Progress 09/01/23 to 08/31/24
Outputs
(N/A)
Impacts
What was accomplished under these goals?
Objective 1. Increase efficacy of Bt proteins for use against ACP 1) Major activities completed / experiments conducted: The screening of bacterial pesticidal proteins (BPP) <55 kDa in size in bioassays for ACP toxicity was continued. Constructs were made with Mpp51Aa1 modified at two additional sites with GBP4 and at 6 sites with GBP6. Psyllid gut binding was confirmed for both of these peptides. Tpp78Aa1 was modified at 5 sites with GBP6. Eight Mpp51Aa1 constructs made by combining mutation sites were developed. All modified pesticidal proteins were expressed. A new "sandwich" feeding assay was developed to assess the toxicity of pesticidal proteins against first instar nymphs. Single dose bioassays were conducted to test the toxicity of 6 BPP against first instar ACP. LC50 values were determined using this assay for BPP toxic to psyllids at a single dose. 2) Data collected: ACP survival was determined in bioassays with BPP. 3) Summary statistics and discussion of results: A new BPP with ACP toxicity, Tpp78Aa1 was identified. A new sandwich feeding bioassay developed for toxicity testing with first instar nymphs showed that first instars are significantly more susceptible to Mpp51Aa1, Cry1Ba1 and Tpp78Aa1. 4) Key outcomes or other accomplishments realized: The increased susceptibility of first instar relative to adult ACP demonstrated through use of the new sandwich feeding assay supports our earlier results showing the almost complete loss of psyllid progeny on citrus plants bioengineered to express BPP. Objective 2. Identify the most effective silencing RNAs against ACP 1) Major activities completed / experiments conducted: Combinations of gene silencing RNAs were tested against ACP by topical feeding and on CTV delivery in plants. 2) Data collected: Psyllid survival was recorded for topical feeding assays; survival, numbers of eggs and egg hatch, and adult emergence were recorded for long term bioassays with CTV delivery of gene silencing RNAs. 3) Summary statistics and discussion of results: A total of 7 out of 29 gene silencing RNAs tested during the course of this project are considered to result in high psyllid mortality, 11 resulted in moderate mortality and 11 in low mortality. CTV delivery of gene silencing RNAs resulted in reduced nymph and adult ACP survival, decreased number of eggs, egg hatch and adult emergence. 4) Key outcomes or other accomplishments realized: The screen of gene silencing RNAs has been completed, and those resulting in high psyllid mortality identified. Objective 3. Assess the combined impact of silencing RNAs and Bt proteins 1) Major activities completed / experiments conducted: Selected dsRNAs were used in combination with BPP at LC50 doses in topical feeding assays to assess the combined impacts on ACP. CTV was engineered for expression of Mpp51Aa1. Valencia plants bioengineered to express Cry1Ba1 were infected with CTV expressing gene silencing RNAs. The impact on ACP progeny adult emergence was assessed. 2) Data collected: Psyllid survival was recorded for topical feeding assays and on CTV infected Cry1Ba1 Valencia. 3) Summary statistics and discussion of results: The combination of gene silencing RNAs and BPP resulted in greater psyllid mortality than use of the individual components, with up to 100% mortality achieved in topical feeding assays. Presence of CTV-delivered gene silencing RNAs in Cry1Ba1 Valencia plants decreased adult psyllid emergence in long term bioassays relative to Cry1Ba1-expressing Valencia plants alone. 4) Key outcomes or other accomplishments realized: The combined use of gene silencing RNAs with BPP under in vitro or in planta conditions resulted in increased psyllid mortality relative to use of either of the individual components alone. Objective 4. Identify the optimal plant promoter for Bt protein expression in the phloem 1) Major activities completed / experiments conducted: Plants from each of the six treatments (three promoters, two pesticidal proteins) plus vector only control were maintained. Western blot analysis was used to confirm protein expression to assess the relative expression levels of each promoter. Whole plant bioassays were conducted to assess the impacts of transgenic plants on ACP survival 2) Data collected: Total eggs laid, egg hatch, survival of nymphs and adult emergence were recorded for up to 90 days. 3) Summary statistics and discussion of results: BPP expression was highest for the 35S promoter which drives expression throughout the plant relative to AtSuc2 and CsPP2, which are both phloem-specific promoters. However, the survival of progeny psyllids approached zero for BPP expressed by all three promoters. Egg laying and egg hatch were not impacted irrespective of BPP used or promoter used to drive expression. Significant drops of up to 100% were seen in the numbers of progeny psyllids that attained adulthood with most mortality occurring during the first and second instars. The combination of two BPP expressed had the greatest impact on progeny psyllids. 4) Key outcomes or other accomplishments realized: Significant results were obtained for all three plant promoters indicating that other considerations should be considered for identification of the "optimal" promoter. For example, the use of a phloem-specific promoter would reduce risk to non-target organisms that ingest other parts of the plant.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Tavares, C.S., Stelinski, L.L., Bonning, B.C. 2024. The sandwich feeding assay for use with first instar nymphs of the Asian citrus psyllid, Diaphorina citri confirms the highest susceptibility of this life stage to bacterial pesticidal proteins. Journal of Invertebrate Pathology 207: 108208 doi: 10.1016/j.jip.2024.108208
