Progress 01/15/18 to 01/14/23
Outputs
Target Audience:scientific community, biotechnology industry, citrus growers, regulatory agencies, graduate students Changes/Problems:Due to the effect of COVID19, we have requested one year no cost extension. This has not changed the scope of work, but allowed us to complete the proposed work. What opportunities for training and professional development has the project provided?This project provides opportunities for training and professional development of 7 graduate students, 7 undergraduates and 5 postdocs including technological training, paper writing, attending online meetings and workshops. How have the results been disseminated to communities of interest?We have published 36 manuscripts on HLB and citrus genome editing. We have optimized the gene editing technology for citrus. Now, we are able to generate homozygous and biallelic mutations. We also developed non-transgenic citrus genome editing technology which are being used to generate genome modified citrus plants. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multiple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. We have completed yeast two hybridization (Y2H) for the 30 effectors including five defense-suppressing effectors and identified their targets in Valencia sweet orange. Overall, more than 100 putative targets have been identified using Y2H. We investigated the spatiotemporal Las colonization in different tissues post ACP transmission. We show that one of Las-secreted proteins, SDE15, suppresses plant immunity and promotes Las multiplication. We investigated flagella, one of the important virulence factors of Las. The expression of flagellar genes was higher in psyllids than in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both 'Ca. L. asiaticus' infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to 'Ca. L. asiaticus'. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with 'Ca. L. asiaticus'. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. We found that HLB is a pathogen-triggered immune disease. Las infection of Citrus sinensis stimulated systemic and chronic immune responses in phloem tissue including reactive oxygen species (ROS) production as indicated by H2O2, callose deposition, and induction of immune related genes. Systemic cell death of companion and sieve element cells, but not surrounding parenchyma cells, was observed following ROS production triggered by Las. Death of companion cells was often observed before that of sieve elements. ROS production triggered by Las localized in phloem-enriched bark tissue. The H2O2 concentration in exudates extracted from phloem enriched bark tissue of Las infected plants kills citrus protoplast cells in vitro, which was suppressed by uric acid (a ROS scavenger) and gibberellin. RNA-seq analysis showed that gibberellin induces the expression of genes encoding H2O2 scavenging enzymes, but inhibits RBOHD that encodes a NADPH oxidase for ROS production. Foliar spray of HLB positive citrus with antioxidants and gibberellin significantly reduced both H2O2 concentrations and cell death in phloem tissues induced by Las and reduced HLB symptoms. In summary, HLB is an immune-mediated disease while mitigating ROS via antioxidants and promoting plant growth can reduce cell death of phloem tissue caused by Las, thus controlling HLB. We have conducted RNAi analyses of 7 putative susceptibility genes including callose synthase genes. CTV RNAi was used to investigate 11 putative HLB susceptibility genes and identified one promising target. Objective 2. We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 89%, representing a significant improvement in citrus genome editing efficacy. We have optimized non-transgenic citrus genome editing based on CRISPR. We have developed citrus base editors that allow us to precisely edit certain residues of target genes in citrus. 2.2 CRISPR-SpCas9 was successfully used to generate homozygous and biallelic citrus mutants in the T0 generation. Currently, the improved CRISPR technology is being used to generate mutants for putative HLB susceptibility genes including CsACD2. Cas12a ribonucleoprotein (RNP) was successfully used to edit the genome of protoplasts. We have generated embryogenic tissue cultures for Hamlin, Valencia, and Rio Red in Florida, Washington navel, Lisbon lemon, and Tango mandarin in California, and Rio Red grapefruit in Texas. Genome editing of the putative HLB susceptibility genes including CsACD2 identified in objective 1 for the different varieties are ongoing. Objective 3. For the genome modified citrus generated using Cas12a and SpCas9 in objective 2, we have conducted the off-target analyses. The mutants are being grown in the greenhouse for evaluation on HLB resistance/tolerance and effect on citrus horticultural traits. Some genome modified plants have been planted in an APHIS approved field site for evaluation. Objective 4. We conducted focus groups and online surveys with consumers to develop a better understanding of how consumers react to information about production methods (including CRISPR) when deciding how much they are willing to pay for products such as orange juice. Findings from the focus group included that consumers initially reluctant to accept GM or CRISPR products were willing to consider such products after being provided detailed information on the safety procedures in effect for developing products (including the length of time needed to develop such products). Results from the online survey showed that there still exists a negative preference for GM or CRISPR products in comparison to conventional products. However, there was less distance in willingness to pay between conventional and CRISPR products than conventional and GM products. Our study found higher risk perceptions