Recipient Organization
DELAWARE STATE UNIVERSITY
1200 NORTH DUPONT HIGHWAY
DOVER,DE 19901
Performing Department
Biological Sciences
Non Technical Summary
Potato virus Y (PVY) is one of the most serious challenges facing potato production worldwide. PVY infection of potato causes decreases in tuber yield and quality. In the United States, potato tuber yield loss may be up to 80% and more than 40% of U.S. seed lots have detectable levels of PVY, despite costly seed certification programs.The lack of resistance and the popularity of several widely planted varieties that are symptomless carriers of PVY have facilitated an increase in PVY incidence and contributed to the emergence ofpotato tuber necrotic ringspot disease (PTNRD), which causes reductions in yield and quality and may cause a total crop loss. Recent research has identified the C-terminal region of PVY HC-Pro protein as the PTNRD determinant; loci controlling PTNRD, HR, and other PVY symptoms have also been mapped to the central region of potato chromosome IV. To elucidate the mechanism of disease production, therefore, we will use next generation sequencing capabilities and sophisticated bioinformatics approaches to uncover key factors playing a role in the production of PVY induced disease in potato. Such information is key to designing control strategies to contain this virus in potato, and indeed other crop plants.Additionally, we will develop a control method based on genetic engineering. This is important because of challenges associated with introduction of natural resistances into cultivated potato varieties. Hence, we have shown in preliminary work thattrans-acting small interfering RNAs (tasiRNAs), a widespread and deeply conserved phenomenon in diverse land plants, can be employed in the control of PVY. In this procedure, tasiRNAs-derived from the virus are processed in the plant and these tasiRNAs degrade complementary viral RNAs, thereby controlling the virus. We will also characterize natural resistance to PVY as found inRandNgenes--which are found in a limited number of potato varieties--by molecularly characterizing potato varieties exhibitingRandNgene resistances to PVY strains. Identification of genes implicated inRandNgene resistance, will be conducted by investigating specific types of RNAs (including small RNAs or mRNAs) produced during PVY infection.This project also has an important workforce development component, notably, training of minority scientists.Indeed, HBCUs, including DSU, play key roles in expanding access to excellence for minority students in STEM. Department of Education statistics show that between 2005 and 2010, HBCUs enrolled only 14% of African American undergraduate students, yet 35% of Black recipients of STEM PhDs had received their BS degree at an HBCU. In STEM disciplines, a key reason for low retention and graduation rates amongst minority students is unpreparedness, frequently due to lack of experiential learning opportunities. To contribute to addressing this problem, each year, at least two undergrads will be trained in genomics and biotechnological approaches. Furthermore, to improve the writing skills of these students, we will establish a scientific and technical writing module within selected inquiry-based lab projects. We will also provide an 11-week summer research-training program in genomics and bioinformatics every year for DSU undergraduate students at the Donald Danforth Plant Science Center. Additionally, two MS students will be trained in genomics and next generation sequencing data analysis. Their thesis research will be on various components of this proposal.Importantly, the data obtained, and techniques developed will be made available in a freely accessible, public database for the wider research community.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The goals of this project are to uncover the molecular basis of PVY strain pathosystems in natural resistance conferred by R and N genes, respectively, as well as through trans-acting siRNA (tasiRNA)-engineered resistance. This will be achieved through tthree objectives, which are to:Determine key factors regulating plant-virus interactions in the establishment of PVY strain-specific pathosystems in potato using deep sequencing and data analysis capabilities;Characterize the nature of tasiRNA-mediated resistance by identifying and molecularly characterizing regulators of this resistance in N. tobacum and in potato. We will also compare efficiencies of TAS1 and TAS3 pathways in generating tasiRNA-mediated resistance.Identify and determine the role of key regulators found to be involved in PVY infectivity and resistance using virus-induced gene silencing (VIGS), short tandem target mimic (STTM) and/or molecular sponges (SPs).We will also train undergraduate and graduate students, as well as postdoctoral scientists.
