose differential expression was able to explain the observed metabolite changes in a numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype by forming less but heavier nodules on P. vulgaris roots.
Project description:250 adult T. urticae females from the London strain (grown on acyanogenic P. vulgaris cv. Prelude bean plants) were transferred to cyanogenic P. lunatus cv. 8078 bean plants. Thirty-five generations after the host transfer, total RNA was extracted from mites growing on both bean species (London and London-CYANO strain) and used in in a genome-wide gene expression microarray (Sureprint G3 microarray, Agilent) experiment to assess significantly differentially expressed genes (FC ≥ 2 and FDR-corrected p-value < 0.05) between mites grown on P. vulgaris (cv. Prelude) bean plants (London strain) and mites grown for 35 generations on P. lunatus (cv. 8078) bean plants (London-CYANO strain).
Project description:To understand the molecular basis for differences of common bean wild-type and mutant in sulphur amino acid content, transcripts were profiled at four developmental stages of seeds from wild-type SARC1 and major seed storage protein-deficient line SMARC1N-PN1 using a CustomArray 90K array. Microarray data confirmed that transcripts of storage and sulphur-rich proteins and sulphur-metabolism related genes were differentially expressed between the lines. The common bean (Phaseolus vulgaris) mutant line, SMARC1N-PN1 and its wild type, SARC1 used in the microarray experiment were grown in the field in London, ON, in 2009. Four developmental stages of seeds, based on fresh seed weight, were harvested. The stages of seeds used are Stage IV M-bM-^@M-^S cotyledon, 25 mg seed weight; Stage V M-bM-^@M-^S cotyledon, 50 mg seed weight; Stage VI M-bM-^@M-^S maturation, 150 mg seed weight, corresponding to the most active phase of reserve accumulation; and Stage VIII M-bM-^@M-^S maturation, 380 mg seed weight, corresponding to the onset of desiccation, were harvested for total RNA extraction. Four biological replicates for Stage IV and V and 3 biological replicates for Stage VI and VIII.
Project description:The pod is the main edible part of Phaseolus vulgaris L. (common bean). The commercial use of the pods is mainly affected by their color. Consumers seem to prefer golden pods. However, planters suffer economic losses because of pod color instability. The aim of the present study was to identify the gene responsible for the golden pod trait in the common bean. ‘A18-1’ (a golden bean line) and ‘Renaya’ (a green bean line) were chosen as the experimental materials. Genetic analysis indicated that a single recessive gene, pv-ye, controls the golden pod trait. A candidate region of 4.24-Mb was mapped to chromosome A02 using bulked-segregant analysis coupled to whole genome sequencing. In this region, linkage analysis in an F2 population localized the pv-ye gene to an interval of 182.9-kb between the simple sequence repeat markers SSR77 and SSR93. This region comprised 16 genes in this region, comprising 12 annotated genes from the P. vulgaris database, and 4 functionally unknown genes. Combined with transcriptome sequencing, we identified Phvul.002G006200 as the potential candidate gene for pv-ye. Sequencing of Phvul.002G006200 identified a single nucleotide polymorphism (SNP) in pv-ye. This SNP is located in the coding region and is responsible for substituting a glutamic acid with an glutamine at position 416 of the pv-ye protein (E416Q). A pair of primers covering the SNP was designed and the fragment was sequenced to screen 316 F2 plants with the ‘A18-1’ phenotype, based on the different site. Our findings showed that the among the 316 mapped individuals, the SNP cosegregated with the ‘A18-1’ phenotype. The findings presented here could form the basis to reveal the mechanism of the golden pod trait in the common bean at the molecular level.
Project description:250 adult T. urticae females from the London strain (grown on acyanogenic P. vulgaris cv. Prelude bean plants) were transferred to cyanogenic P. lunatus cv. 8078 bean plants. Thirty-five generations after the host transfer, total RNA was extracted from mites growing on both bean species (London and London-CYANO strain) and used in in a genome-wide gene expression microarray (Sureprint G3 microarray, Agilent) experiment to assess significantly differentially expressed genes (FC M-bM-^IM-% 2 and FDR-corrected p-value < 0.05) between mites grown on P. vulgaris (cv. Prelude) bean plants (London strain) and mites grown for 35 generations on P. lunatus (cv. 8078) bean plants (London-CYANO strain). 4 replicates for one comparison: mites of the London strain grown on P. lunatus for 35 generations (London-CYANO) compared to mites of the London strain grown on P. vulgaris bean plants (London)
Project description:Common Bacterial Blight (CBB) is a major threat to bean crops caused by Xanthomonas citri pv. fuscans (Xcf). The pathogenicity of Xcf is known to be dependent upon a functional Type 3 Secretion System (T3SS), that allows the injection of numerous Type III Effectors (T3Es) into plant cells. We generated a transcriptomic dataset to compare the response of susceptible and resistant cultivars of Phaseolus vulgaris to the inoculation of the virulent strain Xcf CFBP4885 or its avirulent T3SS-defective hrcV mutant (CFBP13802). This dataset is a valuable resource to investigate the role of T3Es in subverting the cellular functions of bean.
Project description:Common bean (Phaseolus vulgaris L.) is a relevant crop cultivated over the world, largely in water insufficiency vulnerable areas. Since drought is the main environmental factor restraining worldwide crop production, efforts have been invested to amend drought tolerance in commercial common bean varieties. However, scarce molecular data are available for those cultivars of P. vulgaris with drought tolerance attributes. As a first approach, Pinto Saltillo (PS), Azufrado Higuera (AH), and Negro Jamapa Plus (NP) were assessed phenotypically and physiologically to determine the outcome in response to drought on these common bean cultivars. Based on this, a Next-generation sequencing approach was applied to PS, which was the most drought-tolerant cultivar to determine the molecular changes at the transcriptional level. The RNA-Seq analysis revealed that numerous PS genes are dynamically modulated by drought. In brief, 1005 differentially expressed genes (DEGs) were identified, from which 645 genes were up-regulated by drought stress, whereas 360 genes were down-regulated. Further analysis showed that the enriched categories of the up-regulated genes in response to drought fit to processes related to carbohydrate metabolism (polysaccharide metabolic processes), particularly genes encoding proteins located within the cell periphery (cell wall dynamics). In the case of down-regulated genes, heat shock-responsive genes, mainly associated with protein folding, chloroplast, and oxidation-reduction processes were identified. Our findings suggest that secondary cell wall (SCW) properties contribute to P. vulgaris L. drought tolerance through alleviation or mitigation of drought-induced osmotic disturbances, making cultivars more adaptable to such stress. Altogether, the knowledge derived from this study is significant for a forthcoming understanding of the molecular mechanisms involved in drought tolerance on common bean, especially for drought-tolerant cultivars such as PS.
Project description:Background: MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21 and no phasiRNAs have been identified for the legume model Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs of this organism and, particularly, new protagonists of the symbiotic nodulation events. Results: We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs, known to play crucial roles in the well-establishment of nodules, and novel miRNAs specific of the common bean suggesting a specific action of these sequences. In parallel, we identified 125 loci that potentially produce phased small RNAs and 47 of them present all the characteristics to be triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study. Conclusions: We provide here a set of new small RNAs, which contribute to the broader scene of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we draw up here the probable functional regulation associated with the sRNAome and particularly in N2-fixing symbiotic nodules. Degradome sequencing from Phaseolus vulgaris seedling