Progress 02/01/18 to 01/31/19
Outputs
Target Audience:During this reporting period, we have met with citrus stakeholdersthat will be served by the information obtained from thisproject. We participated in six extension and outreach events to shareknowledge with the citrus commuity at formally-organized events. The audience at the events included citus growers, citrus nurseris,industry leaders, members of the Citrus Reasearch and Development Foundation (FL) and California Citrus Mutual, and fellow citrus researchers. Several extension talks were given at formal extension and eduction events. These included: Pelz-Stelinski, K. S. 2018.Antimicrobial strategies for controllingCLas and the Asian citrus psyllid. 27thAnnual Citrus Expo. 15-16 August, Ft. Myers, FL. Pelz-Stelinski, K. S. 2018.Developing new bactericide technologies. Growers Education Session. 28 August, Sebring, FL. Pelz-Stelinski, K. S. 2019.Advances in suppression of the Asian Citrus Psyllid Vector and Bacteria (Huanglongbing Pathosystem).Florida Citrus Show. 23-24 January, Ft. Pierce, FL. Several surveys were conducted to assess stakeholder knowledge and interest in this project. THe first wasconducted on April 3, 2018 at the Florida Citrus Grower's Institute in Avon Park FL. This is the largest grower meeting hosted annually by University of Florida, IFAS Extension and the Citrus Research & Development Foundation For Florida citrus growers. The overall attendance of this meeting was historically low and we were only able to receive 38 survey respondents during this event.Another survey was conducted at the International Citrus Business Conference during March 27-28 in Daytona Beech, Florida. The goal here was to obtain information from the processing and business sector of the industry. We obtained 56 responses to this survey.A final survey near project completion was conducted to measure possible changes in knowledge and perception of antimicrobial use among clientele in response to our outreach efforts during the project and in response to the changing industry needs. The survey was conducted during the Citrus Expo program on 15-16 August, 2018 at the Lee Civic Center in North Ft. Meyers, FL. In total, we were successful in collecting data from 43 respondents. Changes/Problems: We received a six monthno cost extension for this project in July 2018.Dr. Altman was struck with a severe illness; which limited his participation in the project. Dr. Altman's postdoctoral scientist,Dr. Carlos Angele, pursued the objectives of identifying PPMO penetration into cells. As previously reported, wetransferred remaining funds from Dr. Altman's program to UF to facilitate completion of work verifying the efficacy of PPMOs in insect cell lines. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Development of the web site, "Science for Citrus Health" has continued. It went live as a UC ANR web site in May 2015,http://ucanr.edu/sites/scienceforcitrushealth/; it has had over 2800 visits to date. The web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease.Peggy Lemaux (UC Berkeley), Beth Grafton-Cardwell (UC Riverside) and Lukasz Stelinski (U Florida) meet monthly to collaborate on the website. Postcards announcing the web site have been distributed to citrus growers at meetings in Florida and California. The web site has a section called research snapshots that describe approaches that are currently being pursued nationwide to manage HLB (both engineered and non-engineered approaches).Our team has worked with researchers to write up descriptions of their research strategies and accomplishments and we then translate that material into language that can be understood by the general public. These Research Snapshots are divided into four new technologies categories, Early Detection Techniques (5 entries), Established Orchard solutions (7 entries), Replant solutions (6 entries) and psyllid control tactics (4 entries). In addition, we have a section called Existing Tools (2 entries) for currently applied technologies.The antimicrobial technology for HLB management snapshots are presented in both the established orchards and psyllid sections because several delivery methods are being considered.Each of the snapshots is written in a form that can be used as a fact sheet to be passed out at extension events and utilized by the media. To date, there have been over 1000 downloads of the snapshots, with most of the individual snapshots reaching more than 100 downloads each. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. The objective of this project is to develop antisense oligonucleotide gene-silencing technologies to reduce CLas in plants and to selectively eliminate CLas or symbionts in ACP. Targeted manipulation of bacteria and ACP provides a unique opportunity for vector/pathogen management because these synthetic molecules are stable and specific, reducing the potential for non-target effects. Multiple genes can be targeted, reducing the potential for resistance. Two antisense oligonucleotide technologies, PPMOs and FANAs, were evaluated in 2018. 2. In 2018, Altman's group (Yale) demonstrated that the EGS-PPMO technology can be used effectively to target bacteria in insects without the development of resistance. This confirmsthat EGS-PPMO can penetrate into insect cells that house either CLas or endosymbiotic bacteria (Objective 1).Pelz-Stelinski's group (UF) andHunter's group (USDA)confirmed that the EGS-PPMO and FANseffectively silence essential genes and reduce populations of CLas in plants (Citrus and non-ACP host, Periwinkle) (Objective 1) and in ACP (Objective 2.1), and that antisense FANAs are more effective than streptomycin at reducing CLas in plants. Reduced inoculation of CLas in citrus was observed when ACP were treated with antisense molecules in an artificial diet prior to feeding on healthy citrus. Pelz-Stelinski also demonstrated the reduction of ACP symbionts (Objective 2.2) after feeding on citrus treated with specific antisense oligonucleotides. Ingestion of these molecules reduced ACP feeding and survival. 