This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program 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 technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of October 12, 2016, a total of 9,309 plants have passed through inoculation process. A total of 293,895 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant). Research results will soon be available in which we are comparing success rates using ACP colonies on lemon versus citron, and using ACP colonies from greenhouses versus walk-in chambers.
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. T 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. Publications from this project 1. Jia H, Zhang Y, Orbovic V, Xu J, White F, Jones J, Wang N. (2016) Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J. doi: 10.1111/pbi.12677. 2. Jia, H., Orbovic, V., Jones, J.B. and Wang, N. 2016 Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating Xcc.pthA4:dCsLOB1.3 infection. Plant Biotechnol. J., 14: 1291 1301. doi: 10.1111/pbi.12495. 3. Jia H, Wang N. 2014 Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 9(4):e93806. doi: 10.1371/journal.pone.0093806
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.
Core Citrus Transformation Facility (CCTF) moved to temporary location in the last week of July. The staff tried to minimize the impact of moving on the operation of the lab. We ended up canceling only half week worth of experiments. However, the location of the lab itself presented a challenge and has negatively affected productivity. Within the last two months, the death rate of micro-grafted positive transgenic shoots more than doubled. Some of previously grafted and established plants also exhibited signs of poor growth and few of them died. I have instructed the staff to do grafting in lab in different building and keep grafted shoots out of CCTF. We also consulted with the member of Mature Tissue Transformation who does grafting and got some advice from him. The orders continued to come to CCTF in high numbers. Within this quarter we have received 12 more orders. Altogether, we received 50 orders since the beginning of the year. Resistance to canker and HLB continue to dominate the orders placed at CCTF. CCTF produced 60 plants within the last quarter. Out of those plants, five were Carrizo citranges, three Swingle citrumelos, two Pineapple sweet orange, and 50 Duncan grapefruits. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. Those plants are very tall now and will require additional care soon. If they are supposed to be propagated by cuttings, that should be done very soon, since this type of propagation is not very successful when done during the months of November, December, and January.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,cuttings have been initiated for selected transgenic lines. Molecular analyses has been completed and a wide variation in transgene response is being observed. Due to yet unexplained reasons, transgenic lines expressing the ASAL transgene are performing better horticulturally than lines with expressing the APA transgene. Rooted cuttings are expected to be available in the fall.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of September 2016, we have processed more than 40,850 grower samples. For the 2016 calendar year to date, we’ve received 3,531 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 54% 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 85,000 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 July through September 2016, there were 306 growers samples processed in addition to research samples and psyllids. 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.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of September 2016, we have processed more than 40,850 grower samples. For the 2016 calendar year to date, we’ve received 3,531 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 54% 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 85,000 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 July through September 2016, there were 306 growers samples processed in addition to research samples and psyllids. 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.
Production from the mature transformation pipeline during the last quarter was interrupted due to the move to the packinghouse while the AC in #103 is repaired. We lost time due to unforeseen delays in connecting utilities & other issues (no hot water, mold, AC issues, electrical problems, & autoclave issues), the physical move itself, & the set-up in the temporary laboratory. Presumably, we will lose some time moving back to #103 at the end of October. We continue to provide transgenics to Drs. Dutt, Grosser, McNellis, Mou & Wang. An additional 12 independent, transgenic mature Valencia and Hamlin scions (some events were duplicated to equal 15) were shipped to Dr. McNellis at Penn State University this quarter. These plants were small & had not undergone the secondary graft, because in vitro plants are easier & cheaper to ship than large citrus trees. Apparently they transplant very well. Ten transgenic Hamlin & Valencia scions (with duplicates & triplicates) were produced for Dr. Mou that also had not undergone secondary grafting. Secondary grafts have been performed on all plants for Drs. Grosser, Dutt, and Wang, to enhance the growth of the transgenics. Six additional transgenics were transferred to Dr. Wang. Approximately 21 transgenics have been produced for Dutt & Grosser since the last report, of which 16 had rooted & were transferred. Dr. Hao Wu presented a talk at the ASHS meetings in Atlanta, GA. His talk was entitled, “Biolistic transformation of citrus”, ASHS Annual Meeting, Atlanta, August 7-11, 2016. Recently we introduced Kurhaski and Glen Navel cultivars for Drs. Grosser and Dutt through shoot-tip grafting (STG). Kurharski is a rootstock similar to Carrizo but it has some nematode tolerance, and Glen Navel sweet orange is pollen sterile, so it will provide a contained system to prevent transgene flow. Some of the budwood from FDACs in Chiefland was contaminated with the yeast endophyte, so it was essential that STGs be conducted on all introduced material prior to tissue culture. Mandarin & pummelo are being introduced for Dr. Wang. Phosmannose isomerase (PMI) selection works well after biolistics in immature citrus & it significantly decreases the number of escapes compared with nptII selection. Using PMI selection after biolistics, we were able to produce an additional 10 immature transgenics while significantly decreasing the number of nontransformed escapes. We are still investigating whether PMI will be useful for mature citrus transformations. Initial observations indicate that mannose is toxic to mature shoot development, but tests are being conducted to determine the effect of mannose after the shoots have formed on sucrose medium.