led to lower willingness to pay for CRISPR products (specifically orange juice), while risk preference led to higher willingness to pay for similar products. Four distinct market segments were identified in the data. These included groups that: focused on the consistency of the product over other attributes (i.e. no-pulp); price insensitive shoppers (higher income and preferred Florida orange juice); a group that was less interested in orange juice in general (but when they were, they strongly preferred Florida as well); and a group that purchased orange juice regularly and was less concerned about attributes (interestingly, this group was the only that preferred GM to GE, suggesting they were averse to newer technologies). Overall, our results showed there is a market for CRISPR products, though willingness to pay will still lag conventional products. Objective 5. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. A video outreach project was developed. We also published multiple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Yuan X, Chen C, Bassanezi RB, Wu F, Feng Z, Shi D, Li J, Du Y, Zhong L, Zhong B, Lu Z, Song X, Hu Y, Ouyang Z, Liu
X, Xie J, Rao X, Wang X, Wu DO, Guan Z, Wang N. Region-Wide Comprehensive Implementation of Roguing Infected
Trees, Tree Replacement, and Insecticide Applications Successfully Controls Citrus Huanglongbing. Phytopathology. 2021
Aug;111(8):1361-1368.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Pandey SS, Xu J, Achor D, Li J, Wang N. Microscopic and transcriptomic analyses of early events triggered by Candidatus Liberibacter asiaticus in young flushes of HLB-positive citrus trees. Phytopathology. 2022 Nov 30. doi: 10.1094/PHYTO-10-22-0360-R. Epub ahead of print. PMID: 36449527.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Huang Y, Zhu F, Koh J, Stanton D, Chen S, Wang N. Proteomic and bioinformatic analyses of proteins in the outer membrane and extracellular compartments and outer membrane vesicles of Candidatus Liberibacter species. Front Microbiol. 2022 Sep 26;13:977710.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ribeiro C, Xu J, Hendrich C, Pandey SS, Yu Q, Gmitter F, Wang N. Seasonal transcriptome profiling of susceptible and tolerant citrus cultivars to citrus Huanglongbing. Phytopathology. 2022 Aug 24. doi: 10.1094/PHYTO-05-22-0179-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Huang X, Wang Y, Wang N. Base Editors for Citrus Gene Editing. Front Genome Ed. 2022 Feb 28;4:852867. doi: 10.3389/fgeed.2022.852867. PMID: 35296063; PMCID: PMC8919994.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Jia H, Wang Y, Su H, Huang X, Wang N. LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants. Cells. 2022 Jan 18;11(3):315. doi: 10.3390/cells11030315.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Huang X, Wang Y, Wang N. Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System. Front Plant Sci. 2022 Jan 11;12:769907.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ma W, Pang Z, Huang X, Xu J, Pandey SS, Li J, Achor DS, Vasconcelos FNC, Hendrich C, Huang Y, Wang W, Lee D, Stanton D, Wang N. Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin. Nat Commun. 2022 Jan 26;13(1):529.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Pandey SS, Hendrich C, Andrade MO, Wang N. Candidatus Liberibacter: From Movement, Host Responses, to Symptom Development of Citrus Huanglongbing. Phytopathology. 2022 Jan;112(1):55-68.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Jia H, Omar AA, Orbovi? V, Wang N. Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit. Phytopathology. 2022 Feb;112(2):308-314.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2022
Citation:
Answiya Neupane 2022 Effect of HLB on leaf growth and development
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Barber MS Student Thesis: 2021 Antimicrobial Activity of Niclosamide against Bacterial Phytopathogens of Economically Important Crops
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Yixiao Huang Student Dissertation 2022 THE OUTER MEMBRANE PROTEOME OF CANDIDATUS LIBERIBACTER SPECIES AND ITS FUNCTIONAL STUDY IN THE CITRUS HUANGLONGBING PATHOSYSTEM
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Hu B, Rao MJ, Deng X, Pandey SS, Hendrich C, Ding F, Wang N, Xu Q. Molecular signatures between citrus and Candidatus Liberibacter asiaticus. PLoS Pathog. 2021 Dec 9;17(12):e1010071. doi: 10.1371/journal.ppat.1010071. PMID: 34882744; PMCID: PMC8659345.
|
Progress 01/15/21 to 01/14/22
Outputs
Target Audience:scientific community, biotechnology industry, citrus growers, regulatory agencies, graduate students Changes/Problems:Due to the effect of COVID19, we have requested one year no cost extension. This will not change the scope of work, but allow us to complete the proposed work. What opportunities for training and professional development has the project provided?This project provides opportunities for training and professional development of 7 graduate students, 7 undergraduates and 5 postdocs including technological training, paper writing, attending online meetings and workshops. How have the results been disseminated to communities of interest?We have published 8 manuscripts on HLB. We have optimized the gene editing technology for citrus. Now, we are able to generate homozygous and biallelic mutations. We also began to generate non-transgenic genome modified citrus plants. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multiple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work as outlined in the timeline. We will test the genome modified citrus against HLB and other horticultural traits. We will conduct genome editing for newly identified target genes. We will conduct acceptance study and extension as planned. We will collect data for further analyses.