3. We have produced evidence supporting the use of antisense oligonucleotides for use in the management of the HLB pathosystem (insect vectors and pathogen). A field component was not within the scope of work approved for this project; however, we demonstrated the efficacy of these synthetic molecules in cell culture, insects, and in greenhouse assays. The results indicate that CLas can be reduced in plants more effectively with PPMO or FANA oligonucleotides than with the antibiotic currently registered for use in citrus to reduce CLas infections. The ability to design antisense molecules that are specific to bacteria renders thisapproach flexible for agricultural pathosystems. Objective 1. 2018, Altman's group (Yale) investigated thedesign of External Guide Sequences (EGS), which guide PPMO molecules into bacterial cells, that base pair with essential mRNA and resembles the minimum RNA structure requirements to be recognized and cleaved by RNase P is a valuable tool to prevent the proliferation of antibiotic-resistant bacteria.RNase P of cleavage ptRNATyrand DptRNAArgwas observed, demonstrating the versatility of the ribozyme to cleave different substrates that have similar structure and fulfill the minimal structural requirements to be recognized by the ribozyme. . Preliminary results evidence the effectiveness of M1RNA to cleave other RNA substrates that may derive in the cleavage of messenger RNA of essential genes. The interruption of essential genes in pathogens may reduce the cell viability.Together results validate the versatility of the reaction for use with ACP endosymbiont cell cultures. This means that the EGS-PPMO technology can be used effectively to target bacteria in insects without the development of resistance. The efficacy of PPMOs in suppressingCLas titer inCitrusplantswas tested by delivering the oligos through root infusions into trees harboring the bacterium. Plantsplants treated with the PPMOs showed a reduction inCLas titer compared to controls. Two days post-treatment, whereas water-infused and streptomycin-infused trees had 44.2 and 27.5% increase inCLas titer, the trees exposed to PWgyrA-14 and CLgyrA-14 decreasedCLas density by 20.2 and 31.3%, respectively; seven days post-treatment, CLgyrA-14-treated plants loweredCLas titer by 40%, whereas plants infused with water or streptomycin increasedCLas titer by 37.4 and 50%, correspondingly. DeliveringLigAFANA through root infusion toCLas-infected trees, significantly reduced the quantity of the bacterium. Treatments effectswere expressed as percentage change inCLas titer. Supression was persistent during the 30 day sampling period, with the highest Liberibacter repression observed two and seven days post treatment (81% and 51.6%, respectively). Root infusion application of streptomycin sulfate into infectedCitrusplants did not significantly reduced theCLas titer.Citrusplants infused with water significantly increased theirCLas quantity by 67% and 89% after 14 and 30 days, respectively. Objective 2. DigyrAtranscripts were significantly reduced by 68% when S2-wDi cells were incubated with PWgyrA-14, compared to untreated cells (U= 9.0, n1=n2= 9,p= 0.006). CLgyrA-14 reduced the expression ofwDigyrAby 55% when contrasted to its expression in non-treated cells (two sample K-S test,p<0.05).There was a significant difference in the density of viable wDi between the treatments (F[3, 8]= 997.1,p<0.0001). Compared to untreatedWolbachiacells, both PPMO treatments greatly reduced the number of viable wDi cells. The results suggested that the RNase P-mediated repression of the DNA gyrase subunit A gene is lethal for theWolbachiacells. A 70% repression of theCLasgyrAtranscript was observed when adult psyllids where fed the artificial diet containing CLgyrA-14, compared to untreated psyllids (t(16) = -2.638,p=0.018). There was no significant difference in the amount ofCLasgyrAmRNA, between psyllids that were fed PWgyrA-14 and untreated psyllids (t(16) = 1.226,p=0.238) The bactericidal effect of PPMOs was tested by quantifyingCLas relative density inD. citriaduts, after feeding psyllids with a solution containingPWgyrA-14 or CLgyrA-14 for seven days. Dunnett's test showed a significant 82% reduction ofCLas titer in adult psyllids that were treated with CLgyrA-14, compared to untreated insects. No difference inCLas density was observed betweenD. citriadults exposed to the PWgyrA-14 and untreated psyllids. The results showed that degrading theCLasgyrAmRNA was detrimental for the bacterium titer insideD. citri. This study demonstrated the efficacy of FANA ASOs in reducing the amount of bacterial mRNA, in bothin vitroandin vivoexperiments, causing a deleterious effect in bacterial density.D. citriadults were fed an artificial diet containingLigA­-FANA, at a concentration that significantly reduced the expression of theCLasLigAgene. Seven days post-treatment, quantitative PCR was used to estimateCLas copy number in the psyllids, by targeting the bacterium 16S rRNA gene.CLas density was significantly lower in the psyllids that ingestedLigA-FANA. The results showed that degrading theCLasLigAmRNA was detrimental for the bacterium density. 4) These results evidenced the potential of FANA oligos as an environment-friendly alternative for the management of HLB, different than RNAi. Both FANA ASOs and RNAi are post-transcriptional gene silencing technologies, that involve the binding of complementary oligonucleotides to target RNA through base paring. However, there are intrinsic characteristics of FANA ASOs that may position them as a better choice than RNAi for controlling HLB. The clientele support for genetic engineering andantimicrobial technologies as tools used in current citrus production practices did not change between 2017 and 2018 surveys. Overall, "genetic engineering" as a mode of citrus crop protection from pests and diseases appears slightly more appealing to clientele than more specified use ofantimicrobial technology for disease management. Grower knowledge in areas of antimicrobial technology appeared to have increased as research and development of these technologies progressed over the course of this project.
