We are working on a method for rapid and efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System (PMDIS). Currently we are evaluating several sets of plants that we have injected with the HLB bacterium-containing extracts using different conditions. In these inoculations we tested different types of tissue (stems, leaves, seed coats within an infected citrus plant or HLB-infected psyllids) that can serve as resources of the HLB bacteria for preparation of the inoculum, different composition of extraction buffers for preparation of the bacterial suspension and different parameters of injection. We are also evaluating how age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as types of flushes being inoculated affect efficiency of inoculation. Plants that have been injected are now being tested by PCR with HLB bacterium-specific primers to evaluate what proportion of plants became infected. We are constantly increasing number of inoculations in which we test different conditions. Those plants are being maintained in the greenhouse and monitored for the disease development. Some successful infections of citrus plants using PMDIS were achieved, however infection rates were less than those seen upon graft-inoculation of plants with HLB-containing tissue. Currently we are working on improvement of PMDIS-based inoculation procedure. We included plants of many species into our experiments: citrus, tobacco, periwinkle, papaya. Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University who uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease sent us some of his nozzles that he uses with his injection device. We are testing those nozzles to see if they would increase the efficiency of our inoculation procedure. Dr. Gonzalez also shared his injection protocol with us, so now we are looking to see if we can improve our method by doing some modifications. Besides working with the HLB bacterium, we are interested to try our injection system with Liberibacter crescens culture. We already obtained a USDA-APHIS permit for working with this bacterium. Liberibacter crescens culture was received from Dr. Triplett’s lab and has been maintained. Currently we are starting to perform injections using this culture and utilizing several different plant hosts.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. In March 2013, twelve different isolates from plants identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, were inoculated into receptor plants in a greenhouse at Ft. Pierce. From the twelve isolates which were inoculated into receptor plants in the greenhouse, nine isolates have been established. The isolates which were not recovered came from citrus relatives that are not highly graft compatible with citrus. The nine isolates which have been recovered and established have been grafted into plants for the cross protection trial. DNA extracts from these nine isolates are being sequenced.
The objective of this project is to develop Fixed-Quat antimicrobial technology as an alternative to traditional Cu bactericides/fungicides. Fixed-Quat has been engineered to minimize phytotoxicity of native Quat compounds (i.e. Quarternary Ammonium compounds) while maintaining its superior antibacterial properties. A series of eight Fixed-Quat composite gel materials have been synthesized using two EPA approved Quat materials. Antibacterial efficacy was evaluated extensively against E. coli using various standard antibacterial assays such as microplate alamar blue assay, growth curve and CFU. Lowest bactericidal concentration of Fixed-Quat was estimated to be ~8 ppm of Quat; the effective concentration could be lowered further through optimization of the synthesis protocol. Even at this concentration (8 ppm of Quat), the antibacterial efficacy of the newly synthesized Fixed-Quat formulations were twice as better as Kocide 3000 at 100 ppm metallic Cu concentration. To test the feasibility of industrial scale production levels, concentrated Fixed-Quat formulations containing active Quat ingredient at 23000 ppm has been prepared with two EPA approved Quat ingredients. Evaluation of antibacterial efficacy of Fixed-Quat formulations against X. alfalfae (a citrus canker surrogate) is under progress. Additional material characterization data, phytotoxicity effect studies and rain-fastness study results will be available within next two reporting periods. Two most promising Fixed-Quat formulations will be delivered for field trials by summer of this year.
