Agriculture & Natural Resource
Non Technical Summary
Common bean (Phaseolus vulgaris L.) is the most important grain legume in the world and is a $1.2 billion crop in the United States. Common bean and other legumes are affected greatly by many biotic and abiotic stresses, and specifically common bean yields are negatively impacted by the bean rust fungal pathogen Uromyces appendiculatus, which may cause losses from 18-100%. We plan to use cutting-edge molecular genetics and epigenomics techniques to better understand the resistance and susceptibility mechanisms to this important disease. Differences in DNA methylation and gene expression will be compared among a rust-resistant wild type, susceptible wild type, and two susceptible mutants derived from the resistant wild type. This work builds on our previous common bean molecular genetics and genomics work in the area of disease resistance and comparative genomics along with the development of a reference DNA methylome project that is underway. This project will help us determine the effect of genome mutations affecting disease resistance on global DNA methylation and transcription simultaneously, under normal and biotic stress conditions. Additionally, the proposed project will also contribute to understanding of disease resistance mechanisms in soybean (Glycine max) and lima bean (Phaseolus lunatus) which are closely related legumes of economic significance in the United States. This project will further allow for the training of researchers from many levels at Delaware State University, the University of Arkansas-Pine Bluff, and at Mayville State University.
Animal Health Component
Research Effort Categories
Goals / Objectives
Objective 1: Develop reference leaf methylomes and transcriptomes from four genotypes of common bean.Objective 2: Develop biotic stressed leaf methylomes and transcriptomes from four genotypes of fungal rust-infected common bean and determine the methylomic and transcriptomic changes between resistant and susceptible genotypes.Objective 3: Expand the reach of the project through development of community genomics resources and integrated research-education and outreach programs.
Objective 1:Genotypes and Growth Conditions: Bean genotypes that will be used for reference methylome work are the cultivar Sierra (resistant wild type), the cultivar Olathe (susceptible wild type), and two susceptible mutants derived from Sierra via fast-neutron-bombardment, crg and ur3-Δ3. We will follow current protocols from the PI's laboratory in achieving these steps. Germinated seeds will be planted in a growth chamber, with day/night temperatures of 28/20°C and 14/10 h photoperiod. Two-week-old plant primary leaves will be collected and stored in a -80?C freezer until DNA and RNA isolation.Whole Genome Bisulfite Sequencing: DNA isolation from leaves will be conducted using a cetyl trimethylammonium bromide (CTAB) method. Extracted DNA will be used for whole genome bisulfite sequencing. Bisulfite conversion of genomic DNA will be performed using the EZ DNA Methylation-Lightning™ Kit from Zymo Research™. Unmethylated cytosines (C) undergo deamination and are ultimately sequenced as thymines (T), whereas 5-methylcytosines (5mC) are unaffected by bisulfate conversion and remain sequenced as cytosines (C). The resulting bisulfite-converted DNA will then be prepared with an Illumina® HiSeq SBS Kit and sequenced on an Illumina® HiSeq 2500 platform at the sequencing center at the Delaware Biotechnology Institute (DBI) in Newark, DE, where the PI is an affiliated faculty. Reads will then be mapped back to the reference genome (G19833, http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvulgaris). Bioinformatic analysis will be conducted using Integrative Genomics Viewer (IGV, 29) and the CLC Workbench software that is used for sequence analyses.Transcriptome Sequencing (RNA-Seq): RNA will be extracted from leaves with TRIzol® reagent, from Life Technologies™, according to the manufacturer's instructions. Extracted RNA will then be prepared for Illumina® sequencing with a TruSeq® RNA Sample Preparation Kit v2 and sequenced on an Illumina® HiSeq 2500 platform. Reads will then be mapped back to the reference genome (G19833, Phytozome). Bioinformatic analysis will be conducted using IGV as well as CLC Genomics Workbench.Validation Using Real-Time PCR (RT-PCR): As a confirmation that the RNA-seq data accurately reflects transcript levels, selected genes will be verified using real-time PCR (RT-PCR). Some genes that will be incorporated as a control include Cytochrome P450, WRKY7, and other randomly selected genes that show significant differential expression in the transcriptome results.The same RNA that will be submitted for RNA-seq will be reverse-transcribed using with Oligo dT using Superscript III First Strand Synthesis System (Life Technologies, 18080-051) according to manufacturer's instructions. Real-time PCR (RT-PCR) will be conducted on a 7500 Real-time PCR system Applied Biosystems platform with Power SYBR®Green PCR master mix (Applied Biosystems, 4309155). The ΔΔCT method will be used to analyze differential expression, in reference to the constitutively expressed gene cons7.Bioinformatic analysis: In addition to IGV and CLC Genomics Workbench mentioned above, Eukaryotic Orthologous Groups (KOG) Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Analysis will also be used. KOG analysis will be used to determine the classification of genes that are of significant differences in expression, as determined by RNA-seq analysis.These research protocols are well established in the PI and Co-PI's laboratories.Objective 2:All of the above procedures and analyses methods described in Objective 1 will be conducted for Objective 2, except that in this case, we are inoculating the plants with bean rust. Two-week-old plant primary leaves will be rust-inoculated with a solution of water, approximately 20,000 spores per milliliter, and 0.01% Tween 20 along with appropriate mock-inoculated control plants.Bioinformatic analysis: Bioinformatic analysis of the resulting data will be carried out as per the procedures described above, and the unstressed reference transcriptome and methylome data from Objective 1, will be compared to the stressed data from Objective 2. This analysis will reveal differences between plants treated with bean rust, as well as the differences between resistant and susceptible genotypes with regards to DNA methylation and gene expression.Objective 3:Students from high schools, undergraduate and graduate students will work with the PI and Co-PIs and research associate to develop and interpret the data generated by the first two objectives. Students and technical staff will work together to communicate the results at conferences and in publications. Epigenomics Web Site and Databases: In order to develop a portal so that collaborators, as well as external community members, can access project details, we will implement a common bean epigenenomics web site at DSU. Personnel from the DSU Center for Integrated Biological and Environmental Research will be involved in developing, maintaining and updating the web site. Previous and future results can be maintained on this portal.