Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Plant Science
Non Technical Summary
Virus diseases are among the major biotic constraints to legume production in the tropic and subtropical Americas. Common bean (Phaseolus vulgaris L.) is susceptible to virus infection and one of the most widely cultivated legumes in the world. Tepary bean, Phaseolus acutifolius (L. Gray) is a legume native to the southwestern United States and Mexico where it has been grown by the native peoples since pre-Columbian times. It is more drought-resistant than the common bean Phaseolus vulgaris (L.) and is grown in desert and semi-desert conditions. In a previous study, our lab has shown that BCMV is seed-borne with percentage of seed transmission ranging from 58-70% and is readily detected in bean seed lots. Isolates of BCMV are known to remain viable in common bean seeds for up to 30 years, and under field conditions the infected seedlings originated from BCMV-seed can serve as the source for virus spread by several aphid species in a non-persistent manner. Although yield losses of 53-83% in symptomatic bean attributed to BCMV, symptomless BCMV infections can result in >50% yield reduction in certain cultivars. Analogous detailed studies are lacking for tepary bean, however, our research has confirmed that BCMV, similarly, causes symptomatic and asymptomatic infections of cultivated tepary bean. The project goal is to apply functional genomics analysis to determine whether a naturally-occurring potyvirus, Bean common mosaic virus, contributes to the ability of tepary bean to withstand extreme drought/heat conditions of desert climate niches where it is endemic. The hypothesis is that BCMV infection is detrimental to tepary bean 'health'; the null hypothesis is that the BCMV virome has no effect on 'health' and performance of virus-infected tepary bean plants. The research outcomes are expected to increase our understanding of: (1) functional genomic profiling of tepary bean exposed to drought/heat stress; (2) dual-host-virome profiling of drought and heat stressed, BCMV-infected tepary bean; (3) determining whether wild tepary bean harbor BCMV or other plant viruses (virome), initially, by RT-PCR and then by NGS 'discovery' for select isolates; if present, do genome sequences and/or virome population structures differ among 'cultivated' and 'wild' tepary bean. How host-virome interactions play out, in the face of abiotic stress is a primary interest. Genes of interest are those involved in stress responses, as are the genomic regions disease resistance or tolerance to BCMV.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Develop and apply genomic tools
Project Methods
The study is designed to learn whether naturally-occurring potyviruses transmitted through seed contribute to withstanding drought conditions, or if virus infection is detrimental or has no effect on health and performance of tepary bean plants.(1) Characterization of selected tepary bean lines and/or accessions harboring potyviruses (compared to virus-free, experimental control plants) in response to water and/or heat-stress (field and/or growth chamber).Abiotic and biotic stressExperiments will utilize tepary bean genotypes naturalized to southern Arizona, or from the GRIN germplasm collection, and/or improved genotypes (USDA-ARS, Dr. Tim Porch), and selected wild tepary bean seed available through the Index Seminum, Desert Legume Program (DELEP) http://www.ars-grin.gov/cgi-bin/npgs/html/stats/site.pl?DLEG.The cultivated tepary genotypes, germplasm accessions, or wild genotypes will be grown under contrasting irrigation regimes, well-watered and water-limited conditions, either in pots in a growth chamber and/or in research plots located in southern Arizona.In field plots, seed will be sown in mid-June to early July, with planting synchronized with the beginning of the monsoon season. Standard agronomic practices for the desert Southwest at the UA farm (Maricopa Agriculture Center, Maricopa), will be practiced. Field plots will use a randomized complete block design with three replications per the two irrigation levels.In growth chamber or field experiments, water will be applied according to frequency of irrigation so that the water-limited treatment receives approximately half that the well-watered level. In addition, plants subjected to the above water regimes will be exposed to two temperature regimes, simulating heat stress or more moderate growth conditions.Field phenotypingPlants will be phenotyped for various traits to capture their differential response to the hot, arid environment. Traits will include initial emergence rate and plant height on a weekly basis. The time of bloom (dates), number of flowers, number of pods set, number of seed per pod, biomass, and harvested seed mass will be collected for plants in the experimental plots.For field studies, additionally, aerial imagery data will be collected using drones carry high-resolution cameras.Data will be analyzed to derive the following traits: normalized difference vegetation index (NDVI) or green normalized difference vegetation index on a per plot basis to include with the analyses.For longitudinally collected data such as weekly plant heights, a logistic growth model can be fit to the data to derive empirical parameters that also serve as phenotypic data for additional analyses.