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Tavares, C.S., Mishra, R., Kishk, A., Wang, X., Ghobrial, P.N., Killiny, N., Bonning, B.C. 2024. The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium. Journal of Invertebrate Pathology 204:108122. doi: 10.1016/j.jip.2024.108122
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
B.C. Bonning, invited seminar. Gut binding peptides to enhance bacterial pesticidal proteins and inform resistance mechanisms. Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico. August 19, 2024
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Stelinski, L.L. 2024. Integrating antimicrobials into a broader management plan for ACP and HLB. Citrus Expo. 21-22 August, 2024. Florida State Fairgrounds, Tampa, FL.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Killiny, N. RNA interference in Asian citrus psyllid, from basic research to application. Entomological Society of America Annual Conference. National Harbor, MD, November 8th, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Kishk, A., Mishra, R., Tavares, C. S., Bonning, B. C. and Killiny, N. The toxicity of Bacillus thuringiensis bio-insecticidal proteins, Mpp51Aa1 and Cry1Ba1, against the healthy and Candidatus Liberibacter asiaticus-infected Diaphorina citri. Entomological Society of America Annual Conference. National Harbor, MD, November 11th, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rashidi, M. and Killiny, N. Identification of arylphorin in Diaphorina citri and determination of its role in response to Bt-bioinsecticidal proteins. Entomological Society of America Annual Conference. National Harbor, MD, November 5th, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Choaa El Mohtar, Cecile J. Robertson, Turksen Shilts, Carmen T. Bierman, Maria J. Robinson. Using Citrus tristeza virus (CTV) to limit ACP survival and reproduction on citrus. IRCHLB Riverside CA, March 26-29, 2024 Journal of Citrus Pathology. O3-B-6 https://escholarship.org/uc/item/33r648r0
|
Progress 09/01/22 to 08/31/23
Outputs
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
What opportunities for training and professional development has the project provided?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. How have the results been disseminated to communities of interest?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. What do you plan to do during the next reporting period to accomplish the goals?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.
Impacts
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) <55 kDa in size in bioassays for ACP toxicity was continued. A total of 20 alanine mutants of Mpp51Aa1 were generated and tested for toxicity in bioassays as an additional approach to improving toxicity of this pesticidal protein. Conditions for expression and purification of Cry1Ba1 modified with GBP4 were optimized. The in vivo gut binding of peptides identified from screening a phage library in ACP nymphs was assessed using fluorescence microscopy. 2) Data collected: ACP survival was determined in toxicity bioassays for GBP4-modified Mpp51Aa1 and for alanine mutants of Mpp51Aa1. Fluorescence intensity was recorded for nymph gut binding peptide-mCherry fusion proteins. 3) Summary statistics and discussion of results: GBP4 modification of Mpp51Aa1 did not result in increased toxicity against ACP. Sites within Mpp51Aa1 that resulted in increased or decreased Mpp51Aa1 toxicity to ACP on mutagenesis were identified. Only one of three GBP4 modified Cry1Ba1 proteins expressed well. The gut binding of two nymph gut binding peptides has been confirmed. One out of nine BPP screened resulted in ACP toxicity. 4) Key outcomes or other accomplishments realized: Alanine mutagenesis revealed key sites within Mpp51Aa1 for ACP gut binding that will facilitate modification for increased ACP toxicity. Objective 2. Identify the most effective silencing RNAs against ACP 1) Major activities completed / experiments conducted: The impact of CTV delivery of selected ACP gene silencing RNAs was assessed in planta. 2) Data collected: The number of progeny adult psyllids following introduction of parent psyllids on to CTV-infected plants was determined. 3) Summary statistics and discussion of results: Variation in adult ACP emergence was noted across replicate plants for both test and control plants (healthy, CTV infected, CTV-GFP). However, several promising CTV constructs expressing gene silencing RNAs that reduced emergence of progeny adults were identified. 