Progress 01/15/18 to 01/14/24
Outputs
Target Audience:The target audience focus was directed toward citrus producers and other stakeholders associated with the citrus industry, field/crop managers, extension specialists/agents, departments of agriculture, researchers at universities and in industry, post-docs and graduate students attending conferences and workshops in the fields of entomology, plant pathology, virus-vector biology, plant protection, insect ecology, agronomy, biopesticide use or development, and/or molecular diagnostics use or development. Results were disseminated through presentations at professional meetings, abstracts and proceedings, and publications. Changes/Problems:The ACP biological system is difficult to work with and fraught with problems associated with the biology of the vector and of the pathogen-citrus host pathosystems. Much more time than anticipated was required to establish effective ACP feeding systems for nymphs and adults. The large number of PoP genes screened (~200 targets) required enormous coordination within and between the collaborating labs, and the teams rose to the occasion despite hardships and the inability to purchase molecular and disposable reagents and supplies during and even after the pandemic. Hurricane damage to greenhouses/colonies in Florida during the project resulted in major set-backs and lost experiments. The CoVID pandemic disrupted work in both labs and after post-docs left the AZ lab to take jobs, it was difficult to recruit new young scientists to complete the work left underway. One additional no-cost extension was requested but denied, despite the extraordinary circumstances. The PI and co-PI and authors of the publications, accepted/in press (with publications fees to pay), and those to be submitted are committed to completing, submitting, and publishing during 2024. What opportunities for training and professional development has the project provided?At least ten post-doctoral associates and two graduate students were trained during this project in Arizona and Florida. Technicians and technical staff in the Brown and Qureshi labs learned new techniques, methodologies, and skills. Our collaborators lab staff were also exposed to dsRNA technology for biopesticide development. All of the post-docs and graduate students were placed in positions at universities, in industry, and/or went on for a Ph.D. after completing a M.S. degree. One technician was hired by industry. How have the results been disseminated to communities of interest?The results have been reported at conferences involving the citrus industry and other industry stakeholders and at professional meetings including profession plant pathology and entomology societies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Laser experiments were completed to achieve foliar delivery of dsRNA. Localization of dsRNAs (100, 300, 600 bp) with or without adjuvants to tomato plant vasculature was corroborated by fluorescence confocal microscopy and knockdown (quantitative PCR) in potato psyllid (PoP) by ingestion-access of dsRNA from LASER-dsRNA treated plants. Two 'best' adjuvant/surfactant combinations resulting in cuticle disruption and dsRNA penetration were identified among six evaluated (Thakre et al., 2023). The optimal doses of dsRNA for ACP nymphs and adults were 100 and 200 ng/μL, respectively (Saberi et al., 2023). This is somewhat higher than for potato psyllid adults, at 100 ng/μL (Mondal et al. 2022) but within previously published ranges. Among the more than ~186 targets (potato psyllid fast track system) screened singly and/or in groups, seven groups of targets intended for stacked delivery (combining multiple dsRNA targets) were identified, with five selected as the most highly-promising for ACP screening/testing, In PoP gene knockdown of 50-75% has been achieved with greatly reduced fecundity in female potato psyllid, significant mortality of nymphs and/or adults (nymphal instars are more susceptible to RNAi than adults, but phenotype overall, is gene-specific). Of the seven top groups resulting from the fast-track PoP screen, five were advanced to ACP bioassay/knockdown studies. Testing in ACP was completed for three of the five groups, achieving our final goal of 2-3 effective dsRNA groups for ACP. Three additional groups remain to be tested but could not be completed by the end of the grant. Knockdown of one dsRNA group of gene targets (vATPases) resulted reduced fecundity and/or significant mortality in PoP adults (Mondal et al., 2022; Paredes et al., 2022). Results for this group were equally promising for ACP (Saberi et al. 2024). Knockdown of 3-4 genes involved in sugar metabolism and transport, respectively, resulted in significant mortality of PoP adults and nymphs post-dsRNA ingestion (Arad et al., 2023). One dsRNA group (3) interferred with potato-psyllid mediated "Ca. Liberibacter" transmission (Paredes et al. 2024). Genes involved in cytoskeleton remodeling in the gut involved during CLso-Liberibacter infection (transcriptome analysis: He and Brown et al., 2023) and by knockdown, and reduced Liberibacter load and reduced transmission to the plant by 50% or more and symptom severity was also reduced. This group has not been tested in ACP. The vATPase B,E knockdown in the psyllid gut (Mondal et al. 2022; Saberi et al. 2024) was visualized by confocal microscopy using bDNA-FISH and optimized methods to co-localize CLso in the psyllid gut, in relation to potential knockdown of genes encoding proteins essential for CLso gut invasion (Rastegar et al. 2024 TBS). These sensitive approaches can be used in lieu of the intended/failed PCR amplification assay proposed for detection of dsRNAs delivered in planta. The proposed assay was ineffective because fewer dsRNA molecules are required for knockdown than can be detected by PCR) (pitfall). The alternate, direct approach facilitated characterization of dsRNA knockdown by FISH labeling RNAs in PoP adult guts/filter chamber to localize expression (reduced or absence of) in vivo. In addition, CLso (16S rDNA-FISH labeled) and actin filaments (phalloidin-stained) were visualized in the PoP gut in the vicinity of dsRNA knockdown (clathrin heavy chain, RAC1, others) during CLso invasion of the psyllid host/vector gut. Preliminary global small RNA profiling experiment was conducted for potato psyllid for after exposure to ss/dsRNAs to trigger silencing evident in the siRNA and piRNA pathways simultaneously (potentially, 2X+-enhanced global silencing). Results of preliminary small RNA profiling was promising, suggesting mortality and knockdown assays in vivo will be informative. Three most effective dsRNA groups were selected for cloning and expression in citrus seedlings (from transfected tobacco) using the CTV-VIGS vector (Southern Gardens:M. Irey and UFL: C. El-Mohtar) and represent siRNA and piRNA triggers. Greenhouse screening for ACP knockdown is underway and the Brown lab will quantify CLas accumulation in citrus and analyze ACP small RNAs to evaluate dsRNA efficacy. Three most effective dsRNA groups were selected for cloning and expression in citrus hairy root system, and represent siRNA and piRNA triggers. Laboratory screening for ACP knockdown and analysis of small RNA isolated from ACP are in progress (Mandadi lab, mortality) and the Brown lab will analyze ACP small RNAs to evaluate dsRNA efficacy.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Arad, N., Paredes-Montero, J.P., Mondal, M.H., Ponvert, H., and Brown, J.K. 2023. RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Insecta: Hemiptera) gut physiology and results in high mortality. Front. Insect Sci. 3:1283334. doi: 10.3389/finsc.2023.1283334.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
He, Ruifeng, Fisher, Tonja W., Saha, S., Pelz-Stelinski, K., Willis, M., Gang, D. R., and Brown, J.K. 2023. Differential gene expression of Asian citrus psyllids infected with Ca. Liberibacter asiaticus reveals hyper-susceptibility of fourth-fifth instars and teneral adults to invasion. Front. Plant Sci. 14:1229620. https://doi.org/10.3389/fpls.2023.1229620.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Thakre, N., Carver, M. Paredes-Montero, J.R., M. Mondal, Hu, J. Saberi, E., Ponvert, N., Qureshi, J.A., and J.K. Brown. 2023. UV-LASER adjuvant facilitated delivery of 100-600 base pair dsRNAs to the tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid. Pest Management Sci. Wiley Online Library. doi 10.1002/ps.7952.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Fisher, T.W., Munyaneza, J., and Brown, J.K. 2024. Sub-optimal temperatures lead to altered expression of stress-related genes and increased ⿿Candidatus Liberibacter solanacearum⿿ accumulation in potato psyllid. Front. Insect Sci. 3:1279365. doi: 10.3389/finsc.2023.1279365.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Ibrahim, Y.E., Al-Saleh, M.A., Widyawan, A., El Komy, M.H., Al Dhafer, H.M., and Brown, J.K. 2024. Identification and distribution of the ⿿Candidatus Liberibacter asiaticus-Asian citrus psyllid pathosystem in Saudi Arabia. Plant Dis. 108(4):(in press).
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Saberi, E., Mondal, M., Paredes-Montero, J.R.,Nawaz, K., Brown, J.K., and Qureshi, J.A. 2024. Optimal dsRNA concentration for RNA-interference in Asian citrus psyllid. Insects 15:58. https://doi.org/10.3390/insects15010058.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Mondal, M., Saberi, E., Qureshi, J.A., and Brown, J.K. 2024. Increasing penetrance in the RNAi-recalcitrant Bactericera cockerelli?by simultaneously targeting multiple genes essential for a global biological process. J. Econ. Entomol. (submitted).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Saberi, E., Qureshi, J.A., and Brown, J.K. 2024. Differential expression of ⿿Candidatus Liberibacter solanacearum chromosomal and prophage genes in different developmental stages of potato psyllid host Bactericera cockerelli. Sci. Reports. (accepted).
