We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. We have so far cloned six full-length cDNAs of citrus genes that potentially regulate SA, ET, and/or JA defense signaling. Agrobacterial strains containing these constructs were placed on the pipeline of citrus transformation in the co-PI Dr. Bowman’s laboratory. We also transformed Arabidopsis to overexpress these genes and to eventually test their defense function in Arabidopsis. We harvested T0 transformed seeds for some constructs and our initial screening of these constructs has yielded several transgenic plants for each construct. Arabidopsis transformation and screening have been continued during this past quarter. In addition, we are in process of cloning additional citrus genes. Six new full-length cDNAs of different citrus genes were cloned into the entry vector and will be further moved to the binary vector pBINplusARS for both Arabidopsis and citrus transformation and eventually for defense tests with the corresponding transgenic plants. In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
In the past year, much attention has been drawn to the CRISPR/Cas9 system. In light of the system’s simplicity and efficiency, and since it has proven to work transiently in citrus (Hia and Wang, 2014), we have decided to use the CRISPR/Cas9 system in conjunction with our CPP transient expression experiments. It is our hope that we can use a modified version of CRISPR/Cas9, in order to activate and suppress target citrus genes depending upon their regulatory role. For example, we intend to suppress the expression of citrus terminal flowering protein (CiTFL), which is responsible for negatively regulating flowering during citrus adolescence, in order to reduce juvenility. The CRISPR/Cas9 system may allow us to perform this as well as other beneficial modifications, without the insertion of a foreign transgene. The focus of our work for this quarter has been primarily concerned with cloning of citrus optimized versions of a nuclease-free Cas9 (Cas9m4) with either an activator domain, an ecdysone receptor (EcR-B), or a suppression domain, Krueppel-associated Box (KRAB). These Cas9 vectors will be cloned into two different reporting vectors for transient expression assays with CPPs and, if necessary, agroinfiltration. The other aspect of our work has been concerned with designing proper sgRNAs, essential for the CRISPR/Cas9 system, for CiTFL and citrus phytoene desaturase (CiPDS), for use as a visual control vector. We intend to be done with most of the cloning by next quarter and intend to produce some transient expression data.
An experiment involving ciFT3 transgenic tobacco plants and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol(PBZ) is currently being monitored. The effects of the chemicals are evident due to the variation in phenotype observed. The transgenic line being used is a T2 that has shown to be homozygous for the ciFT3 transgene from previous GUS assay experiments. PBZ produced a short phenotype with succulent leaves and transgenic plants flowered earlier in comparison to the other treatments. Flower buds appeared 39 days after germination. Controls are being monitored for flowering and are expected to flower around the 150 day mark. The outcome of this experiment will be used to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages by perhaps using one of the compounds. RNA will be extracted at three different time points from each treatment to determine the relative expression of ciFT3 when treated with the compounds. New cDNA concentrations from the 12 month collection period of citrus tissue was used to run a new set of quantitative real time PCR amplification curves for the FT1, FT2, FT3 FLD, FLC, ELF5, and AP1 genes. Statistical analysis is being conducted to find any correlation in expression and flowering time. Work has proceeded designing a transcription activator-like effector (TALE) system inducible by methoxyfenozide that will chemically activate the naturally present FT3 gene in citrus. An 18 monomer TALE has been constructed based on the endogenous FT3 promoter region common to ‘Duncan’ Grapefruit, ‘Carrizo’ Citrange, Pummello, and Poncirus citrus. Progress is currently being made to assemble this FT3 TALE into a plasmid with a FMV promoter, a VP16 activation domain and the inducible ecdysone receptor-based expression system. The efficacy of this construct will be evaluated by co-transforming tobacco with the chemically inducible system and with a plasmid that contains the endogenous citrus FT3 promoter followed by a GUS reporter gene.
Experiment described in the last report continue as the main focus of the project. In addition, we have formed a collaboration with researchers from UF’s Department of Chemistry and the IRREC (Mingsheng Chen, Brent Sumerlin, and Zhenli He) who work with nano-particles chemically assembled using amphiphilic polypeptides (polymers). Our groups seek to develop a method using a combination of encapsulating nano-particles and cell penetrating peptides (CPPs) to deliver cargos to selected tissues (such as the phloem). The first step, undertaken this quarter, is to elucidate the potential toxicity effects of different nano-particles using a combination of tissue culture techniques and fluorescence microscopy. Then, we will examine combinations of benign polymers and CPPs that we have found to be effective in transferring cargo to citrus.
