Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 3110 - Insects | 1060 - Biology (whole systems) | 50% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 25% |
| 215 - Biological Control of Pests Affecting Plants | 4030 - Viruses | 1040 - Molecular biology | 25% |
Citrus greening or Huanglongbing (HLB) disease is associated with plant infection by several species of phloem-limited bacteria designated "Candidatus Liberibacter"."C. Liberibacter asiaticus" (CLas) is currently the only species associated with U. S. HLB.This bacterium is transmitted by Diaphorina citri (Asian citrus psyllid).Delivery of molecules that modulate CLas transmission by D. citri has been possible since 2014, with the applications of citrus tristeza virus (CTV) for virus induced gene silencing (VIGS) in D. citri, but challenges remain at the forefront of this research. The citrus industry desperately needs additional strategies to block transmission by D. citri and modulate D. citri gene expression from the research community, in particular the identification of psyllid genes that regulate CLas transmission and strategies to deliver molecular cargo into citrus for HLB mitigation, such as molecules that can block CLas transmission. In this project, we will be working on two different molecular delivery strategies that can be used to modulate D. citri gene expression, alone or in combination. One strategy involves a tiny plant virus-like RNA called citrus yellow vein associated virus (CYVaV). The second strategy is a novel plant SymbiontTM, developed to deliver molecules such as antimicrobial peptides, to citrus trees. Research will focus on the possible synergy between these two delivery strategies. Research will also use genomics methods to identify psyllid genes that regulate CLas transmission.
Solutions to solving citrus green disease (HLB) is perhaps the most pressing need in U.S. agriculture.The HLB disease complex, the Asian Citrus Psyllid (ACP) and 'Candidatus Liberibacter asiaticus' (CLas) bacterium, has devastated Florida citrus production.The societal impact is massive as thousands of people have lost their jobs, farms and processors have gone bankrupt, and consumer industries inter-connected to citrus industry workers have also been negatively economically impacted. The causative microbe and its insect vector, CLas and ACP, are now found in California and Texas citrus trees.Growers have no options to block transmission of CLas by ACP, and tools are desperately needed in this area.Furthermore, transmission blocking tools must be tailored to the unique needs of the citrus growing states and the needs of juice, fresh fruit, and organic growers. Solutions must include protecting existing and newly planted trees, as well as delivery in an economical format suitable to each industry sector.Our target is ACP-CLas interactions.As an insect-vector borne pathogen, methods to block transmission represent a dead end for the bacteria and a novel solution for huanglongbing in all areas where the insect vector is found. The identification of psyllid genes that block CLas transmission has been a major focus of my laboratory (and others) for the past 8 years. Moreover, my lab haspublished studies that show some ACP transmit CLas very efficiently and others not at all. We showed that the ability or inability to transmit the pathogen is passed on from parents to offspring for several generations. The chromosomal length assembly of the psyllid genome, a novel method to directly sequence CLas from ACP, and a streamlined bioassay to measure CLas acquisition by psyllids are recent advances that make this project possible.Delivery of molecular signals that modulate CLas transmission by ACP is also now possible.Drs. Heck, Shatters and Simon are working on two different molecular delivery strategies that can be used to modulate ACP gene expression, alone or in combination. Dr. Simon discovered a tiny plant virus called Citrus yellow vein associated virus (CYVaV). Her team developed this virus into a stable infectious clone that delivers silencing signals to the plant phloem under a grant funded by the ECDRE program. Drs. Heck and Shatters developed a novel plant SymbiontTMto deliver molecules, such as peptides and silencing signals into the tree. Commercialization of these strategies is underway by both teams. It is unknown whether SymbiontsTMcan deliver viruses such as CVYaV into citrus, and that will be a focus of this work.This base of knowledge and the existing framework of our collaborations provide a means for us to translate these innovative findings into tools that growers can use for huanglongbing management. The four major research questions that will be addressed in this grant proposal include:
Objective 1.Use CYVaV to target 100 psyllid genes for silencing.To successfully target mRNAs within the psyllid, small interfering RNAs (siRNAs) must be developed that are capable of hybridizing well to the mRNA.These will be identified using a co-infiltration assay, where CYVaV carrying specific inserts complementary to regions throughout the ACP mRNA in question is syringe co-infiltrated intoNicotiana benthamianaalong with an expression cassette that transcribes the mRNA.Quantitative PCR will be used to quantify the mRNA after 5 days.Inserts are simultaneously tested for stability by vacuum infiltration ofN. benthamianaand CYVaV+insert examined for stability by first PCR and the batch sequencing over the span of 120 days.The top inserts will be tested in citrus for their ability to block CLas transmission.Objective 2.Can different CYVaV VIGS vectors be stacked in trees?If this is possible, then an entire CYVaV vector can be devoted to inserts that target the psyllid, which would be compatible with the vector being developed by the