SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

<p>Abstract</p> <p>Background</p> <p>Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the <it>Manduca sexta </it>larvae are necessary and sufficient to elicit the herbivory-specific responses in <it>Nicotiana attenuata</it>, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.</p> <p>Results</p> <p>We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC <it>N</it>-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the <it>M. sexta</it>-<it>N. attenuata </it>interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the <it>M. sexta</it>-<it>N. attenuata </it>interaction.</p> <p>Conclusions</p> <p>The analysis of the early changes in the transcriptome of <it>N. attenuata </it>after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.</p

Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago

Identification of genes involved in resistance to Didymella pinodes in pea by deepSuperSAGE genome-wide transcriptome profiling

Póster presentado en la First Legume Society Conference, celebrada en Novi Sad (Serbia) del 9 al 11 de mayo de 2013.Didymella pinodes, causing ascochyta blight, is one of the most important pea pathogens. Despite the devastating consequences of this disease, very little is known about the mechanisms of resistance in the host. We employed the open2architecture transcriptome profiling technique deepSuperSAGE, coupled with next2generation sequencing, to identify pea2specific genes involved in the resistance to this important disease in the resistant Pisum sativum ssp. syriacum accession P665.Two deepSuperSAGE libraries were constructed from leaf RNA of infected and control plants, yielding a total of 17,561 different UniTags. 70% of them could be assigned to known sequences from pea or other plants. 509 UniTags were significantly differentially expressed (p < 0.05; fold change ≥ 2, ≤ 2) in inoculated versus control plants. This study provides a detailed picture of all expressed genes and metabolic pathways differentially regulated during D. pinodes3Pisum sativum interaction and contribute to the identification of candidate resistance genes. Protease inhibitors, antifungal compounds, strengthening of host cell walls, detoxification of D. pinodes toxins and repair of cell walls could contribute to resistance. Ethylene, ABA and indole232acetic acid pathways were up2, while the GA pathway was down2regulated.Peer Reviewe

Identification of Genes Involved in Resistance to Didymella pinodes in Pea by deepSuperSAGE Transcriptome Profiling

Didymella pinodes, causing ascochyta blight, is the most destructive foliar pathogen of dry peas. Despite the importance of this pathogen, very little is known about the mechanisms or genes that control host plant resistance against the fungus. Here we employed deepSuperSAGE genome-wide transcription profiling to identify pea genes involved in resistance to D. pinodes in the wild, resistant Pisum sativum ssp. syriacum accession P665. Two deepSuperSAGE libraries were constructed from leaf RNA of infected and control plants. A total of 17,561 different UniTags were obtained. Seventy per cent of them could be assigned to known sequences from pea or other plants. 509 UniTags were significantly differentially expressed (P < 0.05; fold change ≥2, ≤2) in inoculated versus control plants. Of these, 78 % could be assigned to known sequences from pea or other plants, and 58 % to proteins with known function. Our results suggest that a battery of genes contribute to resistance against D. pinodes in the wild pea accession P665. For example, genes encoding protease inhibitors are activated, and the corresponding proteins may contribute to a lower penetration success. The production of antifungal compounds and strengthening of host cell walls may interfere with the spread of the pathogen. In addition, detoxification of D. pinodes toxins and repair of cell walls could also reduce the damage produced by this devastating necrotroph. Hormones orchestrate metabolic adaptation to D. pinodes infection, since ethylene, ABA and indole-3-acetic acid pathways were up-, while the gibberellic acid pathway was down-regulated. © 2013 Springer Science+Business Media New York.Financial support by AGL2011-22524 and GEN2006-27798-C6-6-E/VEG projects is acknowledged. S.F. was funded by FP7-PEOPLE-2011-IEF-300235 and a JAEDoc contract.Peer Reviewe

Precision medicine for hepatocelluar carcinoma using molecular pattern diagnostics: Results from a preclinical pilot study

The aim of this study was to design a road map for personalizing cancer therapy in hepatocellular carcinoma (HCC) by using molecular pattern diagnostics. As an exploratory study, we investigated molecular patterns of tissues of two tumors from individual HCC patients, which in previous experiments had shown contrasting reactions to the phase 2 transforming growth factor beta receptor 1 inhibitor galunisertib. Cancer-driving molecular patterns encompass - inter alias - altered transcription profiles and somatic mutations in coding regions differentiating tumors from their respective peritumoral tissues and from each other. Massive analysis of cDNA ends and all-exome sequencing demonstrate a highly divergent transcriptional and mutational landscape, respectively, for the two tumors, that offers potential explanations for the tumors contrasting responses to galunisertib. Molecular pattern diagnostics (MPDs) suggest alternative, individual-tumor-specific therapies, which in both cases deviate from the standard sorafenib treatment and from each other. Suggested personalized therapies use kinase inhibitors and immunefocused drugs as well as low-toxicity natural compounds identified using an advanced bioinformatics routine included in the MPD protocol. The MPD pipeline we describe here for the prediction of suitable drugs for treatment of two contrasting HCCs may serve as a blueprint for the design of therapies for various types of cancer