Publications
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Progress 02/01/16 to 01/31/19
Outputs
Target Audience:The target audience were citrus growers throughout the United States, the agro-chemical industry foucused on citrus porduction, and consumers of citrus products. Four survey replicates have been completed in Florida that measure citrus grower knowledge and perception of antimicrobial and genetic engineering technologies. These surveys have also identified certain grower concerns and areas that may require focus during education efforts. Furthermore, a specific and complementary survey has been conducted within the Florida citrus juice and beverage industry. Outreach has also been established through the Science for Citrus Health website highlighting the initiation of this project and what it may bring to fruition. An initial grower survey was conducted to measure respondent knowledge of antimicrobial technology application for HLB management. The survey was conducted during the Citrus Expo program on 15-16 August, 2017 at the Lee Civic Center in North Ft. Meyers, FL. In total, we were successful in collecting data from 112 respondents. A follow-up second replicate of survey 1 was conducted on April 3, 2018 at the Florida Citrus Grower's Institute in Avon Park FL. This is the largest grower meeting hosted annually by University of Florida, IFAS Extension and the Citrus Research & Development Foundation For Florida citrus growers. The overall attendance of this meeting was historically low and we were only able to receive 38 survey respondents during this event. A third survey was conducted at the International Citrus Business Conference during March 27-28 in Daytona Beech, Florida. The goal here was to obtain information from the processing and business sector of the industry. We obtained 56 responses to this survey. A final survey near project completion was conducted to measure possible changes in knowledge and perception of antimicrobial use among clientele in response to our outreach efforts during the project and in response to the changing industry needs. The survey was conducted during the Citrus Expo program on 15-16 August, 2018 at the Lee Civic Center in North Ft. Meyers, FL. In total, we were successful in collecting data from 43 respondents. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?i) Science for Citrus Health Web Site Development of the web site, "Science for Citrus Health" has continued. It went live as a UC ANR web site in May 2015,http://ucanr.edu/sites/scienceforcitrushealth/; it has had over 2800 visits to date. The web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease.Peggy Lemaux (UC Berkeley), Beth Grafton-Cardwell (UC Riverside) and Lukasz Stelinski (U Florida) meet monthly to collaborate on the website. Postcards announcing the web site have been distributed to citrus growers at meetings in Florida and California. ii) Research Snapshots The web site has a section called research snapshots that describe approaches that are currently being pursued nationwide to manage HLB (both engineered and non-engineered approaches).Our team has worked with researchers to write up descriptions of their research strategies and accomplishments and we then translate that material into language that can be understood by the general public. These Research Snapshots are divided into four new technologies categories, Early Detection Techniques (5 entries), Established Orchard solutions (7 entries), Replant solutions (6 entries) and psyllid control tactics (4 entries). In addition, we have a section called Existing Tools (2 entries) for currently applied technologies.The antimicrobial technology for HLB management snapshots are presented in both the established orchards and psyllid sections because several delivery methods are being considered.Each of the snapshots is written in a form that can be used as a fact sheet to be passed out at extension events and utilized by the media. To date, there have been over 1000 downloads of the snapshots, with most of the individual snapshots reaching more than 100 downloads each. iii) Presentations Presentations were made by Stelinski at the UF IFAS Citrus Nursery Workshop, Citrus Growers' Workshop, Citrus Expo, Citrus Pest Management In Service Training # 31517, and Citrus Growers' Institute reaching approximately 1,200 people. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. ProblemHLB is currently devastating the FL citrus industry, with potential to do similar damage in TX and CA. The impacts of this are felt broadly across the industry, including the fresh fruit, juice and processing markets, as well as citrus nurseries and groves.Peptide conjugated morpholinos (PPMOs) and related FANA oligonucleotides have been usedas bactericidal agents andmay represent a viable strategy for controlling HLB.PPMOs and FANAs can be designed to target any bacterial pathogen while leaving endemic microbiota unaffected. In addition, no evidence exists that suggest that pathogens can evolve to resist these oligonucleotides.2. ResultsPelz-Stelinski determined that PPMO and FANA oligonucleotides could be used to silence genes associate with CLas and symbionts in ACP andCLas inhost plants, leading to significantlyreducedbacterial titers and increased insect mortality.Huntervalidated the results of the Wolbachia assays reported by Pelz-Stelinski. In addition, he developed a method to rapidly screen for antisense targets and evaluated methods forimproving delivery of FANAs to citrus trees to improve efficacy of product on ACP or their