In this quarter, several transgenic rootstocks expressing the AtNPR1 transgene were inoculated with HLB infected Valencia budsticks (November). Most of these were repeats of ones that had failed from the september graftings. As with pervious attempts, the Ct values of the infected budsticks were evaluated prior to stick grafting. Ct values ranged from 24.1 to 27 in the repeat budded set. The objective behind this study was to understand the effect of the transgenic rootstock on HLB. The budsticks have been slow to flush due to the winter. qPCR was performed on both transgenic and control roots as well as leaves but no HLB has been detected so far. it is estimated that it will be several more months before the inoculum can be detected. Transgenic rootstocks budded with non-transgenic valencia scion have also been prepared for planting in the field since we expect the field trial will provide the most conclusive data.
Field variety trials are a simple but effective tool to test plant horticultural performance under different environmental conditions and enhance the commercial adoption of new cultivars. Large-scale, rapid implementation of HLB-tolerant cultivars depends on reliable data, and the Millennium Block project is addressing the need of establishing field plantings to generate regional, updated information for the Indian River Citrus District. The project has mainly two objectives: (i) Assess performance of new grapefruit cultivars with certain rootstocks under HLB endemic conditions in the IR district and (ii) ) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy or standard rootstocks. We are in the process of planting four independent studies at the UF/IFAS IRREC. This report is related to the grove planting operations and initial tree care since the study was recently implemented. We planted approximately 3,600 trees and are awaiting for the remaining trees to become available from the nursery. Slow release poly coated fertilizer applied and tree wraps added. We installed the irrigation controller and activated field valves making irrigation system automation fully operable. Sand media filtration and water meter were purchased and delivered. Installation was approved and is in progress. We applied imidacloprid to prevent leaf minor and psillids, and followed with a spraying schedule as suggested by the certified crop advisor. The grove has been continuously scouted for pests such as orange dogs and ants. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a tree location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field. The group met with the certified crop advisor to develop a spray program schedule based on time of year and conditions to be applied as determined by IPM scouting. Masters student started on Jan/2020.We completed fertilizing the trees for the Spring season. Tree height, tree width in two positions (E-W and N-S) and trunk diameter were measured in three central trees trom each experimental plot. Leaf samples for HLB diagnostic were taken from a pool of trees from each experimental plot and sent to the lab. Fruit phenology started been measured in all experimental plots. A schedule of activitites for all proposed measurements was updated and implemented. We already noticed anedoctal differences in plant growth based on the scion and rootstock combinations.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, with CLas+ ACP feeding, as well as studying them in established greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection continues from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo rootstock grafted with non-transgenic rough lemon. Results from 6 and 18 months in the field show transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. A large second planting of Mthionin transgenics went into the ground in April 2019, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants). The next significant data collection for both plantings will be in April 2020; at 2 years for trees from the initial planting and 1 year for the second planting. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are being made a third MThionin planting.The Mthionin construct has also been extensively transformed into Valencia, Ray Ruby and US-942 to provide transgenic material of these critical varieties. The first 36 putative lines are now in soil and are undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′ using adjusted protocols to improve sensitivity and transmission rates (See section 4). . Further detached leaf assays are being run to compare the relative effectiveness between each generation of chimera constructs and to expand the number of lines tested from each. DLA testing has allowed us to identify lines from several constructs with significant effects on CLas transmission and even increased ACP mortalitly. Recent results include up to 95% mortality in ACP after 7 days feeding on detached leaves of the 3rd generation TBL transgenics and 70% for TPK. Lines from promising constructs have been moved forward into greenhouse studies based on DLA results, as noted below.Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All greenhouse experiments are now using an improved protocol to enhance inoculation. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6 month samples are now collected and being analyzed. We are also emphasizing parallel field trials for all phenotyping efforts. Preparations are now being made for a field planting of ~400 `74′ and `188′ transgenics is scheduled for spring 2020. Completed so far are 196 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. All plants will be ready for planting this fall.Fifteen new transformations, totaling over 5000 explants, have been completed to generate Valencia, Ray Ruby, US-942, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Over 100 new putative transgenic lines of 74-Valenica, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion.The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been bud inoculated with HLB+ RL and are undergoing the first post-inoculation analysis. An additional 370 rooted cuttings were propagated for the third round of ACP-inoculations. From which, the first group of 54 plants large enough to use have been inoculated with the higher pressure 21 day protocol. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer and potential therapeutic effects.The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines) and Carrizo/Mthionin (2 lines) have been returned certified clean. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.
