Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea. Identification of microRNAs and phased small inferfering RNAs in chickpea (Cicer arietinum) by analyzing small RNA sequencing profiles of leaves and flowers using Illumina GAII.
Project description:Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, where samples from mock-inoculated controls acted as references against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and strict selection criteria for differentially expressed genes including a fold change cutoff determined by self-self hybridizations, Students t test and multiple testing correction (P<0.05). Microarray observations were also validated by quantitative RT-PCR. The time-course expression patterns of 756 microarray features resulted in differential expression of 97 genes in at least one genotype at one time-point. K-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, as well as several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on functional validation of the genes of interest. Keywords: time course disease state analysis
Project description:Chickpea (Cicer arietinum L.) seed proteins show a lot of functional properties leading this leg-ume an interesting component for the development of protein-enriched foods. However, both in-depth proteomic investigation and structural characterization of chickpea proteins seed are still lacking. In this paper we report a detailed characterization of chickpea seed protein fraction by means SDS-PAGE, in-gel protein digestion, high-resolution mass spectrometry, and database searching. By this approach twenty SDS gel bands were cut and analysed. While the majority of bands and the identified peptides were related to vicilin and legumin storage proteins, also metabolic functional proteins were detected. Legumins, as expected, were revealed at 45÷65 kDa, as whole subunits with the α- and β-chains linked together by a disulphide bond, but also at lower mass ranges (α- and β-chains migrating alone). Similarly, but not expected, also the vi-cilins were spread along the mass region between 65 and 23 kDa, with some of them identified in several bands. In-depth MS structural characterization allowed to determine that, although chickpea vicilins were always described as proteins lacking of cysteine residues, they contain this amino acid residue. Moreover, similarly to legumins, these storage proteins are firstly syn-thesized as pre-propolypeptides (Mr 50÷80 kDa), that may undergo to proteolytic steps that cut not only the signal peptides but also produce different subunits having lower molecular masses. Overall, about 360 different proteins specific of the Cicer arietinum L. species were identified and characterized, a result that up to date represents the most detailed description of seed’s proteome of this legume.
Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea.
Project description:Understanding the molecular differences in plant genotypes contrasting for heat sensitivity can provide useful insights into the mechanisms that confer heat tolerance in plants. This study is focused on comparative physiological and proteomic analyses of heat sensitive (ICC16374) and tolerant (JG14) genotypes of chickpea (Cicer arietinum L.) when subjected to heat stress at anthesis.Comparative gel-free proteome profiles indicated differences in the expression levels and regulation of common proteins that are associated with heat tolerance in contrasting genotypes under heat stress. The differentially regulated proteins were grouped into three categories based on their involvement in the molecular functions, cellular location and biological processes. Besides the identification of heat shock proteins, other proteins such as acetyl-CoA carboxylase, pyrroline-5-carboxylate synthase (P5CS), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), phenylalanine ammonia-lyase (PAL) 2, ATP synthase, glycosyltransferase, sucrose synthase and late embryogenesis abundant (LEA) proteins were strongly associated with heat tolerance in chickpea. Several crucial proteins such as cystathionine gamma-synthase, glucose-1-phosphate adenyltransferas, malate dehydrogenase, threonine synthase, and non-cyanogenic ß-glucosidase were induced by heat only in the heat tolerant genotype. Based on pathway analysis, we propose that proteins which are essentially related to the electron transport chain in photosynthesis, aminoacid biosynthesis, ribosome synthesis and secondary metabolite synthesis may play key roles in inducing tolerance to heat stress.
Project description:We performed RNA-seq analysis of the root transcriptional response to Fusarium oxysporum f.sp. vasinfectum (FOV) race 4 (FOV4) infection in Gossypium barbadense, also known as Pima cotton. Susceptible Gossypium barbadense inbred lines Pima S-7 (PI 560140) and Pima 3-79 susceptible to Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV)] race 4 (FOV4), and Pima S-6 (PI 608346) which is resistant to FOV4 infection, were used for the preparation of cDNA libraries and further RNA-seq analyses. An isolate of FOV4 (FOV CA-14) from a naturally infested field in Fresno County in the San Joaquin Valley, California was used in this study.
Project description:Size fractionated small RNA from total RNA extracts of Cicer arietinum leaves and from Nicotiana benthamiana infected by Cymbidium ringspot virus were mixed in a ratio of 1000 to 1 in amount, respectively. The RNA was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit Please see www.illumina.com for details of the sequencing technology. Short RNA fractionation and characterization
Project description:Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes and grasspea (Lathyrus sativus L.) ESTs, chickpea responses to treatments with the defence signalling compounds salicylic acid (SA), methyl jasmonate (MeJA), and aminocyclopropane carboxylic acid (ACC) were studied in four chickpea genotypes with ranging levels of resistance to ascochyta blight (Ascochyta rabiei (Pass.) L.). The experimental system minimized environmental effects and was conducted in reference design, where samples from untreated controls acted as references against post-treatment samples. Robust data quality was achieved through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and strict selection criteria for differentially expressed genes including a fold change cut-off determined by self-to-self hybridizations, Students t test and multiple testing correction (P<0.05). Microarray observations were also validated by quantitative RT-PCR. The time-course expression patterns of 715 experimental microarray features resulted in differential expression of 425 genes in at least one condition. The A. rabiei resistant chickpea genotypes showed a more substantial range of defence-related gene induction by all treatments, indicating that they may possess stronger abilities to resist infection. Further, the involvement of SA, MeJA, and ACC signalling was identified for the regulation of some important A. rabiei responsive genes, as well as cross-talk between these pathways. This study also found evidence to suggest the involvement of A. rabiei-specific signalling mechanisms for the induction of several genes that were previously implicated in A. rabiei resistance. Overall, this study characterised the regulatory mechanisms of many chickpea genes that may be important in defence against various pathogens, as well as other cellular functions. Although the size of the microarray was limited, the results provided novel insights to the molecular control of chickpea cellular processes, which may assist the understanding of chickpea defence mechanisms and allow enhanced development of disease resistant cultivars. Keywords: time course defence-signalling teatment analysis
Project description:‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to cold stress. Two groups of a tolerant and susceptible accession were challenged with cold stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaves and flowers/buds/early pods tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 221 transcripts was affected in response to cold stress in all the genotypes and tissue types studied. The DE transcripts in response to cold stress fell into various functional categories, indicating a broad response. Sixteen out of the 221 DE transcripts were consistently expressed in cold tolerant/susceptible genotypes. All these transcripts were repressed and none was found to be consistently induced in response to cold-stress. Most of the putative genes were identified in leaves of tolerant genotypes, and included a beta-galactosidase (DY475141) transcript that was possibly indicative of disaccharide (e. g. sucrose) retention with the effect of protecting cell membranes during cold stress. Several protein synthesis/modification and energy/metabolism transcripts were also repressed (e.g. DY475282, DY396371 and DY475555), which was likely due to the impairment of photosynthesis and respiration at low temperature. Other consistently repressed transcripts in tolerant genotypes included putative signalling (DY396262, DY475384 and DY396307) and defence-related proteins (CV793589 and DY396343), which may be involved in the repression of cell death mechanisms that are absent in tolerant genotypes. In susceptible genotypes, a putative superoxide dismutase precursor protein (DY475397) and sorting nexin protein (DY475523) were the only known transcripts to be consistently repressed. Keywords: Chickpea, Cold stress, Tolerant, Susceptible, cDNA microarray