Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 3110 - Insects | 1150 - Toxicology | 25% |
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 3110 - Insects | 1020 - Physiology | 20% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1040 - Molecular biology | 15% |
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 4010 - Bacteria | 1040 - Molecular biology | 15% |
| 212 - Pathogens and Nematodes Affecting Plants | 4030 - Viruses | 1040 - Molecular biology | 15% |
| 903 - Communication, Education, and Information Delivery | 999 - Citrus, general/other | 3030 - Information and communication | 10% |
The Asian citrus psyllid (ACP) is one of the most serious citrus pests in the U.S. and in citrus growing countries worldwide. In addition to feeding on the phloem of the citrus plant, ACP also transmits the pathogenic bacterium that causes citrus greening or huanglongbing, which has resulted in severe economic losses in global citriculture. Reduction of ACP populations is a first line of defense against the spread of this devastating disease. To this end, citrus growers have repeatedly applied chemical insecticides. Unfortunately, this strategy has resulted in the evolution of insecticide resistance in ACP and has also removed natural enemies that might otherwise help to keep ACP populations in check. Clearly, more sustainable control measures are needed as alternatives to the intensive use of chemical insecticides, to mitigate economic losses.The bacterium, Bacillus thuringiensis (Bt) produces pesticidal proteins that damage the insect gut epithelium allowing the bacterium to replicate within the host insect. These pesticidal proteins (in the absence of the bacterium) are used successfully for the suppression of several insect pests of major agricultural importance. The specificity of these proteins is a major advantage allowing for deployment in conjunction with biological control programs. A second and completely different strategy for insect pest control is the use of gene silencing. In this case, the production of proteins that are essential for the insect to survive, is reduced or stopped by silencing the gene. The use of both Bt pesticidal proteins and gene silencing RNAs in combination results in even greater efficacy than use of either approach alone.Having identified Bt-derived pesticidal proteins active against ACP and promising candidate silencing RNAs, the goal of this project is to identify the very best combination of Bt pesticidal proteins and silencing RNAs for the final product for grower use. The final product will be transgenic ACP resistant citrus or "trap plants". Trap plants, which are highly attractive to ACP can be planted around citrus groves, to attract and kill ACP before they reach the valuable citrus trees. Information about this novel, sustainable approach for management of ACP will be delivered through multiple sources to stakeholders to facilitate adoption in the longer term of by citrus growers.
The $11 billion citrus industry in the U.S. is threatened by the spread of a bacterial pathogen that causes citrus greening. This pathogen is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri, and management of ACP is a key line of defense against the spread of this devastating disease. As current measures are inadequate, our long-term goal is to enable an environmentally benign approach for citrus growers to control ACP within an integrated pest management framework.The overall goal of this application is to identify the optimal components for an innovative approach, the use of the bacterium Bacillus thuringiensis (Bt)-derived pesticidal proteins and ACP gene silencing in combination for generation of a highly effective product for grower use.To this end, the objectives of the research are:
On completion of this project we will be ideally positioned to produce transgenic citrus and/or "trap plants" expressing both Bt pesticidal protein and gene silencing RNAs for deployment to citrus growers. Outreach activities will facilitate public understanding and future grower adoption of Bt- and gene silencing-based ACP control technologies.
