Progress 09/15/23 to 09/14/24
Outputs
Target Audience:Sanju Kunwar, Ana Redondo, Leigh Archer, Fernando Alferez, Ute Albrecht, Ozgur Batuman (2023). Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 12thInternational Congress of Plant Pathology. Lyon, France. August 20-25, 2023. Ozgur Batuman, Sanju Kunwar, Ana Redondo, Denise Manker, Meagan Iott, Thomas Knobloch, Stephane Brunet, and Jeremy Dufour (2023). Novel Plant Defense Inducers for Managing Huanglongbing (Citrus Greening) and Citrus Canker Diseases. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023. Batuman, O.,May 15-17, 2023: Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 18thBiennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O.,May 15-17, 2023: New Antimicrobials and Systemic Acquired Resistance (SAR) Inducers for Managing Citrus Canker Disease. 18thBiennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O.,May 15-17, 2023: The Asian citrus psyllid as a bioindicator of citrus tristeza virus in Florida groves. 18thBiennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O. Trunk Injection Not a Silver Bullet for HLB. Citrus Industry News - January 2023:https://citrusindustry.net/2023/01/13/trunk-injection-not-a-silver-bullet-for-hlb/. Batuman et al. Novel strategies for HLB resistance/tolerance in citrus by gene editing. Citrus Expo. August 16-17, Tampa, FL. Batuman, O. HLB and its management in citrus groves. Presentation to citrus Advisory Board. June 9, 2023 (Virtual). Batuman O. Citrus Pathology Program for Huanglongbing (HLB) management. Presentation to USDA/Cornell Research Group. June 1, 2023 (Virtual). Batuman, O. IPCs' beneficial effects on citrus diseases and challenges they create. UF-IFAS In-service training to extension Agents. May 3, 2023; Immokalee, FL. Batuman, O. Emerging viral diseases of citrus and management of their insect vectors. UF-IFAS In-service training to extension Agents. Mar 28, 2023; Immokalee, FL. Batuman, O. Citrus Pathology Program and Projects. Presentation to specialty crop county extension agents. Apr 27, 2023; Clewiston, FL Batuman, O. Plant pathogens that may exacerbate the HLB effects on citrus trees. UF-IFAS Citrus Seminar Series for citrus stakeholders Jan 10, 2023; Immokalee, FL. Mou, Z., Triplett, E., and Dawson, W. Make transgenic Huanglongbing tolerant citrus lines availalbe to the citrus industry.Citrus Expo. August 16-17, Tampa, FL (abstract). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graguate student and two undergraduate students were trained in experiemntal design, basic molecular biology skills such as vector construction, Agro-mediated transformation, and callus and suspension cell transformation. They were also trained in data collection and analysis. How have the results been disseminated to communities of interest?We routinely communicated with stakeholders including citrus growers, industry, state and federal regulators, students and the public through different approaches including publications, meeting abstracts, discussions, presentations, and demonstrations. We paid special attention to informing the stakeholders the project progresses and the potential strategies to mitigate the HLB disease. What do you plan to do during the next reporting period to accomplish the goals?Our priority is to propagate the RNAi rootstock lines and test their effects on the scion. We have some RNAi rootstock plants generated from cutting and will graft them with the HLB-susceptible sweet orange 'Hamlin'. The other priority is to produce transgene-free CRISPR trees of the candidate genes. We will focus on the constructs that have shown promising results. Additionally, we will continue engaging stakeholders in our projects as we have done previously.
Impacts
What was accomplished under these goals?
