The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we continued propagating the transgenic lines that overexpress Arabidopsis defense genes and inoculated the previously generated progenies. The new progeny plants are growing in the greenhouse. The progenies obtained in the last quarter have been inoculated with Las-infected psyllids for two months and moved back to the greenhouse for symptom development. HLB symptoms on the plants have been carefully monitored and recorded. For objective 2, we performed the second round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results indicated that several lines of the HLB-resitant/tolerant transgenic lines are not chimeric. If these lines are confirmed to be HLB-resitant/tolerant in objective 1, they will be able to be propagated by grafting for industry use. For objective 3, we continued propagating the gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB and inoculated previously generated progenies. The new progeny plants are growing in the greenhouse. As for the transgenic progenies, those obtained earlier were inoculated with Las-infected psyllids and are currently in the greenhouse for symptom development.
Cell-penetrating peptides (CPPs) are a class of peptides (short stretchers of amino acids, the building blocks of proteins) known to translocate across most organic membranes. CPPs are currently revolutionizing the pharmaceutical and medical industries where CPPs are being investigated as vehicles for the delivery of therapeutic compounds and other cargos (proteins, RNA, DNA, antibiotics). Surprisingly, CPPs have also been found to function in plant cells to ferry cargos across cell membranes. The goals of this project were 1) To determine if CPPs could be systemically transported in citrus. 2) To develop a CPP transformation protocol without Agrobacterium in citrus. 3) To evaluate the use of CPPs as delivery tools for disease therapies and study the role of defense genes. Experiments with CPPs alone were unsuccessful with DNA, although protein could be transported. The addition of CRISPR/Cas to the systems appears to be successful. Importantly, plants altered with this system are not being regulated as transgenic.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,most of the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 40% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from the larger lines have been made and are being rooted in the mist bed for future challange with Diaprepes. A number of other potential root specific promoters have been identified from the phytozome database. qPCR gene expression analyses on non-transgenic leaves, flowers, fruit, phloem, seeds and roots have identified some that can potentially be used in the future for root specific gene expression. Results from some of these studies will be presented in the World Congress on In vitro Biology in the summer.
We hypothesized that groves suffering most from HLB were exhibiting stress associated with high soil concentrations of bicarbonate since they support lower fibrous root density compared to groves with lower bicarbonates (< 100 ppm) in irrigation water and/or soil pH (< 6.5). To confirm this relationship, we surveyed 41 grove locations in Highlands and Desoto Counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density was significantly related to well water pH > 6.5 and to soil pH > 6.2. Records from these blocks revealed that yields from groves under high bicarbonate stress declined 20% from 2011 to 2013, in contrast to ridge groves with low bicarbonate stress that increased 6% in production even though HLB incidence had accelerated. Yield losses were correlated with less fibrous root density which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicated that bicarbonate impairs the root s ability to take up important nutritional cations including Ca, Mg and K as well as micronutrients, especially Mn and Fe. Similar data for citrus were lacking; hence there was a need for more specific information for the effects of HLB, bicarbonates and their interaction for trees on the commonly grown rootstocks in Florida (i.e. Swingle and Carrizo) under field conditions. For evaluation in replicated plot trials, acidification of irrigation water and soil treatments that reduce bicarbonate were applied to quantify their effects on root health. Irrigation water acidification was targeted to soil pHs of 7.5, 6.0, 5.0 and 4.0 without or without sulfur application to soil to maintain the pH targets. In response to acidification leaf nutrient concentrations have been maintained in the optimum or high range. Greater leaf concentrations of Mg, Ca, Fe, Mn and Zn in plots were measured at target soil pH of 4.0 and 5.0, but no differences were found with or without sulfur amendment. Lower irrigation applications and few fertigation applications resulted in reduced soil acidification in the irrigated zone. Survey for root density, tree nutrient status and yields was implemented in groves that received water/soil acidification treatments to reduce soil pH and bicarbonate concentrations in comparison to unmanaged groves that were either untreated or experienced little or no bicarbonate stress based on the status of irrigation water and/or liming history. Acidification of irrigation water in central ridge and south central flatwoods Valencia orange groves on Swingle rootstock maintained soil pH below 6.5 on the flatwoods and 6.0 on ridge. Over the last 2.5 seasons of survey, root density as an index of root heath was sustained. Phytophthora populations remained below the damaging level in ridge groves and in flatwoods increases to damaging levels were coincident with the fall root flush dropped back to non-damaging levels for remainder of the season. Compared to 2014-15, yields in the ridge blocks have increased up to 4% and on the flatwoods increased up to 20%. Soil and tree responses to acidification required several seasons to become completely manifested. The final goal is to perform an economic analysis of bicarbonate management in terms of irrigation water and soil acidification costs versus benefit from gains in tree health and productivity so that growers may prioritize their expenditure on practices that mitigate losses of tree productivity due to HLB. With a complete sets of 2015-2016 yield data from acidified grove blocks, a cost benefit analysis will be performed.