Objective 1: Increase efficacy of Bt pesticidal proteins for use against nymph and adult ACP. We will assess the specificity of ACP gut binding peptides, and then use selected peptides to modify Bt pesticidal proteins for increased gut binding. Optimal sites for insertion of peptides into the pesticidal protein will be determined. Binding affinity of the modified toxins will be assessed by pull down assay and microscale thermophoresis for determination of Kd values. Bioassays will be conducted using established procedures to assess toxicity against ACP, and Bt pesticidal protein-mediated damage to the gut epithelium examined by transmission electron microscopy.Objective 2: Identify the most effective dsRNA constructs against ACP We will test the effect of silencing selected genes implicated in psyllid physiology and feeding behavior on nymph mortality and adult survival. The impact of gene silencing on transcript abundance and psyllid survival will be assessed using both in vitro (topical feeding) and in planta (Citrus tristeza virus, CTV) delivery systems.Objective 3: Assess the impact of the combined action of dsRNA and Bt toxins We will test whether transgenic plant delivered Bt pesticidal proteins and CTV-delivered dsRNA in combination will result in more effective ACP control than use of either approach alone. The impact of CTV-delivered candidate silencing RNAs on pesticidal protein efficacy will be assessed by graft infection of CTV into pesticidal protein-expressing transgenic or control Valencia sweet orange. Nymph and adult ACP toxicity assays will be conducted and changes in ACP behavior (including settling, feeding) assessed.Objective 4. Identify the optimal plant promoter for pesticidal protein expression in the phloem We will compare two phloem specific promoters with the constitutive CaMV 35S promoter for expression of bacteria-derived pesticidal proteins in planta. Relative transcript and protein abundance will be determined. Importantly, adult ACP bioassays will indicate the impact of phloem expression relative to constitutive expression of these pesticidal proteins.Outreach: Information on Bt- and dsRNA-based technologies will be delivered to stakeholders, building on an existing web-based framework. Citrus grower-focused outreach activities will facilitate grower adoption of this multifaceted approach for ACP suppression.
Target Audience
The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry.The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry.
Changes / Problems
Nothing Reported
Training & Professional Development
Trainees during this reporting period were two undergraduates, one graduate student and two postdoctoral researchers. Trainees received mentorship from Bonning and Killiny including good scientific practice, presentation skills, critical assessment of the published literature. The graduate student and postdoctoral researchers presented during the quarterly research update meetings with all laboratories involved in the project, and during the twice yearly advisory board meetings with two industry mentors.
Dissemination Streams
Details of project progress have been relayed to stakeholders via five research manuscripts, multiple presentations at theSecond Congress of the International Society for Citrus Huanglongbing and phloem-colonizing bacterial pathosystems (ISCHPP), held in October 2022 in Florida, at the annual meeting of the Entomological Society of Americaheld in Vancouver in 2022, to citrus growers at the Citrus Expo held in Florida in 2023,and at the annual meeting of the Society for Invertebrate Pathology, held in Maryland in 2023.
Next Reporting Steps
Objective 1. Cry1Ba1 modified with GBP4 at three sites will be tested for toxicity. The screen for additional ACP-active pesticidal proteins will be completed with an additional 2 to 4 proteins to be tested. A combination of amino acid changes in Mpp51Aa1 will be tested for enhanced ACP toxicity, based on the results of the alanine mutagenesis. Objective 2. Five selected dsRNAs will be tested in combination with Mpp51Aa1 or Cry1Ba1 to evaluate the impact of the combined strategies on ACP mortality. Objective 3. Bioassays to assess the impacts of CTV-delivered ACP gene silencing RNAs in conjunction with pesticidal proteins in vitro and expressed by transgenic Valencia will be conducted. Objective 4. The amounts of pesticidal protein expressed in transgenic plants by different plant promoters will be determined. Bioassays on these plants will be continued to determine the optimal promoter for use in Valencia. Outreach Presentations on the use of bacterial pesticidal proteins in combination with ACP gene silencing RNAs are planned for the coming year. <br><br>
<br>What was accomplished under these goals? Objective 1. Increase efficacy of Bt proteins for use against ACP 1) Major activities completed / experiments conducted: Bioassays with Mpp51Aa1 modified with GBP4 were conducted. The screening of bacterial pesticidal proteins (BPP) <br><b>Publications</b><br>
Target Audience
The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry.
Changes / Problems
A no-cost extension has been approved to offset the six month delay in hiring of personnel for this project.
Training & Professional Development
During this reporting period, two undergraduate students, one graduate student and two postdoctoral researchers received training, professional development opportunities and mentorship from project investigators Bonning and Killiny. In addition to gaining experience with laboratory techniques, the graduate and postdoctoral trainees also had opportunity to present their research results during the quarterly update meetings. They also received feedback on biannual written reports provided to the advisory board. The undergraduate students learned about pesticidal protein expression and purification and conducted ACP bioassays.