Impacts
What was accomplished under these goals?
Objective 1. We have completed yeast two hybridization (Y2H) for the 30 effectors including five defense-suppressing effectors and identified their targets in Valencia sweet orange. Overall, more than 100 putative targets have been identified using Y2H. We investigated the spatiotemporal CLas colonization in different tissues post ACP transmission. The spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development, and provides the knowledge supporting that young leaves should be the focus of HLB management. Here we show that one of Las-secreted proteins, SDE15, suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit suppresses the hypersensitive response (HR) induced by Xanthomonas citri subsp. citri (Xcc) and reduces the expression of immunity related genes. SDE15 also suppresses the HR triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homologue of Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) based on Y2H, co-IP and pulldown assays. SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB. In addition, we have confirmed at least five more putative HLB susceptibility genes. We investigated flagella, one of the important virulence factors of Las. The expression of flagellar genes was higher in psyllids than in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control. 1.5. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both 'Ca. L. asiaticus' infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to 'Ca. L. asiaticus'. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with 'Ca. L. asiaticus'. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. 1.6. We found that HLB is a pathogen-triggered immune disease. CLas infection of Citrus sinensis stimulated systemic and chronic immune responses in phloem tissue including reactive oxygen species (ROS) production as indicated by H2O2, callose deposition, and induction of immune related genes. Systemic cell death of companion and sieve element cells, but not surrounding parenchyma cells, was observed following ROS production triggered by CLas. Death of companion cells was often observed before that of sieve elements. ROS production triggered by CLas localized in phloem-enriched bark tissue. The H2O2 concentration in exudates extracted from phloem enriched bark tissue of CLas infected plants kills citrus protoplast cells in vitro, which was suppressed by uric acid (a ROS scavenger) and gibberellin. RNA-seq analysis showed that gibberellin induces the expression of genes encoding H2O2 scavenging enzymes, but inhibits RBOHD that encodes a NADPH oxidase for ROS production. Foliar spray of HLB positive citrus with antioxidants and gibberellin significantly reduced both H2O2 concentrations and cell death in phloem tissues induced by CLas and reduced HLB symptoms. RNA-seq analyses of CLas infected and healthy C. sinensis demonstrates that CLas infections induce the expression of genes encoding NADPH oxidases and triggers downregulation of antioxidant enzyme genes, supporting that CLas causes oxidative stress. CLas also stimulate the expression of immune related genes. In summary, HLB is an immune-mediated disease while mitigating ROS via antioxidants and promoting plant growth can reduce cell death of phloem tissue caused by CLas, thus controlling HLB. 1.7. We have conducted RNAi analyses of 7 putative susceptibility genes. Objective 2. 2.1. We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 89%, representing a significant improvement in citrus genome editing efficacy. 2.2 CRISPR-SpCas9 was successfully used to generate homozygous and biallelic citrus mutants in the T0 generation. Currently, the improved CRISPR technology is being used to generate homozygous mutants for putative HLB susceptibility genes including CsACD2. Cas12a ribonucleoprotein (RNP) was successfully used to edit the genome of protoplasts. We have generated embryogenic tissue cultures for Valencia sweet orange in Florida, Navel sweet orange in California, and Rio Red grapefruit in Texas. Genome editing of the putative HLB susceptibility genes including CsACD2 identified in objective 1 for the three different varieties are ongoing. Objective 3. For the genome modified citrus generated using Cas12a and SpCas9 in objective 2, we have conducted the off-target analyses. The plants are being kept in the greenhouse for analyses of horticultural traits and resistance against HLB. Some genome modified plants have been planted in an APHIS approved field site for evaluation. Objective 4. We have conducted surveys as planed regarding the consumer acceptance of genetically modified citrus. Objective 5. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multiple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Yuan X, Chen C, Bassanezi RB, Wu F, Feng Z, Shi D, Li J, Du Y, Zhong L, Zhong B, Lu Z, Song X, Hu Y, Ouyang Z, Liu X, Xie J, Rao X, Wang X, Wu DO, Guan Z, Wang N. Region-Wide Comprehensive Implementation of Roguing Infected Trees, Tree Replacement, and Insecticide Applications Successfully Controls Citrus Huanglongbing. Phytopathology. 2021 Aug;111(8):1361-1368. doi: 10.1094/PHYTO-09-20-0436-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Hu B, Rao MJ, Deng X, Pandey SS, Hendrich C, Ding F, Wang N, Xu Q. Molecular signatures between citrus and Candidatus Liberibacter asiaticus. PLoS Pathog. 2021 Dec;17(12):e1010071. doi: 10.1371/journal.ppat.1010071.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li J, Kolbasov VG, Lee D, Pang Z, Huang Y, Collins N, Wang N. Residue Dynamics of Streptomycin in Citrus Delivered by Foliar Spray and Trunk Injection and Effect on 'Candidatus Liberibacter asiaticus' Titer. Phytopathology. 2021 Jul;111(7):1095-1103. doi: 10.1094/PHYTO-09-20-0427-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Pandey SS, Hendrich C, Andrade M, Wang N. Candidatus Liberibacter: From Movement, Host Responses, to Symptom Development of Citrus HLB. Phytopathology. 2021 Oct 5;. doi: 10.1094/PHYTO-08-21-0354-FI.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Vasconcelos FNC, Li J, Pang Z, Vincent C, Wang N. The Total Population Size of 'Candidatus Liberibacter asiaticus' Inside the Phloem of Citrus Trees and the Corresponding Metabolic Burden Related to Huanglongbing Disease Development. Phytopathology. 2021 Jul;111(7):1122-1128. doi: 10.1094/PHYTO-09-20-0388-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Pandey SS, Nogales da Costa Vasconcelos F, Wang N. Spatiotemporal Dynamics of 'Candidatus Liberibacter asiaticus' Colonization Inside Citrus Plant and Huanglongbing Disease Development. Phytopathology. 2021 Jun;111(6):921-928. doi: 10.1094/PHYTO-09-20-0407-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ribeiro C, Xu J, Teper D, Lee D, Wang N. The transcriptome landscapes of citrus leaf in different developmental stages. Plant Mol Biol. 2021 Jul;106(4-5):349-366. doi: 10.1007/s11103-021-01154-8.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wang N. A promising plant defense peptide against citrus Huanglongbing disease. Proc Natl Acad Sci U S A. 2021 Feb 9;118(6). doi: 10.1073/pnas.2026483118.
|
Progress 01/15/20 to 01/14/21
Outputs
Target Audience:scientific community, biotechnology industry, citrus growers, regulatory agencies, graduate students Changes/Problems:COVID19 causes a lot of challenges and slows down our progress, but we are able to move the project forward as planned. What opportunities for training and professional development has the project provided?This project provides opportunities for training and professional development of 5 graduate students and 5 postdocs including technological training, paper writing, attending online meetings and workshops. How have the results been disseminated to communities of interest?We have published 9 manuscripts on HLB. We have optimized the gene editing technology for citrus. Now, we are able to generate homozygous and biallelic mutations. We also began to generate non-transgenic genome modified citrus plants. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters. We have also filed one patent. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work as outlined in the timeline. We will continue to confirm the HLB S genes, further optimize the genome editing technology, and edit the confirmed S genes and test the genome modified citrus against HLB and other horticultural traits. We will conduct acceptance study and extension as planed.
Impacts
What was accomplished under these goals?