In September 2012, FireWall (Agrosource, Inc.) was granted an EPA Section 18 registration for control of citrus canker in Florida grapefruit. The FireWall label restricted use to no more than two applications per season. As a condition for FireWall registration, EPA required monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in commercially treated groves. The objective of the survey was to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey was conducted in 3 or 4 grove locations each season in November-January. A report of the survey results were submitted each January to FDACS to be forwarded to EPA. No Xcc resistant to streptomycin were recovered from commercial groves during the 3 year survey. Greenhouse assays of sweet orange seedlings injection-infiltrated with Xcc consistently demonstrated that streptomycin, the active ingredient in Firewall, moves trans-laminar and controls Xcc infection in expanding leaves. Furthermore, this assay demonstrated that streptomycin moves upward into the next leaf flush where there is detectable activity against Xcc inoculated 10 weeks after spray application to initial flush. These assay results confirm streptomycin moves upward and explain the consistent performance of streptomycin against canker in field trials. Penetration of leaves by streptomycin insures that it is protected from wash-off by rainfall, weathering and degradation by UV light.
The objective of this project is to use new technologies to accelerate the elimination of graft transmissible pathogens in germplasm accessions for use in citrus breeding in Florida. These new technologies include the application of cryotherapy (freezing the buds in liquid nitrogen followed by recovery of the treated buds by grafting onto seedling rootstocks) and the use of mini-plant-indexing which allows the biological indexing for graft transmissible pathogens using young seedling indicator plants, 60-75 days old seedlings. During the current reporting period we continue to maintain/evaluate thirty scion selections (five replicate plants of each) that had been cryo-treated at the National Center for Genetic Resources Preservation (NCGRP) in Fort Collins. One hundred fifty, budeyes of CLas-infected selection USDA 1-23-130 were sent to the NCGRP in Ft. Collins. 100 buds were processed and 50 buds were recovered following cryo-treatment and grafted onto seedling rootstock (Carrizo). In the previous report we identified nine promising scion selections that were chosen for cryopreservation. All nine of these selections have been placed into cryopreservation at the Germplasm Preservation Lab. Plants recovered from cryopreservation will be sent to Ft. Pierce for evaluation and pathogen testing. Our permit expired during the last reporting period and must be renewed prior to return of plant material to Florida. During the current reporting period four additional promising scion selections were identified in the USDA citrus project. These selections will be cryopreserved within the next few weeks. To date, there is total of 16 USDA advance scion selections that have been cryo-preserved. These selections can otherwise only be maintained as whole plants. Currently, greenhouse space is at a premium for HLB research and therefore, use of greenhouse space for germplasm preservation has become minimal. To maintain advanced selections in the field means likely infection with Liberibacter, and subsequent HLB. The cryopreservation process is proving to be an efficient means to preserve citrus germplasm.