Dissemination Streams
Information related to this project was relayed to a variety of stakeholders via an updated Project Snapshot on the Science for Citrus Health web site, an article in the Citrus Industry Magazine, two primary research manuscripts, and two presentations at the annual meeting of the American Phytopathological Society. A presentation was also given to Florida citrus growers at the 2022 Citrus and Specialty Crop Expo that included details of this project.
Next Reporting Steps
Objective 1. Peptide modified Mpp51Aa1 will be tested for enhanced toxicity against ACP. Cry1Ba1 will be peptide-modified at three sites and tested for toxicity. The screen for additional ACP-active pesticidal proteins will continue. Objective 2. The selected dsRNAs will be tested in combination with Mpp51Aa1 or Cry1Ba1 to evaluate the impact of the combined strategies on ACP mortality. Objective 3. Bioassays will be conducted to assess the impacts of one or more CTV-delivered ACP gene silencing RNAs alone or in conjunction with pesticidal proteins expressed by transgenic Valencia. Objective 4. Additional transgenic plants will be regenerated and micrografted. Lines with pesticidal protein expression confirmed will be evaluated in ACP bioassays. Outreach Multiple presentations are scheduled including at the 2022 Citrus Juice and Beverage Conference and Citrus & Specialty Crop Expo. <br><br>
<br>What was accomplished under these goals? Objective 1. Increase efficacy of Bt proteins for use against ACP 1) Major activities completed / experiments conducted: We identified ACP gut surface proteins bound by selected ACP gut binding peptides (GBP) 3 and 4. We used in silico analyses with reference to the published literature to identify sites within the pesticidal proteins Cry1Ba1 and Mpp51Aa1 for modification with GBP4. We repeated the phage display screen for isolation of ACP nymph GBP. Pesticidal proteins 50% mortality, seven resulting in 30-50% mortality, and eight with low mortality were identified. 4) Key outcomes or other accomplishments realized: Gene silencing of targets resulting in high mortality, and targets expected to synergize the action of pesticidal proteins were selected for testing in combination with Bt pesticidal proteins. Objective 3. Assess the combined impact of silencing RNAs and Bt proteins 1) Major activities completed / experiments conducted: Long term (90 day) bioassays were conducted to examine the impacts of transgenic plants expressing Mpp51Aa1 or CTV-delivered gene silencing RNAs on adults including fecundity and on nymph survival. Plants were infected with two CTV vectors delivering either gene silencing RNA or Mpp51Aa1. The stability of the vectors and protein expression was monitored in the mixed infection by RT-PCR and western blot respectively. Two new CTV vectors were constructed for delivery of single gene silencing RNAs and vector stability assessed. Four CTV producing silencing RNAs along with WT CTV and a negative control targeting GFP, were introduced into four transgenic Valencia lines expressing Cry1Ba1 to address the combined impacts of the two approaches. 2) Data collected: ACP mortality was monitored in bioassays. The stability of CTV vectors in planta was assessed by observation of RT-PCR product sizes in agarose gels. Mpp51Aa1 expression in planta was assessed via western blot. 3) Summary statistics and discussion of results: Both transgenic plants and CTV delivered gene silencing RNAs significantly impacted ACP populations in long term bioassays. The CTV vector expressing Mpp51Aa1 was outcompeted by the CTV vector producing the gene silencing RNA on coinfection of the plant. Stable CTV vectors were developed for expression of two new ACP gene silencing RNAs. 4) Key outcomes or other accomplishments realized: The impacts of Mpp51Aa1 or selected gene silencing RNAs on the next generation of ACP over the long term were characterized. Infection of transgenic Valencia expressing Cry1Ba1 with CTV vectors delivering gene silencing RNAs will allow for assessment of impacts of the combined bioactives against ACP. Objective 4. Identify the optimal plant promoter for Bt protein expression in the phloem 1) Major activities completed / experiments conducted: Plants were micrografted from each of the six treatments (three plant promoters, two pesticidal proteins) plus vector only control. Transcription levels were assessed for plants that survived acclimatization to the greenhouse from five of the treatments, and western blot conducted for detection of Cry1Ba1 for one. 2) Data collected: GUS selection was used for identification of transformed plants. Transcript levels were determined relative to wild type. 3) Summary statistics and discussion of results: A total of 4-23 micrografted plants were generated for each of the six plant constructs. Pesticidal protein transcription was confirmed in most cases. Cry1Ba1 expression was confirmed for all plants of one construct tested. 