Objective 1. 1.1 We have completed yeast two hybridization (Y2H) for the 30 effectors including five defense-suppressing effectors and identified their targets in Valencia sweet orange. Overall, more than 100 putative targets have been identified using Y2H. Confirmation of the targets is ongoing using co-immunoprecipitation and BiFC assays. We are conducting genome editing of the identified HLB S gene of Valencia sweet orange and Duncan grapefruit. 1.2 We investigated the spatiotemporal CLas colonization in different tissues post ACP transmission. At 75 day-post-ACP-removal (DPR), CLas was detected in roots of all trees, but in the mature leaf of only one tree, of the nine plants that were successfully infected via ACP transmission, consistent with the model that CLas moves passively from the source to sink. CLas was detected in 11.1%, and 43.1% mature leaves, which were unfed by ACPs during transmission, at 75, and 365 DPR, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates CLas is capable of both passive and active movement with passive movement being dominant. At 225 DPR, leaves fed by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and highest CLas titer, followed by that of leaves emerged post ACP removal, and mature leaves not fed by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes at most three years for CLas to infect the whole tree. Overall, the spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development, and provides the knowledge supporting that young leaves should be the focus of HLB management. 1.3 Here we show that one of Las-secreted proteins, SDE15, suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit suppresses the hypersensitive response (HR) induced by Xanthomonas citri subsp. citri (Xcc) and reduces the expression of immunity related genes. SDE15 also suppresses the HR triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homologue of Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) based on Y2H, co-IP and pulldown assays. SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB. In addition, we have confirmed at least five more putative HLB susceptibility genes. 1.4 We investigated flagella, one of the important virulence factors of Las. We analyzed the flagellar genes of Las. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control. 1.5. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both 'Ca. L. asiaticus' infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to 'Ca. L. asiaticus'. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with 'Ca. L. asiaticus'. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. Objective 2. 2.1. We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. 2.2 CRISPR-SpCas9 was successfully used to generate homozygous and biallelic citrus mutants in the T0 generation for two selected genes. Currently, the improved CRISPR technology is being used to generate homozygous mutants for putative HLB susceptibility genes including CsACD2. Here we further developed the non-transgenic genome editing of citrus technology via transient expression of Cas9- sgRNA DNA, RNA or ribonucleoprotein (RNP) into protoplasts by PEG-mediated transfection or into embryogenic cells of suspension culture by particle bombardment. Citrus protoplast or embryogenic cells appear to be recalcitrant to transformation using different approaches. Only transient expression of CRISPR/Cas9 DNA in protoplast cells led to efficient transformation and genome editing mutation rate of 78% was observed. We have finally generated non-transgenic genome modified mutations. The plants are under regeneration. We have generated embryogenic tissue cultures for Valencia sweet orange in Florida, Navel sweet orange in California, and Rio Red grapefruit in Texas. Genome editing of the putative HLB susceptibility genes including CsACD2 identified in objective 1 for the three different varieties are ongoing. Objective 3. For the genome modified citrus generated using Cas12a and SpCas9 in objective 2, we have conducted the off-target analyses. The plants are being kept in the greenhouse for analyses of horticultural traits. Some genome modified plants have been planted in an APHIS approved field site for evaluation. Objective 4. We are conducting surveys as planed regarding the consumer acceptance of genetically modified citrus. Objective 5. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multiple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Clark, K., Pang, Z., Trinh, J., Wang, N., and Ma, W. 2020. Sec-delivered effector 1 (SDE1) of Candidatus Liberibacter asiaticus promotes citrus Huanglongbing. Mol Plant Microbe Interact. 33:1394-1404
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Franco JY, Thapa SP, Pang Z, Gurung FB, Liebrand TWH, Stevens DM, Ancona V, Wang N, Coaker G. 2020 Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression. Mol Cell Proteomics. 19:1936-1951.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Huang, X., Wang, Y., Xu, J., and Wang, N. 2020. Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations. Plant Mol Biol 104:297-307
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Pang, Z., Zhang, L., Coaker, G., Ma, W., He, S.Y., and Wang, N. 2020. Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease. Plant Physiol 184:792-805
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Andrade, M., Li, J., and Wang, N. 2020a. Candidatus Liberibacter asiaticus: virulence traits and control strategies. Tropical Plant Pathology. 45: 285-297
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Andrade, M.O., Pang, Z., Achor, D.S., Wang, H., Yao, T., Singer, B.H., and Wang, N. 2020b. The flagella of 'Candidatus Liberibacter asiaticus' and its movement in planta. Mol Plant Pathol 21:109-123
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Qiu, W., Soares, J., Pang, Z., Huang, Y., Sun, Z., Wang, N., Grosser, J., and Dutt, M. 2020. Potential Mechanisms of AtNPR1 Mediated Resistance against Huanglongbing (HLB) in Citrus. Int J Mol Sci 21:2009. doi: 10.3390/ijms21062009.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Thapa, S.P., De Francesco, A., Trinh, J., Gurung, F.B., Pang, Z., Vidalakis, G., Wang, N., Ancona, V., Ma, W., and Coaker, G. 2020. Genome-wide analyses of Liberibacter species provides insights into evolution, phylogenetic relationships, and virulence factors. Mol Plant Pathol. 21:716-731.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wang, N. 2020. A perspective of citrus Huanglongbing in the context of the Mediterranean Basin. Journal of Plant Pathology. 102:635640
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Progress 01/15/19 to 01/14/20
Outputs
Target Audience:scentific community, citrus growers, regulatory agencies, graduate students Changes/Problems:There are no major changes/problems. What opportunities for training and professional development has the project provided?This project provides opportunities for training and professional development of 5 graduate students and 5 postdocs including attending meetings and workshops. How have the results been disseminated to communities of interest?We have published 8 manuscripts on HLB, we havebeen giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters. We have also filed one patent. What do you plan to do during the next reporting period to accomplish the goals?We will continue our work as outlined in the timeline. We will continue to confirm the HLB S genes, further optimize the genome editing technology, and modify those S genes and test the genome modified citrus against HLB and other horticultural traits. We will conduct acceptance study and extension as planed.