Goal and Objectives: The project goal is to create the highest quality genome sequence assembly resources for five commercially and biologically important citrus varieties using breakthrough technologies in sequencing and assembly, to provide the research community with the best possible tools to support development of HLB-resistant cultivars by genome editing or other techniques. The specific objectives are:1. Sequence sweet orange, grapefruit, Clementine (all sensitive), lemon and LB8-9 Sugar Bell® (both tolerant) genomes using PacBio and develop chromosome scale assemblies using proximity ligation technology. Sequence RNA transcript libraries using Oxford Nanopore technology, yielding full-length sequences, to aid annotation, assembly, etc.2. Annotate genomes, phase haplotypes, compare structural and relational genomics among the five genomes to develop a package of tools for researchers to effectively utilize sequence resources for gene discovery, gene sequence retrieval, genome editing, and ultimately the creation of HLB-tolerant or -resistant citrus cultivars.3. Make these resources freely available to the research community through USDOE-JGI’s Phytozome portal, and other genome sequence platforms.4. Extract full length, putative HLB-responsive gene sequences from all genomes, make comparisons of sequences, transcripts and protein structures as they may relate to host-pathogen interactions and disease development or its suppression, and predict candidate sites for guide RNA designing and gene editing.Timeline Year 1: Objective 1: Produce suitable plant materials for PacBio sequencing technical requirements, isolate high-quality genomic DNA samples for sequencing, generate PacBio genome sequence data, and assemble PacBio sequence reads as data are received. Objective 2: Prepare RNA libraries for transcriptome sequencing, generate full-length transcript sequence data on Nanopore sequencers.Results 1. We have produced the plant materials described, for Valencia orange (S, for sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, for tolerant), and Lisbon lemon (T).2. We isolated high-quality DNA (high molecular weight DNA, or HMWDNA) as required for sequencing on the PacBio Sequel II platform. We modified previously used procedures to meet the stringent quality standards required for this platform, in collaboration with Dr. Shana McDevitt, Director of the UC Berkeley Vincent J. Coates Genomics Sequencing Laboratory, This involved several iterations back and forth, with QC provided by Dr. McDevitt’s group, and modifications to the protocol by Gmitter’s lab, until stringent QC standards were achieved.3. We generated the PacBio raw sequence data for the 5 genomes listed above. The first genomes were finished running in late October, and preliminary output from the first genome run showed that >250 Gbp of sequence were produced, yielding potentially > 83X coverage of the genome, very near our target of 85X coverage. The other four have been completed, as well.4. Preliminary analyses and assemblies have been carried out. For four of the five genomes, the results have exceeded the quality of any other publicly available citrus reference genomes, and this is before the further steps using proximity ligation technology to finalize at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we will sequence further.5. We have prepared materials from two genomes for the Dovetail Omni-C proximity ligation sequencing, the next step toward assembling to yield a chromosome scale full length assembly. They were sent to Dovetail Genomics on 18 February 2020.6. We have begun preparing the samples for transcriptome sequencing, which will be used to annotate the genomes (i.e. to define and identify all the genes within the assembly).Conclusions1. We completed all genome sequencing work under Year 1, Objective 1; the exception is that we need to resample and generate more sequence data for the Ruby Red grapefruit.2. We have not yet generated the full-length transcript sequence data, as proposed for Year 1, Objective 2, though we have begun the process. This goal was compromised because budgetary uncertainty beyond year 1 precluded hiring a post-doc to carry out the work.3. Omni-C sequencing for ligation proximity and chromosome scale assembly is underway for 2 genomes.4. Once funding is assured, we will move quickly to meet the benchmarks in the Project Timeline. A post-doctoral candidate has been recruited to begin working on the project in late April. A no-cost extension was approved for year 1, to end in July 2020, and all tasks for year 1 (and some for year 2) will be completed by that time.
