Progress 03/15/10 to 03/14/13
Outputs
Target Audience: Researchers in the fields of Plant Biology, Entomology and Bioinformatics. Especially focused toward those who study plant/insect interactions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The Co-PI, Dr. Fescemyer has become one of the leading scientists in fall armyworm transcriptome analysis and has been invited to present his results at several international meetings. "Explorations in integrative biology with butterflies and agricultural moth pests," Workshop on Adaptation to a Biotic Environment in Lepidoptera, Feb. 14-15, 2013, Paris, France "Explorations in Integrative Biology with Butterflies and Agricultural Moth Pests," Butterfly Genetics Group, University of Cambridge,September 11, 2012Cambridge, United Kingdom. How have the results been disseminated to communities of interest? Dr. Fescemeyer reported findings at the two following international conferences: "Explorations in integrative biology with butterflies and agricultural moth pests," Workshop on Adaptation to a Biotic Environment in Lepidoptera, Feb. 14-15, 2013, Paris, France "Explorations in Integrative Biology with Butterflies and Agricultural Moth Pests," Butterfly Genetics Group, University of Cambridge,September 11, 2012Cambridge, United Kingdom. Dr. Luthe presented findings in these venues: TARI (Taiwan Agricultural Research Institute) - “Maize’s Multipronged Defenses Against Caterpillars and Other Herbivores” (October 2012) National Chung Hsing University (Taichung, Taiwan) -- “Lessons from Mother Nature: What We Have Learned from Caterpillar Resistant Maize”(early October 2012) Second International Meeting on the Insect Midgut (Guangzhou, China) -- “Maize’s Multipronged Defenses Against Caterpillars and Other Herbivores” ( September 24 to 28, 2012) University of Toledo -- “Maize’s Multipronged Defenses Against Caterpillars and Other Herbivores” (December 7, 2012) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. In objective one, next generation sequencing, multiplex ID tagging and orthology-based computation will be used to produce a functionally annotated sequence for transcripts expressed in each tissue, life stage and strain of fall armyworm examined. Response: We have accomplished this goal and are in the process of preparing the publications. 2. In objective two, expression profiling with next generation sequencing will test the hypothesis that eating foliage from the lepidopteran resistant corn inbred line Mp708 alters transcription of genes involved in nutrient utilization, development and growth of fall armyworm plant host strain larvae. Response: This has been accomplished and the results were published by Fescemyer et al. in 2013. 3. Constructing ArmywormBase to house all data from this project in a public, Web-accessible form is the third objective. Please see the section on "Products" for information regarding the database.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Fescemyer, H. W., G.V. Sandoya, T.A. Gill, S. Ozkan, J.H. Marden and D.S. Luthe. 2013. Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut. Insect Biochemistry and Molecular Biology 43: 280-291.
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Progress 03/15/11 to 03/14/12
Outputs
OUTPUTS: Sequencing and ortholog-based functional annotation of the transcriptome in several tissues and life stages of the fall armyworm (FAW), Spodoptera frugiperda, is Aim 1 of the project. Toward completion of this aim, we used mRNA-seq on the paired-end Illumina HiSeq platform to sequence larval cDNA libraries deriving from RNA separately isolated from labial (silk) salivary glands, fat body, midgut and whole bodies of both the corn and rice plant host strains. As negotiated in year one of this project, a unified transcriptome is being generated by combining our sequence data with that from the French National Institute for Agricultural Research (INRA) which consists of 454 and single-end Illumina sequence derived from several tissues and life stages of the corn strain. Progress on Aim 3 of this project involves continued development of ArmywormBase and a joint USA and French database (e.g., SPODOBASE or LepidoDB) to house the unified transcriptome in annotated form for public access via the WEB. Most of the effort in year two was toward completion of Aim 2 which involves identifying larval genes whose expression level is influenced by eating foliage from the lepidopteran resistant corn inbred line Mp708. A manuscript is in preparation to report results of an experiment conducted in year one that used additional support from The Huck Institutes of the Life Sciences at Penn State in performing SOLiD RNA-seq to determine gene transcript expression profiles for midguts from corn strain larvae fed whorl tissue from susceptible Tx601 or resistant Mp708 maize inbreds. A second more extensive mRNA-seq experiment was completed. This experiment resulted in transcriptome sequences from tissues of corn and rice strain larvae (described above), data for testing differential expression due to plant host strain, host plant (foliage from Bermuda grass, susceptible Tx601 maize, or resistant Mp708 maize) fed to the larvae or their interaction, and data for differentiating sequence differences between the plant host strains. Posters presented at scientific meetings: 1) SOLiD RNA-Seq Reveals Differences in Midgut Expression Profiles Between Larvae that Ate Resistant of Susceptible Corn Foliage, H.W. Fescemyer, G.V. Sandoya, J.C. Vera, J.H. Marden and D.S. Luthe, Bioinformatics and Genomics Retreat, September 16-17, 2011, University Park, PA; 2) Gene Expression