Bacterial pathogens rely on the secreted “effector” proteins as essential virulence factors to cause disease. The HLB-associated bacterium Candidatus Liberibacter asiaticus (Las) possesses the Sec secretion system, which is a general protein secretion machinery that delivers effectors from the pathogen cells into the phloem of infected plants. Our research interests have been primarily in the Sec-delivered effectors (SDEs) produced by Las and our long-term goals are: 1) using SDEs as detection markers for Las diagnosis; 2) using SDEs as molecular probes to understand HLB pathogenesis. Previously, we identified ~30 SDEs from Las through bioinformatic analysis of the Psy62 genome; these SDEs were then analyzed on their gene expression levels in infected citrus trees. Results from these analyses allowed us to focus on three SDEs, which are highly expressed in infected tissues of various citrus varieties. Building on these previous results, this project aims to characterize the citrus targets of these three SDEs. Our central hypothesis is that SDEs contribute to HLB development by manipulating their host target(s) in citrus. Understanding how SDEs modulate citrus physiology and immunity will provide important mechanistic insight into HLB pathogenesis. This knowledge will then facilitate the development of sustainable control strategies. We proposed to pursue three objectives at the beginning of this project and we are happy to report that all of these objectives have been successfully accomplished. 1) Identify citrus proteins associating with three selected Las effectors using yeast two hybrid screens. We used the yeast two hybrid (Y2H) screening approach and identified the citrus targets of each of the three SDEs. For this purpose, we constructed a normalized citrus cDNA library containing more than 3 millions of primary clones using HLB-infected RNA samples. This library was then subjected to a next generation sequencing-based screening using each SDE as the bait. 2) Confirm the effector-host target interactions using a series of in vitro and in vivo assays. We confirmed the SDE-citrus target interaction using a series of biochemical assays including targeted Y2H and in vitro co-immunoprecipitation. We first analyzed the potential SDE targets identified from the Y2H screening for promising candidates that are: 1) differentially expressed in HLB-infected citrus; and 2) potentially contributing to immunity and/or HLB symptom development. The full-length cDNA of these genes were then cloned in various plasmid vectors for experiments to confirm their interaction with the SDEs. In average, we examined the interaction of each SDE with ~10 candidate targets. Through this process, we further focus our research on one SDE, which specifically interacts with a class of enzymes that have known function in plant defense. Intriguingly, these targets are also manipulated by effectors produced by other bacterial and fungal pathogens to promote virulence. 3) Design control strategies aiming to enhance the resistance/tolerance of citrus to HLB based on effector activities and the functions of their targets. We are designing chemical treatment strategies based on the enzymatic activity of the citrus targets that we identified for one SDE. This work will continue beyond the funded period of this project.
In January 2016, we continued with the planned treatments both in the greenhouse and field. We followed the proposed plan of work with the following experiments. I: Effect of drenching application of SL on HLB-infected trees. Soil in potted Valencia trees was drenched with SL at the predetermined concentration. Tree characteristics were noted and changes recorded on a bi-weekly basis. The second drenching treatment was applied in February as planned and data continued to be collected. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). This is a repeat of the experiments involving spraying SL on Valencia potted trees in the greenhouse. Tree characteristics were noted and changes recorded on a bi-weekly basis. Second spray treatment was applied in February and data on flowering, flushing and fruit set recorded biweekly. III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. This treatment has been postponed. IV: Effect of spray application of SL on other varieties of citrus in groves. ‘Midsweet’ was selected as a second variety to be tested for SL effect on tree health. Tree physical characteristics and fruit drop are being monitored throughout. Data on fruit drop was completed after application of spring treatment. V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds were applied to trees. So far, diluted sucrose solutions have been effective in enhancing new flush in trees with advanced stages of HLB. Sucrose has been applied to trees in a monthly basis and some recovery has been observed. For all treatments, data is being collected on the appearance of new growth, flowering, fruit drop and vegetative growth in general.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. 1. To test for he unlikely but increasing possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. Samples of nectar, honey, pollen, albedo, flavedo and flowers were collected in spring time and now being analyzed. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees. The trees have been placed in a greenhouse and continue currently under observation. At the moment, symptoms have appeared and PCR will be performed soon. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. 4. Trees have been grafted for a substantial amount of time and some started showing HLB symptoms. However, given that analysis of this objective destroys the trees, more time is needed to be certain that HLB has taken root.
