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An Integrative Transcriptome, Proteome and Metabolome Approach for Better Understanding Plant - Pathogen Interactions in Grape

Sheikh, Mehboob
Florida A&M University
Start date
End date

The proposed project addresses: CBG priority areas- e) Global Food Security and Agricultural Biosecurity, USDA priority areas- 1. Plant Health and Production and Plant Products, and NIFA challenge areas- 1.Plant health and production and plant products; c. Food safety; d. Food security. The project has six key objectives

(1) determine genetic variation in expression of disease tolerance-related genes, proteins and metabolites, and their identity among grape genotypes with diverse disease tolerance characteristics,

(2) determine variation in the molecular and cellular responses of tolerant and susceptible genotypes to anthracnose and PD infection,

(3) isolate and characterize proteins/enzymes and metabolites associated with disease tolerance to assess their role in plant defense,

(4) conduct in vitro bioassays to determine antimicrobial activity of plant extracts from tolerant and susceptible genotypes,

(5) develop small RNA libraries of tolerant and susceptible genotypes, and healthy and infected tissue to determine regulation of defense related genes, and

(6) strengthen linkages among FAMU faculty to enhance multidisciplinary research and provide hands-on experiential learning to students and faculty across the campus in various transcriptomics, proteomics and metabolomics experimentations and bioinformatics analysis. Outcome from the proposed research will not only help identify the components responsible for disease tolerance but also better understand their regulation during host-pathogen interactions for improving disease tolerance in crop plants. The information, material and expertise gained from this project will be extended to identify, isolate and characterize important plant and animal genes to promote collaborative research and to understand the biology of host-pathogen mechanism by applying latest high throughput techniques at other 1890 institutions there by strengthening ongoing multi-institutional collaborative projects. This project will also add immense sequence information to the database that can be used as reference data sets for large scale RNA-Seq and proteomic projects in grapes and other crops with similar disease instance. This project will also provide opportunity for student experiential learning where they will learn experimental design, sample collection and analysis, and core bio-techniques such as preparation of buffers, RNA and protein isolation, PCR, library construction, bioinformatics data analysis, statistical tools, metabolite analysis, etc. A series of workshops and presentations will be conducted during the field days and consumer meetings, and brochures and pamphlets will be developed to demonstrate our findings and increase awareness among the consumers about disease management. The outcome of this research will be disseminated to the scientific community, grape growers and consumer groups through presentations at the conferences and publications in peer reviewed journals.

More information

Grape production offers a significant economic opportunity for small farmers in the United States, especially in Florida, due to ever increasing demand for grape products because of its tourist industry and perceived health benefits. Florida imports >90% of grape products to meet tourist industry demand and hence, Florida is keenly interested in increasing domestic grape production to meet growing demand and improve local economies. The major hurdle in growing grapes in Florida or any warm-humid parts of the U.S is disease. Most of the major diseases of grapes including fungal diseases such as black rot (Guignardia bidwellii), downy mildew (Plasmopora viticola), powdery mildew, anthracnose (Elsinoe ampelina), several kinds of blights and fruit rot, and bacterial disease such as Pierce's disease (Xylella fastidiosa) persist in the South and cause severe economic damage. The southern grape industry is based primarily on muscadine and hybrid bunch grape species because of their tolerance to Pierce's disease, which precludes growing of California-type bunch grapes. Although, native muscadine grapes (Vitis rotundifolia) are tolerant to most diseases they yield inferior berries and poor quality wines. Because of the superior quality and consumer preference for bunch grape products, southern grape industry prefers to grow bunch grapes (Vitis vinifera). However, they are highly susceptible to diseases and a major issue in South. Among grape diseases, anthracnose caused by E. ampelina and Pierce's disease (PD) caused by a xylem-inhabiting bacterium, Xylella fastidiosa are major concern to Southern grape industry. Although extensive research has been conducted on diseases of European bunch grapes (Vitis vinifera), limited research exists on anthracnose and Pierce's diseases which are of key interest to southern grape growers, especially to Florida farmers. Therefore, our study is focused on understanding the complex biological interactions that impact disease tolerance in grapes grown under warm/humid southern climate using systems approach and employing power of transcriptomics, proteomics and metabolomics to better understand the molecular and cellular events that occur in the cell between tolerant and susceptible genotypes, and during pathogen infestation. We propose to perform transcript profiling employing Illumina system, protein profiling using iTRAQ labeling and sequencing system, and metabolome profiling using biochemical and instrumental analysis for comprehensive understanding of disease biology in Florida grown bunch grapes to determine progression of gene, protein and metabolome expression that are associated with disease tolerance which is essential for developing disease tolerant grape suitable to South. The information, material and expertise gained from this project will be extended to identify and characterize important genes, proteins and metabolites for enhancing ongoing research, and building expertise on campus in latest molecular and cellular technologies. This effort will also add immense sequence information to the database which is of great value to national and international researchers.