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Paredes-Montero, J.R., Nawaz, K., Rastegar, M., Mondal, M., Saberi, E., Qureshi, J., and Brown, J.K. 2024. Knockdown of actin cytoskeleton pathway genes results in reduced accumulation of ⿿Candidatus Liberibacter solanacearum in potato psyllids and reduced transmission.(in preparation).
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Saberi, E., Mondal, M., Paredes-Montero, J.R., Brown, J.K., and Quereshi, J.A. 2024. Evaluation of vacuolar ATPase genes as potential RNAi targets in Asian citrus psyllid, Diaphorina citri (to be submitted).
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Rastegar, M., Paredes-Montero, J.R., Mondal, M., Ponvert, N., and Brown, J.K. 2024. Visualizing psyllid transcripts and localizing ⿿Candidatus⿿ Liberibacter solanacearum in potato psyllid alimentary canal by bDNA-FISH (to be submitted).
|
Progress 01/15/22 to 01/14/23
Outputs
Target Audience:Citrus growers in Arizona, California, and Florida through research conference and educational-extension presentations Stakeholders, researchers, and other private sector interested in delivery of dsRNA/RNAi for ACP management Pathologists, entomologists, and epidemiologists with interest basic and apllied Liberibacter research Industry cooperator deploying CTV-VIGs vector for dsRNA delivery Federal and state agencies pursuing research in the area of therapeutic dsRNA and delivery methods Graduate students, and post-docs interested in RNAi technology for sprayable and expressed dsRNAs for pest/vector control Changes/Problems:Because the CTV-vector studies were delayed by the CoVID pandemic, we are behind in this objective (1b.) and would like to follow up on the first year citrus plants in which dsRNAs are expressed, which is anticiapted to be late sumer-fall 2023 and 2024. Because of the delay in identifying new post-docs (two took jobs and left the lab in 2022 and another in March 2023), funding will be available to support 1-2 post-docs for another year. However, a no-cost extension for 2024-2025 would be needed, and I'm not certain this is permitted because this year is our second no-cost extension. Please advise if an exception due to coVID might be a possibility. Unfortunately, if not, this may mean unspent funds would need to be returned. With an additional year of funding, the hairy root plants-chimera system could be exploited as well, to analyze plants in which additional dsRNA groups or pairs of dsRNA already evaluated in sucrose feeding chamber assays (in progress to complete all the groups). The study system (collaborative with K. Mandadi) allows for expression of dsRNAs in citron roots and translocation to grafted non-transgenic scions/citrus for Asian citrus psyllid mortality and transmission assays and knockdown in ACP. These data will serve to identify the best candidates to mobilize into the CTV-delivery system (Southern Gardens), and narrow down the groups to the several most likely to produce highest mortality, reduced fecundity, and transmission abatement (all in progress). What opportunities for training and professional development has the project provided?One undergraduate, 1 M.S. graduate student (completed her M.S. thesis), and 4 post-doctoral Associates were involved in the project. Three post-docs have taken their first career job. How have the results been disseminated to communities of interest?Presentations reporting project results have been delivered at virtual and in-person at pathology and entomology professional conferences, at grower-specific conferences in Florida. Dr. Qureshi presented workshops and extension programs in FL that included information about RNAi potential for HLB management and the concept of evaluting non-target effects for beneficial insects/arthropods. The webite completed last year is available to the public and provides general background and concepts underlying dsRNA and the evolution of its conception as a biopesticide. What do you plan to do during the next reporting period to accomplish the goals?1. Complete ACP screening of Groups 2-7 and of ss/dsRNA 'dual punch' approach (Patent Application) for efficacy against adult ACP. 2. Select the best targets (stacked groups of 3-4 and/or, singles, pairs) for CTV-vector expression for cloning and carry out molecular analysis of transfected citrus plants (transgene and small RNA) and ACP (knockdown) to mobilize the best targets for mortality/reduced fecundity and transmission abatement in citrus (collaborative with Southern Gardens). 3. Select the best stacked groups of 3-4 and/or, singles, pairs) for hairy root transformation-expression and bioassay with ACP nymphs/adults; carry out molecular analysis of transfected citrus plants (transgene and small RNA) and ACP (knockdown) to mobilize the best targets for mortality/reduced fecundity and transmission abatement in citrus (collaborative w/ Mandadi, TAMU). This accelerated screening will faciliate selection of the best targets for CTV-expression and serve as preliminary data to prepare a grant proposal that will focus on expression/analysis of the best ss/ds and dsRNAs in CTV and/or in transgenic citrus. Bringing the CTV delivered dsRNAs and ss/dsRNAs to fruition in transfected (non-transgenic plants after hairy root screening) will be the main focus of the proposed project, however, in time, transgenic citrus may become a lucrative product for some sectors. 4. Complete basic analysis of select dsRNA groups that interact in the same pathway by confocal microscopy, fluoresence in situ microscopy of CLso and localization of cellular proteins labeled by FISH-RNA probes and/or antibody probes. Of particular interest are those targets that when knocked down result in reduced transmission by perturbing CLso interactions with the cytoskeletal pathway (in progress). 5. Prepare and submit manuscripts and present results at relevant conferences in 2023.
Impacts
What was accomplished under these goals?
Objective 1: (a) The laser experiments were completed, and a manuscript has been submitted. Localization of dsRNAs (100, 300, 600 bp) with or without adjuvants to the tomato vasculature was corroborated by fluorescence confocal microscopy and knockdown (quantitative PCR) in potato psyllid (PoP) by ingestion-access of dsRNA from LASER-adjuvant-dsRNA treated tomato plant bioassay. The best adjuvant/surfactant combination for cuticle disruption and dsRNA penetration were identified among the six evaluated (Thakre et al, submitted). (b) CTV-constructs are in progress for Group 1 and 2 targets for ACP analysis. The first has been inoculated to N. benthamiana and is infectious, so ready for transfection of citrus (orange). The second is in the cloning stage and is expected to be ready for transfection shortly. Transfected citrus leaf samples will be collected for analysis (CTV infection; UFL lab/Southern Gardens) and ACP knockdown by qPCR amplification (Brown lab). Plants from multiple events will be be maintained and evaluation optimally, carried out for 1-2 years under field conditions (Southern Gardens). Additional dsRNA targets will be provided when Groups 3-6 ACP assays are completed (in progress). Only targets with the highest efficacy based on knockdown and phenotype will be cloned into the CTV vector and transfected into citrus plants for expression. Objective 2: Small RNA sequencing will be used to accomplish this objective. Small RNAs from potato psyllid fed on single-stranded and double- stranded dsRNAs to Group 1 targets have been sequenced and results show that the ss/dsRNA is processed into predictable lengths, indicating that both the pi-RNA and siRNA pathways have been triggered. Analogous experiments will be conducted to demonstrate the first proof of concept for dual-pathway RNAi in ACP. Objective 3: The optimal doses of dsRNA for ACP nymphs and adults was found to be 100 and 200 ng/µL, respectively. This is slightly higher than for potato psyllid adults, at 100 ng/µL (Mondal et al. 2022; this project). Objective 4: This objective has been completed for three gene targets (Mondal et al., 2022), with results extrapolated to an analogous study was completed for ACP this year (Saberi et al., submitted). Objective 5. Seven top groups and or pairs, single targets have been identified in PoP and efficacy in ACP has been completed for two of the seven groups. The goal was 2-3 groups. Objective 6: Preliminary global small RNA profiling experiment has been conducted for potato psyllid for after exposure to ss/dsRNAs to trigger silencing evident in two pathways simultaneously (potentially, 2X+-enhanced global silencing), the siRNA and piRNA pathways. This preliminary small RNA profile analysis was highly promising, suggesting it is worthwhile to conducted replicated mortality and knockdown assays (in progress). Objective 7: Continuing according to plan; J. Qureshi, UFL co-PI (see extension and conference presentations re: Products).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Qureshi J. A. 2022. Advancing citrus protection against Asian citrus psyllid from traditional to protected production systems. Symposium Management of Invasive Pest Insects in the Caribbean and Floridas Pathways 26th International Congress of Entomology, Helsinki, Finland. 17-22 July 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Qureshi J. A. 2022. Managing Asian citrus psyllid and the pest complex of citrus using integrated approaches 10th International IPM Symposium, Denver, Colorado, 28 February to 3 March 2022. Invited.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Qureshi, J. A. 2022. Biological control of invasive Asian citrus psyllid in commercial citrus orchards. Symposium Biological Control of Major Invasive Pests: Achievements and Current Development. Joint Annual Meeting of the Entomological Society of America and Entomological Society of Canada, Vancouver, BC, Canada, 12-17 November 2022.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Qureshi, J. A., A. A. Khan. 2022. Preying on Asian citrus psyllid and other pests. Citrus Industry Magazine, 103 (1) 12-14. https://citrusindustry.net/2022/01/24/preying-on-asian-citrus-psyllids-and-other-pests/