Simon lab and Silvec containing inserts that target CLas, host gene expression, and the putative helper virus, Citrus vein enation virus.To test if different CYVaV VIGS vectors can be stacked, we will (i) co-infiltrate CYVaV vectors with similar sized inserts in similar places; and (ii) infiltrate one vector, and then 30 days late graft in the second vector (and then switch the order).Q-PCR with insert-containing primers will be used to quantitate the levels of the two vectors over a period of 90 days.Objective 3. Can CYVaV vectors be expressed using SymbiontsTM?Drs. Heck and Shatters developed a novel technology, called SymbiontsTM, to deliver genetically encoded molecules into the tree. Small clusters of autonomously growing plant cells that are made by expressing plant growth regulator genes fromAgrobacterium tumefasciensare inoculated onto the side of a tree and express any gene of interest. We will develop SymbiontTMvectors to express CVYaV and monitor whether CYVaV can be systemically delivered to citrus from the site of SymbiontTMattachment. If SymbiontTMdelivery works, we will also test SymbiontsTMfor CYVaV stacking. The symbiont containing one will be attached to a citrus tree that the Simon lab/Silvec has already infected with the other and both CYVaV will be followed for the length of the grant.SymbiontTMtechnology was developed in partnership with AgroSource, Inc. This objective will involve developing a new partnership between AgroSource and Silvec Biologics, the company commercializing CYVaV technology.Objective 4. Identify additional psyllid genes involved in CLas transmission using genome wide association studies. CLas is uncultivable and studies to identify genes involved in CLas transmission by ACP rely on using insects reared on CLas infected trees and comparing their organ and tissue-specific gene expression to those reared on healthy trees. Our research has shown that CLas infected trees change the expression of thousands of ACP genes, so identifying the few with a direct role in regulating CLas transmission is a major challenge. A more direct way to find ACP genes involved in transmission is to leverage the natural variation that exists in ACP for the ability to transmit CLas. The genes involved in CLas transmission can be discovered by comparing the genomes of ACP that transmit well (referred to as vectors) to the genomes of ACP that do not transmit (referred to as non-vectors).We will use high throughput genome sequencing technologies to quantify CLas titer in ACPvector and non-vector insects. Dr. Buckler is an expert in genome wide association studies in plants, and he will guide research to use genome wide association studies to identify ACP genes that regulate CLas transmission.The function of a subset of genes will be tested using RNA silencing by CYVaV vectors.
Target Audience
The target audience for this research includes stakeholders in the citrus industry, including growers, processors, nursery owners, packing houses, grower organizations, and Silvec Biologics, the partnering biotech start-up company who will advance this research through commercialization. DOE ORISE and high school students in the TST Boces New Visions Life Sciences Program also benefitted from the research. The general public is a also target audience of the research.
Changes / Problems
Nothing Reported
Training & Professional Development
Training: 1. A PhD student completed her degree at Cornell University. 2. An ORISE scholar gained training in computational biology and is applying to graduate school to advance their career. 3. A postdoctoral fellow gained experience in vector entomology, RNAi and project management 4. A new collaboration was establiished with Dr. Osgur Batuman, at the University of Florida, to expand the genomic analysis to include insect infecting viruses.
Dissemination Streams
Results have been reported in peer-reviewed publications, university seminars, at grower meetings, scientific conferences and other industry events.
Next Reporting Steps
The GWAS analysis will be completed this year. Manuscripts are in preparation describing the CYVaV symbiont work, RNAi, the haplotyping research and the GWAS analysis. We are in a discussion with the company about advancing the Silvec-USDA partnership to advance products towards commercialization help citrus growers manage citrus greening disease.
Target Audience
The target audience of our effort includes the general public,citrus growers, private industry partners,plant pathologists, and university partners.
Changes / Problems
A change for next year will be the addition of a sub-award with Cornell University. A request for this sub-award is in process. The work is all going according to plan and no major changes or problems in the approach are noted.
Training & Professional Development
The Heck Lab is situated on the campus of Cornell University, in Ithaca, NY. Dr. Heck has been partnering with Cornell professor, Dr. Marc Fuchs, to provide training to postdocs and graduate students conducting research on the project. A new sub-award will be developed in the next FY to solidify these training opportunities as a part of the project. Paperwork for that is underway with NIFA, Cornell and USDA ARS.Graduate and postbac students have already been trained in computational biologyapproaches. A postdoc on the project has been trained in molecular biology and molecular virology. Trainees on the project are preparing papers for submission to peer-reviewed journals. They participate in weekly lab meetings and are expected to write written weekly reports detailing their research projects and career development goals. A postdoc on the project is interested in writing an independent fellowship and will get mentorship on writing a research proposal.