Pure cultures of Elsinoe ampelina (anthracnose) and Xylella fastidiosa (PD) will be used for inoculation/infection studies. Tissue samples will be collected at different time intervals following inoculation. Total RNA will be extracted from anthracnose- and PD-infected and control samples. mRNA will be purified and cDNA will be synthesized and libraries will be prepared for RNA-sequencing. RNA-Seq will be performed using the HiSeq2000 Illumina system. Sequence data obtained will be electronically assembled into a set of unique non-overlapping continuous sequences. RNA's from control and treated samples will be used for Real-Time PCR to validate expression of selected genes from RNA-Seq data. Identification and characterization of differentially expressed proteins among infected and control samples will be performed by iTRAQ labeling. Proteins will be isolated from leaf and xylem tissue using sucrose-phenol extraction method. Proteins (100ug) from healthy and infected tissue will be precipitated and resulting pellets will be air dried, reduced, alkylated, trypsin-digested and labeled using 8-plex iTRAQ reagent kit (ABSciex, CA). The healthy samples will be labeled with iTRAQ tags 113, 114, 115 and infected samples will be labeled with tags 116, 117, 118, 119 and 121. The peptide mixture will be combined, lyophilized, fractionated by HPLC and sequenced using Orbitrap LTQ mass spectrometer. The MS/MS Data will be analyzed for protein identification and quantification by software tools ProteinPilot and Peaks Studio 5.3 (BSI Inc, ON, Canada). Total soluble sugars will be estimated using anthrone method, and amino acids, phytoalexins and phenolics composition will determined by HPLC to identify variation in their accumulation pattern between infected and healthy plants. Chitinase and stilbene synthase enzyme analysis will be performed to determine differences in enzyme activity between infected and control plants. Antifungal and antibacterial activity of the leaf and xylem tissue extracts from susceptible and tolerant, and healthy and infected tissue will be determined to asses' genetic variation in their expression. Small RNA will be isolated and purified from low molecular weight RNA enriched by 0.5 M NaCl and 10% PEG8000 precipitation. Small RNAs will be ligated sequentially to chimeric oligonucleotide adapters. Reverse transcription will be preformed after ligation with adapters followed by PCR. Library preparation and sequencing will be done as describe earlier for RNA-Seq. The major outcome/product of this project will be the identification of gene/s/proteins/metabolites that confer resistance/tolerance to fungal and bacterial diseases such as anthracnose and PD, respectively as well as deriving timeline of their expression during disease progression. The project will generate transcriptome, proteome and small RNA profiles of healthy and infected tissue which would help determine regulation of disease tolerance genes. The outcome of this project can be easily measured based on our success in identifying key genes, proteins and metabolites, and demonstration of their association with disease tolerance and susceptibility.

2012/09 TO 2013/08
Target Audience: The target audience of the project were scientists and grape growers interested in developing/ growing disease-tolerant grape cultivars. The research was presented at FAMU field days where growers and clientele were made aware of the research findings and its advantages to the grape community. The unique proteins and genes identified in this project will aid in developing grape cultivars tolerant to anthracnose and Peirce’s diseases. Results of this research will benefit grape breeders and aid in screening grape breeding population and advanced breeding lines for disease tolerance using proteins and genes as biomarkers. Two African American undergraduate students and one graduate student were trained in gene and protein technologies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project personnel was involved in training three undergraduate students, two high school students and one graduate student in independent projects, prepare reports, analyze results and presentations. Further this project also enhanced project scientist experiences and helped strengthen research in protein and gene technologies, advance ongoing research in grape biotechnology. Besides this project helped establish collaborative research between CAFS and College of Biological Sciences and enabled training students in agricultural and biological sciences. The resources of this project also helped project personnel attend professional meetings, learn latest developments in the field, and network with other scientists to enhance ongoing research on the campus. Undergraduate Students Babich Remy, Kambiranda DM. Katam R, Basha SM, Lekan M Latinwo. 2013. Leaf Proteome analysis of different Florida Hybrid bunch grapegenotypes. Symposium on Functional Biology"" Jones Hall 109, Department of Biology, FAMU, August, 1, 2013. Chibanguza K, Kambiranda DM, Katam R, Basha SM, Lekan M Latinwo. 2013. Identification of Proteins Associated with Pierce’s Disease Tolerance in Xylem Tissue of Vitis Species. Presented at the ARD conference, Jacksonville, Florida April 4-8. Tilghman S, Kambiranda D, Basha SM. 2012. Characterization of Xylem Sap Components Associated with Pierce’s Disease Tolerance. Presented at NSF-REU student summit, FAMU, Biology. How have the results been disseminated to communities of interest? The results of this research werepresentedat the Florida Horticultural Society and Florida Grape Growers’ Association meeting where grape growers, hobbyist wine makers and public interested in growing grapes participate. In addition, the project data was also shared with grape growers, consumers and community during field days, seminars and workshops. What do you plan to do during the next reporting period to accomplish the goals? Metabolite analysis will be carried out using HPLC among anthracnose-tolerant and -susceptible genotypes of Florida hybrid bunch grapes to determine if tolerant varieties produce unique metabolites responsible for anti-fungal activity. Metabolite analysis will also reveal genetic variation among diverse grape genotypes. Shotgun proteomics will be performed using control and anthracnose infected leaf samples from tolerant and susceptible Florida hybrid bunch grape genotypes. Monitoring proteome changes among tolerant and susceptible genotypes will reveal if genotypes resistant to anthracnose disease produce unique proteins and how they overcome the infection. Xylem sap proteome of tolerant and susceptible Vitis species will aid in identifying novel proteins. We propose to use high throughput shot gun proteomics to increase the coverage of the proteome and identify low abundance proteins in sap that may play a role in defense response against Xylella fastidiosa. Further, susceptible and tolerant genotypes will be inoculated with X. fastidiosa, and transcriptome analysis using RNA-seq will be performed. This study will pave way towards further understanding the nature and function of Vitis xylem sap proteins/genes and their role in plant defense.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Natural Toxins
Viruses and Prions
Bacterial Pathogens
Chemical Contaminants
Food Defense and Integrity