Impacts
What was accomplished under these goals?
Objective 1. We have completed screening of 20 putative Las effectors and 2 of them repressed plant defense. 1.1. We have completed yeast two hybridization (Y2H) for the 20 effectors including two defense-suppressing effectors and identified their targets in Valencia sweet orange. Overall, more than 50 putative targets have been identified using Y2H. Confirmation of the targets is ongoing using co-immunoprecipitation and BiFC assays. Meanwhile, we have conducted CTV-mediated gene silencing of 15 putative HLB susceptibility genes in collaboration with Dawson lab. Sweet orange plants carrying the CTV constructs were inoculated with Las via grafting. Interestingly, gene silencing of one of the putative HLB susceptible genes led to significant HLB tolerance. The plants showed mild HLB symptoms, similar growth as non-inoculated plants whereas the growth of control plants was significantly reduced and showed severe HLB symptoms. We are characterizing the putative mechanism of the HLB S gene. We are conducting genome editing of the identified HLB S gene of Valencia sweet orange and Duncan grapefruit. In addition, we also overexpressed the HLB S gene in Valencia sweet orange and will inoculate them with Las once they are one year old. 1.2. Here we show that one of Las-secreted proteins, SDE15, suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit suppresses the hypersensitive response (HR) induced by Xanthomonas citri subsp. citri (Xcc) and reduces the expression of immunity related genes. SDE15 also suppresses the HR triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homologue of Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) based on Y2H, co-IP and pulldown assays. SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB. 1.3. We investigated flagella, one of the important virulence factors of Las. We analyzed the flagellar genes of Las. No flagellum was observed for Las incitrusand dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control. This study suggested that we can utilize some flush specific promoters against Las infection. We are investigating whether the flagellum is involved in Las movement in planta and whether it induces plant immune responses by interacting with specific receptors. 1.4. Here, we investigated another important virulence factor of Las, the type IVc tight adherence (Tad) pilus. The Tad loci are conserved among members of Rhizobiaceae, including 'Ca.L. asiaticus' andAgrobacteriumspp. Ectopic expression of the 'Ca.L. asiaticus'cpaFgene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype incpaFmutant ofA. tumefaciens, indicating CpaF of 'Ca.L. asiaticus' was functional and critical for bacterial adherence mediated by Tad pilus. We are investigating whether the Tad pilus is involved in Las movement in planta and whether it induces plant immune responses by interacting with specific receptors. Objective 2. In this objective, we have been optimizing the citrus genome editing via the CRISPR technology and meanwhile, we are editing several putative HLB susceptibility genes including CsACD2 identified in Objective 1. 2.1. Recently, CRISPR-Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. To further expand the tool box for genome editing of citrus, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit acitrusgenome for the first time. LbCas12a was successfully used to modify two genes in Duncan grapefruit via Xcc-facilitated agroinfiltration. Finally, no potential off-targets were observed. Therefore, CRISPR-LbCas12a can readily be used as a powerful tool forcitrusgenome editing. 2.2 CRISPR-SpCas9 was successfully used to generate homozygous and biallelic citrus mutants in the T0 generation for two selected genes. Currently, the improved CRISPR technology is being used to generate homozygous mutants for putative HLB susceptibility genes including CsACD2. 