4) Key outcomes or other accomplishments realized: Confirmation of transcription and translation of pesticidal proteins by the transgenic plants tested to date will allow for comparison of three promoters for delivery of pesticidal proteins from two different structural classes. Outreach 1) Major activities completed / experiments conducted: An article describing the project has been published in Citrus Industry Magazine including challenges and opportunities associated with the technology. A new fact sheet / snapshot describing the project was generated for the Science for Citrus Health web site including potential deployment strategies such as the use of trap crops. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: Availability to growers of additional materials providing information on the project will enhance the likelihood for adoption in the long term. <br><br><b>Publications</b><br>
Target Audience
The target audience for this project is comprised of pest management researchers, and stakeholders associated with the citrus industry.
Changes / Problems
Nothing Reported
Training & Professional Development
During this reporting period, two undergraduate students, one graduate student and three postdoctoral researchers received training, professional development opportunities and mentorship from project investigators Bonning and Killiny. In addition to training in laboratory techniques, the graduate student and postdoctoral researchers received feedback on draft reports and presentations for quarterly research updates. The undergraduates received training in psyllid maintenance and psyllid bioassays.
Dissemination Streams
During this reporting period, information related to the project has been relayed to pest management stakeholders via a webinar, held in February 2021 and coordinated by Science for Citrus Health, a magazine article and presentation at the Florida Citrus Show.
Next Reporting Steps
Objective 1. An Mpp-type, Bt pesticidal protein and a three domain protein will be modified with the newly identified gut binding peptides and tested for enhanced toxicity in adult and nymph bioassays. Objective 2. Selected dsRNAs will be tested in combination with Bt pesticidal proteins to evaluate the impact of the combined strategies on ACP mortality. Objective 3. Bioassays will be conducted to assess the impacts of one or more CTV-delivered ACP gene silencing RNAs alone or in conjunction with Bt pesticidal proteins delivered either by CTV or by transgenic plants. Objective 4. GUS selection for screening of constructs will be continued. Early Valencia 1 and Hamlin will also be used for optimized transformation rate. Outreach Update Science for Citrus Health Snapshot as the project develops. Interview one of the team for a podcast. <br><br>
<br>What was accomplished under these goals? Objective 1. Increase efficacy of Bt proteins for use against ACP 1) Major activities completed / experiments conducted: Following screening of a phage display library for additional ACP gut binding peptides (GBP) for modification of pesticidal proteins, four peptide-mCherry fusions were expressed and fed to ACP. Dissected guts were examined by fluorescence microscopy to identify which peptide bound to the gut surface under in vivo conditions. UV-crosslinking was used to determine which ACP brush border membrane vesicle proteins were bound by selected peptides. Bioinformatics analysis was conducted to identify proteins predicted to be on the gut surface. Protocols for assessment of the toxicity of pesticidal proteins against ACP nymphs were optimized. 2) Data collected: Corrected fluorescence readings were determined for 5 to 10 guts from insects fed on each test or control treatment. The gut surface proteins bound by selected peptides were identified from LC-MS/MS data including SequestHT scores for each protein. ACP nymph survival was determined. 3) Summary statistics and discussion of results: The in vivo binding of ACP GBP3 and GBP4 was confirmed. The gut surface binding partners of GBP15 were identified. The new ACP nymph bioassay was optimized to allow for determination of LC50 values for Bt pesticidal proteins of interest. 