(1) Produce non-GMO CRISPR citrus trees of the target genes identified by CTV-RNAi In this year, we focused on establishing transgene-free gene editing in embryogenic calli and suspension cultures, as they are easier to regenerate plants than protoplasts. We targeted theCitrus sinensisphytoene desaturase geneCsPDSfor visible identification of the resulting albino pdsmutants. Guide RNAs (sgRNAs) were produced through in vitro transcription from a synthetic transcription template. Cas9-EGFP fusion proteins were incubated with the gRNAs in a nuclease reaction buffer, and the resulting Cas9-EGFP/gRNA complex was subsequently encapsulated with Lipofectamine. Embryogenic calli and suspension cultures of the sweet orange cultivar 'Hamlin' were transinfected with the encapsulated Cas9-EGFP/gRNA complex, and conditions for high cell viability were determined. Cells were harvested at 30- and 60-day intervals after transfection to evaluate the editing efficiency. Mismatch assays revealed cleavage products indicative of induced insertion or deletion mutations (indels) at the target sites. Twomonths later, the actively dividing 'Hamlin' sweet orange colonies were transferred to a fresh medium supplemented with maltose for embryo regeneration, resulting in the development of embryos with the typical albino phenotype, which is indicative of putative genome editing. Sanger sequencing revealed that the albino embryos carried mutations in theCsPDSgene, which was often a single-base deletion or insertion. This protocol is currently being used to edit the candidate genes. For Agrobacterium-mediated transformation, four co-base editing constructs targeting the citrus ALS gene (for tolerance to the herbicide imazapyr) and the candidate genes were transformed into citrus explants. A large number of regenerated shoots were selected with imazapyr. Both the ALS and candidate genes in the imazapyr-tolerant shoots were sequenced to assess the editing efficiency. Editing events were identified in the ALS gene with a low efficiency, but no editing events were found in the candidate gene. We are optimizing the conditions to increase the editing efficiency in the ALS gene to make the imazapyr selection more effective. Meanwhile, three twinPE (priming editing) constructs targetingCsPDS and one candiate gene were transformed into citrus explants. Fifteen stable transgenic plants were obtained for the CsPDS gene, but no albino phenotypes were observed. Sequencing the target site did not find editing events, suggesting that the designed constructs are not effective. We are propagating the twinPE transgenic plants and will analyze the progeny plants for editing events. (2) Produce intragenic miRNA rootstocks that will silence the target genes in the scion New cuttings from the previously generated RNAi rootstock lines were made and about one third of the cuttings developed roots after a long time (more than 10 months) with repeated auxin treatments. We will graft these rootstock plants generated from cuttings with the HLB-susceptible sweet orange 'Hamlin'. Since cuttings from the RNAi lines are difficult to root, we planted the parental RNAi rootstock plants into the field in July 2024. The rootstock lines were produced from mature explants and will produce fruits/seeds in the coming year. We will use seeds to propagate the RNAi rootstock lines to test their effects on HLB. Moreover, 19 new RNAi rootstock lines, including six lines silencing two genes, were produced. We have backed up most of the lines and will plant the parental lines into the field in the Spring of 2025 for seed production. Additionally, several RNAi constructs targeting different candidate genes are in the transformation pipeline for generation of more RNAi plants. (3) Evaluate HLB tolerance of the newly generated citrus trees Three RNAi rootstocks grafted with 'Hamlin' were planted in the field in July 2024 for HLB tolerance evaluation. (4) Engage stakeholders in project development and execution process We continued educating stakeholders including citrus growers, industry, state/federal regulators, as well as students and the public about the progress of the project and current HLB control strategies through various outreach activities. We focused on transgene-free CRISPR citrus plants and RNAi rootstocks that express small RNAs to silence candidate genes in the scion for HLB tolerance and disseminated the ideas to a broad range of audience. Various discussions, presentations, and demonstrations on transgene-free CRISPR citrus plants and other HLB management strategies were conducted in several events held in Florida and elsewhere in 2023-2024. Potential strategies for controlling HLB in Florida citrus groves were also discussed with the stakeholders.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Mahmoud LM, Dutt M (2024) Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome. Plant Cell Reports 43 (7):171.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Harris, F.M., and Mou, Z. (2024). Damage-associated molecular patterns and systemic signaling. Phytopathology 114, 308-327.
