In the period leading to June 30, we finished obtained raw data from transcription assays reporting function of CLas transcription factors. We analyzed results from RNA transcription assays on the Sm (deletion) visRvisN mutant carrying a plasmid with CLas visNR. The overexpressed CLas visNR caused upregulation of 38 genes with a cut-off of 1.5X. Among these, most dramatically changed was expression of rem (SMc03046, a response regulator for motility during exponential phase growth) and a number of flagellar synthesis genes which went up by 4 to 8X. We also isolated, assayed and analyzed genes responsive to the CLas lsrB gene. The LsrB proteins are LysR-type transcriptional regulators. The S. meliloti ortholog of LsrB protein has previously been shown (Tang and Cheng, 2013) to function in symbiosis and to regulate the lrp3-lpsCDE operon. Our deletion mutant of Sm lsrB grows poorly and is highly susceptible to destruction by the detergent DOC. The cloned CLas lsrB gene partly complements this function, to make Sm lsrB mutants a bit more resistant to DOC. However, our transcription results indicated that CLas LsrB does not function well in S. meliloti as a transcription factor. Specifically, expression of lrp3-lpsCDE was unchanged by the presence of CLas LsrB. On a global level, CLas LsrB only changed expression of 4 genes, and by a maximum of 1.2 fold either up or down Transcript analysis for the CLas CtrA regulator expressed in wild type S. meliloti showed that CLas CtrA upregulates a number of cell-division related genes including minCDE (2 to 3X change). Several genes related to native CtrA regulation were down-regulated by CLas CtrA protein. This result was unexpected, given that no genes showed decreased expression due to the native Sm ctrA gene. The phrR regulatory gene exists in two copies in S. meliloti, with the two proteins (PhrR1 and PhrR2) being 56% identical to each other. PhrR1 protein is 59% and PhrR2 is 48% identical to the CLas PhrR protein. As background for the CLas expression studies, a S. meliloti double mutant of phrR1 and phrR2 genes had to be constructed. This mutant (or even the phrR1 single mutant) grew very poorly. The cloned CLas phrR gene did not restore growth to the S. meliloti mutant, and when expression of CLas PhrR protein was induced with IPTG, it somewhat inhibited growth of the wild type control as well; for example, the cells expressing CLas PhrR protein yielded smaller colonies. The overexpressed PhrR transcription factor did not induce high levels of any gene (maximum upregulation was 1.2X) although it had slightly stronger negative effects on a small number of gene targets. Assembling all the data, we began to select which target genes would be used to construct chromogenic (lacZ) and fluorescent (GFP) fusions for the robotic assay.
This is a three-year continuing project, terminated in Aug. 31, 2016. The overall objective was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. Based on our optimized nano delivery system and our screened effective antimicrobials from our previous funded projects (CRDF#584 and #617), a total of 14 antimicrobials were formulated in nano emulsions and applied on the HLB-affected potted plants in the greenhouse by foliar spray and bark-painting, including two agricultural antibiotics {Validoxyamine (VA) and Zhongshengmycin (ZS)}, seven antimicrobial compounds {Sulfadimethoxine Sodium (SDX), Silver Nitrate (SN), Silver Phosphite (SP), EBI-602, Actidione (ACT), p-Cymene (PCY) and Carvacrol (Carv)}, two antibiotics {(Oxytetracycline (OXY) and Streptomycin (Strep) }, combination of ACT and VA (Act+VA), and two positive controls {(Ampicillin (AMP) and Penicillin (Pen) }. The results indicated that the nanoemulsion formulation enhanced the therapeutic efficiency of the above antimicrobials against Las bacterium. We also screened two adjuvants and optimized one formulation to improve the effectiveness of Pen by foliar spray. More than 180 HLB-affected citrus trees were treated by combining thermotherapy, chemotherapy and nutrients. The thermotherapy was carried out by steam at 125~128 F for 120 seconds or 180 seconds, respectively. The chemotherapy treatments included EBI-602, Silver nanoparticle and CARV, using Pen as the positive control. The Nutrient treatment was additional micronutrient nutrition beyond the normal fertilization. According to the Field Trial Tree Evaluation Methods developed by CRDF, we investigated tree canopy, tree health, fruit drop and fruit quality as well as Las bacterial titers by real-time PCR. The tree canopy decline index (DI) was compared between the treated and control plants. The two-year results showed that PEN was the more effective to control Las bacterium than EBI-602, silver nanoparticle or CARV. Thermotherapy and additional nutrition promoted citrus growth and vigor, especially in the severe HLB-affected trees, whereas Las bacterial titers returned to original levels after a short-term decrease by heat-treatment. The disease severity index (SDI) decreased by 6% after application with PEN, followed by EBI-602 (4%), CARV (3.5%) and silver nanoparticle (1.3%). The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) decreased the fruit drop by 10~20 %, increased the fruit and juice weight by 3~13 %, and decreased the ratio of brix to acid by 0.2~5.0 %. However, this project was terminated in Aug. 31, 2016. Thereby, we could not get the second year data of fruit drop, fruit quality and yield. In addition to keep on the field trials of our previous enhanced projects, more than three-year s results indicated that PEN was also the most effective antimicrobial in eliminating the Las bacterium by gravity bag infusion. Due to the larger molecular weight and less solubility in water, VA, SDX, PCY and CARV were not very effective when applied by gravity bag infusion. The outcomes of this project will have potentials to go forward to solve the immediate problems that Florida citrus faces. A total of three publications have been published in Crop Protection, PLoS ONE and Journal of Applied Microbiology.