The project has been granted a no cost extension into July 2020; year 1 activities, and some of year 2 objectives, have been nearly completed. A comprehensive overview of progress since project inception through April 2020 follows below. Timeline Year 1: Objective 1: Produce suitable plant materials for PacBio sequencing technical requirements, isolate high-quality genomic DNA samples for sequencing, generate PacBio genome sequence data, and assemble PacBio sequence reads as data are received. Objective 2: Prepare RNA libraries for transcriptome sequencing, generate full-length transcript sequence data on Nanopore sequencers.Results 1. We have produced the plant materials described, for Valencia orange (S, for sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, for tolerant), and Lisbon lemon (T).2. We isolated high-quality DNA (high molecular weight DNA, or HMW DNA) as required for sequencing on the PacBio Sequel II platform. We modified previously used procedures to meet the stringent quality standards required for this platform, in collaboration with Dr. Shana McDevitt, Director of the UC Berkeley Vincent J. Coates Genomics Sequencing Laboratory, This involved several iterations back and forth, with QC provided by Dr. McDevitt’s group, and modifications to the protocol by Gmitter’s lab, until stringent QC standards were met.3. We generated the PacBio raw sequence data for the 5 genomes listed above. The first genomes were finished running in late October 2019, and preliminary output from the first genome run showed that >250 Gbp of sequence were produced, yielding potentially > 83X coverage of the genome, very near our target of 85X coverage. The other four genomes now have been completed, as well.4. Preliminary analyses and assemblies have been carried out. For four of the five genomes, the results have exceeded the quality of any other publicly available citrus reference genomes, and this is before the further steps using proximity ligation technology to finalize assembly at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we will sequence further. WE now have prepared the Ruby Red grapefruit HMW DNA for additional sequencing, but we will have to store it until the UCB Genome Sequencing Laboratory reopens for normal business; currently their resources are devoted entirely to COVID-19 screening. 5. We have prepared materials from two genomes for the Dovetail Omni-C proximity ligation sequencing, the next step toward producing a chromosome scale full length assembly. They were sent to Dovetail Genomics on 18 February 2020. They have completed their work on two genomes, and their data files have been transferred to the collaborators at UCB and JGI. These results are being integrated with the PacBio sequence data to complete assembly at the chromosome level. Further work on the proximity ligation of the other genomes in on hold until we can work at the UF-CREC labs, to prepare the next round of samples to submit to Dovetail Genomics.6. We had begun preparing the samples for transcriptome sequencing, which will be used to annotate the genomes (i.e. to define and identify all the genes within the assembly). However, this activity is currently on hold, pending reopening of the UF-CREC labs.Conclusions1. We completed all genome sequencing work under Year 1, Objective 1; the exception is that we need to resample and generate more sequence data for the Ruby Red grapefruit. This has been initiated, with our resampling of HMW DNA.2. We have not yet generated the full-length transcript sequence data, as proposed for Year 1, Objective 2, though we have begun the process. This goal was compromised because budgetary uncertainty beyond year 1 precluded hiring a post-doc to carry out the work. Now this also is temporarily on hold, pending the reopening of the UF-CREC labs.3. Omni-C sequencing for proximity ligation has been completed for two genomes, and chromosome scale assembly is underway. 4. A post-doctoral candidate was recruited to begin working on the project in late April; however, his hiring has been put on hold because of COVID-19 travel restrictions. A no-cost extension was approved for year 1, to end in July 2020, and most tasks for year 1 (and some for year 2) will be completed by that time.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, the following activities have been conducted: (1) Preparing and maintaining transgenic plants for field trials in the greenhouse. Transgenic plants prepared earlier for the proposed field trial were maintained in the greenhouse, waiting for transplanting. Newly produced transgenic plants expressing a regulatory gene of systemic acquired resistance were regularly watered and fertilized. A group of transgenic rootstocks were produced. We plan to graft scions onto these rootstocks in the coming year. (2) Expressing and purifying the extracellular domains of a group (10) of citrus homologs of an Arabidopsis disease resistance gene that encodes a receptor-like kinase with the extracellular domain binding nicotinamide adenine dinucleotide. It has been shown that overexpression of this receptor-like kinase increases resistance to bacterial pathogens. The citrus genome carries more than ten homologs of this receptor-like kinase. We have cloned in the last quarter the extracelllar domains of the closest ten homologs in an E. coli expression vector. In this quarter, we optimized the protein expression conditions, expressed and purified these fusion proteins from E. coli. These proeins are fused with GFP. We are currently testing the nicotinamide adenine dinucleotide-binding activities of these proteins using Monolith NT.115. The homolog(s) with nicotinamide adenine dinucleotide-binding activity will be the citrus functional homolog and will be used to generate intragenic/cisgenic citrus plants. We have generated transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor.s
The fall cover crop mix planted in early November at both locations has had excellent germination and growth. This mix included daikon radish, coker, Wrens grain rye, and dove millet. Sunnhemp, alyce clover, sesbania, dixie crimson clover, and yellow sweet blossom clover were included in the mixes for the nitrogen-fixation treatments (1/2 of the rows). Germination counts and biomass samples have been collected, and are currently being analyzed.