During the course of this project we found that PAMP-triggered immunity (PTI) plays an important role in citrus resistance against canker and HLB. Furthermore, there was a correlation between the level of resistance observed in different genotypes (from most susceptible to most resistant: ‘Duncan’ grapefruit, ‘Navel’ sweet orange, ‘Sun Chu Sha’ mandarin, ‘Nagami’ kumquat) and the extent and intensity of the PTI in terms of transcriptional gene induction of defense genes. We also found that PTI induced by flg22 restricted growth of Xcc in planta only in the resistant genotype (‘Nagami’ kumquat). Flg22 from CLas also induced defense gene expression reprogramming. Given these exciting results we used genomic resources available online to identify the receptor gene for flg22 (named FLS2). We will test whether the ectopic expression of FLS2 from resistant kumquat is capable of increasing the resistance in a susceptible genotype (grapefruit). We will clone the FLS2 gene from kumquat into an expression vector (we have all the necessary plasmids available) and perform transient expression experiments to compare defense gene expression levels and bacterial growth that would indicate an increase of resistance in grapefruit.
In the previous reports, we have reported the development of Soft Nanoparticles (SNPs) using two essential oils, EO A and EO B. The formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter Crescens (L.Crescens) and both the formulations and the controls showed > 90 % inhibition at 1, 5 and 10 % (v/v) dilution. Phytotoxicity of select formulations were performed at 1:1, 1:10 and 1:20 dilutions and all formulations showed low phytotoxicity when applied at 1:20 dilutions. The developed formulations were tested for their stability with adjuvants by a technique similar to the well-known ‘jar test’. Cohere and Cling are spreader-sticker type adjuvants that are frequently added to pesticide spray tank before spraying to enhance pesticide penetration. It was seen that most of the formulations were stable. Based on the efficacy, phytotoxicity and stability tests, formulations for EO A and EO B have been short-listed for possible field trial. SNPs have also been developed and characterized with Thyme Oil. The droplet size ranged from 3 to 18 nm and the oil loading ranged from 1 to 20% (w/w) using agriculturally approved surfactants. The surfactant loadings are similar with those in formulations with EO A and EO B. Addition of a co-surfactant greatly enhanced the oil loading to up to 20% (w/w). Selected formulations were also tested for stability with the adjuvants and most of the formulations were stable and the addition of adjuvants changed the particle size of the SNPs marginally but was still in the required range (5-16 nm). Future plans include testing of selected formulations of thyme oil for antibacterial activity / efficacy against the surrogate bacteria as well as phytotoxicity studies. To separate the efficacies of the essential oils from the surfactants, emulsions and microemulsions have been developed with low and ultra-low surfactant and oil loadings. Formulations have also been developed with different surfactants that are known to be safe and used in pharmaceutical formulations (e.g. Pluronic’ F127 and Tween’ 80). These formulations will be tested for their inhibition efficacy on the surrogate bacteria. These experiments will assist in understanding the efficacy of the oil in SNP as well as in determining the minimum amount required for L. crescens inhibition. Dye doped SNPs have also been developed to understand their foliar uptake. In addition, experiments are being planned to quantify the effectiveness of the adjuvants (Cling and Cohere) performance on citrus leaves.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the lasA1 and lasA2 genes. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana and citrus. We have obtained transgenic lines with these constructs. These transgenic Arabidopsis lines were verified by PCR and RT-PCR and their segregation in T2 and T3 were analyzed. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and/or overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. We also expressed LasA1 protein using the Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E. coli. Purified LasA1 protein are used for antibody production and crystallization study. Immunoprecipitation and elution of FLAG-tagged autotransporters from Agro-infiltration in Nicotiana benthamiana yielded several protein candidates, indicating LasA1/LasA2 interacted with mitochondria and chloroplast proteins. It is worth noting that we are able to detect LasA1 protein from Las-infected plant tissue using Western blot. The successful generation of this antibody will enhance our research in several aspects. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein has also been generated.
