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.
Citrus trees transformed with a chimera AMP (thionin-D4E1) and the thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had lower Las titer(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. Root samples from control plants and transgenic Carrizo expressing chimera and thionin were taken nine months after grating inoculation. Our results showed transgenic Carrizo expressing thionin significantly inhibited Las growth (0.5% of control level) compared to control and transgenic Carrizo expressing chimera. Antibody against thionin will be produced for Western detection. 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. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees 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 will be isolated and Las titer will be tested. 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 titer 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.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of March 2016, we have processed more than 38,000 grower samples. For the 2016 calendar year to date, we’ve received 917 samples from growers, which is on track towards a calendar year total very close to 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 46% of submitters in 2016. Additionally, nearly 43,800 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 81,800 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 March 2016, there were 855 growers samples processed and 1,071 research. Since the start of the current grant in July 2015, the lab has received 3,093 growers samples, which is even higher than the expected increases in sample volume. 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.
Liberibacter crescens strain BT-1, has recently been cultured under laboratory conditions and is the model system for our studies. We had previously reported that we had initiated studies using the well-studied R2 tailocin to design fusions between N-terminal tail fiber region of the R2 tailocin and C-terminal portions of tail spike from BT-1 prophages. Tailocins are protein assemblages that function like the tails of phages, by adsorbing to the bacterial cell and then puncturing the cell envelope. Unlike phages, tailocins do not have a capsid and thus inject no DNA, instead relying on the membrane puncturing activity to kill the cell. Like phages, tailocins use tail fibers to recognize specific receptors on the target cell surface. Tailocins are thus potent and specific lethal nanoparticles. The assembly of active tailocin requires chaperones specific to the C-terminal and the availability of chaperone can limit tailocin production. In order to understand the protein folding necessary to assemble functional hybrid tailocins, we are fusing different N-terminal region-encoding portions of the tail fiber gene of the well-studied tailocin R2 to a series of C-terminal regions of the P2 tail fiber. This is being accomplished in- trans using a broad host vector with a tac promoter. This series of experiments will allow us to understand the fusion(s) points necessary to construct active tailocins. Using the developed overlay system that incorporates several modifications of medium BM7, we are testing broad host phages, tailocins and environmental samples to identify phages active against L. crescens.
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 will be used to generate canker resistant plants.
Previously, we have shown that the Genome of Candidatus Liberibacter asiaticus (CLas) possess luxR gene that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing”. However, the genome lacks the second component; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we have only one objective: study the effect of AHL-producing citrus plants on the pathogenicity of CLas. We have selected different Lux-I genes from different bacteria expressing structurally different AHLs. Due to the difficulties we faced in growing different bacteria to extract DNA for LuxI isolation, we changed our strategy to synthesizing these genes (G-Blocks). Synthesized genes include 1- Agrobacterium tumefaciens N-(B-oxo-octan-1-oyl)-L-homoserine lactone(Hwang et al., 1994) TraI Accession number#L22207.1 2- Pseudomonas fluorescens Six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-L-homoserine lactone(3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-L-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-L-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL)(Khan et al., 2005) PhzI Accession number#AAC18898.1 3-Rhizobium leguminosarum bv. viciae 3-OH-C14:1-HSL (Wilkinson et al., 2002, Edwards et al., 2009) CinI Accession number#AF210630 4-Serratia liquifaciens N-(butyryl)-L- homoserine lactone (BHL) (Oulmassov et al., 2005) SwrI Accession number#U22823.1 5- Vibrio fischeri OHHL (Schaefer et al., 1996) LuxI Accession number#AAD48474.1 We already started the first step of expressing these genes by inserting them in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
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