Recipient Organization
USDA-ARS, GENETICS AND PRECISION AGRICULTURE UNIT
810 HIGHWAY 12 EAST
MISSISSIPPI STATE,MS 39762
Performing Department
(N/A)
Non Technical Summary
This project addresses the ECDRE priorities "A cure for HLB-infected trees and strategies for maintaining their productivity" and "A delivery system for therapeutics, nutrition, and other HLB solutions." In this proposal, we will first explore all potential uses of NMV-M/CFL as a biocontrol agent to control HLB. We will understand how this virus suppresses HLB and identify the best citrus varieties (scion and rootstock combinations) which can tolerate high viral and Las titers for application both in new plantings and existing HLB-affected groves. Second, we will explore our newly developed NMV-M/CFL-based expression vector and optimize its antimicrobial or anti-psyllid expression and delivery system with known antimicrobial peptides or siRNA. Concurrently, we will conduct active outreach and extension activities to disseminate our project results to growers, stakeholders, and the public.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Goals / Objectives
This standard research and extension project address the ECDRE priorities, "A cure for HLB-infected trees and strategies for maintaining their productivity" and "A delivery system for therapeutics, nutrition, and other HLB solutions". The overall goal of this proposal is to develop new control measures for HLB by using a newly discovered citrus virus with cross-kingdom antibacterial protection characteristics. We will capitalize on this discovery by characterizing the virus life cycle including its vector, mode of transmission and host range (Objective 1) and determining the molecular specifics underlying the HLB suppression effects (Objective 2). Since the HLB-suppressive effects caused by the virus are affected by the genetic background of the citrus hosts, we will expand our screening for the best combination of virus-rootstock/scion (resistobiome) for field trials (Objective 3). We will optimize the newly developed NMV-M/CFL expression vector, and generate constructs that express previously characterized antimicrobial peptides, such as the stable antimicrobial peptide, and siRNAs for psyllid and HLB control (Objective 4). Project results will be disseminated to stakeholders and the public through active outreach and extension (Objective 5). The selected combinations of virus-rootstock/scion are expected to be the most eco-friendly and cost-effective solution to HLB and provide immediate deployment without regulatory constraints. The stable NMV-M/CFL vector and expression system will be useful not only for the control of HLB but also for functional studies of citrus genome.
Project Methods
2.1 Methods for Objective 12.1.1 We will conduct transmission studies using citrus flatid planthopper, glassy-winged sharpshooter leafhopper, potato leafhopper, and determine their mode of transmission if any one of them can transmit the virus from citrus to citrus or to other related crops. We will determine their acquisition and incubation threshold times by periodically monitoring the virus titers in the insects after feeding on the NMV-M/CFL source plants and their infested plants using RT-qPCR. We will focus on the host range of citrus species and identify the most efficient propagation methods for maintaining NMV-M/CFL-infected citrus plants.2.1.2. For visualization of the virus and Las co-infection or the virus alone at subcellular location, we will use fluorescence in situ hybridization and transmission electron microscopy. Since the virus infection attenuates the damage of phloem and vascular system by suppressing HLB symptoms, we will also examine the healthy state of citrus vascular system that is co-infected with Las and the virus using above-mentioned methods.2.1.3. Sampling of field sites with citrus germplasm collections in CA and TX, as well as representative orchards within each growing region will be performed to assess NMV-M presence. The optimized RT-qPCR detection method will be used for testing NMV-M.2.2 Methods for Objective 22.2.1 We will profile the transcriptomes of leaf samples with appropriate biological replications. Preparation of mRNA and cDNA synthesis will follow the current Illumina directional protocol. Samples will be sent to an outside service for RNA-seq. We will check the quality of RNA reads using FastQC. We will use Salmon for read mapping and counting. DESeq2 will be used to identify differential expression at the allelic level. A list of significantly dysregulated genes will be further analyzed using co?expression network analysis. The differentially expressed genes will be functionally annotated using KEGG orthologs.2.2.2 First, we will synthesize and clone the full-length coding sequences of coat protein 1 and 2 of NMV-M/CFL, respectively. We will express each of these proteins fused with HIS or GST tags in Escherichia coli strain BL21. HIS- or GST- fused proteins will be individually purified using a nickel nitrilotriacetic acid (Ni-NTA) resin binding column (Qiagen) and a GST binding column (Qiagen), respectively, according to the manufacturer's instruction. Immunoblotting with anti-HIS and anti-GST antibodies will be carried out. Secondly, N. benthamiana and