Measurements of the abundance of specific N-cycling genes has begun for soil samples collected at the last annual collection. After one year of cover crops, the abundance of nitrification genes (ammonia-oxidizing archaea and bacteria) significantly increased in treatments with cover crops when compared to the grower standard control, suggesting an increase in soil nitrogen availability. Measurements are in progress to determine the abundance of nitrogen-fixing genes (important with legumes since they are related to changes in the content of ammonium in soils) and denitrification genes (often linked to changes in soil organic carbon, organic matter, and nitrogen losses from the soils). Multivariate statistical analyses are also in progress to evaluate the influence of abiotic (soil properties) on biotic (microbial gene abundance) variables in treatments with and without cover crops.
Dataloggers and soil moisture probes continue to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019 and Feb 2020. Data on these measurements are currently being analyzed and will continue in Fall 2020. Initial analysis of yield, trunk area and canopy volume found no significant differences after 1 year of cover crops for one location. However, preliminary data indicates a greater canopy volume and trunk area at the second field site in treatments with the cover crop + legume mixture compared to other treatments. There were no significant differences in soil phosphorous, potassium, or magnesium concentrations between treatments at either location after 1 year of cover crops. Additional nutrient measurements are still in progress.
Weed density measurements from both study locations were collected in Aug 2019 and Jan 2020. Preliminary analysis of weed density data suggests that cover crops significantly reduced (up to 84%) weed pressure in treated row-middles when compared to non-treated controls. Moreover, cover crops improved the biodiversity in the treated row middles. Annual biomass data collection was also performed in both study locations in Jan 2020. Cover crop and weed biomass was harvested from the plots and bagged. In the lab they were sorted and weighed out to gather information on total weed/cover crop biomass from the treatment/control plots.
The first harvest since cover crops have been planted will occur in Spring 2020 (likely end of March). A project meeting with the grove managers will be held after harvest to discuss the next planting of cover crops. Some root rizhotrons and data loggers were damaged at one of the sites by hogs and some heavy equipment. These problems will be rectified in the next quarter to ensure continuity of of measurements in some treatments.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEP thA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEP thA4 -CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEP thA4 -CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the citrus U6 promoters and testing their efficacy in driving sgRNA and crRNA in genome editing of citrus. We have significantly increased the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. One manuscript is in preparation for publication.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Twelve new rootstock crosses were made in spring 2019 and seeds were harvested and planted into the calcareous soil/ Phytophthora screen, the first stage of the gauntlet screening protocol. Seeds were harvested from many of the UFRs for distribution to nurseries. Seeds from various unreleased rootstock candidates, showing good performance (HLB tolerance, good yields and fruit quality, some tree size controlling with more efficient canopies) in several field trials throughout the industry were harvested to be available for future trials with interested industry partners in Florida, as well as for new trials in other citrus production areas. New hybrids from 2018 crosses for rootstock improvement were field planted. 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Spring crosses for this objective were numerous. We harvested fruit from more than 2 dozen interploid crosses, including several designed to produce sweet orange-like fruit, using HLB tolerant parents selected by our program previously. In addition, selected HLB and canker tolerant pummelo breeding parents were crossed with diploid and tetraploid grapefruit, to produce new grapefruit hybrids with enhanced tolerance of HLB. Embryo rescue has been completed and plant production is underway. Over 100 new somaclones have been regenerated in vitro from EV1 and EV2, for further selection. Four new grapefruit hybrid selections were made in this autumn 2019 that produce fruit very similar to grapefruit in appearance, color and flavor, but with improved fruit quality attributes and substantially more tolerant trees, and these have been entered into the DPI Parent Tree Program for cleanup. Finally, the OLL-20 sweet orange was approved for release to the Florida citrus industry. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have explored some new approaches to quantifying tree responses to HLB, in addition to the previously used subjective approaches. Specifically, we have begun measuring photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. This work is ongoing, and it will improve the precision with which we can define HLB tolerance genes. We have evaluated fruit quality of the more tolerant types of sweet orange-like hybrids, as well as mandarins and grapefruit hybrids, and identified some worthy of further evaluation as potential new cultivars. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Using the quantitative data described in 3. above, we are preparing to conduct additional GWAS to validate previously identified or to identify new genomic regions associated with HLB tolerance and/or sensitivity. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments have been begun.
