Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Agricultural Biology
Non Technical Summary
The potato industry in the US is under threat from newly emerged potato viruses that render potatoes unmarketable by causing necrotic arcs, rings and spots on or inside the potatoes. The two major tuber necrotic viruses of economic importance include aphid-transmitted Potato virus Y (PVY) and soil-borne plasmodiophorid, Spongospora subterranean-transmitted Potato mop-top virus (PMTV) and nematode-transmitted Tobacco rattle virus (TRV). PVY is managed primarily by eliminating lots with high virus incidence from production and identification of these lots is mainly by visual inspections. However, the PVY strains that are now predominant in the US typically cause mild foliar symptoms, making visual inspections are less effective. PMTV and TRV can last for a decade or more in soil and that is resistant to soil fumigation, so growers lack effective management protocols. We propose to contribute to reducing the impact of the tuber necrotic viruses by developing molecular tools to develop molecular tools that will aid in rapid and early detection of resistance to tuber necrotic viruses in potatoes. Specifically, we propose to (1) accelerate the introgression of PVY resistance to elite cultivars of potato suitable for Colorado and (2) tag PVY, PMTV and TRV with a new type of marker gene, Antirrhinum majus Rosea1 which results in red color anthocyanin pigments in leaf tissues infected with the virus. Further, we will characterize symptom development in potato cultivars after inoculation with Rosea1-tagged infectious clones of PVY, PMTV and TRV.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
Potato viruses are the most important seed potato pathogens and are a significant burden on commercial potato production. They cause costly yield and quality reduction and reduce profit margins for producers. Three vector-borne tuber-necrotic viruses are currently economically important threats for the potato industry. These viruses include the aphid-transmitted potato virus Y (PVY), the plasmodiphora-transmitted potato mop-top virus (PMTV) and the nematode-transmitted tobacco rattle virus (TRV). This project utilizes two advanced molecular techniques for screening advanced breeding potato clones for these necrotic viruses, which will accelerate Colorado potato breeding program. The goals of our study are twofold:To accelerate the introgression of PVY resistance to elite cultivars of potato suitable for Colorado.To develop biotechnological tools that allow for the rapid screening of germplasm for resistance to three tuber necrotic viruses, PVY, PMTV and TRV
Project Methods
Objective 1: Experimental Plan: About fifty to hundred advanced breeding clones provided by Dr. Holm will be screened for PVY resistance. Three to five plants for each advanced clone will be grown in the greenhouse. Plants will be mechanically inoculated with different strains of PVY (PVYN-Wi and PVYO) four weeks after planting. Inoculated plants will be monitored for visual symptoms and ELISA (Enzyme Linked ImmunoSorbent Assay) will be performed 20 and 40-days post inoculation to detect the virus (Ottoman et al. 2009). Genomic DNA will also be extracted from leaf samples and PCRs will be performed to determine the presence/absence of molecular markers for Ryadg, Rysto, Rychc genes. The molecular markers that will be used in the current study are as below:Ryadg presence tested using the markers RYSC3 (Kasai et al. 2000)Rysto presence will be tested using YES3-3A and YES 3-3B markers (Song and Schwarzfisher, 2008)Rychc presence tested using Ry186 (Takeuchi et al., 2008)In order to facilitate rapid screening of germplasm with increased accuracy, samples after PCR will sent to MCLab (San Francisco, CA). Additionally, the Rysto gene will be cloned and sequenced from the advanced breeding clones that test positive for the presence of the gene using the YES3-3A and YES3-3B markers.Objective 2: Experimental Plan: The protocol listed below will used to determine if the Rosea1 marker will be operational in PVY, PMTV and TRV.Collection of Viral clones: Potato leaf/tuber tissue collected from fields that are infected with PVY, PMTV and TRV will be used as sources to clone the virus.Cloning of Rosea1: The Rosea1 transcription factor (GenBank accession number DQ275529.1) has already been cloned (as part o the previous grant) from RNA extracted from Snapdragon (Antirrhinum majus) leaf tissue.Construction of recombinant viral clones tagged with Rosea1:PVY-Ros1: The construction of a PVY recombinant clone tagged with Rosea1 is currently being performed according to Cordero et al., 2017. The full-length clone of Rosea1 will be inserted between the cistrons NIb and CP. Recombinant clones will be constructed for the PVY strains O, Wi and NTN.PMTV-Ros1: The full-length genomic cDNA clones of isolates of PMTV have been cloned and will be transcribed in vitro according Lukhovitskaya et al., 2005. The Rosea1 cDNA will be incorporated to the C terminus of the CRP gene located on RNA3.TRV-Ros1: TRV in addition to being pathogen has been modified to serve as a virus-induced gene silencing vector (Liu et al., 2002). A recombinant TRV virus will be constructed by incorporating the Rosea1 cDNA at the C-terminus of the coat protein (CP) gene located on RNA2 as per details listed in Tian et al., 2014.4.Plant Inoculation: Five-week old tobacco and 6-week old potato plants will be used for mechanical inoculations. Plant inoculation will be carried as per Cordero et al., 2017. Briefly, plasmids containing the recombinant viral clones will introduced into Agrobacterium tumefaciens strain GV3101. Recombinant virus clones will be assembled by first agroinoculating 4-week-old Nicotiana benthamiana plants. For mechanical inoculation of plants, crude extracts from the infected tissues will rubbed onto the leaf surface of tobacco and potato plants.Symptom development: Infected tobacco and potato plants will be monitored daily for a period of 14 -21 days. The formation of red foci will indicate the successful replication of recombinant viruses.Virus diagnosis by PCR: RNA will be extracted from infected leaf tissue (Direct-zol RNA Miniprep Plus kit) will be reverse transcribed (cDNA synthesis using Verso cDNA synthesis kit). The reverse transcribed products will be used as templates to determine the presence of recombinant PVY-Ros1, PMTV-Ros1 and TRV-Ros1 using virus specific primers and will be revealed by agarose gel electrophoresis.Objective 3: Experimental Plan: Inoculation of the tagged-PVY isolates will be done with susceptible, partially resistant, and resistant potato varieties to better understand mechanisms of resistance. Symptom expression and virus location will be recorded on a bi-weekly basis in inoculated plants. In addition, we will evaluate the efficacy of crop oils in slowing down the spread of PVY using the recombinant tagged PVY clones. An APHIS BRS (biotechnology) permit will be obtained for development and use of the virus isolates and all work will be done in secure bio-safe labs, growth chambers, and greenhouse facilities to ensure proper handling of these isolates.