HLB-associated root loss was found to occur in 2 phases with an early 30-50% fibrous root loss that occurs before foliar symptoms develop and stays at this level until HLB-induced leaf drop begins. While early root loss averages 30-50% it varies depending on season and root flush. The length of recovery from root flushes decreases with increasing symptom expression in the canopy. The second phase of root loss begins as significant leaf drop begins in symptomatic sectors of the canopy. This second phase is characterized by 70-80% fibrous root loss and dieback of structural roots, starting from the outer tips and moving inward towards the trunk as canopy decline progresses. Surprisingly, HLB also causes a stimulation of root growth. Root growth is increasingly stimulated by Las as the canopy symptoms increase through mild to moderate decline. Root growth only declines once the canopy is in severe decline. From this it could be inferred that fibrous root lifespan was reduced by Las. Greenhouse rhizotron and field minirhizotron (clear tubes buried under grove trees) demonstrated that fibrous root lifespan was reduced from 9-12 months in healthy trees to approximately 4 months in Las-infected trees. The stimulation of root growth and reduction of root lifespan suggest that root stimulation would have a negative effect on citrus trees and that efforts to improve root health should focus on increasing root longevity and supplying water and nutrients in small frequent applications (spoon feeding). Most of the above studies were performed on Valencia and Hamlin trees on Swingle rootstock. Limited field sampling and greenhouse studies confirmed these results for Carrizo. A survey of Las-induced root loss in commercial and experimental rootstocks demonstrated that most rootstocks suffer the same root loss. In all but one rootstock the percent root loss was identical. Rootstocks with higher healthy root densities suffered higher quantitative root loss, which correlated with increased fruit drop, further supporting the conclusion that inducing root growth is counterproductive to maintaining yield. The only rootstock tested that responded differently to Las infection was UFR-4, which increased root density as symptoms developed and spread through the canopy. It was hypothesized that UFR-4 is susceptible to Las stimulation of root growth, but resistant to Las-induced root dieback. This rootstock was used in the initial rhizotron study, but unexpected technical failures prevented quantification of root longevity and root growth. These technical problems were solved in subsequent rhizotron studies and a second rootstock experiment has been initiated, but is not complete at the time of this report. The resistance of UFR-4 to Las-induced root loss provides a possible resource for studying the mechanism of root dieback. Considering the changes in root growth caused by Las, phytohormone concentrations were expected to be altered. However, in 3 rootstocks tested (Swingle, UFR-2, and UFR-4) no differences in phytohormones were detected when analyzed with metabolomic approaches. Gene expression analysis did show an upregulation of ABA genes in the roots suggesting a substantial increase in concentration and signalling, but only a very small increase in ABA was detected with targetted extraction and quantification. Whether this also occurs in UFR-4, which appears to be resistant to Las-induced root loss, still needs to be investigated to determine if it could be a fast screening marker for resistance to root loss.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. The data has been summarized into one manuscript and submitted. We are currently focusing on generating EBE mutated plants in both alleles and generating plants which do not contain cas9 and sgRNA in the plant chromosome.