endosymbiotic microbes.The results demonstrated delivery of all tested molecules into multiple citrus varieties with no significant difference (cuttings or whole seedlings) and into the psyllids that feed on these treated plants or cuttings.A greater concentration was delivered by soil applications than topically applied sprays. Altman's group conducted the synthesis, purification, and radiolabeling of two different ptRNA targeting Wolbachia, as well as the results of RNAse P cleavage reaction of them. They demonstrated the versatility of the reaction since cleavage is observed in both ptRNA structures analyzed and by experiments done with M1RNA or with S30 extract from S2 cells. Singerman conducted cost benefit analysisof FANA and PPMO technologies. Given that we have no data on yield response, as this objective was eliminated from the project, to the bactericide treatment, we determined estimates for breakeven prices and yield. Thetotal cost of production for processed oranges grown in Florida using the proposed morpholino-based bactericides is currently prohibitive, butdoes not consider the likely reduction in processing cost per gram expected from scale-up of oligonucleotide production.3. Broader Outcomes. The PPMO and FANA technologies, previously unexplored for agricultural use, show clear promise fordelivery of anti-pathogen gene silencing that can reducethe HLB pathogen, CLas, or the microbiota of its insect vector. This should facilitate decreased use of insecticides for disease and pathogen management and provide a tool for reducing disease progression and new pathogen transmission by reducing the prevalence of CLas in citrus trees.4. Project ObjectivesObjective 1:Development of morpholino (PPMO)-EGS to disruptCLasin Citrus plants.4.1.1. Effect of PPMOs onCLasinCitrusplants.Root infusion.CLas infected trees were treated with the PPMOs by root infusions and compared to streptomycinor water. TheCLas titer was reduced in plants treated with the PPMOs compared to controls. Two days post-treatment, water-infused and streptomycin-infused trees had 44.2 and 27.5% increase inCLas titer, whereas the trees exposed to PWgyrA-14 and CLgyrA-14 decreasedCLas density by 20.2 and 31.3%, respectively; seven days post-treatment, CLgyrA-14-treated plants loweredCLas titer by 40%, while water or streptomycin increasedCLas titer by 37.4 and 50%. Confocal microscopy of leaves from PPMO treated cuttings had significantly more signal than leaves and petioles sampled from the control cuttings. Deep scan of cleared leaf tissue revealed systemic delivery of PPMO into cells.4.1.2. Effect of FANA onCLasinplants. DeliveringLigAFANA through root infusion toCLas-infectedCitrustrees significantly reduced the quantity of the bacterium.Suppression persisted for the 30 d sampling period, with the highest CLas suppression at 2 and 4 d post treatment (81% and 51.6%, respectively).4.1.3. Improve deliveryinto citrus trees. Confocal microscopy indicated that tagged PPMO were successfully delivered into citrus cuttings and ACP. Significantly more material was delivered to immature leaves compared to mature leaves. Significantly more material was imbibed by immature ACP than adult ACP. In a third experiment, a surfactant improved delivery to citrus leaves.Key Outcomes:Delivery ofLigAFANA to infectedCitrusthrough root infusions significantly reducedCLas titer for at least 30 days. Being able to decrease the bacterial pathogen quantity in both its host and vector, demonstrates the potential of FANAs for the management of HLB as an alternative to antibiotics. While similar effects were observed for PPMOs, the effects were not as long-lasting.Hunter demonstrated successful delivery of all tested molecules into multiple mature citrus varieties with no significant difference and into the psyllids that feed on these treated plants or cuttings.Greater concentrations of FANAs were delivered by soil applications, than topical sprays. Objective 2: Development of morpholino (PPMO)targetingCLas and bacterial symbionts in ACP.4.2.1.Cleavage Activity of RNAse PCleavage of *ptRNATyrwas assayed with S30 fraction of S2 cells, this experiment demonstrated the presence of RNase P in the extract and that the extract can cleave the substrate as it was done with M1RNA and reconstituted RNase P.4.3.2 Cell culture assays.wDi gyrA PPMO and gyrA-FANA mRNA reduced expression and affected the viability of wDi cellsWe assessed the bactericidal capabilities of PPMOs and FANA ASOs, usingin vitroandin vivoassays, by (1)confirming their capacity to penetrate insect cells, (2) silence bacterial essential genes and (3) evaluating their effect in bacterial viability or bacterial titer. To test the effect of PPMOs on insect symbiont cultures, S2 cells infected withWolbachiafromD. citri(wDi) were incubated with a 5µM PPMO solution for 7d. The wDigyrAtranscript level was significantly reduced by 68% when S2-wDi cells were incubated with PWgyrA-14, compared to untreated cells (U= 9.0, n1=n2= 9,p= 0.006). CLgyrA-14 reduced the expression ofwDigyrAby 55% compared to non-treated cells (p<0.05). ThegyrA-FANA treatment significantly reduced (30%) the amount of the target mRNA [t(16) = -2.60,p=0.019]). The wDigyrAtranscript level was significantly lower in the cells exposed togyrA-FANA, compared to cells treated with the scramble control [t(16) = 2.98,p=0.009].4.2.3 Psyllid assays. A 70% repression of theCLasgyrAtranscript was observed when adult psyllids where fed the artificial diet containing CLgyrA-14, compared to untreated psyllids (t(16) = -2.638,p=0.018). There was no