(2) Characterization of virome populations and Host-pathogen Interactions RNA isolation. Total RNA is isolated from tomato leaves by homogenized in 1 mL Tri-Reagent (Sigma Aldrich, St. Louis, MO, USA) using a Mini-bead beater™ machine (BioSpec Products, OK, USA) in 1.5 mL tube (Eppendorf, Hauppauge, NY, USA) containing twenty 0.5 mm diameter Zirconium beads (RPI Research Products, Mt. Prospect, IL, USA) for 4 min. The homogenate is collected by centrifuged at 10,000g for 10 min at 4oC. The RNA pellets are resuspended in 30 µl of nuclease-free water ddH2O, quantified with a Nanodrop 2000 Spectrophotometer™ (Thermo Scientific, Grand Island, NY, USA), and analyzed by agarose (0.7 %) gel electrophoresis. The cDNA is synthesized from 2 µg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems/Thermo Scientific).Quantitative RT-PCR. The accumulation of BCMV RNA (+ stranded) in tepary bean plants will be determined by quantitative PCR using TaqMan™ Universal PCR Master Mix (Applied Biosystems, catalog no. 4305719). Reverse transcription (RT) is conducted using 1 µg of total RNA and a SuperScript™ III First-Strand synthesis kit (Invitrogen, Carlsbad, CA, USA) in a final volume of 20 µL according to the manufacturer's instructions. The RT reactions are stored at −20 °C and used for qPCR amplification using primers and Taqman probe designed to amplify a 469 fragment of the BCMV coat protein (or other) gene (laboratory SOP). Reactions are carried out in triplicate using the CFX96™ Real-Time PCR System (BioRad Laboratories, Hercules, CA, USA). Expression data are normalized (Method 44, CFX96 Biorad Manual).Large and small RNA sequencing. The BCMV /virome associated with tepary bean will be determined by RNAseq sequencing and assembly of large RNAs. Both large and small RNA reads will be obtained to characterize dual-host pathogen interactions in tepary bean subjected abiotic (drought/heat) and biotic (BCMV) stress. Here, large RNAs will comprise viral mRNAs, viral genomic RNAs (~10 kb), and plant mRNAs, while 21nt to 24nt small RNAs are produced through the RNAi mechanism in plants that target invading viral pathogens.RNA isolation and Illumina sequencing. Total RNA is isolated from leaves with petioles attached, flowers, suckers and seeds using Fruit-mate buffer (Takaram) and TRIzol LS Reagent (ThermoFisher Sci). Briefly 50mg previously frozen (-80C) ground tissue is mixed with 1mL Fruit-mate buffer and homogenized for 2 min in MINI BEADBEATER (BioSpec Products Inc. Bartlesville, OK) using the standard setting. The homogenate is centrifuged at 12,000 rpm in a microfuge at 4 °C for 5 min. The supernatant is collected and centrifuged at 13,000 rpm at 4°C for 5 min. All centrifugation steps are at 12,000 rpm at 4°C. One mL TRIzol LS reagent is added to the supernatant of approximately 800μL, and incubated at room temperature for five min. Chloroform (320 μL) is added, followed by agitation for five min, and centrifugation for 15 min. Approximately 800 μL supernatant is collected and precipitated by the addition of an equal volume of isopropanol. The RNA pellet is collected by centrifugation for 10 min, washed with 75% ethanol, collected by centrifugation, air dried and dissolved in 50 μL RNase-free water. Approximately 2μg RNA is used for RNA-seq and small RNA-Seq library preparation. Sample preparation and sequencing are carried out by Novogene Corp. Inc (Sacramento, CA 95826).Bioinformatics Fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) is used to evaluate the quality of sequences, and trim-galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) is used to remove low quality reads. Using Trinity (Grabherr et al 2011) RNAseq data is assembled de novo and using stand-alone blastn search tool and NCBI database de novo assemble sequence contigs were identified.For the small RNA population, contigs obtained from the large RNA-seq de novo assembly are used as reference sequences for mapping small RNA-seq data using Bowtie2 (Langmead B and Salzberg 2012).Reference BCMV sequences will be downloaded from the GenBank database (>22 genomes are available) and will be used for phylogenetic and comparative analyses of the viromes associated with domesticated/cultivated and selected wild tepary bean isolates (ML, Mr. Bayes). The HC Pro domains encoding three modules (domains) mRNA/proteins involved in suppression of RNA silencing of the plant host (and extent of viral accumulation), and cell to cell movement.The latter BCMV functions are directly involved in susceptibility to infection and disease tolerance and resistance. How host-virome interactions play out, in the face of abiotic stress is a primary interest. Gene-based mapping of the tepary bean genome is underway and transcriptomic resources are available (https://www.ncbi.nlm.nih.gov/nuccore?term=Tepary+bean&cmd=DetailsSearch) and will be used for mapping small and large reads. Among genes of interest are those involved in stress responses, as are the genomic regions disease resistance or tolerance to BCMV.