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Brown, J.K., Fisher, T.W., Rast, T, Paredes-Montero, J.R., and Rastegar, M. 2022. Model for psyllid- Ca. Liberibacter systemic invasion-infection based on -omics, Y2H, TEM/SEM, and confocal microscopy-FISH analyses. Hemipteran-Plant Interactions Symposium Dec 2-5, 2022. Melbourne, Australia.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Flynt, A., Mondal, M., Zanacanela, B., Gurung, A., Brown, J.K. 2002. Opportunities offered by piRNA mediated gene silencing in pest control. SysEB Evolution, Entomological Soc. of America, November 13-16, 2022. Vancouver, B.C. Canada.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Saberi, E., M. Mondal, J. R. Paredes-Montero, J. K. Brown, J. A. Qureshi. 2022. RNAi associated knockdown of CHC, vATPase-A, and Snf7 genes and mortality for Asian citrus psyllid adults. Joint Annual Meeting of the Entomological Society of America and Entomological Society of Canada, 12-17 November 2022, Vancouver, BC, Canada.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Saberi, E., M. Mondal, J. R. Paredes-Montero, J. K. Brown, J. A. Qureshi. 2022. Vacuolar ATPases gene targets of RNAi to control Asian citrus psyllid, Diaphorina citri. Annual Meeting of the Florida Entomological Society, 19-22 June 2022, Gainesville, Florida.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Saberi, E., J. A. Qureshi, J. K. Brown, 2022. Quantitative gene expression analysis on targeted genes of Candidatus Liberibacter Solanacearum in the different developmental stages of potato psyllid Bactericera cockerelli. Annual Meeting of the Southeastern Branch of the Entomological Society of America, 26-30 March 2022, San Juan, PR.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Mondal, M., Carver, M., and Brown, J.K. 2022. Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae). Front Physiol. 13: 931951. doi: 10.3389/fphys.2022.931951.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Widyawan, A., Ibrahim, Y.E., Komy, M.H. E., Al Dhafer, H. M., Brown, J. K., and Al-Saleh, M.A. 2023. Differentiation of Candidatus Liberibacter asiaticus in Saudi Arabia based on tandem repeat variability in genomic locus. J. King Saud Univ. Science 35(1) 102376. 10.1016/j.jksus.2022.102376.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Ibrahim YE, Paredes-Montero JR, Al-Saleh MA, Widyawan, A., He, R., El Komy, M.H., Al Dhafer, H.M., Kitchen, N., Gang, D., and Brown, J.K. 2022. Characterization of the Asian citrus psyllid Candidatus Liberibacter asiaticus' pathosystem in Saudi Arabia reveals two predominant CLas lineages and one Asian citrus psyllid vector haplotype. Microorganisms. Oct;10(10):1991. DOI: 10.3390/microorganisms10101991.
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Arad, N., Paredes-Montero, J.R., Mondal, M.H., and Brown, J.K. 202x. RNAi-mediated knockdown of genes involved in the metabolism and transport of sugars results in mortality in Bactericera cockerelli (Insecta: Hemiptera).(Arad, N. M.S. Thesis,2022) Insect Biochem. Mol. Biol. (to be submitted).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Saberi, E., Paredes-Montero, J.R., Mondal, M., Brown, J.K., and Qureshi, J.A. 202x. Optimized double-stranded RNA concentrations validated by gene knockdown and mortality in Asian citrus psyllid to develop dsRNA-biopesticide for its management. Pest Management Science (submitted Mar 2023).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Thakre, N., Carver, M., Mondal, M., Paredes-Montero, J.R., Hu, A., Saberi, E., Qureshi, J.A., and Brown, J.K. 202x. LASER-adjuvant assisted dsRNA delivery to tomato plant vasculature induces RNA interference in the potato psyllid. Pest Management Science (submitted).
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Paredes-Montero, J.R., and Brown, J.K. 202x. Knockdown of genes involved in the actin cytoskeleton pathway abates CLso accumulation in the potato psyllid. Frontiers in Microorganisms (to be submitted).
|
Progress 01/15/21 to 01/14/22
Outputs
Target Audience:Citrus growers/grower groups in Arizona, California, and Florida, other through research and eductional presentations and Website content Stakeholders interested in delivery of dsRNA to target ACP and HLB Industry cooperators to evaluate laser-delivery of dsRNA Industry cooperators deploying CTV-VIGs vector for dsRNA delivery Federal and state agencies pursuing research in the area of therapeutic dsRNA and other small molecule delivery Undergraduate and graduate students, and post-docs interested in biotechnology directed at RNAi for sprayable and expressed dsRNAs for pest/vector control Changes/Problems:We have applied for a no-cost extension for 2022-2023 because of delays resulting from the SARS-Co2 pandemic that limited productivity significantly in both Arizona and Florida where only emergency workers were permitted in the labs and greenhouses. This was permitted primarily to maintain colonies and plants, carry out development/optimization of essential bioassays and molecular analysis of PoP-CLso samples (already extracted or frozen prior to the lab closure), and continue dsRNA screens with PoP, as was possible. Minimal but sufficient funds may remain by Jan 2023 to carry on the research for perhap another 4-6 months. If so a second no-cost extension will be requested to avoid losing momentum by bridging the gap between the end date and when funds can be secured to continue this promising research, collaboratively with FL and TX collaborators. It is anticipated that molecular analyses (qPCR, CLas genome copy number) will conducted in the AZ lab, where all assays have been/will be (ddPCR) optimized for expedited analysis of both PoP-CLso and ACP-CLas study systems during the course of this project. What opportunities for training and professional development has the project provided?Several undergraduates, 1 M.S. graduate student, and 4 post-doctoral Associates were / are involved in the project on average throughout the project. How have the results been disseminated to communities of interest?Presentations reporting the results have been delivered at virtual and in-person conferences, in grower-specific conferences in Florida, and to the Advisory Committee (AZ, FL, CA members) virtually during 2021, and in-person/hybrid format in 2022. The webite is available to the public and provides general background and concepts underlying dsRNA and the evolution of its conception as a biopesticide. The graphics shown at the website were co-developed by three post-doctoral associates in the Brown Lab, UA. What do you plan to do during the next reporting period to accomplish the goals?1. Complete the knockdown and transmission gene assays using PoP borne and reared on CLso-infected tomato, the assay used in the fast-track screening system (UAZ), followed by ACP-CLas testing using the same approach (UFL). Validation of the best candidates for PoP in the ACP-CLas system will guide the selection of dsRNA target for biopesticide development and evaluation in alternative delivery systems (non-laser, foliar, the CTVvv VIGS vector, and in transgenic citrus plants. 2. To determine dsRNA efficacy to interfere with CLso-transmission, knockdown and mortality phenotypes will be further characterized for the most effective dsRNAs/groups using potato psyllids reared on CLso-free tomato plants, but allowed 48 hr acquisition-access period on CLso-tomato plants, 14 days post-CLso inoculation. These results will provide estimates of dsRNA knockdown in Liberibacter-free psyllids followed by Liberibacter infection as adults, simulating Liberibacter-free dispersing adult CLso-ingestion after arriving to an already-infected plant. A comparison is of interest because both CLso-infected or CLso-free potato psyllids are dispersing/establish on CLso-infected plants, and it is expected that dsRNA biopesticides may be most effective in curbing CLso-infection of 'clean' psyllids because knockdown of genes essential for CLso to accumulate in the psyllid vector prior to or at the time of CLso-ingestion, compared to psyllids borne and reared on Liberibacter-infected plants. A combined strategy of transmission interference + mortality induced dsRNAs is expected to reduce psyllid population sizes and reduce primary infection within those populations when dual-target biopesticides are applied. 3. Confocal electron microscopy studies are underway to localize dsRNA targets pre-and post-treatment in the potato psyllid gut, and visualize key proteins and cellular compartments and/or organelles that participate in Liberibacter early-entry and multiplication stages of the infection cycle, prior to exocytosis. The effect of cytoskeletal remodeling post-CLso attachment/entry in relation to psyllid target knockdown and subsequent accumulation of CLso in the psyllid gut to begin to elucidate functional aspects of CLso-gut invasion and accumulation, to aid in understanding mechanisms of pathogenesis and their potential perturbation by dsRNA biopesticides. 4. Preliminary experiments are underway with collaborators to test Groups 1 and 2 stacked dsRNAs inthe CTVvv VIGS vector, and in transgenic citrus plants. The efficacy of dsRNA knockdown and mortality of ACP post -dsRNA ingestion from biopesticide-delivering citrus plants will be evaluated. Citrus leaves expressing the dsRNAs will be assay to determine the extent of dsRNA expression in psyllids exposed to the dsRNAs (qRT-PCR cDNA assay), mortality, knockdown in psyllids, initally at ~5 and 9 dpi. The extent of CLas accumulation in leaves in dsRNA expressing and control citrus seedlings will be quantified by qPCR amplification and droplet-digital PCR. Adoption of ddPCR to this study system for future bioassay studies is expected to reduce error and increase reproducibility of the assays. 5. Apply for research funding to proceed with the next steps in evaluating the efficacy of best dsRNAs to cause mortality and abate transmission using the stacked platform delivering dsRNAs by CTVvv and in transgenic citrus (initially, in the hairy root system) under greenhouse conditions, and as soon as possible under field conditions.