significant difference in the amount ofCLasgyrAmRNA, between psyllids fed PWgyrA-14 and untreated psyllids (t(16) = 1.226,p=0.238).CLas density was significantly lower in the psyllids that ingestedLigA-FANA, compared to untreated psyllids or insects fed with the scramble control-FANA.To test the capacity of PPMOs in reducingCLas transmission, infected psyllids were treated with CLgyrA-14 and PWgyrA-14. There was a significant difference in the distribution of bacterium titer between the treatments (KW-H (3,180)= 7.66,p= 0.05). Psyllids that were treated with CLgyrA-14 were less successful in transmitting the pathogen into citrus plants, compared to untreated insects (U= 745, n1=n2= 45,p= 0.031). Psyllids that were treated withLigA-FANA were less successful in transmitting the pathogen into citrus plants, compared to untreated insects (U= 720, n1=n2= 45,p= 0.018), with significantly lessCLas detected in the plants exposed to FANA-treated psyllids.Key Outcomes:We demonstrated that this technology was successfully delivered into cells or tissues, reduced the amount of specific bacterial transcripts and had a deleterious effect in bacterial density inside the HLB vector and host. ?
Publications
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience: The target audience includes citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production, and consumers of citrus products. We engaged with the target audience through extension and outreach presentations at industry meetings and development of an informational website. Changes/Problems:As reported in our last annual report, participaion by one of the project PIs, Dr. Altman, has been significantly reduced due to illness. This has reduced the expenditures from his laboratory significantly. We have compensated for this by obtaining oligonucleotides in collaboration with Genetools and AUMBiotech. In addition to conjugating oligonucleotides for the project, Dr. Altman's group was tasked with evaluating the uptake efficiency of PPMOs into bacterial cells. Progress has been on this objective, but has been delayed significantly due to his absence. It is not expected that this will ultimately effect the overall goal of the project, as we have continued to screen oligonucleotides in plants and insects in parallel with collection of the kinetic cellular uptake data. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The results are being disseminated to stakeholders throught the citrus health website. Following the imminent filing of a provisional patent, the results of assays will be disseminated to growers through presentations at citrus extension meetings and in articles in Citrus Industry magazine. What do you plan to do during the next reporting period to accomplish the goals?No changes to the proposed goals are anticipated. We expect to focus more effort toward increasing the efficiency of the most effective molecules, particularly several FANA-ASOs, which have shown the most demonstrable efficacy against plant CLas infection. The goal for the next reporting period is to continue to optimize delivery of CLas-targeting oligonucleotides to plants. The next step, not included in the current scope of work, will be scaling up to evaluate the products in a large-scale greenhouse and/or field trial.
Impacts
What was accomplished under these goals?
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We have developed new methods for screening FANA and ASO oligonucleotides, and evaluated the efficiency of uptake for these molecules in vivo, in vitro, and in planta. Several oligonucleotides have been effective in suppressing CLas in Citrus plants and the insect vector; as well as,the Diaphorina citri endosymbionts Wolbachia and Proftella armature. Several key findings include: 1)Both RNA-based bactericidal agents successfully penetrate into cultured insect cells without the need for transfection reagents, 2) FANAs and PPMOs oligonucleotides silenced CLas target genes in ACP feeding bioassays, and 3) CLas bacterial density was negatively affected by both FANAs and PPMOs. Cumulatively, these findings are expected to have an significant impact on ACP and CLas management. The oligonucleotides are targeted bactericides, which means that they will not bind to off-target bacteria. Up to 60% reduction of CLas was observed in our experiments; this reduction is higher than CLas reduction observed in response to broad-spectrum antimicrobial products currently approved for use in citrus. Objective 1. Progress has been made in optimizing cellular uptake of PPMOs in target cells. The goal is to increase the versatility of RNAse P to cleave RNA that resemble precursor transfer RNA in order to knock down target bacterial populations in citrus plants and the insect vector. The design of External Guide Sequences (EGS) that will base pair with essential mRNA and resembles the minimum RNA structure requirements to be recognized and cleaved by RNase P is a valuable tool to prevent the proliferation of antibiotic-resistant bacteria. Because of the high specificity, EGS technology reduces the risk of side effects in the host cells but also will attack specifically to the desired species. Objective 2. Delivery to Insects (Pelz-Stelinski and Hunter).We assessed the bactericidal capabilities of FANA antisense oligos (FANA) and Peptide conjugated morpholinos (PPMOs) by (1) confirming their capacity to penetrate insect cells, (2) confirming that oligonucleotides silence bacterial essential genes in both cell culture and inside adult ACPs and (3) evaluating their effect in bacterial