2.3. Here we further developed the non-transgenic genome editing of citrus technology via transient expression of Cas9-sgRNA DNA, RNA or ribonucleoprotein (RNP) into protoplasts by PEG-mediated transfection or into embryogenic cells of suspension culture by particle bombardment. Citrus protoplast or embryogenic cells appear to be recalcitrant to transformation using different approaches. Only transient expression of CRISPR/Cas9 DNA in protoplast cells led to efficient transformation and genome editing mutation rate of 5.7% was observed. We further used hiTAIL-PCR analysis to show that the genome modified citrus cells are free of foreign DNA. We also detected low off-target mutations in genome modified citrus cells. Our findings represent a breakthrough in citrus breeding using the CRISPR technology. The non-transgenic genome editing method is being used to mutation the putative HLB susceptibility genes including CsACD2 identified in objective 1. 2.4. We have generated embryogenic tissue cultures for Valencia sweet orange in Florida, Navel sweet orange in California, and Rio Red grapefruit in Texas. Genome editing of the putative HLB susceptibility genes including CsACD2 identified in objective 1 for the three different varieties are ongoing. Objective 3. For the genome modified citrus generated using Cas12a and SpCas9 in objective 2, we have conducted the off-target analyses. The plants are being kept in the greenhouse for analyses of horticultural traits. Objective 4. We are conducting surveys as planed regarding the consumer acceptance of genetically modified citrus. Objective 5. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang, N. 2019. The Citrus Huanglongbing Crisis and Potential Solutions. Mol Plant 12:607-609.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hongge Jia, Vladimir Orbovi?, Nian Wang. CRISPR?LbCas12a?mediated modification of citrus. 2019. Plant Biotechnol J. 17:1928-1937.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Muhammad Junaid Rao, Fang Ding, Nian Wang, Xiuxin Deng & Qiang Xu. 2019. Metabolic Mechanisms of Host Species Against Citrus Huanglongbing (Greening Disease). Critical Reviews in Plant Sciences, 37:6, 496-511
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Christopher Vincent, Myrtho Pierre, Jinyun Li and Nian Wang. 2019. Implications of Heat Treatment and Systemic Delivery of Foliar-Applied Oxytetracycline on Citrus Physiological Management and Therapy Delivery. Front. Plant Sci. 10:41.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Sheo Shankar Pandey p and Nian Wang. 2019. Targeted early detection of citrus HLB causal agent Candidatus Liberibacter asiaticus before symptom expression. Phytopathology. 109:952-959.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Andrade, M.O. p, and Wang, N. 2019. The Tad pilus apparatus of Candidatus Liberibacter asiaticus and its regulation by VisNR. Mol Plant Microbe Interact. 32:1175-1187.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Li, J., Pang, Z., Duan, S., Lee, D., Kolbasov, V., and Wang, N. 2019. The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing. Phytopathology. 109:2046-2054.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Li J, Li L, Pang Z, Kolbasov V, Ehsani R, Carter E, Wang N. 2019 Developing citrus Huanglongbing management strategies based on the severity of symptoms in HLB-endemic citrus-producing regions. Phytopathology. 109:582-592.
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Zhiqian Pang, Li Zhang, Gitta Coaker, Wenbo Ma, Sheng-Yang He, Nian Wang Liberibacter asiaticus targets citrus CsACD2 to promote devastating Huanglongbing disease
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Maxuel Andrade1, Jinyun Li1, Nian Wang Liberibacter asiaticus: virulence traits and control strategies.
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Progress 01/15/18 to 01/14/19
Outputs
Target Audience:citrus growers, scientists, students, extension, consumer of orange juice and fresh fruit Changes/Problems:No. What opportunities for training and professional development has the project provided?This project provides opportunities for training and professional development of 5 graduate students and 5 postdocs. How have the results been disseminated to communities of interest?We have been givingpresentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.? What do you plan to do during the next reporting period to accomplish the goals?We will continue our work as outlined in the timeline. We will continue to confirm the HLB S genes and modify those S genes and test the genome modified citrus against HLB and other horticultural traits. We will conduct acceptancestudy and extension as planed.
Impacts
What was accomplished under these goals?