Impacts
What was accomplished under these goals?
Obj 1: A UV laser beam (355nm) with 0.001mJ laser energy was used to wound tomato leaf mid-veins, to which fluorescently-labelled dsRNAs of 100, 300 and 600 bp sizes were co-applied with seven non-ionic penetrants or two oil adjuvants. Fluorometry showed 100 and 300 bp, but not 600 bp dsRNAs, entered the vasculature without adjuvants. Widespread Max and Purespray Green produced optimal mobility. DsRNAs applied to laser-wounded midveins crossed midvein barriers, and translocated to abaxial phloem cells, xylem vessels, and sieve tubes. Laser-delivered dsRNA was ingested by PoP, resulting in significant knockdown. In citrus leaves, neither UV laser nor laser etching resulted in dsRNA detection in treated leaves. ACPadults failed to ingest dsRNA (qPCR knockdown). The aim was abandoned after four failed tests each using different parameters and two laser methods. Obj 2: By RT-PCR dsRNA was detectable in cDNA, at 10-50 molecules. Obj. 3: Potato psyllid fast-track surrogate system: The minimal effective dsRNA concentration needed for optimal knockdown and persistence were evaluated for teneral adults and 3rd-instar nymphs from 48 hr to 12 days at seven concentrations spanning 0.1 ng/µL to 500 ng/µL dsRNA for vacuolar ATPase subunit A, clathrin heavy chain, and non-fermenting protein 7. The minimal and/or second most effective concentration was 100 ng/µL. Greatest 'penetrance' occurred at 100 ng/µL 3-5 days post-IAP. The 3rd instar was most responsive to RNAi, followed by teneral and mature adults, respectively. Asian citrus psyllid: dsRNA concentrations 10, 50,100, 200 and 500 ng/µL were tested for knockdown of Clathrin heavy chain (CHC), vATPase-A, and sucrose non-fermenting protein 7 (Snf7) in the 3rd-instar and teneral adults. Knockdown was positively correlated with dsRNA concentration. Significant knockdown (P < 0.05) ranged from 12-34% for adults and 18-39% for nymphs over the 10-500 ng/µL range. Adult ACP post-ingestion of vATPase-A, CHC and Snf7 dsRNAs at 200 and 500ng/µL exhibited significant mortality by 47-44, 33-43 and 42-50% respectively, 10 d post-ingestion. The optimal dose for nymphs and adults was 200 ng/µL. Obj. 4. See Obj. 1, 3, 5 Obj. 5. Mortality at > 45% threshold / Go-No Go fast track screening platform: I. Knockdown and mortality were evaluated individually and in combinations for ATF4, C7, and D24 genes, essential for biosynthesis and regulation of ecdysteroids. At 10-d dpi, knockdown of D24, at 39-45%, resulted in 51% PoP mortality. Knockdown of C7, at 38-61%, caused 53% mortality, while dsD24 ingestion resulted in 65% mortality when co-delivered with dsD24 and dsC7. INCOMEC, PREMEC, and SWOLLEN phenotypes, consistent with incomplete ecdysis, were observed in 4-12% of treated psyllids. INCOMEC adults survived several days, but failed to mate or fly. SWOLLEN and PREMEC phenotypes were lethal for immatures. ATF4 knockdown caused no mortality or phenotype. For D24 and C7 significantly greater RNAi penetrance was observed for combined, compared to individual, dsRNAs. The highest knockdown leading to incomplete ecdysis and/or mortality was observe for dsRNA targets in the same or interacting pathway(s). Knockdown of ATF4 was 'rescued'. ACP screening is underway for this group. II. Five genes involved in sugar metabolism and transport in the PoP gut were evaluated: Alphaglucosidase1 (AGLU1), Aquaporin2 (AQP2), Trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2). Silencing AQP2 and TRET1 resulted in 20-30% PoP mortality 9-d dpi, when dsRNAs were delivered individually or co-delivered. The earliest and greatest knockdown occured for AGLU1 with single or stacked targets, at 30-65% mortality at 9dpi. Knockdown with different combinations of targets revealed the greatest PoP mortality post-delivery of all five dsRNAs, at 65.11%. AQP2 and TRET1 exhibited additive effect when co-delivered with TRE1/2 and AGLU1, based on knockdown and mortality. RNAi penetrance was enhanced by stacking dsRNAs. ACP screening is underway for this group. III. The effects of simultaneous knockdown of four of 13 genes whose expression is integral to vacuolar ATPase (V-ATPase)-mediated acidification of intracellular compartments, was compared to knockdown of each gene alone. The hypothesis was that silencing the expression of multiple protein submits of a vital enzyme would result in knockdown equal to or greater-than-additive knockdown, compared to that achieved by silencing individual genes, and effectively perturb proton pump activity. Ingestion-access to stacked V-ATPase subunit dsRNAs resulted in 23-28% and 16-28% knockdown, leading to 52% and 46% mortality and in treated immature and adult psyllids, respectively. Ingestion-access to individual dsRNAs resulted in knockdown ranging from 42-58% and 23-41% in immature and adult psyllids, respectively. Mortality was not observed post-ingestion of individual dsRNAs. Greater knockdown and mortality resulted from stacking the group of four, compared to the single dsRNAs, and showed an additive RNAi effect. Co-delivery of multiple dsRNAs resulted in knockdown of multiple subunits of psyllid vacuolar ATPase underscoring the potential for dsRNA biopesticides that target biological processes or pathways instead of single genes. ACP screening is underway for this group. V. Screening of one more mortality group and four 'transmission groups' is in progess for PoP. See Other Products. Obj 6: Small RNA sequencing has been completed for dose-treated PoP; in progress for ACP. Obj 7: See Products for Outreach results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Paredes-Montero, J.R., Arif, U., and Brown, J.K. 2022. RNA interference-mediated knockdown of genes implicated in the synthesis of ecdysteroids, impairs molting in the potato psyllid, Bactericera cockerelli (Insecta: Hemiptera). Pest Management Science DOI 10.1002/ps.6848.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Mosharrof Mondal1, M., Flynt,A.S., Brown, J.K. 2021. Characteristics of a piRNA loci to be used to trigger piRNA-induced gene silencing in hemiptera
ntomological Society of America-Denver, Oct 31-Nov 3, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Saberi, E., M. Mondal, J. R. Paredes-Montero, J. K. Brown, J. A. Qureshi. 2021. vATPase-A gene silencing through orally delivered dsRNA in Asian citrus psyllid, Diaphorina citri. Entomological Society of America-Denver, Oct 31-Nov 3, 2021. Virtual
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Mondal, M., Saberi, E, Qureshi, J.A., and Brown, J.K. 202x. Increasing penetrance in the RNAi-recalcitrant Bactericera cockerelli?by targeting multiple genes essential to a biological process instead of individual genes
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Mondal, M., and Brown, J.K. 2022. Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemipetra: Triozidae). Frontiers in Invertebrate Physiology (submitted)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Saberi, E. and Brown, J.K. 2022. Differential expression of Candidatus Liberibacter solanacearum chromosomal and prophage genes in different developmental stages of potato psyllid host, Bactericera cockerelli. Phytopathology (revised, res-submitted).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Thakre, N., Carver, M., Mondal, M., Rast, R., Etxeberria, E., Hu, A., Saberi, E., Qureshi, J.A., and Brown, J.K. 202x. Optimization of adjuvants for laser-assisted dsRNA delivery to tomato plants for RNAi in potato psyllid. Pest Management Sci. (submitted).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Saberi, E., Mondal, M., Paredes-Montero, J.R., Brown, J.K., and Qureshi, J.A. 202x. Optimal dsRNA concentrations to achieve RNAi penetrance by oral delivery to adult and third instar Asian citrus psyllids. Pest Management Sci (to be submitted May 2022).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Qureshi J. A. 2021. Integrated citrus pest management. International Workshop. The role of plant protection in sustainable agriculture development. February 21-23, 2021.Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Qureshi J. A. 2021. Managing global pest threats to citrus production. Fifth International conference on agriculture and food science. October 28-30, 2022. Virtual.