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:Mahmoud, L., Killiny, N., Grosser, J.W., Dutt, M. July 31 to August 4, 2023. Lipofectamine-mediated Cas9/gRNA complex delivery into citrus protoplasts. ASHS annual conference,July 31 to August 4, 2023, poster. Batuman, O., May 15-17, 2023: Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O., May 15-17, 2023: New Antimicrobials and Systemic Acquired Resistance (SAR) Inducers for Managing Citrus Canker Disease. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O., May 15-17, 2023: The Asian citrus psyllid as a bioindicator of citrus tristeza virus in Florida groves. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman et al. Novel strategies for HLB resistance/tolerance in citrus by gene editing. Citrus Expo. August 16-17, Tampa, FL. Batuman, O. HLB and its management in citrus groves. Presentation to citrus Advisory Board. June 9, 2023 (Virtual). Batuman O. Citrus Pathology Program for Huanglongbing (HLB) management. Presentation to USDA/Cornell Research Group. June 1, 2023 (Virtual). Batuman, O. IPCs' beneficial effects on citrus diseases and challenges they create. UF-IFAS In-service training to extension Agents. May 3, 2023; Immokalee, FL. Batuman, O. Emerging viral diseases of citrus and management of their insect vectors. UF-IFAS In-service training to extension Agents. Mar 28, 2023; Immokalee, FL. Batuman, O. Citrus Pathology Program and Projects. Presentation to specialty crop county extension agents. Apr 27, 2023; Clewiston, FL. Batuman, O. Plant pathogens that may exacerbate the HLB effects on citrus trees. UF-IFAS Citrus Seminar Series for citrus stakeholders Jan 10, 2023; Immokalee, FL. Mou, Z., Dawson, W., Grosser, J., Orbovic, V., Dutt, M., Levy, A., El Mohtar, C., Batuman, O., Irey, M. pproaches toward Huanglongbing tolerance. Citrus Expo, August 16-17, 2023, Abstract. Mou, Z., Dawson, W. Generation of transgenic Huanglongbing tolerant citrus varieties. Citrus Expo, August 16-17, 2023, Abstract. Sarkar, P., Mou, Z., Levy, A. Overexpression of AtNPR1 enhances tolerance to Liberibacter infection by elevating basal immune responses. 2023 IS-MPMI Congress, July 16-20, Poster. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this year, this project trained one graduate student in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, and protoplast transformation, as well as collection and analysis of data. How have the results been disseminated to communities of interest?Since the start of project, we have been communicating with stakeholders including citrus growers, industry, state/federal regulators, as well as students and the general public through various approaches including publications, meeting abstracts, talks, presentations, and demonstrations. We do our best to inform the stakeholders the project progresses and the potential strategies to mitigate the HLB disease. What do you plan to do during the next reporting period to accomplish the goals?We will conduct the well-designed experiments in the proposal to achieve the project objectives. Our first priority is to obtain non-GMO CRISPR mutants of our candidate genes, and the second priority is to find RNAi rootstocks that can silence the target genes in the non-transgenic scions and provide robust tolerance to HLB.
Impacts
What was accomplished under these goals?