Profiles Underlying the Dynamics of Insect-Plant Interactions and Life-History Variation, H.W. Fescemyer, J.H. Marden and D.S. Luthe, Cornell Symposium on Lepidopteran Biology, October 11, 2011, Ithaca, NY. PARTICIPANTS: INDIVIDUALS AT PENN STATE: Dawn S. Luthe is the PD who directed the project and provided laboratory and greenhouse facilities. German V. Sandoya and Torrence A. Gill are postdocs in the Luthe Lab who contributed qPCR data. James H. Marden is a coPD who provided laboratory facilities and consultation on statistical analysis of SOLiD mRNA-Seq expression profile data. J. Cristobal Vera is a student in the Marden Lab who contributed to reference assembly and annotation. Howard W. Fescemyer is a coPD who performed all technical components, statistically analyzed project data, prepared project results for all presentations given, mainatains a collaboration with French scientists at INRA and Genoscope, and presented project results at the Bioinformatics and Genomics Retreat in September 2011 and the Cornell Symposium on Lepidopteran Biology in October 2011. INDIVIDUALS AT INRA AND GENOSCOPE: Philippe Fournier is unit director of the Unite de Biologie Integrative et Virologie des Insectes at INRA -- Universite Montpellier II. He is our primary collaboartor and liaison with Genoscope and other INRA scientists. Emmanuella d'Alencon is a research molecular biologists in the Unite de Biologie Integrative et Virologie des Insectes at INRA -- Universite Montpellier II. She is working on the transcriptome and genome sequencing of FAW. Fabrice Legeai is a research bioinformatician in the UMR Biologie des Organismes et des Populations Appliquees a la Protection des Plantes at INRA Rennes AgroCampus. He is working with H.W. Fescemyer and E. d'Alencon to assemble and annotate the transcriptome and genome of the FAW. Jean Weissenbach is the Director of Genoscope where sequencing of the FAW genome is being performed. TARGET AUDIENCES: Research scientists in industry (e.g., agricultural biotechnology companies), academics (e.g., land grant colleges and universities), and government (e.g., USDA ARS) are the primary target audiences for knowledge coming from this project. Aim3 is to deliver ArmywormBase as a WEB-accessible transcriptome database for public access to all sequencing data generated by this project. We have begun constructing ArmywormBase using the software and HTML code framework of CinxiaBase. ArmywormBase will specifically house assembled and annotated Illumina sequence generated in the USA by us from various larval tissues. We are collaborating with French INRA and Genoscope colleagues to develop a unified transcriptome generated from USA and French sequence data housed for public access via the WEB on Spodopbase (http://bioweb.ensam.inra.fr/spodobase) or Lepido-DB (http://www.inra.fr/lepidodb). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Aim 2 of the project is to identify genes in FAW whose expression level is influenced by eating foliage from lepidopteran resistant corn inbred line Mp708. We completed an experiment in year one that used the SOLiD mRNA-seq platform to compare gene expression profiles in midguts from corn strain larvae that ate susceptible Tx601 or resistant Mp708 corn foliage. In the past, second year, analysis of mRNA-seq read counts mapped against 7869 contigs in the SPODOBASE reference transcriptome was refined by using the Bioconductor packages edgeR for normalization and DESeq for differential expression analysis. This analysis showed normalized transcript accumulation for 100 mapped contigs to be highly significantly greater (P ≤ 0.005) in midguts of larvae fed whorl tissue from the Mp708 maize inbred (Mp708-midgut) compared with that in midguts of larvae fed whorl tissue from the Tx601 inbred (Tx601-midgut). Differential transcript accumulation for 81 of these 100 contigs was 2-fold or more higher in Mp708-midguts compared with Tx601-midguts. These 100 contigs were functionally annotated via Blast against predicted proteins from the genome of Bombyx mori, UniProt, predicted proteins from the genome sequence of Drosophila melanogaster, and the Gene Ontology (GO) database. The annotations were grouped using DAVID Bioinformatics Resources to reveal three groups of functionally related genes upregulated in Mp708-midguts compared with Tx601-midguts; digestive enzymes, protein components of the peritrophic membrane, and innate immunity. These findings were supported by results from separate experiments using reverse-transcription quantitative PCR (qPCR) to measure differential expression of selected target midgut genes (12 from FAW and for comparison, 6 from the European corn borer, Ostrinia nubilalis) in midguts of larvae fed artificial diet, foliage from resistant Mp708 corn or foliage from susceptible Tx601 or B73 corn. A manuscript in preparation is using these mRNA-seq and qPCR findings because they enable us to conclude how midgut tissues respond to the plant treatments. Foliage of caterpillar resistant corn expresses Mir1-CP cysteine protease, which attacks the peritrophic membrane (PM). Midgut genes showing highly significant treatment differences were only found in larvae that ate resistant corn foliage. These genes tended to be upregulated more than 4-fold. Midguts from larvae that ate resistant foliage have an expression profile that suggests they are trying to counteract the action of Mir1-CP. In contrast to the susceptible foliage treatment, these midguts upregulated genes involved in PM repair, protein digestion by PM bound enzymes, and proteins involved in wounding and antimicrobial reactions.