|
Progress 01/15/20 to 01/14/21
Outputs
Target Audience:Our efforts reached the following target audiences: Citrus producers in Arizona, California, Florida, and Texas; Private sector- various; Extension and outreach peers, Research community; Government colleagues (State and Federal), and the Public. Changes/Problems:Several assays were modified somewhat or replaced by more effective approaches. These are noted in the text. In addition, lab closures in Arizona and Florida prevented anticipated progress, exacerbated by layoffs of lab workers needed for colony upkeep and support. Beginning February 2021, three hires have been completed but the experimentation is behind schedule. We anticipate requesting a no-cost extension next year to make up for the shortfall in research progress. Data collection needed for manuscripts was also delayed and so we are behind in submitting our manuscripts mid-2020 to Dec 2021, as planned. The website materials have been collated but the webmaster has significant back logs to address, including our site. The link is expected to be available in May 2021. What opportunities for training and professional development has the project provided?Post-doctoral Associates; M.S. Graduate Student, Ph.D. student, and undergraduate students Laboratory techniques-molecular, bioassay experiment development and implementation, diagnostic assay development and implentation, dsRNA design pipeline, various bioinformatics tools. How have the results been disseminated to communities of interest?Abstracts at professional meetings, Extension bulletins and presentations, and referred manuscripts have been prepared for 2021 submission. One manuscript has been published (Cicero and Brown, 2021). Growers and Research/Extension Event Presentations 2020-2021 Qureshi, J. Overview of Asian citrus psyllid management including dsRNA. Florida Citrus Extension Agents. RNAi Focus Topic Organized by J.A. Qureshi: The power and promise of RNA interference in controlling Asian citrus psyllid. Killiny, N. 2021. Florida Citrus Show. (invited speaker). What do you plan to do during the next reporting period to accomplish the goals?Complete screening of stacked and single/combo targets in PoP system. Complete screening of stacked and single/combo targets in ACP system. Identify best targets for cloning into CTVvv and other virus vectors for expression in citrus (Southern Gardens). Characterization/localization of selected mRNA transcripts in situ (PoP and ACP) to corroborate effects of RNAi and characterize relevant phenotypes. Bioinformatic screening of ACP and PoP and endosymbiont genomes for potential HGT targets susceptible to RNAi / CRIPSR knockdown.
Impacts
What was accomplished under these goals?
Objective 1 Penetrants and dsRNA laser delivery: Nine penetrants were evaluated for foliar delivery of dsRNA to tomato plants. Three non-ionic penetrants and two seed oil adjuvants enhanced cuticular penetration and uptake of 100-600-bp dsRNAs into tomato phloem and/or xylem. Fluorometric analysis showed 100- to 300-bp ds RNA entered leaf vasculature without surfactants, however, 600-bp dsRNA required organosilicon surfactants for entry and spread. Confocal fluorescence microscopy showed FAM-labeled ds RNAs applied to laser-wounded leaf midveins entered and moved across the midvein barrier, into abaxial phloem cells, xylem vessels, and phloem sieve tubes. Significant levels of gene knockdown confirmed 600 bp dsRNAs were biologically active in planta when applied with the two best penetrants. Potato psyllid knockdown, post-ingestion access to 600 bp dsRNA-treated tomato plants, corroborated dsRNA vascular-delivery, 3-days post-ingestion (dpi). This observation was consistent with confocal imaging of dsRNA 600 bp in phloem and xylem tissues. A protocol was developed for tracking two dsRNAs by qPCR in leaf punches from tomato leaf veins to study the mobility of dsRNA, and an RT-PCR assay developed to quantify dsRNA copy number in laser-wounded leaflets, indicated as few as seven dsRNA molecules were detectable. In four experiments, a commercially-developed light-laser failed to deliver biologically-active dsRNA to 'Valencia' orange for ingestion by third instar or teneral adults, based on phenotype and qPCR knockdown. The Advisory Committee (Annual meeting, February 22, 2021) recommended stopping these experiments and focusing on target testing in ACP to expedite selection of field-ready dsRNAs. Impacts: 1. The use of new tools provided evidence of dsRNA mobility and viability in tomato plants for psyllid knockdown corroborating the viability of laser delivery and biological viability of laser-delivered dsRNA biopesticides for psyllid control. 2. Knowledge that dsRNA flow in the plant is irregular and somewhat unpredictable, is consistent with studies that show that phloem-xylem activities and flow of sap are influenced by plant physiology, vascular structure, and plant type. Thus, the best way to monitor dsRNA mobility and persistence is through psyllid ingestion bioassay and qPCR knockdown quantification. Objective 2 Experiments designed to detect dsRNAs in psyllids, post-ingestion showed dsRNA was detectable shortly after ingestion, becoming undetectable by day 2-3. It was determined that the more interesting question is to understand the location or compartment in the psyllid alimentary canal (or body) where mRNA is expressed and silenced. Impact: This new knowledge underscores the utility of characterizing dsRNA-psyllid interactions for promising candidates, directly, by in situ hybridization to localize mRNA expression in psyllid organs and tissues, instead of tracking dsRNA using molecular methods. Objective 3 Three factors governing RNAi efficacy were evaluated: (i) a range of dsRNA concentrations (0.1, 1, 10, 50, 100, 150, 200 and 500 ng/μL) were evaluated to determine minimal effective doses of dsRNA required for robust RNAi, (ii) developmental stage-specific response to environmental RNAi, and (iii) gene silencing in a time-course study. PoP. The optimal concentration for RNAi penetrance of 3rd larval instar, teneral adults, and mature adults was shown to be 100 ng/ul, with a 2-day ingestion-access to dsRNA. Higher than optimal dsRNA concentrations did not increase gene silencing. ACP. Comparisons of cut-stem and sucrose-feeding assays were carried out for ACP adult and immature instars. The feeding chamber was adopted as the most effective screening system. Knockdown was not significantly different for 3rd instar larvae and adults at 100, 200, 500 and 200-500 ng/ul respectively. The optimal dose at 200 ng/uL, was adopted for dsRNA screens. Significant mortality was obtained for ACP adults post-ingestion of dsRNA_9 (26%), dsRNA_11 (25%), and dsRNAGroup_1 stacked genes (42%) 10 dpi. ACP mortality occurred for 6-10 days post-ingestion for dsRNAs at 22-23%. Impact: This is the first study to systematically evaluate RNAi efficacy factors for PoP or ACP knockdown. For PoP, the minimal effective concentration of dsRNA of 100 ng/μL was adopted for fast-track screening of stacked and single targets to inform the best candidate targets for ACP screens, whereas, for ACP, the optimal dose of 200 ng/uL, was adopted for dsRNA screens. Objective 4 1. Initial screens used cut-stem plant assays, however, results proved highly variable. Results were more consistent using a sucrose feeding chamber, and so was adopted for screening. 2. For the 3rd instar nymph assays, four groups met the criteria for further evaluation as single or different pairs to identify optimal combinations. Fifteen groups were tested for transmission abatement phenotype using 3rd instar nymphs; four were advanced. Twelve groups were tested for oviposition and mortality phenotypes; three were advanced. Impact: 1. Twelve groups or 'stacked' dsRNAs (3-4) showing moderate to high knockdown were identified as lucrative targets to advance into screening with ACP. Selections are based on potato psyllid mortality (55-78%), reduced oviposition (50-55%), and reduced CLso transmission efficiency (45-75%), and are phenotypes consistent with other effective biopesticide. 