Update for this quarter:
Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State were planted.
Fruit were harvested for transgenic gene flow experiment and seed extracted.
Five seedlings resulted from FF-5-51-2 x early-flowering transgenic Carrizo (cross in test site) and one resulting seedling has already flowered.
Gmitter group has begun to collect data on Clementine x C. latipes.
BRS visited site to orient new staff.
Previous two quarters
Previously established at the site:
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside.
11) Availability of the test site for planting continues to be announced to researchers.
Plantings:
1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit.
2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen.
3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection.
4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.
5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.
6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes.
9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.
10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.
11) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.
12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State
13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
Project rationale and focus:
The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations.
Most recent quarter:
Stover lab:
· 5300 for Stover lab – for inoculations to screen antimicrobial transgenes
Stover lab:5300 ACP for inoculating 100 detahced leaves, 90 no-choice small trees and six homogenate assays of peptides.
Bowman lab:
First test group of sweet orange on rootstocks was scored in November 2019 (at 4 mai) and PCR being processed now. Grafted trees for groups 2, 3, and 4 are growing in greenhouse now. Will begin ACP inoculation of group 2 in April.Rootstock liners for groups 5, 6, and 7 are being grown in greenhouse now.
Other users:
· 500 for Dean Gabriel – UF
· 4200 for Yongping Duan – inoculations of resistant Duncan Grapefruit
· 1440 for Randy Niedz – inoculations of Ridge Pineapple and Duncan Grapefruit
Previous quarter:
Over 11,560 ACP infected ACP were used in the last quarter, to screen trees in no-choice inoculation of transgenic citrus and prescreen transgenic events using detached leaf assays
As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field.
In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
Create new candidate hybrids. Sexual hybridization is completed between selected elite parents during spring flowering and seed collected in the fall. During this quarter, seed were collected from crosses of elite rootstock hybrids US-942 and US-897 with pummelos. Selected hybrids will be grown-out for propagation, testing, and establishment of seed trees. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora, along with showing favorable effects on grafted tree yield, fruit quality, and tree size. Propagate and plant new field trials. Replicated multi-year field trials with commercial scions are essential to evaluate performance of rootstocks, both to determine whether each new rootstock should be released for commercial use, and to develop comparative performance information among new and existing rootstocks for a diversity of scions, soils, and management conditions. Most of the rootstock field trials are planted with a single scion representing a common commercial type on each of 40-60 different rootstocks. Adequate replication is considered a critical factor in the USDA rootstock trials, with 6-7 replications the minimum and 12 replications the optimum to provide an acceptable level of reliability for results. Three new rootstock trials with sweet orange scion on promising rootstocks were planted in this quarter. Nursery trees for three additional rootstock trials are being prepared in the greenhouse for planting in spring 2020.Collect data from field trials. Information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Measurements related to cropping are on an annual cycle based on the scion, while measurements of health and tree size are on a schedule determined by the specific conditions and goals of the trial. Cropping data was collected from 6 trials with early-maturing scions during Nov 2019-Jan 2020. Assessments of tree health and measurements of tree size were completed on nine trials during this quarter.Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Some of the newest hybrid rootstocks can be uniformly propagated by seed, but others cannot. As the best rootstocks are identified through testing, seed sources are established and used to determine trueness-to-type from seed. Studies were initiated this quarter to evaluate seed propagation for 25 of the most promising SuperSour hybrid rootstocks.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. I recognize that this information is of great value to citrus growers and nurseries, and am working to create summaries from 38 trials that will be posted onto a website for easy access by Florida growers. It is anticipated that the first stage of that information will be posted to https://citrusrootstocks.org/ soon. Release of superior new rootstocks for commercial use. Several of the 300 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Perforance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2021-22.