Publications
- No publications reported this period
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Progress 03/15/10 to 03/14/11
Outputs
OUTPUTS: Aim 1 of the project is to sequence and functionally annotate the transcriptome in several tissues and life stages of the fall armyworm, Spodoptera frugiperda. Toward completion of this aim, we developed collaboration with the French International Institute for Agricultural Research (INRA) to share transcriptome sequence data and participate with the French National Sequencing Center (Genoscope) to sequence the genome of fall armyworm. This collaboration provides access to INRA's 454 and Illumina based transcriptome sequence derived from several tissues and life stages of the corn strain, thereby allowing us to focus our Aim 1 efforts on assembly and annotation of this sequence. Aim 2 is to examine the corn host plant strain of fall armyworm for larval genes whose expression level is influenced by eating foliage from the lepidopteran resistant corn inbred line Mp708. Using additional support from The Huck Institutes of the Life Sciences at Penn State, we completed an experiment using the SOLiD RNA-Seq platform to determine gene transcript expression profiles for midguts from corn strain larvae that ate susceptible Tx601 or resistant Mp708 corn foliage. A second more extensive experiment is currently under way. This experiment will use the paired-end Illumina mRNA-Seq platform to determine differences in gene transcript expression profiles in fall armyworm tissues collected from both corn and rice strain larvae that ate foliage from Bermuda grass, susceptible Tx601, or resistant Mp708 corn foliage. Now that RNA extraction is done, we are preparing paired-end cDNA libraries from the isolated RNA for Illimina sequencing. Posters presented at scientific meetings: 1) SOLiD RNA-Seq Reveals Differences in Midgut Expression Profiles Between Larvae that Ate MP708 Resistant or Susceptible Corn Foliage, D.S. Luthe et al., Annual USDA NIFA Project Directors Meeting, December 11-12, San Diego, CA; 2) Transcriptomic Determination of Genes Involved in the Nutritional Ecology of Fall Armyworm Plant Host Strains, D.S. Luthe et al., Fourth Annual Arthropod Genomics Symposium, June 10-13, 2010, Kansas City, MO. Oral presentations given in France: 1) Explorations in Transcriptomics, Functional Genomics, and Bioinformatics with a Butterfly and a Moth, H.W. Fescemyer, The French National Sequencing Center, November 2, 2010; 2) Explorations in Integrative Biology with Butterflies and Agricultural Moth Pests, H.W. Fescemyer, INRA -- Universite Montpellier II, November 3, 2010. Aim3 is to deliver ArmywormBase as a WEB-accessible transcriptome database for public access to all sequencing data generated by this project. We have begun constructing ArmywormBase using the software and HTML code framework of CinxiaBase. ArmywormBase will specifically house assembled and annotated Illumina sequence generated in the USA by us from various larval tissues. We are negotiating with French INRA and Genoscope colleagues as to where a grand transcriptome generated from USA and French sequence data will be housed for public access. PARTICIPANTS: INDIVIDUALS AT PENN STATE: Dawn S. Luthe is the PD who directed the project and provided laboratory and greenhouse facilities. Dr. Luthe also presented project results at the Annual USDA NIFA Project Directors Meeting in December 2010. James H. Marden is a coPD who provided laboratory facilities and consultation on statistical analysis of SOLiD mRNA-Seq expression profile data. Howard W. Fescemyer is a coPD who performed all technical components, statistically analyzed project data, prepared project results for all presentations given, presented project results at the Fourth Annual Arthropod Genomics Symposium in June 2010, and developed collaboration with French scientists at INRA and Genoscope. INDIVIDUALS AT INRA AND GENOSCOPE: Philippe Fournier is unit director of the Unite de Biologie Integrative et Virologie des Insectes at INRA -- Universite Montpellier II. He is our primary collaboartor and liaison with Genoscope and other INRA scientists. Emmanuella d'Alencon is a research molecular biologists in the Unite de Biologie Integrative et Virologie des Insectes at INRA -- Universite Montpellier II. She is working on the transcriptome and genome sequencing of fall armyworm. Fabrice Legeai is a research bioinformatician in the UMR Biologie des Organismes et des Populations Appliquees a la Protection des Plantes at INRA