2. Stacking dsRNAs was additive or synergistic, making dsRNA biopesticide strategies ideal for psyllids because of the inherently lower RNAi penetrance, compared to some other insects. Objective 5 Three target groups selected based on 'functional pathway' criteria (identified in fast-track screens) evaluated in ACP 3rd instar nymph and adult screens, were advanced to single and pairwise combination screens based on moderate to high mortality, interruption of immature instar development processes, and reduced oviposition by adult females. The earliest ACP mortality was achieved 2-days post-ingestion of Group_1 stacked dsRNAs, and resulted in significantly higher mortality than single dsRNAs. Screens with three additional groups (2-4) are underway and seven more await screening. Impact: Based on RNAi screens in ACP with the first groups of gene targets, advanced from the fast-track system, the results are highly encouraging, and warrant testing of the 11 additional Groups and 40 single candidates for knockdown with mortality, reduced oviposition, or reduced CLas transmission. Objective 6 SmRNA sequencing for PoP has been completed for several experiments and analysis is underway, and a 5-instar ACP transcriptome library has been determined, annotated, and analyzed based on differential expression profiling (He et al., 2021). The ACP genome, version 3 has been made available (Boyce Thomson Institute, Cornell). Three PoP genomes have been determined and assembly/annotation is underway (Fu et al. TAMU, northwestern), with Central and Western in progress (this project). Impact: Additional resources for functional genomics will facilitate more reliable or enhance target predictions for RNAi biopesticide development. Objective 7 1. Presentations have been delivered to the citrus industry and research community, and educational materials have been produced. 2. Laser experiments were translated to testing stages using light laser-etching device, collaboratively with an industry partner, Premier Citrus, to deliver dsRNA to citrus seedlings at different concentrations and different test parameters. 3. Project Website (link available May 2021) Impacts: Stakeholders continue to show interest and enthusiasm about the potential availability of dsRNA biopesticides as a component of integrated pest and disease management practices; Encouraging experimental results have spurred Federal and state agencies to continue to fund therapeutic delivery systems for control of citrus greening disease.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
He, Ruifeng, Fisher, Tonja W., Saha, S., Peiz-Stelinski, K., Willis, M., Gang, D. R., and Brown, J.K. 202x. Differential gene expression of Asian citrus psyllids infected with Ca. Liberibacter asiaticus reveals hyper-susceptibility of fourth-fifth instars and teneral adults to invasion. Scientific Reports
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Mondal, M., and Brown, J.K. 202x. Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemipetra: Triozidae). Frontiers in Invertebrate Physiology
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Mondal, M., Montero Paredes, J.R., Saberi, E., Qureshi, J.A. and Brown, J.K. 202x.Silencing multiple genes targeting the same pathway or process as an alternative to achieve superior phenotype in an RNAi recalcitrant pest Bactericera cockerelli (Sulc) (Hemipetra: Triozidae). Pest Management Science
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Paredes-Montero, J.R., Arif, U., and Brown, J.K. 202x. RNA interference-mediated knockdown of genes implicated in the synthesis of ecdysteroids impairs molting in the potato psyllid Bactericera cockerelli (Insecta: Hemiptera). Pest Management Science
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Thakre, N., Carver, M., Mondal, M., Rast, R., Etxeberria, E., Hu, A., Saberi, E., Qureshi, J.A., and Brown, J.K. 202x. Optimization of adjuvants for laser-assisted delivery of dsRNA to tomato plants. Pest Management Science
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
Saberi,E. and Brown, J.K. 202x. Differential expression of Candidatus Liberibacter solanacearum genomic and prophage genes in different instars of the potato psyllid host, Bactericera cockerelli. Insects
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Saberi. E., M. M. Hossain, J. R. Paredes-Montero, J. K. Brown, J. A. Qureshi. 2020. Oral delivery of dsRNA to Asian citrus psyllid. Entomological Society of America. Oral presentation (abstract).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Saberi. E., M. M. Hossain, J. R. Paredes-Montero, J. K. Brown, J. A. Qureshi. Optimizing dsRNA concentration for gene silencing in Asian citrus psyllid. Entomological Society of America
|
Progress 01/15/19 to 01/14/20
Outputs
Target Audience:Citrus industry stakeholders, government-state and federal, public Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Objectives 1,4. Junior female post-doctoral associate is lead on this objective in Arizona, and young male post-doc is lead in Florida. Objective 2. none to report Objective 3. Second Junior post-doctoral associate is lead on this objective. Objective 5. Junior post-doc leading Obj. 3 and third junior post-doc are collaborating on this complex, time-consuming objective. Objective 6. A junior post-doc (Obj. 3) is lead on this objective. How have the results been disseminated to communities of interest?Objectives 1,4. A joint-manuscript draft is completed, and undergoing revision for submission within next 1-2 months. A joint second manuscript has been outlined elaborating additional results in tomato to evaluate knockdown in potato psyllid, in relation to laser-delivered dsRNA persistence, in planta, for different sizes of dsRNA in tomato plants, to advance the principle of concept for translation to citrus. Objective 2. No information has been disseminated. Objective 3. A manuscript is in preparation to report the results of these extensive experiments with three gene targets, specifically selected for the different extents of knockdown induced in dsRNA treated psyllids. Objective 5. Two manuscripts will be prepared, one by each junior post-doc as lead. Objective 6. A manuscript reporting the findings for the whitefly dsRNA biogenesis and follow-up experiments has been submitted for review. 6. Bulletins, presentations, and website (in progress). What do you plan to do during the next reporting period to accomplish the goals?Objectives 1,4. Laser dsRNA laser assisted delivery optimization for citrus is progress with 2-NBDG fluorometry studies in Valencia by fluorometry/uptake studies of dsRNA (300bp,100bp). Preliminary results indicated dsRNA100 and 300 enter most readily on abaxial side of leaf with penetrant M. As a control, pinprick wounding will be tested in Valencia and Fukomoto orange to determine if 100 and 300bp sizes of dsRNA are able to enter and move within the citrus vasculature (more complex than tomato). The dsRNA will be monitored in different locations on the plant for persistence over time by quantitative PCR. What is learned from these studies will be translated to testing in citrus by our collaborators in Florida, using the optimal size dsRNA (100-200 bp) delivered to citrus psyllids by laser and stem-cut assay, followed by feeding of immature and adult Asian citrus psyllids on dsRNA, and qPCR quantification of gene knockdown. In Florida, we have established a relationship with a commercial company to test laser-delivery of dsRNAs to citrus plants, and test knockdown in Asian citrus psyllids (benchmark to begin was April but now setback because of the coronavirus outbreak). Selected promising dsRNAs have prepared for testing in Florida, and such ACP testing will continue until all promising targets are evaluated for phenotype and knockdown qPCR analysis. The most promising will be tested by laser ( if preliminary experiments, are promising), and will be cloned into the CTV vector (Southern Gardens), and tested by bark flap injection (Southern Gardens). Objective 2. Experiments with nanoparticle-dsRNA emulsions will be continued to address this objective. Objective 3. Experiments were initiated by our collaborators in Florida to test these parameters for Asian citrus psyllids (interrupted by closure; no data to report yet). Objective 5. Testing additional candidate targets is underway for three new different groups of targets. Characterization will be undertaken for several best performers from different sets, combined. Among the other 40 targets not yet tested individually, selected dsRNAs from groups showing good promise, will be selected to determine which may make the greatest contribution(s) to knockdown. Objective 6. A second group of PoP teneral adults were exposed to selected target gene dsRNAs, and submitted for small RNA sequencing to more closely evaluate miRNA biogenesis with targets resulting in promising knockdown. Further, in this experiment, Clso-infected psyllids were compared to no CLas, to evaluate potential differences occuring in psyllids free of and infected with Liberibacter. Sequence analysis is in progress. Objective 7. We will continue to share progress and implications of our work to the citrus growers in Florida, California, and Arizona. The information provided to the growers and industry partners was highly appreciated and considered useful for making advances in the management of this vector-pathogen complex.
Impacts
What was accomplished under these goals?