During the 3 years of funding, we identified and characterized a regulon from ‘Ca. L. asiaticus’ involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of ‘Ca. Liberibacter asiaticus’, using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. When bound to LdtR, the chemicals inactivate the protein, which disrupts a cell wall remodeling process that is critical for survival of the pathogen when exposed to osmotic stress (i.e. within the phloem of a citrus tree). Several model strains were used to confirm that the newly identified transcription factor (LdtR) and its regulated genes (ldtR and ldtP) confer tolerance to osmotic stress.These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease. The logical subsequent step of our research is the optimization of all necessary parameters to use the chemicals identified directly on field applications. The results were published in PlosPathogens Journal: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004101
Transgene Stacking for Long-Term Stable Resistance: PCR analysis of transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) resulted in identification of 7 lines that contain both transgenes. Agrobacterium mediated transformations to produce transgenic plants containing other combinations of stacked genes are in progress. Transgenic plants with the PCR positive stacked genes are being clonally propagated for further evaluation. Improving Consumer Acceptance: 1. In efforts to develop an intragenic citrus plant, we have in addition to the binary vector for an inducible cre-lox based marker free selection containing the cre gene driven by a Soybean heat shock gene promoter, a binary vector containing the cre gene driven by a citrus-derived heat shock promoter. Citrus rootstock Carrizo has been transformed with both of these constructs and numerous transgenic plants are being regenerated. 2. Hamlin and W Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells. One of the T-DNA contain a grapevine myb gene under the control of a 35s promoter and the other contain T-DNA containing the selectable positive/negative fusion marker cassette. Plants have been regenerated that are purple in color from the anthocyanin production. Pending molecular analysis of the regenerated lines, we speculate one of two possible scenarios: a) The plants contains only the T-DNA of interest or 2) The plant contains both T-DNAs integrated into the genome. Somatic embryos are now being germinated and resulting transgenic plants will be evaluated. Induction of early flowering: The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 has also been produced for testing. Since previously generated FT and AP1 plants flower in vitro but not as young plants in the greenhouse, we are testing the possibility of a synergy when both are present together. Transgenic plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. Propagation of new transgenics for field testing: ‘ Propagation of LIMA-B (AMP) transgenic plants for further study; ‘ Propagation of Carrizo transgenic lines with LIMA gene to test a potential rootstock effect on non-GMO scion. Efforts to establish a new transgenic field site: Working with Dr. Phil Stansly, we have submitted an addendum to our transgenic field permit with APHIS to add an additional field site which would be located at the UF Immokalee Research and Education Center. We plan to plant 400 new transgenic trees at this site after approval. A few hundred trees have also been prepared for planting at the USDA Picos Farm site.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants transformed with citGRP1 were test by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107/CFU. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slightly canker resistance by infiltration test. A series of transgenic scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
This quarter we have made good progress on our transformation approaches: 1. Stable transformations in citrus using new vectors. Transformation experiments were carried out in Duncan grapefruit and carrizo with 5 constructs in a preferred vector backbone, including an empty vector control . 52 carrizo transformants were selected and analyzed by PCR. The genes of interest that were screened for included avrGf1, avrGf2, and NPTII. PCR results showed that 5 of 6 transformed with the avrGf1 gene were positive and 9 of 30 plants were positive for the avrGf2 gene. NPTII was detected in 22 of the 52 carrizo plants. 6 out of 7 Duncan plants were positive for NPTII. These results demonstrate improved transformation efficiency using this vector backbone. Further epicotyl transformation experiments were carried out with both ‘Duncan’ grapefruit (624 segments) and ‘Pineapple sweet orange (136 segments) with 6 constructs. These segments were placed on media without selection (kanamycin). This modification was done to see if transformation efficiency would improve. Rooting experiments and transplanting segments are ongoing. Of 1,507 transformants selected so far, 168 putative transgenic shoots of grapefruit, sweet orange and Carrizo were confirmed as transgenic by GUS assay (35S:GUS construct in vector) this period. To date, although no GUS positive or chimeric shoots have been observed for the sweet orange cultivar, Duncan grapefruit and carrizo citrange had totals of 4 and 134 GUS positive shoots, respectively. We will continue to screen and progress positively transformed plants for further analysis. Pathogenicity tests will be carried out as soon as plants reach adequate size. 2. Stable transformation in test systems Tobacco: N. tabacum lines transformed with the 14 EBE promoter fused to GUS, generated at the UC Davis transformation facility, were tested by GUS assay in the presence and absence of the X. citri effector PthA4, delivered as a 35S expression construct via Agrobacterium. Nine of 10 of the lines showed GUS expression only in the presence of PthA4. This system is now established as functioning and will provide a rapid means of testing the ability for individual and combinations of X. citri effectors to induce the promoter construct. We are in the process of harvesting seed for full scale screening. Tomato: A second test system was designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. Positive transformants of Bonny Best and large Red Cherry tomato cultivars were confirmed using DNA dot blot. T0 transgenic tomato were screened for pathogenicity reaction with X. euvesicatoria strains (8510 avrBs3 transconjugant & Race 9) and X. gardneri, and several Bonny Best and large Red Cherry were found to be resistant. This demonstrates that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance. We are harvesting seed from transgenic plants and are in the process of testing seedlings for resistance.