The use of antimicrobials is one of the few effective treatments against HLB in citrus trees. However, penetration of substances into trees is hindered by the presence of protective layers such as the thick cuticle on leaves, and cork on stems. To overcome the obstacles imposed by the cuticle to increase penetration of externally supplied substances, we have successfully tested laser light. Laser light technology involves the use of low level light energy to disperse the cuticle creating microscopic and superficial indentations of approximately 250 m. In doing so, infiltration of substances into the leaf is greatly enhanced. Once inside the leaf tissue, substances can follow the natural transport pathway through the apoplast, absorbed by phloem cells, and transported throughout the tree. Specific goals are 1. To build and test a more flexible and elaborate laser machine that will allow for more decisive experiments in the greenhouse; 2. Test for the effectiveness of several antimicrobials; 3. Carry out initial field experiments with young trees. The laser machine was delivered and tested under laboratory conditions. A series of initial experiments were conducted to fine tune the machine in terms of laser energy, distance, speed of laser and striking angle. All these parameters have been determined for optimal efficiency. Experiments aimed at quantify the enhanced penetration of applied substances into lasered leaves compared to unlasered leaves were performed with fluorescent deoxyglucose (NBDG) given that a system to measure oxytetracycline has not been refined. Our experiments showed that laser light enhanced penetration of NBDG approximately 4,500 times per 27 mm2. These experiments were performed in both healthy and HLB affected trees with similar results. Additional experiments to test for possible dessication and effect of leaf age were performed. Although dessication of lasered leaves was noticed in greenhouse trees, surprisingly no dessication (leaf curling) was noted in field grown trees eve in the absence of applied wax. Number of laser episodes per leaf was tested in young and mature leaves. Young leaves are physically incapable to sustain any laser treatment whereas mature leaves are capable of weathering only one. Assessment of oxytetracycline penetration is being halted until a quantitative method is refined. At the moment of writing this report, DOC personnel have refined their HPLC and we are ready to complete this last phase of the project.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for six years and new trees are being added every few months. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015. The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al, Plant Disease, 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree (Miles et al., 2016). All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Our significant progresses during this reporting time period are: 1) Using mature shoot segments of Valencia and Washington navel, we have demonstrated that the Kn1 gene can improve transformation efficiencies by approximately 2-fold compared to the control vector, which is much lower than those observed in juvenile citrus transformation. 2) We used an epigenetic modulator in our transformation experiments and observed about a 2- to 3-fold increase in overall transformation efficiency in mature tissues of Valencia and Washington navel oranges. We further demonstrated that the epigenetic modulator produced a 10-fold increase in shoot regeneration efficiency of mature citrus with no transformation when compared to the controls. 3) With expression of a 35S::GUS gene containing an intron as an indicator, we examined Agrobacterium infection and T-DNA integration activities in mature citrus using tobacco leaf discs and juvenile citrus tissues as references. Consistent with the fact that tobacco leaf discs can be efficiently transformed with Agrobacterium, we observed very high levels of transient and stable expression of GUS in the cut edges of tobacco discs. When juvenile citrus tissues were used for Agrobacterium infection, we observed reasonable levels of both transient and stable GUS gene expression. Using mature explants of Valencia, however, we observed extremely low levels of transient and stable expression of the GUS gene. As we have shown that although both the Kn1 and Ipt gene dramatically enhanced transformation efficiencies of juvenile citrus via increased shoot regeneration, they were far less effective at improving transformation on mature citrus tissue. Also, in mature Valencia and Washington navel oranges, we found that using an epigenetic modulator led to about 10-fold increase in shoot regeneration, but only a 2- to 3-fold increase in transformation efficiency (i.e., transgenic shoot production). We hypothesized that after improved shoot regeneration, Agrobacterium-mediated T-DNA integration remained the major challenges to improving mature citrus transformation. We are now working to enhance efficiencies of Agrobacterium-mediated stable T-DNA integration. Combining the various molecular tools we have, we would like to develop a ‘vector’ that is highly efficient and genotype-independent for mature citrus transformation. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been published: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. Two manuscripts are under preparation, reporting some of the results summarized above.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and six PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we will add the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs have been created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs have also been created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and protein expression has been confirmed by immunoblot. These constructs have also been transformed into Arabidopsis for analysis, and two constructs have been provided to the Lake Alfred transformation facility, Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
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