Objectives 1 and 4. Arizona team Leaf cuticular waxes and epidermal cell walls protect plants against desiccation and entry of deleterious substances, but also impede delivery of nutrients or treatments to the plant vasculature. This aim was undertaken to optmize the entry and systemic movement of dsRNA (transcripts) of potato psyllid (PoP), Bactericera cockerelli into tomato plant phloem. Minute wounds were made to the depth of the pallisade cells-mesophyll tissue, and 100-, 300- and 600-bp FAM-labeled dsRNA molecules or 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) were applied and their uptake and vascular loading were monitored by fluorescence spectroscopy. Non-ionic penetrants and oil adjuvants were evaluated for enhancing uptake and delivery of 600-bp dsRNA into phloem and/or xylem tissues. Induction of RNA-interference (RNAi) 'knock down' of expression of tested psyllid genes was evaluated by allowing adult psyllids to ingest dsRNA during phloem-feeding from dsRNA-treated and -untreated (control) tomato plants by qPCR. Fluorometric analysis indicated 100- to 300-bp dsRNA penetrated and was mobile in leaf vasculature without assistance of penetrants tested. Uptake of 600-bp dsRNA required the aid of penetrants, with organosilicones, providing the most rapid and complete entry into the vasculature. Confocal fluorescence microscopy results showed that FAM-labeled dsRNAs applied to laser-wounded leaf midveins resulted in dsRNA entry, penetration across the midvein barrier, and translocation into abaxial phloem cells and xylem vessels, with low-level detection in phloem sieve tubes. Psyllid-based bioassays and qPCR analysis for gene knockdown have confirmed the efficacy of penetrant-assisted laser dsRNA delivery techniques. Florida team: A postdoctoral associate was hired in February 2019. A continous supply of citrus and orange jasmine plants needed for delivery of dsRNA experiments with ACP was initiated. More than one hundred sweet orange Valencia-Swingle and 300 orange jasmine plants were established, and maintained in separate greenhouses. 100 Valencias were graft inoculated with infected HLB plant tissues for ACP-CLas colonies/source plants. A separate room was established for laser and permissions/training needed to use laser technology were addressed. Two greenhouses to maintain plants and seedlings, two additional glasshouses were set up for establishing HLB positive and negative plants and psyllid colonies. Colonies of HLB positive and HLB negative psyllids are maintained in those houses using citrus and murraya plants and psyllids. Equipment was purchased (laser, qPCR and PCR machines, -20C freezer, PCR work station, and centrifuge) to establish essential molecular capacity in the Florida lab. Laser parameters were optimized by testing dsRNA delivery over a range of settings, with and without penetrants. A fluorescent soluble probe (2-NBDG) was used for determining the suitable concentration of chemicals to deliver to the phloem (Sub obj. 2).The position of the probe was monitored using a fluorescent scope. Fluorescence micrographs of laser-perforated areas on citrus leaves after dye application were prepared at 10 min intervals through 3 observations and an additional at 4 hours. The 2NBDG was used to assess delivery efficiency of different penetrants to facilitate dsRNA entry/mobility in tomato plants. Objective 2. We tested several fluorescently-labeled compounds to optimize detection in the psyllid to circumvent auto-fluorescence caused by chitin interference with visualization, with the next steps being in situ hybridization detection and/or confocal imaging. We also initiated experiments using labeled, nanoparticle delivery dsRNA to potato psyllids (PoP). How long dsRNA persists when bound to nanoparticles is not yet known but it dsRNA appeared to be stabilize and released, based on knock-down results. Objective 3. For psyllid adults (PoP) the minimum effective concentration of dsRNA to achieve knockdown is 100 ng/uL. This is much lower than published results for potato psyllid. Increasing the dsRNA starting concentration to saturating levels did not increase knockdown significantly. Knockdown persists for nine days before begining to decline in psyllids. Knock-down of gut dsRNases somewhat increased the efficacy of RNAi in adult psyllids. Tests to evaluate knockdown in replicated experiments involving 3rd, 5th instars, teneral adults and adults have been completed for all instars for three gene targets. Six genes were tested in preliminary studies to identify differential knockdown candidates, to represent the expected variation in RNAi depending on the particular gene target selected. Objective 5. PoP-CLso-fast track surrogate system, We have designed and tested ~50 dsRNAs, in groups of 3 targets, using a pathway basis strategy to optimize 'hard' knockdown and result in mortalty with or without an additional phenotype, and/or reduce CLso titer and subsequent transmission. Ten of these from the best performing groups have been tested individually (teneral adults, immature 3rd instar.For each PoP gene, 50 qPCR primers/probe sets were designed and synthesized for knockdown / qPCR amplification. PoP gene targets showing the best RNAi produced phenotypes: mortality, developmental delay, reduced fecundity, and reduced CLso transmission. ACP-CLas-citrus system: Based on shared nt/AA sequence homology among genes tested thus far (80.5-78.6%) eight ACP dsRNAs have been designed and synthesized, and primer probes, designed. These dsRNAs will be provided to our Florida collaborators for testing in ACP/citrus by delivery via laser (2 targets), and sucrose diet and Muraya rooted cuttings, or Valencia orange cut stem assays. Preliminary testing using these delivery methods has been underway since September 2019. The PoP draft genome has been obtained from our cooperator at TAMU, and Brown laboratory PoP transcriptomes were obtained/curated during previous projects. The ACPv3 curated genome was used for ACP gene sequence validation, using our laboratory ACP transcriptomes. The average size of PoP dsRNAs were ~220 bp. To accommodate CTV-vector insertion and mobility in planta by laser or other delivery methods, ACP dsRNAs have been designed at ~150 bp. 6. Global large and small RNA gene expression profiling of psyllids (PoP) and whitefly (non-target) has been undertaken for insects post-ingestion of 3 target dsRNAs. Data have been analyzed to identify the siRNAs in each profile to evaluate dsRNA biogenesis in the different insects indicating more than one type of RNA molecule may trigger RNAi. If this pans out by further testing, enhancing RNAi may become possible. 7. PI's were invited to present project updates to the Citrus Research and Development Foundation and relevance of RNAi to ACP/HLB management. CRDF is largest grower organization in Florida working on citrus and ACP/HLB, and works with other states including AZ, CA and TX. Citrus Research and Development Board, Directors Meeting, Arcadia, FL. April 23: Brown JK, Qureshi JA, Etxeberria E. 2019. A study system for discovery and development of dsRNA biopesticides as potential components of HLB/ACP management.; Qureshi, J. A. 2019. Psyllid and HLB Management: Biopesticidal dsRNA, laser delivery and impact. Arcadia, FL. Post docs Dr. Esmaeil Saberi works closely with PI's and postdocs from Brown's lab, Drs. Neha Thakur, Dr. Mohamed Mosharrof, and Jorge Paredes. Each presented an update to the project Advisory Board during the Annual meeting November 5-9, 2019 (Tucson). Website: Extension and research articles were prepared to upload to the project website. The link has been established and graphics were developed by post-docs (collaboratively) informing laser delivery, RNAi pathway-mechanisms, and illustration of psyllid dsRNA feeding chamber.The launch is later than expected but will be made available by early summer.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Fisher, T.W., Paredes, J.,He, R., Breci, L., Cicero, J., Willis, M., Gang, D.R., and Brown, J.K. 2019. Proteomic and transcriptomic evidence for cytoskeletal remodeling, endo-exocytosis, and an intracellular lifestyle for Candidatus Liberibacter in the psyllid host.
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
He, Ruifeng, Fisher, Tonja W., Peiz-Stelinski, K., Willis, M., Gang, D. R., and Brown, J.K. 2020. Differential expression profiling of Ca. Liberibacter asiaticus infected psyllid host reveals late instar-specific hyper-susceptibility to invasion and cytoskeletal remodeling and endo-exocytosis pathways.
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Thakre, N., Carver, M., Rast, Tim, Etxeberria, E., Hu, A., Saberi, E., Qureshi, J., and Brown, J.K. Optimization of LASER delivery of dsRNA to vascular tissues of tomato plants to facilitate RNA-interference in the potato psyllid.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Brown, J.K. 2019. Psyllid-Ca.Liberibacter interactions involved in the circulative, propagative transmission pathway: molecular and cellular interfaces. Joint International Citrus Virology Conference and International Research Citrus Workshop-HLB, Riverside CA March 10-15, 2019.
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Progress 01/15/18 to 01/14/19
Outputs
Target Audience:Citrus growers, citrus industry professionals, Extension Specialists and Agents, Private sector nursery and pest control, Public, Scientists, Changes/Problems:The only major change is that Dr. Hu stepped off the project because with his other responsibilites to research and extension, he was unable to participate fully in project activities. Dr. Qureshi can coordinate extension activities with specialists/agents he knows in the citrus-growing states, and Dr. Brown will oversee the website. What opportunities for training and professional development has the project provided?Post-doctoral scientists have collaborated to create a pipeline for all the major steps in dsRNA target selection, cross-checking PoP and ACP databases, dsRNA design, off-target effects-check, bioinformatically, dsRNA synthesis, stability post-synthesis, primer/probe design for qPCR, and testing oral delivery systems. Technical and/or post-doctoral scientists optimized and mastered laser wounding for dsRNA delivery, manipulation of psyllid instars, qPCR analysis. The undergraduate has mastered tomato and citrus plant and psyllid colony maintainance. How have the results been disseminated to communities of interest?A presentation was given in the College of Agriculture 'Frontiers' seminar series describing pathology-related aspects of HLB, the rationale behind RNAi and dsRNA biopesticides, and the biological basis for target selection. The research teams (AZ, FL) prepared PPT presentations and reported project progress to the Advisory Committee during our 1.5-day Annual Progress meeting in December 2018. What do you plan to do during the next reporting period to accomplish the goals?Objectives 1,4. We will continue to document the extent of mobility of dsRNA using the probe and confocal microscopy, using selected penetrants (identified in Yr-1), and test the activity by psyllid feeding at different intervals from the delivery point (in planta, laser wounding), over time (persistence). Presently 2 promising dsRNA candidates are being tested in these studies for knock down assessment. Similar experiments will be carried out with respect to penetrants that enhance dsRNA delivery in citrus leaves/seedlings for the very best penetrants identified in tomato experiments. If the compounds found most promising for tomato are equally promising in citrus, the