4) Key outcomes or other accomplishments realized: New ACP gut binding peptides have been selected for use in modification of selected Bt pesticidal proteins. Development of a novel bioassay method for long term feeding of nymphs with pesticidal proteins will allow for assessment of the relative toxicity of test proteins against nymph and adult ACP. Objective 2. Identify the most effective silencing RNAs against ACP 1) Major activities completed / experiments conducted: A total of 17 ACP gene targets were identified for testing against ACP. dsRNAs were synthesized and delivered to ACP nymphs via topical feeding at doses ranging from 100 to 1000 ng/ul with dsRNA targeting GFP as negative control. Relative transcription levels of targeted genes were determined by RT-qPCR in test and control insects. 2) Data collected: Nymph mortality, adult survival and lifespan were recorded following test and control treatments. Ct values was used to assess the efficiency of transcript reduction in dsRNA-treated ACP.. 3) Summary statistics and discussion of results: The highest nymph mortality was observed at doses between 200 and 500 ng dsRNA. 4) Key outcomes or other accomplishments realized: Gene silencing in ACP provided for the functional characterization of a set of genes involved in ACP physiology and immunity. The collated information on mortality associated with targeting different genes by RNAi will be used for selection of gene candidates to test in conjunction with Bt pesticidal proteins. Objective 3. Assess the combined impact of silencing RNAs and Bt proteins 1) Major activities completed / experiments conducted: Citrus tristeza virus (CTV) vectors were constructed for delivery of two gene silencing RNAs, either alone or in combination. A CTV vector that produces antisense GFP sequence was used as a negative control. Budwood of five transgenic Valencia lines expressing a Bt pesticidal protein was increased for use in bioassays with CTV delivered silencing RNAs. RT-PCR was conducted to assess the stability of CTV RNAi vectors expressing gene silencing RNAs. Bioassays were set up with plants infected with CTV expressing a Bt pesticidal protein and ACP gene silencing RNAs. 2) Data collected: The stability of CTV vectors in planta was assessed by observation of RT-PCR product sizes in agarose gels. ACP population numbers were monitored overtime in CTV bioassays. 3) Summary statistics and discussion of results: Stable CTV vectors were developed for expression of multiple ACP gene silencing RNAs. A CTV vector for delivery of both silencing RNA and a Bt pesticidal protein is under construction. 4) Key outcomes or other accomplishments realized: Production of CTV vectors for delivery of gene silencing RNAs and / or the Bt pesticidal protein will allow for assessment of impacts of individual or combined bioactives against ACP. Objective 4. Identify the optimal plant promoter for Bt protein expression in the phloem 1) Major activities completed / experiments conducted: Valencia sweet orange callus and epicotyl explants were transformed with six Agrobacterium constructs for expression of Bt pesticidal proteins under the control of one of three promoters, and vector only control. For juvenile transformation, three co-incubation experiments with Valencia explants were done, and shoots tests for GUS activity. Mature Valencia stems were co-incubated with one expression vector. 2) Data collected: GUS selection was used for identification of transformed plants. 3) Summary statistics and discussion of results: Following juvenile transformation, 152 shoots were tested for GUS activity. A total of 1000 explants were co-cultured with Agrobacterium for mature transformation. None of the screens to date have resulted in GUS-positive plants. Screening is ongoing. 4) Key outcomes or other accomplishments realized: Similar to our prior experience with these pesticidal proteins, transformation rates are very low. The use of lines with greater transformation efficiency is under consideration. Outreach 1) Major activities completed / experiments conducted: The Science for Citrus Health Research Snapshot related to this project was translated into Spanish. A webinar featuring project PI Bonning was held February 24, 2021 after which a survey of attendees was conducted. 2) Data collected: The number of attendees and responses to specific survey questions was recorded. 3) Summary statistics and discussion of results: A total of 163 participants attended the webinar, including from 7 countries overseas. 97 (53%) responded to the survey. 4) Key outcomes or other accomplishments realized: Overall, the webinar was well received and considered relevant to pest management work by the majority of attendees. <br><br><b>Publications</b><br>