viability or relative density. Fluorescence microscopy demonstrated that both FANA and PPMO transfected into insect cells 24 hours post treatment, without any delivery agent. A fluorescently labeled FANA was detected in ACPs digestive tracts after feeding from previously treated citrus leaves. Incubating either a Wolbachia-specific FANA or a PPMO with insect cells containing ACP Wolbachia (wDi), significantly reduced the expression of a wDi essential gene by 30% and 55%, respectively. The viability of isolated wDi cells was greatly reduced when treated with either the Wolbachia-specific FANA or the PPMO, compared to untreated cells. Oral delivery by an artificial diet of a CLas-specific FANA or a PPMO, significantly silenced the expression of two CLas essential genes by 33% and 77%, respectively, in adult ACPs. Delivery of the CLas-specific FANA through excised citrus leaves, that take up the oligo for 24 hrs, was also effective in suppressing the target transcript in psyllids (60% reduction). CLas relative density was significantly lower in the ACPs fed with either the CLas-specific FANA or the PPMO, compared to untreated insects. Objective 2. Delivery to Plants (Hunter and Pelz-Stelinski). Experiment 1: Determine if FANA ASOs are effectively delivered to plants through new citrus growth absorption ('in planta assay'). Oligonucleotides were systemically delivered to plant tissues using the 'in planta' bioassay. Results were determined by confocal microscopy of leaf and petiole tissues. Confocal microscopy of leaves from treated cuttings had significantly more signal than leaves and petioles sampled from the control cuttings. Deep scan of cleared leaf tissue revealed systemic delivery of PPMO into cells. Experiment 2. Determine if PPMOs are systemically delivered to plant tissue when roots are immersed in PPMO treated DI water. Treatments were plied to root tips that were either cut or uncut. PPMOs were successfully delivered to the upper leaves of citrus seedlings using naked root immersion methods. Fluorescently labeled PPMOs were detected by microplate reader in leaf tissue collected from PPMO treated seedlings 12d post treatment; however, PPMOs were not detected 7d post treatment. Experiment 3. Determine if PPMOs are systemically delivered to plant tissue when applied as a soil drench. PPMOs were successfully delivered systemically to potted plants when applied as a soil drench. Upper leaves sampled from PPMO treated plants had significantly higher microplate readings compared to leaves sampled from the water treated Murraya paniculata. Experiment 4. Determine if PPMOs are systemically delivered to plant tissue when applied as a foliar spray to the lower leaves of a citrus plant and detected in untreated leaves of upper tree canopy. PPMO fluorescent probe was only detected on surface of treated leaves in initial trials. This was due to the solution drying too fast for the plant leaves to absorb much of the PPMO. Even with repeated wetting, there was no significant delivery of the PPMO using the topical spray method. Next, surfactant was added to the treated solution which could assist PPMO absorption into citrus leaves. This final method produced successful delivery and detection of fluorescently labeled PPMOs in treated leaves. Experiment 5. Determine if PPMOs can be systemically delivered to plants by amending soil with PPMO treated granular clay absorbents. Results: PPMOs were detected in leaves of citrus seedling treated with PPMO+clay amended soil 90 to 95d post treatment. Clay absorbent potted plants had consistently greater detection values for PPMO over time, than the control (water only) treatment leaves. Experiment 6. The objective of this experiment was to determine if PPMOs and FANA ASOs targeting CLas are effective in suppressing plant pathogenic bacterium, CLas. In this experiment, two injections were made on replicate trees, one month apart. Infected citrus was injected with water, PPMO, or a mix of four FANA oligonucleotides (CLIBASIA). The percentage reduction of live CLas was significantly reduced in plants treated with PPMO (76%) and FANA (65.5%) after 30d post-treatment. Subsequent sampling at two and seven months post treatment indicated that the percentage of live CLas increases over time, following the initial decline in CLas. There was high variability among the CT values in response to treatments, indicating a need to develop additional, consistent methods for validating efficacy. Dr. Hunter has performed binding efficiency assays to determine whether an in vitro assay can be used to pre-screen efficacy of potential PPMO and FANA oligos. Confocal microscopy and microplate spectrometry show successful delivery of PPMOs in both plants and insects; however, target binding efficiency and activity against bacteria post treatment or ingestion is not indicated by these tests. This data is confirmation that PPMOs and FANA are successfully suppressing the gene target and may be used to indicate if the treatment dose should be increased or if a mix or two targets may be more successful. Binding efficiency of PPMO's have been shown to be reduced with the increase in the number of single nucleotide polymorphisms, SNPs, in the target sequence. Increased binding of PPMO to target bacteria means less available target. Quantitative qPCR analyses detecting indicatedh significantly greater increases in Ct values among PPMO oligonucleotide treated samples (CLas Ct=36, Wolbachia Ct=40, Proftella Ct=28) compared with untreated controls (CLas Ct=26, Wolbachia Ct=22, Proftella Ct = 17).