Project Methods
We will examine the molecular basis of PVY strain pathosystems in susceptible potato, as well as in potato with natural resistance and/or engineered resistance. Our focus will be on PTNRD, which threatens the potato industry in the US. The methods to be used in executing the work are summarized below.1.To determine key factors regulating plant-virus interactions in the establishment of PVY strain-specific pathosystems in potato, we will analyze potato varieties with different levels of resistance to PVY. To this end, recent research has identified the C-terminal region of PVY HC-Pro cistron as the PTNRD determinant; loci controlling PTNRD, HR, and other PVY symptoms have also been mapped to the central region of potato chromosome IV. Therefore, to determine whether the PVY pathosystem is controlled by this chromosome region,we will inoculate tubers of healthy 'Eva' (extreme resistance), 'Ranger Russet' (moderate resistance), and 'Desiree' (susceptible) and incubate for1 h at20°C and10°C, respectively; at least 10 tubers will be inoculated for each variety and virus strain.sRNA and RNA-seq libraries will then be prepared from infected tuberswill be deep sequenced.In this process, replicated (3X) libraries will be sequenced on the Illumina HiSeq 2500 platform. Following sequencing, adaptor sequences will be trimmed and t/r/sn/snoRNAs removed. vsiRNAs, siRNAs, miRNAs, phasiRNAs, and vsiRNAs, as well as mRNAs will be identified using automated workflows available in Co-PD Meyers' lab.Other queries or algorithms, many of which are built into the Meyers lab website (http://mpss.danforthcenter.org) can identify host-derived heterochromatic siRNAs and phasiRNAs in an automated manner.For each PVY strain, differential levels of respective sRNAs or mRNAswill be determined based on the following outcomes:a.Significant increase or reduction in 'Eva' compared with 'Desiree'; these could represent factors involved in PTNRD production;b.Significant increase or reduction in 'Desiree' compared with 'Eva'; these could be involved in virus susceptibility, such as targets of HC-Pro.2.To characterize the nature oftasiRNA-mediatedresistance, we will use both the TAS1 and TAS3 gene pathways. Here,6 to 8 constructs, each containing 4 to 10 tasiPVYs from different conserved regions of PVY genomes will be cloned into the modifiedTAS1- andTAS3-containing plasmids forN. tabacumstable transformation.As a control, we will transformN. tabacumwithTAS1andTAS3constructs in which mutations are introduced in the tasiPVY site to whichmiRNA"seed region" (2nd- 9thnucleotide) binds. We willmake a syn-tasiPVY construct with a target sequence for miR6024 or miR6027, both abundant 22-nt miRNAs that are conserved in theSolanaceaeand that target nucleotide-binding domain and leucine-rich repeat (NLR)-encoding genes and are broadly expressed in diverse tissues. For each tasiPVY construct, 20 to 30 transgenicN. tabacumlines will be assessed for virus resistance. Thus, tasiPVY plants and control plants will be mechanically inoculated with different PVY strains and responses recorded over time. TAS-ELISA and northern blot hybridization and/or qRT-PCR will be used to quantify virus levels. These analyses will identify transgenic lines, and thus most efficient constructs showing a strong resistance to specific virus strains or to all strains assessed. The most efficient constructs will be used to transform potato variety 'Desiree' and the resistance of transgenic potato examined as forN. tabacum. To characterize the nature of tasi-RNA mediated resistance, we will analyze sRNAs and mRNAs in transgenic plants and compare with non-transgenic plants as described above.3.To determine the role of key regulators found to be involved in PVY infectivity and resistance, we will use gene RNA based knockdown approaches. Thus, virus-induced gene silencing (VIGS), short tandem target mimic (STTM) and/or molecular sponges (SPs) approaches will be used to determine the role of miRNA targets in PVY infection; correspondingly, VIGS will be used to determine the role of protein-coding genes in tasiPVY-mediated PVY resistance.To silence miRNAs, we will use STTM containing short sequences mimicking miRNA target sites, separated by a linker. The miRNA target mimic sequences contain the binding sites of the miRNAs targeted for silencing plus three unique nucleotides (CUA) placed between the 10thand 11thnucleotides of the miRNA, resulting in formation of a "bulge" of the miRNA::STTM duplex. Thus, the STTM binds the miRNA but no cleavage occurs, instead, the miRNA is sequestered, resulting in accumulation of natural targets of the miRNA. Virus-based microRNA silencing (VbMS) system in which the STTM targeting multiple miRNA targets are inserted in viral vectors have been shown to be a robust system for silencing multiple miRNAs from one construct. We will thus insert STTM into the TRV2 vector, which will be transformed intoA. tumefaciens,cultures of which will be co-infiltrated cultures of TRV1 to leaves ofN. tabacumand/orN. benthamianaas described; control plants will be inoculated with TRV1 and the TRV2 without STTM. To confirm that the assay is working, we will target a known miRNA. Two weeks post infiltration, plants will either be mock inoculated (control) or challenge inoculated with PVY in upper non-infiltrated leaves.As an alternative to STTM and VbMS, we will employ miRNA sponges (SPs), which have been used to knockdown miRNAs in plants. A miRNA sponge is an artificial RNA containing many copies of miRNA binding sites that can bind to cognate miRNAs without being cleaved by them. Thus, similar to STTM, a miRNA SP is a decoy that sequesters miRNA away from their natural targets, thereby derepressing target gene expression. Plant SPs may contain up to 15 copies of miRNA binding sites (linked by a 4 nt spacer) that contain mismatches at the cleavage site for effective knocking down of the target miRNAs. Because SPs can contain multiple miRNA binding sites, many miRNAs can be knocked down and would enable functional analysis of members of the same miRNA family.In addition to monitoring PVY symptom development, we will carry out ELISA, northern blot analysis and/or RT-PCR detections of the virus. To make sure that the appropriate miRNA is overexpressed or down-regulated, we will determine relative miRNA levels in these plants using qRT-PCR as we described recently. We will also assess the level of predicted miRNA targets using qRT-PCR and relate the results to those of their miRNA triggers.These experiments will determine whether or not the specific miRNA is involved in PVY infection.To silence differentially regulated protein-coding genes, we will silence genes usingthe TRV VIGS vector. Thus, we will clone 300-500 bp of the 3'UTR of each gene of interest into the TRV vector and introduce this intoN. tabacumplants using agroinfiltration. To determine whether silencing affects plant response to PVY infection, we will inoculate upper leaves of plants with PVYNTNafter two weeks and monitor phenotypes for 3 weeks. In each of these experiments, we will inoculate control plants with the VIGS vector containing GFP so as to evaluate the effect of VIGS vector alone on subsequent infection with PVYNTN. These analyses will be repeated to confirm results. Virus titer will be determined by RT-PCR. Levels of silencing of target genes will also be determined using qRT-PCR.