(1) Produce non-GMO CRISPR citrus trees of the target genes identified by CTV-RNAi We used both protoplast transformation and Agrobacterium-mediated tissue transformation to generate non-GMO CRISPR events. For protoplast transformation, different methods were used to overcome cellular challenges to increase the transformation efficiency and resulted in more viable cells with integrated donor DNA in the citrus genomes. Lipofection (or liposome transfection), polymers, dendrimers are techniques for delivering genetic material into a cell via liposomes, which can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Dendrimers are a class of synthetic polymers with spherical and hyperbranched structures as well as a high density of surface functional groups. These polymers have been widely used as carriers for the delivery of drugs, nucleic acids and proteins. We developed a protocol using lipofectamine and poly-amidoamine (PAMAM) dendrimers to deliver foreign DNA into protoplasts. We also developed a protocol of using cationic lipid nanoparticles to mediate delivery of in vitro assembled Cas9/gRNA complex ribonucleoproteins (RNPs) into citrus protoplast using cationic lipid nanoparticles (Lipofectamine). One-step synthesis of gRNA template was designed to synthesize in vitro transcription of gRNA targeting the citrus phytoene desaturase (PDS) gene. Different amounts of in vitro translated NLS-Cas9-EGFP fusion protein was incubated with 250, 500 or 1000 ng of gRNA. We recorded high transfection efficiency with 2 µg Cas9 protein and either 250 or 500 ng gRNA complex that had been encapsulated in 2 µl lipofectamine. Protoplasts were subsequently incubated with the RNP complex for 2, 4, 8, 16, 24 and 36 hrs. Viable EGFP-expressed cells were observed after 24 h incubation, whereas the longer incubation with the RNP complex caused severe cell damage. T7EI mismatch assay resulted in cleavage products indicative of induced insertion or deletion mutations (indels) at the target sites. These results showed that lipofection-mediated transfection approach can be an effective method for the use of the DNA-free Cas9/gRNA technology for genome editing in citrus protoplasts. This method is being used to target the candidate genes. For Agrobacterium-mediated transformation, we tested an intron-containing Cas9 vector in the absence of antibiotics selection and found that 100% of the edited shoots contained the T-DNA, though the editing efficiency reached ~5%, indicating that it is difficult to obtain non-GMO CRISPR events using the highly efficient intron-containing Cas9. We then built multiple base editing constructs targeting the citrus ALS gene for tolerance to the herbicide imazapyr. We are using these constructs for co-editing of our target genes under the selection of imazapyr. We have also made twinPE (prime editing) constructs to test if this new gene-editing strategy works in citrus. (2) Produce intragenic miRNA rootstocks that will silence the target genes in the scion We previously produced 24 Carrizo rootstock lines expressing a hairpin RNA targeting one of our candidate genes (hpRNAi). We used two strategies to test if the small RNAs move between rootstock andscion for gene silencing. Thefirst strategy is to check the effect of the Carrizo rootstock on a susceptible sweet orange (Hamlin) scion. The second strategy is to check the effect of the hpRNAi Carrizo as scion on a susceptible rootstock of sweet orange or grapefruit. We first took cuttings and tried to root the CarrizohpRNAi lines but failed. We are using a modified protocol to promote rooting of the hpRNAi lines. Meanwhile, we grafted Hamlin sweet orange onto the original hpRNAi lines that are on non-transgenic rootstocks. Initial RT-PCR revealed that the target gene is downregulated in the Hamlin scion tissues, indicating that the small RNAs moved from the Carrizo hpRNAi tissues into the Hamlin scion. We are confirming this result with different approaches. For the second strategy, we have successfully budded the Carrizo hpRNAi lines on grapefruit rootstock. We will test the silencing effect in the grapefruit rootstock after the Carrizo shoots are established. We have also built several miRNA constructs targeting different candidate genes. Transformation of rootstocks with these constructs are ongoing. (3) Evaluate HLB tolerance of the newly generated citrus trees Nothing to report. (4) Engage stakeholders in project development and execution process Outreach activities were conducted to inform stakeholders including citrus growers, industry, state/federal regulators, as well as students and the public about the progress of the project and on HLB control strategies. Both using transgene-free CRISPR citrus plants and using intragenic rootstocks expressing microRNA to silence target genes in the scion for HLB resistance or tolerance are exciting topics for a broad range of audience. These topics, together with other HLB management strategies, were discussed, presented, and demonstrated virtually or in person in multiple events held in Florida and elsewhere in 2022-2023. Various potential strategies for mitigating HLB in Florida citrus groves were also communicated with the stakeholders.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Sanju Kunwar, Ana Redondo, Leigh Archer, Fernando Alferez, Ute Albrecht, Ozgur Batuman (2023). Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Ozgur Batuman, Sanju Kunwar, Ana Redondo, Denise Manker, Meagan Iott, Thomas Knobloch, Stephane Brunet, and Jeremy Dufour (2023). Novel Plant Defense Inducers for Managing Huanglongbing (Citrus Greening) and Citrus Canker Diseases. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023.
- Type:
Websites
Status:
Published
Year Published:
2023
Citation:
Transgene-free citrus using CRISPR CAS9 (project website; Last Modified: Tue, 27 Sep 2022): https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/
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