4) Key outcomes or other accomplishments realized: Effective ACP gene silencing RNAs delivered using the CTV vector have been identified. Objective 3. Assess the combined impact of silencing RNAs and Bt proteins 1) Major activities completed / experiments conducted: The impact of CTV infection of transgenic Cry1Ba1-expressing plants on ACP progeny adult emergence was assessed. Long term (90 day) bioassays were conducted to examine the impacts of transgenic plants expressing Cry1Ba1 on adults including fecundity and on nymph survival. Two CTV vectors without gene silencing RNAs were introduced into transgenic Valencia lines expressing Cry1Ba1 to address the impact of CTV infection of transgenic plants on ACP. 2) Data collected: ACP mortality was monitored in bioassays. 3) Summary statistics and discussion of results: In preliminary assays, CTV infection (without gene silencing RNAs) of Cry1Ba1 transgenic Valencia resulted in reduced ACP adult emergence relative to controls. 4) Key outcomes or other accomplishments realized: Transgenic plant infection with CTV may reduce ACP survival even without gene silencing RNAs, but research into this question is ongoing. Objective 4. Identify the optimal plant promoter for Bt protein expression in the phloem 1) Major activities completed / experiments conducted: Plants from each of the six treatments (three promoters, two pesticidal proteins) plus vector only control were propagated. Additional plants were generated for expression of both pesticidal proteins in the same plant for comparative purposes. Transcription levels were assessed for all plants that survived acclimatization to the greenhouse from five of the treatments. Bioassays were conducted to assess the impacts of transgenic plants on ACP survival 2) Data collected: Pesticidal protein transcript levels were determined relative to WT. ACP survival on transgenic plants was assessed. 3) Summary statistics and discussion of results: Pesticidal protein transcription was confirmed in most cases. While transgenic expression of Mpp51Aa1 did not impact the survival of parent ACP, the emergence of progeny adults was significantly impacted in 60% of plants tested to date. 4) Key outcomes or other accomplishments realized: Generation of multiple sets of transgenic plants expressing one or both pesticidal proteins under the control of different plant promoters for continued assessment in bioassays. Outreach 1) Major activities completed / experiments conducted: Presentations were given at the Citrus Expo meeting at which emphasis was placed on the use of transgenic technology for management of citrus greening disease. An informational flier was generated about the Science for Citrus Health website to increase grower traffic to this site. 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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kishk, A., Tavares, C., Mishra, R. Bonning, B.C., Killiny, N. Influence of Candidatus Liberibacter asiaticus infection on the susceptibility of Asian citrus psyllid, Diaphorina citri to Bacillus thuringiensis bioinsecticidal proteins, Mpp51Aa1 and Cry1Ba1. J. Invertebr. Pathol. doi: 10.1016/j.jip.2023.107972
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Mishra, R., Narayana, R., Ibanez-Carrasco, F., Achor, D., Shilts, T., El-Mohtar, C., Orbovi?, V., Stelinski, L., Bonning, B.C. 2023. Bacterial pesticidal protein Mpp51Aa1 delivered via transgenic citrus severely impacts the fecundity of Asian citrus psyllid, Diaphorina citri. Applied and Environmental Microbiology doi: 10.1128/aem.00723-23
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Blackburn, M.B., Sparks, M.E., Mishra, R., Gundersen-Rindal, D.E., Bonning, B.C. 2023. Genomic sequencing of fourteen Bacillus thuringiensis isolates: insights into geographic variation and phylogenetic implications. BMC Research Notes 16(1):134. doi: 10.1186/s13104-023-06411-1
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Orbovic, V., Ravanfar, S.A., Achor, D., Shilts, T., Ibanez-Carrasco, F., Banerjee, R., El-Mohtar, C., Stelinski, L., Bonning, B.C. 2023. Cry1Ba1-mediated toxicity of transgenic Bergera koenigii and Citrus sinensis to the Asian citrus psyllid Diaphorina citri. Frontiers in Insect Science vol 3: doi: 10.3389/finsc.2023.1125987
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Tavares, C.S. , Bonning, B.C. 2022. Mpp51Aa1 toxicity to Diaphorina citri nymphs demonstrated using a new, long-term bioassay method. Journal of Invertebrate Pathology, 1 95:107845. doi: 10.1016/j.jip.2022.107845
|
Progress 09/01/21 to 08/31/22
Outputs
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. What opportunities for training and professional development has the project provided?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. How have the results been disseminated to communities of interest?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. What do you plan to do during the next reporting period to accomplish the goals?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.