Publications
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:The target audience is citrus growers throughout the United States (Florida, Texas, and California), the agro-chemical industry focused on citrus production, and consumers of citrus products. Changes/Problems:At the time this was started, Dr. Altman was struck with a severe illness and has spent the past six months in hospitals and rehabilitation centers. This has limited planned communication and spending with Dr. Altman's program. We have been able to work around this by utilizing Genetools and AUM Lifetech for synthesisof PPMO and FANA molecuyles, respectively, and through communication with Dr. Altman's postdoctoral scientist (Dr. Angele). Thus, we remain on track with respect to the goals of the project. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Through this NIFA-sponsored project, and RNAi-based strategy projects, we are developing extension and education programs for growers and end-users, which address the use of genetic tools to lessen the impact of HLB on citrus. We developed an extension outreach plan that enhances existing extension educator programming in citrus. We have three primary outreach approaches. (i) Development of Powerpoint Presentations, which can be tailored to specific venues and locations. Presentations will provide a general education on the genetic methods used to modify plants, viruses, and insects and cover state and national regulatory requirements and consumer attitudes. To bring the latest information on the various approaches to combat citrus greening, we will work directly with researchers involved in these efforts to obtain detailed information on precisely what and how they are being done and the current status of their efforts. (ii) Creation of Fact Sheets that cover general topics of classical and modern methods of genetic modification and how these are being used to modify various crop plants. Another will cover how genetic engineering and other mitigation approaches can be used to address the citrus problem. (iii) Citrus outreach website (http://ucanr.edu/sites/scienceforcitrushealth/ ), which is specifically designed to provide grower information on huanglongbing/citrus greening and genetic strategies for control and detection. It includes the following sections: About Us, Background, Research, Outreach, News and Links. What do you plan to do during the next reporting period to accomplish the goals?Our first goal, which is only partly finished is to show that, using PPMOs (RNase P) and FANA ASOs, we can stop the growth of CLas and ACP endosymbionts. We have ordered small scale PPMOs to enable Dr. Angele (postdoc in Dr. Altman's laboratory) to validate inhibition of bacterial growth on a small scale in the laboratory. Small-scale experiments will indicate that nucleotide matching exactly in the sequence related to the E. coli gyr sequence on bacteria with up to three mismatches in the gyr sequence. We have also ordered FANA and PPMO with novel gene targets for evaluation in a second round of bioassays. The next phase of our project will be to optimize the concentration and specificity of the molecules evaluated during 2016, and replicate results of the feeding assays and small tree trials. Specifically we will: Increase the concentration of the PPMO and FANA oligos in ACP feeding assays. Increase exposure time of the wDi cells to FANA oligos. Repeat the experiment with a scramble control-PPMO to confirm that changes in gene expression is due to the action of RNase-P. Test different delivery method of the PPMO into the psyllids (injection, cut plant feeding bioassay). Complete longevity and transmission bioassays with ACP Large scale PPMOs and FANAs vetted during the previous year have been ordered to facilitate large-scale plant and insect bioassays, including assays to evaluate inhibition of CLas transmission by psyllids.
Impacts
What was accomplished under these goals?
This project proposes to eliminate huanglongbing (HLB) using a novel, gene-based bacterial therapy strategy that targets the HLB pathogen and Asian citrus psyllid symbionts. In this study, we successfully tested the delivery into cell lines, plants, and insects (Hemiptera) with two RNA silencing and regulation products. Specifically, we used self-delivering FANA antisense oligonucleotides (FANA ASOs) that knock down the target mRNA by cleaving the target mRNA (after RNase H mediated cleavage of the mRNA target FANA ASOs are instantaneously recycled to target other copy numbers of the target and thus can target multiple copy numbers) and morpholino oligonucleotides (PPMO) that use a cell penetrating peptide conjugate for deliveryand acts as translational blockers (since morpholinos are independent of RNase H mechanism, one morpholino can only target one copy number of a gene). FANA ASOs provide a superior RNA silencing alternative to existing RNAi strategies that have low efficacy, big delivery challenges and high manufacturing costs. Further, FANA ASOs are known to be non-toxic, are highly stable and provide a highly target specific gene knockdown. The capability of FANA ASOs to efficiently self-deliver without the use of any formulation, conjugate or carrier, in contrast with conjugated PPMOs, make them very attractive for topical spray applications. Topical sprays onto citrus leaves, root absorption, and tree trunk injections using FANA ASOs and PPMOs were evaluated. Both FANA ASOs and PPMOs were designed against a range of targets, including the Asian citrus psyllid (ACP), ACP endosymbionts (Wolbachia and Proftella armature), and the citrus greening pathogen,CandidatusLiberibacter asiaticus (CLas). We observed successful increase in insect mortality and reduced bacteria within the insects and/or citrus trees. This is the first evidence for successful delivery of FANA ASOs and PPMO by plant delivered targeting