In our work through March, we ramped up the RNA expression assay portion of our project. For study of LdtR, we compared RNA transcripts from S. meliloti (deletion)ldtR bearing a plasmid with Plac (IPTG-inducible) Ca. Liberibacter asiaticus ldtR with a WT control bearing the empty vector. Cells were induced for 1 hours at 0.5 mM IPTG to induce expression of the regulator genes. These RNAs were converted to cDNA for hybridization to gene chips. Background information on ldtR and ldtP genes showed that the Sinorhizobium LdtR protein binds to its own promoter and to that for ldtP. As shown by Pagliai et al, ldtR and ldtP are conserved in S. meliloti; their version of CLas LdtR, encoded by a sequence amplified from Ca L. asiaticus strain psy62, binds to a characteristic promoter motif including promoters for LdtR and LdtP. Our transcription assays showed that in S. meliloti, the expression level of the Sm ldtP gene does not go up in response to th plasmid-borne CLas LdtR. We did see high variation in results among three biological replicates. We found that overall 8 genes went up by 1.5-fold or more. Some of these are ctrA controlled, some are cell cycle related, and some related to transcription effects of a (deletion) podJ mutation or of NCR-peptide exposure. Of these 8 genes, only 2 had CLas orthologs. We observed that ldtR is less motile than WT and expression of CLas LdtR may further reduce motility. Tests for stress sensitivity showed there is no differential growth for the engineered strain on plates containing the detergent deoxycholate at concentrations of 0.1% or 0.2% (the highest level tried). As reported in January, we constructed mutations for the visR and visN genes. The VisR-VisN proteins function as a heterodimer to constitute a LuxR-type transcription factor. We found the (deletion)visR visN double mutant has a motility defect. A plasmid carrying the cloned CLas visNR genes was able to complement the mutant defect of the S. meliloti (deletion)visR visN double mutant. Preliminary transcription results show that the CLas visNR plasmid activates expression of several dozen S. meliloti genes.
During this reporting period (April, May and June, 2016), we analyzed over two dozen newly transformed plants from Dr. Janice Zale’s program (University of Florida Mature Citrus Transformation Facility, Lake Alfred, FL). These plants are ‘Hamlin’ sweet orange and ‘Carrizo’ rootstock lines. However, we were not able to detect the FLT-antiNodT fusion protein expressed in any of these lines by protein immunoblotting. This contrasts with our results in ‘Duncan’ grapefruit, where nearly all the independently transformed lines expressed detectable levels of full-length FLT-antiNodT fusion protein. Further molecular analyses during the present funding period indicated that the transgene encoding the FLT-antiNodT fusion protein was not detectable in the ‘Hamlin’ and ‘Carrizo’ lines, although the markers for transformation (kanamycin resistance and green fluorescent protein) were detectable. Unfortunately, these results suggest that these plants are not actually transformed with the FLT-antiNodT fusion protein transgene. However, our original and proposed activity, transforming ‘Duncan’ grapefruit and testing for HLB resistance, was successful and is still in progress. Plants are continuing to be propagated for testing for HLB resistance at in Dr. Tim Gottwalds’ lab at Ft. Pierce. When sufficient plants are ready, these will be transferred into an HLB transmission greenhouse and exposed to Asian citrus psyllids carrying Candidatus Liberibacter asiaticus. During the present funding period, protein samples from the Ft. Pierce trees were analyzed by protein immunoblotting and these trees were found to be expressing moderate to high levels of FLT-antiNodT fusion protein, which is very promising. Although Dr. McNellis had planned to send rooted clones of the seven ‘Duncan’ lines at Penn State to Dr. Gottwald’s lab at Ft. Pierce, the permit for such transfer was not approved. Transporting citrus back to Florida is unlikely to ever be approved. Dr. McNellis then pursued an alternate plan to transfer plants to Ft. Detrick, Maryland, to Dr. Bill Schneider’s lab, for testing for HLB resistance. Dr. Schneider has agreed to participate in this collaboration, and Dr. McNellis has applied for a USDA APHIS permit for this plant transfer. This application is still under review by USDA APHIS.
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.
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