Our progresses we have made for this project: 1) We have used the Kn1 gene to drastically improves shoot regeneration efficiently from transgenic cells of citrus. We have successfully used a maize knotted1 (KN1) gene to enhance genetic transformation efficiencies of juvenile tissues of six citrus varieties, Pineapple, Hamlin, Sucarri, Valencia, Carrizo and Eureka lemon via Agrobacterium-mediated infection. Our results demonstrate that expression of the KN1 gene improved transformation efficiencies from 3- to 15-fold compared to a control vector, 3- to 11-fold relative to the highest transformation efficiencies previously reported for the same citrus varieties. Stable incorporations of T-DNA into our transgenic plants have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of KN1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to transform, we have demonstrated that the kn1 gene can be an effective molecular tool for enhancing the genetic transformation of juvenile citrus tissues. Using mature shoot segments of Valencia and other cultivars as explants, we also found that the KN1 gene can improve transformation efficiencies compared to the control vector BUT an increase in efficiency is lower than what has been observed in juvenile citrus tissues. 2) We have demonstrated that manipulation of auxin transport can significantly enhances shoot regeneration of citrus. We have observed that the apical ends of epicotyl segments regenerated more shoots than the basal ends, and we therefore hypothesized that auxin transport and/or endogenous auxin concentration may play a key role in shoot regeneration of citrus explants. We tested some auxin transport modulators and identify one modulator that improved shoot regeneration. However, when the modulator was included in the transformation experiment, the transformation efficiency did not improve (i.e., number of transgenic shoots produced per explant). We hypothesize that the auxin modulator may inhibit Agrobacterium infection or T-DNA integration. 3) We have shown that an epigenetic modulator may be used to enhance shoot regeneration and transformation of mature citrus tissue. When we used an epigenetic modulator in transformation experiments with mature tissues, we observed increases in transformation efficiency of several citrus cultivars including Valencia and Washington Navel oranges. We have further demonstrated that the epigenetic modulator can lead to increases in shoot regeneration efficiency of mature citrus tissues when compared to the controls. 4) We have demonstrated that low Agrobacterium infection and T-DNA integration efficiencies are limiting factors for mature citrus transformation. As described above, we have developed some tools for enhancing shoot regeneration from mature citrus tissues. However, when these tools were used in mature citrus tissue transformation, the increase in transformation efficiency was lower than in juvenile tissues. We have further shown that the Agrobacterium infection and DNA integration are a major factor limiting transformation efficiency of mature citrus tissues, which provides a basis for our future experimentation to improve transformation efficiency of mature citrus tissues. We have published one manuscript reporting that Kn1 can drastically improve genetic transformation efficiencies of six citrus cultivars including a lemon cultivar: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. The second manuscript reporting the effects of poplar transport of endogenous auxin and an auxin transport modulator on citrus regeneration and transformation will be submitted in 2-3 weeks. The third one is currently under preparation.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,all the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 35% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from all the better performing lines have been made and are being rooted in the mist bed for future challenge with Diaprepes. A number of other potential root specific promoters are being evaluated. Several have been cloned and transformation vectors are being produced. Results from our studies have been presented in the World Congress on In vitro Biology.
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 have been working on five transgenic lines (HAM 13-3, HAM 13-29, and DUN 57-25, DUN 205-25c, and DUN 207-8) that exhibit tolerance to HLB. HAM 13-3: we had some difficulty with propagation of this particular line. We have repeated the propagation and are waiting for the progenies to become old enough for HLB test. HAM 13-29: we have generated 21 progenies. After HLB inoculation, 11 of these plants have no HLB symptoms and others exhibited mild symptoms. DUN 57-25: four progenies have been made and tested. Three plants have no HLB symptoms and one plant was crappy and discarded. We are continuing propagating this line. DUN 205-25c: this transgenic plant became dark green and the leaves are thick and extremely curly. We are propagating this plant. DUN 207-8: eight progenies have been generated. These plants have been inoculated with HLB. For objective 2, we confirmed the chimerism of the transgenic plants using real-time quantitative PCR. Results showed that HAM 13-3, HAM 13-29, DUN 57-25, DUN 205-25c, and DUN 207-8 are homogeneous (not chimeric). For objective 3, three Ray Ruby grapefruit putative mutants (#3, #6, and #93) showed tolerance to HLB. Seven, 10, and 10 progenies were generated for mutant line 3, line 16, and line 93, respectively. The progenies were tested for HLB resistance/tolerance. Unfortunately, none of these mutated plants exhibited resistance/tolerance to HLB.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of June 2016, we have processed more than 40,500 grower samples. For the 2016 calendar year to date, we’ve received 3,288 samples from growers, which is on track for a calendar year total exceeding 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 48% of submitters in 2016. Additionally, more than 44,100 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 84,750 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from April through June 2016, there were 2,370 growers samples processed in addition to research samples and psyllids. This quarter completed the first year of the grant, with a grand total of 5,401 growers samples being processed from July 2015-June 2016. These numbers are significantly higher than the expected increases in sample volume, which may indicate that the second half of the grant could see even higher numbers of growers samples submitted. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
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. Five putative transgenics have survived and passed a PCR screen, and these will soon be grafted onto rootstocks. 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 have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to coexpress Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. 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, a GFP reporter has been added into each CRISPR construct. The constructs with GFP will be re-verified in N. benthamiana and sent for transformation.