strategies to manage agricultural arthropod pests and plant pathogens. Overview: During the past year, PPMOs and FANAs targeting CLas and the ACP symbionts, Proftella armature and Wolbachia, were designed and synthesized (objective 1) for subsequent screening bioassays. Screening of molecules was conducted in cell line bioassays (Wolbachia), psyllid artificial feeding assays (Wolbachia and ACP targets), small plant cutting assays, and whole plant assays (objective 2). Obj. 1 (Altman) At Yale, gene targets were examined and used to develop suitable PPMO products targeting CLas, Proftella, and Wolbachia, using the methods outlined in the original research proposal. The last step of synthesis of the conjugate, joining the CPP with the MO to produce a PPMO construct was conducted in collaboration with Genetools). Synthesis and distribution of the PPMOs occurred during the initial six months of the project. The main part of the work was recruiting Dr. Hong Moulton (Genetools) to make PPMOs against selected bacteria (CLas--Candidatus Liberibacter asiaticus; ACP and an ACP Wolbachia strain) on a large scale for testing. This was done and we succeeded in showing that specific bacteria with a fluorescent nucleotide at one end (CLas) of a PPMO could be detected in an infected sapling (see below). This important result opened the door for specific cleavage of the bacteria and the demonstration that our method of inactivating bacteria in infected saplings was possible. Experiments with Wolbachia have been initiated to validate protocols for Wolbachia cell line growth. Dr. Pelz-Stelinski has supplied all the necessary details for cell growth and culture. In addition, design and synthesis of PPMOs and FANAs with target sequences with greater homology to CLas and Wolbachia has been initiated. These products should be available for small scale bacterial growth and psyllid fitness bioassays by late February 2017. Obj. 2 (Pelz-Stelinski and Hunter): Relative quantification in cell culture. PPMOs with the External Guide Sequence (EGS) targeting an endosymbiont gene repressed the expression of a Wolbachia (wDi) DNA gene in cell culture. As previously demonstrated, the EGS is able to associate to the target mRNA if it has three or less mismatches. The downregulation was observed only after seven days of exposure regardless of the concentration of PPMO used. It seems that the PPMO needs more than three days to penetrate the membrane of the insect cells, before it can permeate the bacterial cell membrane. PPMO suppression of the wDi gene after seven days: 1 µM: 1.96-fold; 5 µM: 3.45-fold; 10 µM: 2.4-fold. The PPMO significantly reduced the density of wDi in S2 cells, compared with the control treatment. PPMO concentrations of 1 and 5 µM lower wDi cell density after 24 and 72 h, respectively. After seven days of exposure to the PPMO the wDi cell density decreased in a dose-response manner. ALEXA FLUOR 647-tagged control FANA oligos were incubated with Wolbachia cell lines to evaluate uptake during 24 h of exposure. No transfection agent was required for the incorporation of the oligos into the insect cells. Microcopy imaging confirmed that the oligonucleotides successfully invaded both the host cell line nucleus and the Wolbachia chromosome. These results confirmed that FANA molecules could successfully target intracellular bacteria. Subsequently, five FANA oligos targeting Wolbachia (wDi) genes were evaluated in cell culture bioassays. Four of the five FANA oligos significantly repressed the expression of the wDi gene in cell culture after 24 (FANA-2) or 48 h of exposure (FANA-3, FANA-4, FANA-5). The oligo FANA-3 seems promising: FANA-3 significantly reduced the expression of a wDi gene by 1.9-fold after two days of exposure and increased its suppression by 3-fold by the third day. PPMO/FANA treatments targeting endosymbiont of Asian Citrus Psyllid, Candidatus Profftella armature. In addition to endosymbiont cell line bioassays, we evaluated FANA oligos specifically targeting ACP to determine whether these molecules could be successful administered to insects in feeding assays. Expression of an ACP gene was significantly reduced (1.3 and 1.8 fold, respectively) when insects were fed on artificial diet containing two different FANA oligos. Efficacy of a PPMO targeting Proftella in artificial feeding assays was also evaluated with psyllids in plant feeding assays. In artificial feeding assays 2.31 times more live Proftella were retained in ACP exposed to control (untreated) diets, as compared with ACP that fed on PPMO augmented diets. Further, PPMO exposed psyllids retained 30% (or 3.3 times) more dead Profftella as compared with psyllids exposed to untreated diet. PPMO/FANA treatments targeting CLas. Successful Delivery methods thus far include root absorption, tree trunk injection, and cut stem absorption of both PPMO and FANA molecules. These are the first evidence of delivery and activity of PPMO and FANA in plant (citrus). Preliminary data supports that these molecules can be used to suppress CLas under experimental conditions, using potted citrus trees, in glasshouses. Method of delivery was direct tree trunk injections. In PPMO treated citrus seedlings, half of PPMO treated trees showed a decrease in CLas titer after 60 d, while the second had a lower level of increase compared to untreated controls. In contrast, CLas titers increased untreated controls after 60 d. Live/dead analysis of CLas using PMA-qPCR indicated untreated infected control trees showed an increase 2.1 times greater than the PPMO treated group trees.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Hunter, W. J. Metz, A. Sandoval Majoica, S. Altman, M.J. Boyle4 V. Aishwarya, G. McCollum, and K. Pelz-Stelinski. 2017. FANA and Morpholino treatments for targeting pathogens and insects in citrus.
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