information will be provided to the FL team to begin testing them for laser-delivery of dsRNA by early-summer or sooner. Objective 2. We have initiated and will continue testing best methods for fixation and embedding/sectioning of teneral and immature instar (2-3) psyllids post-dsRNA ingestion (labeled) as an approach to visualize-localize and semi-quantify dsRNA ingested by psyllids. Objective 3. Evaluation will continue to determine the minimum/maximum (saturated) dsRNA concentration required for knockdown. Targets are assessed singly and stacked, over a range of concentrations found for other insects to show strong knockdown potential. Targets are selected based on potential interactions in similar pathways (metabolic, developmental, cellular or cytoskeletal remodeling/perturbations upon CLso/CLas invasion), predicted to be involved in psyllid viability (mortality) or bacterial gut invasion/circulation/salivary gland association. Objective 5. We will continue to select (bioinformatically, literature) candidates beyond what we have identified in previous studies (transcriptomics, proteomics, yeast-2 hybrid and co-IP), and test them. We will continue to evaluate prospective psyllid targets for gene knock down using sucrose-feeding delivery (controlled amount, measurable amounts, ingested) and/or plant root uptake (concentrations required to achieve efficacy observed for sucrose delivery) of dsRNA delivery systems. These experiments comprise four replicated studies with immature instars 2-3 and teneral adult potato psyllids, carried out by two independent teams (two replicates each) and different delivery methods (sucrose or in planta/post-root uptake delivery). The initial assesment of efficacy will be determined by qPCR. Promising targets will be further tested by 'phenotype-bioassays' if knockdown is sufficiently substantial. Two tests will be used for phase II assessment of phenotype: immature and adult dsRNA ingestion (infected and uninfected psyllids) and mortality tabulation, or CLso transmission, post-treatments. This is a massive undertakting, involving all members of the team. Objective 6. We have not carried out profiling of psyllids exposed to dsRNA during this project. However, we are analyzing the results of large and small RNA profiling experiments conducted in the last few months of the NuPsyllid project, and are learning to interpret the patterns. This work is cooperative with Dr. Alex Flynn (U Southern MI), an expert in RNAi mechanisms. We also have done some preliminary testing of polymer-coated dsRNA provided by polymer chemists at USM to assess whether stability and efficacy of dsRNA is improved. So far we have only tested the stability of the polymer-dsRNA complex to withstand selected penetrants with which we have succeeded in increasing the rate of dsRNA penetration into phloem cells (for some, dsRNA enters the xylem as well). Other aspects relevant to objectives 1-6 Virus vectors for dsRNA delivery: The CTVvv vector will not be tested until promising dsRNAs are confirmed and optimized, and then construction can begin (end of Yr-2). Another possible vector, the iRNA (Anne Simon, U-MD) is under consideration for expression of dsRNA hairpins, and experiments are expected to be initiated in several months. The Simon lab has shown that the infectious RNA replicates and accumulates to a high titer in plant phloem (and tomato, others), while not having a pathogenic effect on plants (early work by Weathers, 1960, 1963). It also can be engineered to accomodate a 300-500 bp insertion (so 2-3 hairpins can be stacked if desired). This approach may be more effective than using the CTVvv and/or laser wounding for delivery. We are keeping our options open. Whether the iRNA virus-like molecule occurs in citrus trees in Florida, Arizona, or Texas is not known. The iRNA has been detected in the citrus collection in Riverside CA. A survey of mother stocks in Arizona will be carried out this year, in cooperation of the AZ citrus growers and nursery tree producers in Yuma, AZ. In FL ACP colonies (CLas infected and uninfected) have been established in Immokalee FL, and a qualified post-doc has been hired (February) with experience in molecular biology and manipulation of the CLso/CLas study systems. The colonies will be tested by qPCR to track titer over time in preparation for the initial dsRNA testing, anticipated to begin mid-2019. The experimental/laboratory laser (identical to that purchased for the AZ lab) has been moved to the Qureshi lab from its initial home in Lake Alfred where it was tested and methods optimized during the first year, and optimization of laser wounding was accomplished. The expertise has been passed along to the post-doctoral Associate and he is currently establishing his system using citrus seedlings and ACP, while targets are further explored using the fast track PoP system in AZ. We are in contact with a company that makes a laser for the purpose we have proposed, to deliver dsRNA, and are discussion possible collaborative arrangements to test delivery of dsRNA in citrus seedlings with their equipment and previous knowledge. We are in contact with our formal industry partner, Southern Gardens, and will visit there in April to discuss logistics, capacity, possible experimental designs. We have designed the splash page and made several figures and one fact sheet on RNAi technology/biopesticides and have discussed the website design with the UA-CALS design team. We expect to launch the initial website by mid-summer 2019.
Impacts
What was accomplished under these goals?
Objective 1: The laser and probe systems were ordered / purchased, and our fluorescent microscope was outfitted with a new camera setup for monitoring fluorescently labeled dsRNA. The systems were installed in a dedicated room (signed, red light indictor) and calibrated. Users were certified by UA biosafety trainers. A standardized approach was instituted for design of qPCR primer and probe combinations, and developing a standard curve using internal baseline genes for plants and psyllids. Ten candidate penetrant compounds were obtained from suppliers and evaluated for the ability to deliver dsRNA at different laser settings and different concentrations/volumes, over a range of 100-2000 bps. Mobility was monitored using the probe and by confocal microscopy. Two combinations of penetrants facilitated rapid dsRNA delivery into vascular tissues e.g. abaxial and/or adaxial phloem and/or xylem. These penetrants will be evaluated for dsRNA uptake by citrus seedlings. Objective 2: We were not entirely satisfied with the preliminary approach reported in the proposal, and so decided to return to the drawing board. We initiated testing methods for quantification of dsRNA and visualization in the gut, if this is possible. Approaches initiated involve fixation and embedding/sectioning of teneral adults and immature instars (2-3) post-dsRNA ingestion (labeled) with in situ visualization-localization and semi-quantifiable estimation of dsRNA using labeled probes and fluorescent light microscopy. Confocal imaging is a back-up method however, chitin can be problematic due to autofluorescence. Several dsRNA labeling methods were tested for direct incorporation but these approaches incorporated on a few labels per dsRNA. We are evaluating in situ hybridization to achieve high-resolution detection and visualization ofpsyllid-ingested dsRNA. Objective 3: We have spent a fair amount of effort improving the delivery of dsRNAs to psyllids by modifying our adult feeding system and devising one for immature instar feeding. Oral delivery has been used to test a range of 5 'doses' in replicated experiments with qPCR analysis (4 replicates). Experiments are in progress to test different 'effective' concentrations of dsRNA, with 10 targets tested so far, in combinations of 1, 2, and 3 dsRNAs. After dose is established, time course assays will be carried out to determine efficacy by gene knockdown, followed by mortality or transmission bioassay in accordance with predicted gene function. Objective 4: We have tested several gene targets by applying dsRNAs with penetrants to laser-wounded sites, and allowed psyllids access to feed on different leaves, in relation to delivery site. Current experiments involve removing psyllids from leaves located at different tiers in time course and qPCR analysis. Initial results show that dsRNAs are stable and 'active' in plants e.g. ingestion and gene knock down. Objective 5. Another substantial effort has gone into establishing a multi-step process for the design of dsRNA considering multiple alleles in both PoP and ACP databases, confirming annotation of gene function using the nr and SwissProt databases and KEGG pathway analyses. In the next step possible non-target effects are considered using in silico analysis (BLASTn) to arthropod sequence db. If all specifications are met, gene fragment is cloned, confirmed by sequencing, archived, and used for dsRNA synthesis and primers/probe design for qPCR. Also, gene targets are grouped by pathways or associations, and then tested in groups of three. If stacked dsRNAs show promise based on qPCR knockdown, the candidates proceed to bioassay testing.If promising, individual targets are tested to determine which dsRNAs confer knockdown in CLso uninfected adults and immature 2nd-3rd instars. The best dsRNAs are assessed in planta by laser delivery and tested for efficacy and persistence in tomato/CLso/PoP and in parallel, in the citrus/CLas/ACP system, respectively. Best citrus candidates will be evaluated at Southern Gardens with hand-held or a tractor mounted laser, and cloned into CTVvv and iRNA vectors. Objective 6: We have not initiated experiments yet to profile small RNAs (sRNAs) and 'large' or messenger RNAs (mRNAs) in psyllids because no target is sufficiently validated to do so. However, previously, experiments were carried out to evaluate this approach for characterizing the RNAi pathways in psyllids [NuPsyllid project]. The sRNAs and mRNAs reads were mapped to psyllid transcripts (in-house PoP db) (in progress). Objective 7: Due to unexpectedly heavy commitments, Dr. Hu (co-PI) has stepped off the project. Dr. Qureshi will take responsibility for all extension activities. Dr. Brown will oversee and maintain the project website. Dr. Qureshi's activities in extension and outreach activities have been primarily in Florida this year, related to HLB management practices where he is incorporating information about dsRNA biopesticides for IPM. Dr. Qureshi is organizing an RNAi workshop, inviting speakers to discuss development, testing, commercial use, and regulation of biopesticidal dsRNAs. Participants from AZ, CA, FL, and TX will attend. Similar workshops will be presented during annual or seasonal citrus-related conferences in AZ, CA, and TX during 2020-2021. Brown and Qureshi will present a research/extension project seminar to the Citrus Research & Development Foundation Board in Arcadia, FL in April 23, 2019. The website splash page has been designed and draft fact sheets/figures discussing RNAi technology/biopesticides have been prepared. The design has been discussed with the UA-CALS website team, and website is slated to be launched by mid-summer 2019.
Publications
- Type:
Other
Status:
Accepted
Year Published:
2018
Citation:
Circulative, propagative transmission up close: Ca. Liberibacter spp. exploits endo/exocytosis and cytoskeleon remodeling pathways in the psyllid vector. CALS Frontiers in Life Science Research Seminar, University of Arizona, Tucson, AZ. April 13, 2018.
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