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Random mutagenesis of EFR ectodomain to gain elf18-CLas responsiveness did not produce gain-of-function mutants. Using chimeric elf18 peptides of wild-type and CLas sequences, we could determine that both the N- and C-terminal regions of elf18-CLas were responsible for the lack of recognition by EFR suggesting that elf18-CLas is impaired in both binding and activation. In order to identify complex EFR mutants that respond to elf18-CLas, we are developing a FACS-based screen. We fused promoters that are expressed at a low basal level and PAMP inducible to nuclear-localized GFP and tested by Agrobacterium-mediated transient expression in N. benthamiana and in Arabidopsis protoplasts. All constructs gave significant basal expression, which is presumably due to unrestricted expression in these transient systems. Therefore, it is necessary to produce transgenic lines of these reporter construct. Modelling of the EFR/elf18/BAK1 complex has been performed, based on the available FLS2/flg22/BAK1 structure. Sites have been selected for targeted mutagenesis to improve recognition of elf18-CLas. We have also examined the interaction with BAK1 and identified two regions which may be involved in elf18 binding. Mutagenesis constructs of these regions is currently being screened for elf18-CLas response. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Reciprocal XA21 and EFR chimeric receptors have been produced and transformed into Arabidopsis for functional testing. Since it is now known that axYS22-Xac is not the ligand of XA21, we tested the response of the EFR-XA21 chimera to elf18, in order to determine the function of the XA21 cytoplasmic domain in dicots. These constructs performed in a similar manner to wild-type EFR. In addition we tested XA21, EFR, XA21-EFR and EFR-XA21 for pathogen resistance to Pseudomonas syringae pv. tomato DC3000 COR-. Interestingly, all constructs provided an elevated level of resistance, indicating that the chimera of XA21-EFR was functional and that XA21 was capable of perceiving a potential ligand Pseudomonas. A manuscript describing this work is currently being prepared for submission to PNAS. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs including EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore Lab for transformation. These were transformed into E.coli DH5. cells and Agrobacterium tumefaciens strain Agl1. Confirmation of clones was done using PCR (EFR and XA21 designed primers) and restriction analysis (NcoI and SpeI restriction enzymes). Initial transformation experiments have been carried out with a total of 100 ‘Pineapple’ sweet orange segments. Germination rate for sweet orange is 5%, which is extremely low. ‘Pineapple’ sweet orange and Duncan grapefruit seedlings are on germination media and transformation experiments will be initiated in May 2014.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we performed another NAD+ treatment experiment, in which NAD+ was either infiltrated into the citrus leaves or sprayed on the surface. After 24 hours, the NAD+-treated leaves were inoculated with the canker bacterial pathogen Xanthomonas citri subsp. citri. Canker symptoms were analyzed two weeks later. Compared with the Actigard treatment, the NAD+ treatment did not provide strong protection to the plants in this experiment. It is possible that time between NAD+ treatment and canker bacteria inoculation was too short. We are design another experiment in which the NAD+ induction time will be longer. We are also preparing citrus plants for root treatment with NAD+. For objective 2, in last quarter, about 20 independent lines have been generated for each construct. The transgenic seedlings have been growing in the greenhouse. We have performed molecular characterization of the transgenic plants. DNA was extracted from each individual plants and PCR was performed to confirm the presence of the transgene. Only a fraction of the putative transgenic seedlings contain the transgene. We are currently testing the expression levels of the transgene in these plants. We will also generate more transgenic lines using the constructs.
The goal of this project is to understand the biology of HLB by identifying key host components and processes involved in disease development. We use secreted proteins (also called effectors) from the causative agent, Candidatus Liberibacter asiaticus (CLas), as molecular probes because they have been considered key virulence proteins of bacterial pathogens. Our previous research using bioinformatic and experimental approaches identified four CLas effectors that are highly expressed in infected trees. We will isolate the direct citrus targets of these effectors, which will reveal important information of HLB pathogenesis. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first two quarters of this project, we cloned the four CLas effector genes into the Y2H bait vector, transformed them into the yeast strain AH109, and confirmed that the effectors are highly expressed in the yeast without self activation activities. Therefore, these constructs are appropriate for Y2H screens. In the third quarter (Year 1) of this project, our main efforts include: 1) Construct citrus cDNA libraries that will be used for Y2H screening. We collected RNA samples from asymptomatic and symptomatic tissues of HLB-infected sweet orange leaves. These RNA samples were mixed with RNA extracted from healthy tissues to ensure that we would be able to cover as many genes as possible. The RNA has been sent out for cDNA library construction by a company. We have been working with the company to normalize the library in order to minimize the influence from the over-representative transcripts of highly expressed, housekeeping genes, which may bias the screening later on. 2) Make gene expression constructs that produce fusion proteins with each CLas effector gene tagged to a gene encoding the yellow fluorescence protein (YFP). These fusion proteins have been transformed to plant cells to determine the localizations of these effectors in plants using microscope.
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 two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants 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. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. As of April 9, 2014, a total of 5,314 transgenic plants have passed through inoculation process. A total of 106,250 bacteriliferous psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. As an offshoot of the research, damage by western flower thrips was so severe that research was conducted to validate damage by this pest to developing flush and facultative predation on ACP, which led to the following publication: Hall, D. G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection. 60: 66-69. A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