citrus cDNA libraries will be prepared using young leaves. The respective plasmids with the viral ORF and the cDNA libraries will be co-transformed into Saccharomyces cerevisiae strain Y2HGold. The initial Y2H screening will be performed on SD/-Leu/-Trp/-His/media using Cp1 or Cp2 as bait. Certain Las pathogenicity genes of interest will be individually tested against P16, Cp1 and Cp2 as bait. Then, the positive library clones will be identified via DNA sequencing and subsequent BLAST searches. These plasmids will be subsequently used to repeat the Y2H experiment to confirm interaction with the respective viral proteins.2.2.3. Expressions of selected genes will be verified by RT?qPCR using primers designed to the sequences of interest. We will also use RT?qPCR to extend the test to other citrus plants that are co-infected with Las and the virus and display enhanced HLB tolerance.2.3 Methods for Objective 32.3. We will screen more citrus varieties of scions and rootstocks, such as navel orange, mandarin, lemons, and trifoliate hybrids using graft-based inoculation of Las and NMV-M/CFL. Individual selections will be propagated via grafting on selected rootstocks either from seedlings or from cuttings. The inoculation will be either co-inoculated with Las and NMV-M/CFL simultaneously or first with Las and then with NMV-M/CFL. The HLB symptoms and suppression effects by NMV-M/CFL will be recorded periodically. The titers and their dynamics of Las and NMV-M/CFL will be quantitatively measured by multiplex RT-qPCR (Duan, newly developed method).2.3.2. The field trials of Valencia sweet orange and pumelo bud-sport lines are in their sixth years at the USHRL Picos Farm. Some of them are severely impacted and the other are much less damaged by HLB. These plants will be inoculated with NMV-M/CFL via multiple grafting or agroinfiltration inoculation of the NMV-M/CFL infectious clone upon APHIS approval. A total of 18 trees with 3 replicates of 6 trees for each citrus variety will be inoculated. The disease suppression, growth restoration, and the quantification of titers of Las and NMV-M/CFL will be carried out as described above.2.3.3. NMV-M/CFL-infected scion and rootstock plants will be subject to field trials at the USHRL Picos Research Farm using a randomized complete block design with 18 replicates per scion/rootstock combination. These trials will include 10 selected bud-sport scion lines and two rootstocks. We will score tree height, trunk diameters, and canopy volume, appearance, and density using standard procedures after six months and then annually. For disease evaluations, we will collect leaf samples every six months and conduct multiplex RT-qPCR to quantify Las and NMV-M/CFL titers. Meanwhile, we will document disease index.2.4 Methods for Objective 42.4.1. We have demonstrated that N. benthamiana allows NMV-M/CFL-GFP to replicate and assemble into virions with recombinant NMV-M/CFL and the mgfp-5 molecule (Figure 4). We will clone the full-length SAMP gene under the promotor of the coat protein, which is expected to produce the protein to a high level. GFP will be cloned in the same way as a control to monitor the level of expression and the systemic movement.We will evaluate the expression of the inserts in N. benthamiana plants both at RNA level using RT-qPCR and at protein level using Western blot analysis with antibodies against the SAMP peptide and GFP.. If successfully constructed, these expression constructs will be transferred to citrus plants to check their infectivity behavior again. After successful expression of the antimicrobial peptide or siRNA in citrus, we will evaluate their effects against Las or psyllids in a control greenhouse using graft-based inoculation.2.4.2. In addition to expressing proteins such as antimicrobial peptides and GFP, NMV-M/CFL will also be an excellent vector for expressing small interfering RNAs (siRNA). We will utilize the NMV-M/CFL vector to express hairpin RNAs that target negative regulators of citrus immune responses. The recombinant viral vectors will also be tested first in N. benthamiana to examine the efficiency of siRNA formation, and then will be introduced into citrus plants for HLB resistance testing.2.5 Methods for Objective 52.5.1. Effective communication and dissemination of project outcomes with stakeholders is key to the acceptance and adoption of potential HLB therapies. Team members of this project have strong relationships with growers and stakeholders in FL, CA, and TX and are in close communication with stakeholder groups. To engage growers and stakeholders, we will organize meetings in each state to directly inform them of project results and incorporate visits to field trials and greenhouse experiments.2.5.2. Results will also be communicated to stakeholders, researchers, and the public through multiple channels, including presentations at trade//stakeholder meetings, industry newsletters and magazines, scholarly journals and conferences, and social media.2.5.3. We will request an in-person investigatory meeting with the EPA and FDA. Along with a letter of request, we will provide an outline of our research, objectives, relevant data marked "confidential" and the proposed method(s) of application or use of the NMV-M/CFL for the agency's review.