Objective 1. Our hypothesis is that Las requires specific secreted effectors that directly or indirectly activate key host S genes or their products, which subsequently facilitate pathogen multiplication and trigger disease symptoms of HLB. To test this hypothesis, we will conduct functional characterizations of critical virulence effectors of Las and the associated key HLB S genes using mutagenesis, yeast two hybrid assays, ectopic expression, and RNA interference techniques. For the predicated SDEs, we have tested whether they suppress programmed cell death (PCD). For the SDEs suppressing PCD, we identified their targets (HLB susceptibility genes) using yeast two hybrid assay. Yeast two-hybrid, in vitro protein pull-down and in vivo bimolecular fluorescence complementation assays showed that SDE15 interacts with ACD2 (ACCELERATED CELL DEATH 2), a repressor of plant PCD and that it enhances the red chlorophyll catabolite reductase (RCCR) activity of ACD2 to remove porphyrin-related molecules, accumulation of which causes PCD. SDE15 promotes the chlorophyll break-down in planta and contributes to the development of yellowing symptom associated with HLB. We have conducted RNAi for 12 putative target genes. The silenced plants have been inoculated with Candidatus Liberibacter asiaticus (Las) to further confirm their involvement of HLB disease development. Objective 2. We hypothesize that HLB resistance can be obtained by modifying key citrus HLB S genes to be unresponsive to Las. We will alter key S genes using CRISPR-Cas9/sgRNA genome editing technology to develop HLB resistant citrus varieties. In this objective, we have been optimizing the non-transgenic genome editing of citrus using the CRISPR technology and generating biallelic mutants for the putative HLB S genes. We have generate tissue cultures formajor citrus scion varieties in Florida, California, and Texas and two rootstock varieties. In our study, we employed CRISPR-LbCpf1, derived from Lachnospiraceae bacterium ND2006, to edit the citrus genome. First, LbCpf1 was successfully used to modify the CsPDS gene in grapefruit Duncan via the Xcc-facilitated agroinfiltration. Next, LbCpf1 driven by either 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4-CsLOBP) of CsLOB1. CsLOB1 is the canker susceptibility gene induced by the corresponding pathogenicity factor PthA4 of Xanthomonas citri, via binding to EBEPthA4-CsLOBP. Totally, seven 35S-LbCpf1-transformed Duncan plants were generated, designated as #D35s1 to #D35s7, and ten Yao-LbCpf1-transformed Duncan plants were created, designated as #DYao1 to #DYao10. LbCpf1-directed EBEPthA4-CsLOBP modifications were observed in three 35S-LbCpf1-transformed Duncan (#D35s1, #D35s4 and #D35s7). However, no LbCpf1-mediated indels were observed in the Yao-LbCpf1-transformed plants. Importantly, transgenic line #D35s4, containing the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Therefore, CRISPR-LbCpf1 can be readily used as a powerful tool for citrus genome editing. Here, we conducted nontransgenic genome editing of citrus via the transient introduction of Cas9-sgRNA DNA, RNA or ribonucleoprotein (RNP) into protoplast by PEG-mediated transfection or into embryogenic cells in a suspension culture by particle bombardment. Citrus protoplast or embryogenic cells appear to be recalcitrant to transformation using different approaches. Only the transient expression of CRISPR/Cas9 DNA in protoplast cells led to efficient transformation, and a genome editing mutation rate of 5.7% was observed. We further used hiTAIL-PCR analysis to show that the genome-modified citrus cells are free of foreign DNA. We also detected low off-target mutations in genome-modified citrus cells. Our findings represent a breakthrough in citrus breeding using CRISPR technology. We are currently in the process of regenerating citrus from the genome modifed cells. Objective 3. We hypothesize that select modified citrus varieties will have HLB resistance and acceptable horticultural traits. We will evaluate the genome-modified citrus varieties for HLB resistance, tree growth and development, canopy development, and other related horticultural traits. This objective will be conducted once the genome modified plants are planted in the field. Objective 4. Acceptance of genetically modified citrus will be determined by the availability of educational material about the genome modification process. We propose to provide educational material and assess public sentiment to genome edited products through customer responses to educational material. We are conducting surveys as planed regarding the consumer acceptance of genetically modified citrus. Objective 5. Extension and outreach with stakeholders are hypothesized to facilitate the development and delivery of products to end-users. We propose to provide extension and educational outreach to stakeholders and consumers. We have been giving presentations at the extension events on the collaborating states of FL, CA and TX including the Florida Citrus Expo, Grower Day, Florida Citrus Growers' Institute, California Citrus Growers Education Seminar Series, the UC Riverside Citrus Day and the Texas Citrus Showcase. We also published multple extension materials regarding genome editing in industry magazines and the Cooperative Extension newsletters.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Hongge Jia, Vladimir Orbovi?, Nian Wang CRISPR-LbCpf1-mediated modification of citrus
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Sheo Shankar Pandey and Nian Wang 2019 Targeted early detection of citrus HLB causal agent ⿿Candidatus Liberibacter asiaticus⿿ before symptom expression
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Zhiqian Pang, Li Zhang, Xiaobao Ying, Jinyun Li, Shuo Duan, Sheng-Yang He, Nian Wang SDE15 of Candidatus Liberibacter asiaticus suppresses programmed cell death to facilitate intracellular bacterial growth
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Nian Wang The citrus Huanglongbing crisis and potential solutions
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Shuming Wang, Hang Su, Yuanchun Wang, Xiuping Zou, Nian Wang Efficient nontransgenic genome editing in citrus via the transient expression of CRISPR/Cas9 DNA
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