Candidatus Liberibacter asiaticus has not been cultured. However, Liberibacter crescens, has been cultured under laboratory conditions. The focus of our project has been to develop a detection system for bacteriophages (phages) and/or phage components (tailocins) using L. crescens strain BT-1 as a model system. We have accomplished the development of the assay system, which we have used to conduct screening of phages and broad host tailocins. Liberibacter is a member of the Rhizobiaceae. It is our experience that phylogenetically related microorganisms can share common surface components, such as phage receptor sites. Bioinformatic and structural analyses indicates that there is high homology in the surface structures of Rhizobium spp., Agrobacterium spp. and Liberibacter spp. Therefore, one of our strategies has been to search for naturally occurring phages active against Rhizobium spp. or Agrobacterium spp. that may also show activity against Liberibacter spp. We are continuing our screening of both Rhizobium spp. and Agrobacterium spp. phages against BT-1 using modifications of the assay, since growth condition can affect phage susceptibility. Strain BT-1 harbors two prophages (LC1 and LC2) that we have determined are not inducible by UV or oxidative stress. This indicates that the prophages are defective. The phages are predicted to be podophages, because their genomes exhibit no separate tail structure gene. We will continue to construct fusions between N-terminal tail fiber region of a broad host tailocin and the putative C-terminal portions of tail spike from BT-1 prophages to obtain active tailocins against Liberibacter.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance (The manuscript for this research will be published in Frontiers in Plant Biology). Antibody against thionin will be produced for investigating the correlation of thionin expression and HLB resistance. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
No new plantings in last quarter, but land is prepared for additional UF trees. 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.).
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and are being used to generate canker resistant plants. We have conducted multiple tries of genome editing using protoplast. Currently, we are optimizing the condition to conduct genome editing using protoplast. We also tested different sgRNAs to generate deletion in the coding region of CsLOB1.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermo-therapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (April 2016 to June 2016), three foliar sprays of the antimicrobial chemicals (Pen, Pcy, Carv and EBI-602) were applied at a two week interval for all treated trees from March to May, 2016. For gravity bag infusion, two refill applications of Pen, SD and SDX were conducted at two week interval in April, 2016. According to the Field Trial Tree Evaluation Methods developed by CRDF, we investigated tree canopy, tree health, fruit drop and fruit quality and Las bacterial titers by real-time PCR. Fruit quality tests were done on the field trail of combination of chemotherapy, thermo-therapy and nutrient therapy. A total of 50 fruit were harvested from the 3 trees in each trial replicate. Tests were run on 20 fruit from each sample for size, peel color, puncture resistance, fruit weight, juice weight, brix and acid. The tree canopy decline index (DI) was compared between the treated and control plants. Eight mature leaves with petioles from each of the treated and control trees were sampled around the canopy for PCR test. The preliminary results indicated that:1) the integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop, increase the fruit and juice weight, and decrease the ratio of brix to acid; 2) compared to the control plants, all antimicrobials reduced the Las bacterial titers, especially PEN. 3) Both SD and Pen reduced the DSI through two years application; 4) two new adjuvants (Bio and MF200) improved the effectiveness of Pen by foliar spray; 5) Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep on our application and prepare the final reports. One papers has been published in
In the second quarter of 2016 Core Citrus Transformation Facility (CCTF) continued to operate without interruption although prospective moving date for the lab was June 17th. Eventually the date was pushed back to July 21st so facility is still in its old location. Due to the very high number of orders placed in the last quarter and increased work load, I have hired one more employee who was trained in the lab during the month of April. This new employee is working full time. However, another employee was taken back from 1.0 FTE to 0.4 FTE at her own request. The number of orders placed at the CCTF remained high. We have received 12 orders within the last 3 months. Seven of those orders were paid in advance although no material associated with transformation was received. Customer just wanted to secure the place in our work schedule for time when they are ready to send us plasmid constructs. The plants produced within the last quarter are almost all from the experiments associated with orders placed within last 9-12 months. We produced 67 plants: nine Carrizo citranges, six Swingle citrumelos, and 52 Duncan grapefruits. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
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