Impacts
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 <55 kDa in size were screened in bioassays for ACP toxicity. 2) Data collected: The gut surface proteins bound by GBP3 and 4 were identified from LC-MS/MS data including SequestHT scores for each protein. ACP survival was determined in toxicity bioassays. 3) Summary statistics and discussion of results: The gut surface binding partners of GBP3 and 4 were identified. Ten GBP4-modified Mpp51Aa1 were produced for testing. Nine nymph GBP were isolated. Two new pesticidal proteins with ACP toxicity were identified. Two others resulted in feeding deterrence. 4) Key outcomes or other accomplishments realized: The suite of peptide-modified Mpp51Aa1 will allow for assessment of toxicity relative to wt Mpp51Aa1, and identification of optimal sites within the protein for modification. Selected nymph GBP can then be used for modification at these optimal sites. Objective 2. Identify the most effective silencing RNAs against ACP 1) Major activities completed / experiments conducted: A cumulative total of 27 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/µl with dsRNA targeting GFP as negative control. 2) Data collected: Nymph mortality was recorded. 3) Summary statistics and discussion of results: Six gene targets resulting in >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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ravanfar, S.A., Achor, D.S., Killiny, N., Shilts, T., Chen, Y., El-Mohtar, C., Stelinski, L.L., Bonning, B.C., Orbovi?, V. 2022. Genetic modification of Bergera koenigii for expression of the bacterial pesticidal protein Cry1Ba1. Frontiers in Plant Science 13:899624. doi: 10.3389/fpls.2022.899624
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Rashidi, M., Killiny, N. 2022. In silico characterization and gene expression analysis of Toll signaling pathway-related genes in Diaphorina citri. Insects-MDPI
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Bonning, B.C. Stelinski, L.L. 2022. A two-pronged approach to suppress psyllids. Citrus Industry Magazine. 103(8): 10-12. August 15, 2022.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Bonning, B.C., Stelinski, L.L., Lemaux, P.G. 2022. Two-pronged approach to suppress the Asian citrus psyllid vector of HLB. https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/Psyllid_Management/Two-Pronged_Approach_to_Suppress_the_Asian_Citrus_Psyllid_Vector_of_HLB/ Updated August 22, 2022
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Killiny, N. 2022. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease. Annual meeting of the American Phytopathological Society, Plant Health 2022, held August 5-10, Pittsburgh, PA, USA. (Oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
El Mohtar C., Shilts, T., Robertson, C., Dawson, W.O. 2022. Mapping Effect of Insertion Position and Length of Truncated RNA on Citrus Tristeza Virus Induced Gene Silencing (CTV-IGS). Annual meeting of the American Phytopathological Society, Plant Health 2022, held August 5-10, Pittsburgh, PA, USA. (Poster presentation)
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Progress 09/01/20 to 08/31/21
Outputs
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
What opportunities for training and professional development has the project provided?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. How have the results been disseminated to communities of interest?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. What do you plan to do during the next reporting period to accomplish the goals?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.
Impacts
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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mishra, R., Guo, Y., Kumar, P., Canton, P.E., dos Santos, C., Banerjee, R., Kuwar, S., Bonning, B.C. 2021. Streamlined phage display protocols for identification of insect gut binding peptides highlight peptide specificity, Curr. Res. Insect Sci. 1: 100012 https://doi.org/10.1016/j.cris.2021.100012
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Sankovitz, M., Alonso, B. Rivera M, Stelinski L., Garc�a-Figuera S., Lemaux P.G., Grafton-Cardwell B. HLB Research featured on website: Science for Citrus Health. https://citrusindustry.net/2020/10/06/hlb-research-featured-on-website/ (Article in Citrus Industry News magazine about the website)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Webinar hosted by Science for Citrus Health, 2-24-2021 "Emerging Technologies to Manage Asian Citrus Psyllid and HLB".
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Nabil Killiny, 2021, The power and promise of RNA interference in controlling Asian citrus psyllid. Presentation at Florida Citrus Show, May 12-13, 2021. Havert L. Fenn Center, Ft. Pierce, Florida.
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