Rennes AgroCampus. He is working with H.W. Fescemyer and E. d'Alencon to assemble and annotate the transcriptome and genome of the fall armyworm. Jean Weissenbach is the Director of Genoscope where sequencing of the fall armyworm genome is being performed. TARGET AUDIENCES: Research scientists in industry (e.g., agricultural biotechnology companies), academics (e.g., land grant colleges and universities), and government (e.g., USDA ARS) are the primary target audiences for knowledge coming from this project. Aim3 is to deliver ArmywormBase as a WEB-accessible transcriptome database for public access to all sequencing data generated by this project. We have begun constructing ArmywormBase using the software and HTML code framework of CinxiaBase. ArmywormBase will specifically house assembled and annotated Illumina sequence generated in the USA by us from various larval tissues. We are negotiating with French INRA and Genoscope colleagues as to where a grand transcriptome generated from USA and French sequence data will be housed for public access. Possibilities are Spodopbase (http://bioweb.ensam.inra.fr/spodobase), Lepido-DB (http://www.inra.fr/lepidodb), or InsectaCentral (http://insectacentral.org). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Aim 2 of the project is to identify genes in fall armyworm whose expression level is influenced by eating foliage from lepidopteran resistant corn inbred line Mp708. Using additional support from The Huck Institutes of the Life Sciences at Penn State, we completed an experiment using the SOLiD RNA-Seq platform to compare gene expression profiles in midguts from corn strain larvae that ate susceptible Tx601 or resistant Mp708 corn foliage. Larvae of the corn host plant strain were fed throughout development on susceptible or resistant corn foliage. Midgut minus food bolus was dissected from last instar feeding phase larvae. Total RNA was isolated from individual midguts and pooled into 3 replicates per plant treatment. We prepared six uniquely tagged cDNA libraries from mRNA. All libraries were pooled and sequenced in one full SOLiD run. The unique tags enabled sorting of sequence reads obtained by replicate and plant treatment. Reads were filtered to remove any with a base whose quality is greater than 95% accuracy. A reference was prepared using Sanger ESTs from Spodobase. We assembled these ESTs into a reference of 7869 contigs and annotated them against UniProt with our own PipeMeta. High quality SOLiD reads were mapped against these reference contigs with NextGENe. Normalized, RPKM expression level was quantified and used in t-tests. A volcano plot was prepared using data on all contigs to which reads had mapped. This plot enabled us to resolve contigs showing very highly significant differences in expression level with respect to plant treatment. It also enabled us to detect whether expression level was higher when larvae ate susceptible or resistant corn foliage. The orthogonal annotations linked each contig to a functional gene, thereby allowing us to derive conclusions about how midgut tissues responded to the plant treatments. Foliage of caterpillar resistant corn expresses Mir1-CP cysteine protease, which attacks the peritrophic membrane (PM). Midgut genes showing highly significant treatment differences were only found in larvae that ate resistant corn foliage. These genes tended to be upregulated more than 4-fold. Midguts from larvae that ate resistant foliage have an expression profile that suggests they are trying to counteract the action of Mir1-CP. In contrast to the susceptible foliage treatment, these midguts upregulated genes probably involved in PM repair, protein digestion by PM bound enzymes, wounding and antimicrobial reactions, protein translation, and hydrolytic defense targeting Mir1-CP. We used quantitative real time PCR to verify that intestinal mucin and chitin deaceytlase 1 are genes highly upregulated only in midguts from larvae that ate resistant foliage. Both genes are likely involved in repair of the PM, thereby lending support to the hypothesis that mucins in the PM are a target of the Mir1-CP cysteine protease. These results ascertain efficacy of RNA-Seq with SOLiD next generation sequencing in transcriptome expression profiling to discover with limited genomic resources how a broad group of genes in a caterpillar respond to detrimental nutritional effect of eating resistant corn foliage.
Publications
- No publications reported this period
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