The project has five objectives:
(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants
(2) Graft CTV- and HLB-free buds onto rootstocks
(3) Generate a large number of vigorous and healthy citrus trees
(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses
(5) Collect the field trial data
In this quarter, the following activities have been performed:
(1) To ensure the quality of the citrus plants for the field trial, protein levels in the propagated plants were analyzed. Leaf tissues were collected for total protein extraction. After extraction, total protein was separated in SDS-PAGE gel. The proteins were transferred onto a nitrocellulose memebrane. The blot was probed with an antibody against the overexpressed protein. It was found that all progeny plants express good amount of the transgenic protein. The plants were carefully managed by regular watering and fertilization. This batch of plants will be transplanted into the field at the end of April or early May of 2020.
(2) Purify antibody against a major citrus defense protein. One boost was conducted to the rabbits. Serum was extracted form both rabbits and purified with protein A beads. The purifed antibody was tested using the citrus protein transiently expressed in Nicotiana benthamiana. It was also tested using total protein extracted from the sweet orange ‘Hamlin’. The titer of the antibody has been improved by the boost. The antibody can now be used to analyze other citrus materials.
(3) Analyze the transgenic plants already planted in the field. The size of the plants in the field at Ft Pierce USDA ARS were measured. Leaf samples were collected from each plant. Total protein and RNA have been extracted. Assays of the transgenic protein levels and CLas titers are ongoing.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require regulatory approval.
Based on two year’s graft-inoculation assays in greenhouse with two HLB bacterial isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees.
The first group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown at our research farm, Picos Farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infected plants. It is worth noting that the new HLB isolate from Picos Farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest, graft-based greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. Prelimilnary data showed some of the selections are better than the others with either lower disease index or better canopy growth. Some of the selections showed much lower infection rate (less than 20%) than the control (40%) and poor performers (40-50%) after planting in Picos research farm for 26 months. Among the Scott’s seedling selections, one of the 4 selections displayed its outperformance than the others with the lowest disease rate of ca. 13.0% and better growth canopy. All the plants were verified by Las-specific qPCR assays, and there were no significant difference among infected genotypes in term of Las bacterial titers. It is worth noting that some of the plants propagated via cutting (without a rootstock) showed some promising outcome with less disease index. However, there was no significant difference observed among the three rootstocks in the trials. Some of the genotypes planted in Nov. 2017 (about 10%) bears fruits, and the fruits from these genotype selections were collected, and they are going to be evaluated for juice quality via taste panels and instruments. In summary, we have yet oberved at least two selections (one from bud sport) that are significantly better than the others (including control grapefruit plants).
The second group of the propagates on the three different root stocks mentioned above (Ca. 750 plants) were planted in Scott Groves in two lots, with this experiment fully planted by mid-September 2019. All of the propagates have been tested for the presence of Las via qPCR. All the 700 plus plants in Scott grove grow as expectedand and will be further evaluated.
Objective: A single objective of this project is to assure the presence of active site that will provide un-interrupted service for production of transgenic citrus plants to researchers involved in fight against huanglongbing (HLB) and citrus canker. Through its services, Citrus Transformation Facility (CTF) offers a place for groundwork for the scientific community. For the laboratories without transformation capabilities, CTF makes their projects possible by producing transgenic plants. CTF staff also participate in other research projects that had to do with production of transgenic citrus plants and when needed, offer advising and training services.
Major accomplishments per objective: The uninterrupted operation of CTF that resulted in production of transgenic citrus plants is the major accomplishment for the 2019. Throughout the whole year, the facility was open and ready to accept the orders and start working on them almost immediately. Altogether, the CTF received 25 orders during last year. Placed orders included requests for transgenic Duncan grapefruit, Valencia orange, Mexican lime, and Indian curry leaf plant (Murraya koeinigii). The number of produced transgenic plants is 246 (Table 1). We have produced additional 17 plants (10 Duncan and seven Valencia) that were designated as transgenic and included in one of our quarterly reports. However, upon additional testing we decided those plants are not carrying the genes they were supposed to and we deducted them from the final count. Those plants that were produced belong to following cultivars: Duncan grapefruit, Mexican lime, Valencia sweet orange, Pomelo plants, Kumquat plants, Pineapple sweet orange Carrizo citrange, and plants of Indian curry leaf plant. All of the plants produced by CTF were the result of research that has a goal of fighting the HLB disease. These plants have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants, they produce chemicals that can kill Asian Citrus psyllids. All plants stayed in the state of Florida where further tests will be conducted to test desired traits resulting from introduction of transgenes.