Dissemination Streams
The team is developing a project brief for the Science in Citrus Health website. That is in progress and expected to be published in the summer of 2023. This website is geared towards growers and other interested members of the public. Dr. Heck and her trainees have plans to present this research at a variety of conferences this year, including the 2023 Entomological Society of America meeting, the 2023 International Society for Plant Pathology meeting in Lyon, France and the 2023 American Phytopathological Society meeting in Denver.
Next Reporting Steps
In the next reporting period, research will proceed as outlined in the proposal. The biggest milestone weexpect to complete is the construction of a Symbiont vector expressing CYVaV and evaluating Symbiont delivery of CYVaV. This aspect of the research will have aheavy focus on plant molecular biology and plant virology. <br><br>
<br>What was accomplished under these goals? A major thrust of research was developing gene silencing tools to block CLas transmission.Considering the project timeline, we did a literature search for RNAi targets inD. citri, knockdown of which either had a negative effect onD. citriand/or affected the insect's ability to acquire CLas. Since we are going to insert siRNAs into CYVaV, target genes that were knocked down by small RNAs (siRNA/ miRNA) in previous studies were particularly of interest. Based on this and our coooperator Silvec's experience of developing CYVaV constructs targeting psyllidgenes,V-ATPase subunit E(V-ATPase E) was selected as one of our targets.Inhibitor of apoptosiswas also chosen because silencing it by feeding led to high mortality within five days anddelivery via feeding is closer to what we plan to deliver siRNAs.Arginine kinasewas chosen as our target for the same reason. For the novelty of our project, we chose to target a highly abundant peptide inD. citriwhich we found using a peptidomics method developed in the Heck Laboratory (tentatively named DcLpeptide).In addition to its abundance, the peptide has striking structural similarities to neuropeptides (secretion signal, small sequence repeats flanked by dibasic cleavage sites, C-terminal amidation), suggesting that this peptide may play an important role inD. citriphysiology. Therefore, by targeting it using RNAi we would not only evaluate its potential as a target but unravel its role inD. citri. Work is in progress to make 10 CYVaV constructs per target gene. Research is also ongoing to develop a CLasacquisition and transmission assay that allows us to quantify the impact of gene silencing in nymphs on CLas acquisition and transmission. The method is expected to be broadly useful to the citrus greening research community. A paper is in preparation describing this method. To test whether CYVaV vectors can be stacked in trees, we are using CYVaV vectorswith different-sized inserts.V1 (with 61nt insert) and V2 (with 198nt insert), were infiltrated intoN. benthamianaat the same time. Four weeks after infiltration, symptomatic leaves were collected for the presence of CYVaV vectors. Among the 12 tested plants, sevenshowed exclusive infection of V1 vector, oneshowed exclusive infection of V2 vector, and fourwere infected with both vectors. Interestingly, a V1/V2 recombination vector was also detected in the dual infection. These interesting data show that CYVaV vectors can be stacked in trees but that recombination between different viral vectorscan occur, which may limit commercial deployment of a stacked system until further research is done to minimize recombination events.We have also infiltrated CYVaV vectors with similar-sized inserts as well. These experiment arestill ongoing. To test whether CYVaV can be delivered using Symbiont technology, intellectual property agreements were developed between the USDA ARS and Silvec Biologics. This took some time, but we were successful in developing an MTRA that allowed the research to proceed while protecting the intellectual property of both parties involved in the agreement. The team hasdesigned Symbiont-expressingCYVaV constructs. The construction of theconstruct is ongoing. Dr. Simon and her team at UMD is funded on another NIFA grant to evaluate siRNAs expressed by CYVaV that target the CLas bacterium. Research on this project is also helping to support this objective. Additionally, Dr. Simon has made some interesting observations about CYVaV infection of citrus that lead us to hypothesize the virus may help to alleviate foliar symptoms of HLB.We obtained APHIS PPQ permits to recieve CYVaV-infected citrus and will infect these trees using insect inoculations and monitor for disease progression. APHIS BRS permits are in progress to recieve the genetically modified CYVaV-infected material. To determine whether natural variation in ACP genes contributes to CLas transmission, we sequenced over 500 ACP collected from four grove sites in Florida at 7x depth and phenotyped each insect for CLas titer using qPCR. Genome wide association analysis is ongoing in collaboration with the Buckler lab at the USDA ARS in Ithaca, NY.Briefly, all reads from the individual samples were mapped to the Diaphorina citri v. 3 genome. Mean read depth plots and SNP depth plots were produced to allow fro informed SNP filtering and inputation and direct comparison of the Manhattan plots to regions of dense read depth or SNP abundance. An association model was developed that accounted for field sample site, relatedness, and other possible confounding variables. The Manhattan plot reveals a few key peaks that represent SNPs signficantly associaetd with CLas acquisition. The genomic regions under these peakswill be further analyzed using linkage disequilibrium and gene ontology analysis to identify psyllid genes that may be candidates for regulating CLas acquisition and transmission. <br><br><b>Publications</b><br>