Table 1. Plants produced by CTF in 2019
Cultivar Number of plants produced
Duncan grapefruit 155
Mexican lime 32
Valencia sweet orange 20
Pomelo 21
Kumquat 4
Pineapple sweet orange 4
M. koenigii 10
Number of co-incubation experiments done with explants of different cultivars and appropriate bacterial strains was 150. About 150,000 explants were used in those experiments. In only one experiment all the explants were contaminated and in three others there was a partial loss of material. The data from 136 experiments were collected in 2019. One hundred and ten experiments included green fluorescent protein (GFP) as a reporter gene and because of that we inspected under the microscope about 100,000 shoots and buds that sprouted from treated explants for the presence of GFP fluorescence. Altogether there were 1914 transgenic shoots and buds but 1293 were chimeric and 621 were exhibiting GFP fluorescence in all tissues. We have also preformed about 950 PCR reactions with primers specific to LOB gene and to GUS gene during selection of Duncan grapefruit shoots positive for gene carried by JJ8 binary vector. Additional 925 PCRs were done with Valencia shoots using primers specific for sequences carried by the JJ7 vector. Furthermore, 570 GUS assays were also done with samples from Valencia shoots in search of those transformed with sequences from the JJ7 vector.
In February of 2019, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for supply of seeds that lasted until November. In September, the crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. Since CUPS-produced fruit do not tolerate well storage conditions of cold room in CREC’s packinghouse, we lost more than 50% of fruit in a short period of time. At the end of January of 2020 when the second half of Duncan grapefruit gets harvested from CUPS, we will concurrently use them for experiments and extract seeds that will be stored. In order to secure sufficient supply of Duncan seeds, we will purchase half of the box of fruit in February 2020. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.
Major shortcomings, unfinished business: High majority of work done in CTF on production of transgenic citrus plants includes GFP as a reporter gene. Numbers reported in the above section best describe why. Almost all plants produced in the 2019 were selected based on the GFP fluorescence. All of the PCR reactions and GUS assays performed last year for orders that did not use GFP, lead to production of just a few plants. These tests also resulted in some false positives I described above. CTF is at the point where we can relatively easy satisfy the orders for some citrus cultivars and produce about 10 transgenic plants with desired gene within nine months if the GFP is a reporter gene. GFP is a powerful tool that researchers are holding on to, because it helps them get the results (transgenic plants) fast. CTF has no leverage to steer people away from using GFP as a selection tool in the process of production of transgenic citrus plants. Such an effort would also be counterproductive until equally efficient reporter gene is available.
The flux of employees working in the CTF remained high. Two employees who worked at CTF in the beginning of 2019 have left. One of these employees was funded from the USDA grant where there was money left over upon his departure. We are presently in the final stages of hiring a replacement. In the spring of 2019, one OPS employee was hired on a temporary basis for six months. This person left the facility on November 1st although some funds remained available. New employee was already hired as a replacement.
The opportunities going forward: Future opportunities for the CTF reflect the needs of Florida Citrus Industry. The most important thing that CTF can do is to participate in fight against HLB and citrus canker by producing trees with increased tolerance and/or resistance to these diseases regardless of methodology used.
Researchers using CRISPR for editing of citrus genome are still trying to produce homozygous plants that do not have in their cells any “leftovers” from the process of genetic modification. Even when this gets accomplished, that should be just the beginning of the use of this technology in the improvement of citrus. This was, and still is, the great opportunity for the CTF to play its role by producing citrus plants with edited genes for the benefit of all stakeholders in the citrus industry.
The CTF was the first site where cisgenic citrus plants were produced. These plants contain only the DNA from citrus even after genetic modification. Since it is not known whether CRISPR can be used successfully in all efforts for improvement of elite citrus cultivars, introduction or modification of genes from same or related species remains as valid approach. CTF is ready for such efforts at any given time.
Publications from this project
1) Jia, H., Orbović, V., Wang, N. (2019) CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnology Journal, doi: 10.1111/pbi.13109
2) Song, G., Prieto, H., Orbović, V. (2019) Agrobacterium-mediated transformation of tree fruit crops: Methods, progress, and challenges. Frontiers in Plant Science, 10:226.
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