The first manuscript, “Citrus Huanglongbing Disease Detection Using Narrow Band Imaging and Polarized Illumination”, was accepted on December 8th, 2013, for publication in the Transactions of the ASABE as a Full-length article. The vision sensor which was designed and developed for the real-time HLB detection purpose was first tested and calibrated in a simulation of an in-field condition at the laboratory using the dataset created on September 26th, 2013. Then it was examined under a real situation in a citrus grove (CREC) on November 1st, 2013. Two datasets for the in-lab experiment and one dataset for the in-field experiment were created, which contained HLB negative, HLB positive, and zinc deficient leaf samples. The HLB infection status of all the samples were confirmed by a qrt-PCR test. The preliminarily experiment showed that the optimum distance between the sensor and the leaf was 80 cm. Two simple image descriptors including gray values’ mean, and standard deviation were used for clustering purpose. The results showed that the sensor highlighted the starch accumulation in the HLB affected leaf very clearly and differentiated it from visually analogous symptoms of zinc deficiency. The HLB detection accuracies ranging from 96.7% to 100% were achieved for the in-lab and the in-field experiments. Compared to our previous study, the HLB detection accuracy increased significantly in both zinc and non-zinc deficient leaf samples. The second journal manuscript, ‘An Optimum Method for Real-Time In-Field Huanglongbing Disease Detection Using a Vision Sensor’, was prepared based on these results to be submitted for the Transactions of the ASABE. Also another abstract, ‘A Vision Based Sensor for Huanglongbing Disease Detection under the Field Condition’, was written and submitted for the 2014 ASABE Annual meeting to be held in Montreal, Canada in July 2014. In another study, the sensor performance was evaluated in two experiments. In the first experiment, the improvement in HLB identification achieved using the narrow band illumination and polarizing filters was compared with a natural imaging condition. A separability index was introduced to compare the sensor and RGB camera images of the sample in four classes of HLB positive, zinc deficient HLB positive, HLB negative, and zinc deficient HLB negative. The results showed that the proposed vision sensor significantly increased the classification accuracy. In the second experiment, the effect of starch accumulation on the images of citrus leaves and ground leaves were compared. The results showed that the starch in the ground leaves were more distinctive rather than un-ground leaves. A third journal manuscript, ‘An Evaluation of a Vision Based Sensor Performance in Huanglongbing Disease Identification’, was prepared based on these results and it is under the final revision. Also another abstract, ‘A Study of How the Starch Accumulation in Huanglongbing Affected Citrus Leaves Reflects the Polarized Light’, was written and submitted for the 12th International Conference on Precision Agriculture (ICPA) to be held in Sacramento, California in July 2014.
Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. We have surveyed three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We selected the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus. We have received the sequencing result for the first batch of samples and are almost done with analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Now we are aligning the reads from DNA samples to sweet orange genome and C. clementina genome (V1.0), about 30%-40% reads could not mapped on these three citrus genomes. Those unmapped reads which are not citrus sequences are being used for metagenomic analysis. We also analyzed the RNA-seq data. Totally 2383 citrus genes were down-regulated while 2017 genes were up-regulated by citrus blight. Meanwhile, two methods were used to analyze these differentially expressed genes: GSEA (Gene set enrichment analysis) which is Gene ontology based method and Mapman-Mapman pathway based method. Root samples were collected again from 12 trees in the selected citrus grove at St. Cloud in March 2014. Interestingly, further test in April indicated that two previous healthy trees became citrus blight positive. Further analysis of those trees are being conducted. All the sequencing data have been uploaded to public database. We analyzed the hormones in the blight diseased trees and healthy trees. Quantitative reverse transcription PCR was used to further compare the gene expression of selected genes of citrus. We sampled for the third time and further analysis of those trees are being conducted. Metagenomic analysis of the sequenced samples is being conducted. We are in the process of sampling fourth time for metagenomic analysis.
Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. We have surveyed three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We selected the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus. We have received the sequencing result for the first batch of samples and are almost done with analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Now we are aligning the reads from DNA samples to sweet orange genome and C. clementina genome (V1.0), about 30%-40% reads could not mapped on these three citrus genomes. Those unmapped reads which are not citrus sequences are being used for metagenomic analysis. We also analyzed the RNA-seq data. Totally 2383 citrus genes were down-regulated while 2017 genes were up-regulated by citrus blight. Meanwhile, two methods were used to analyze these differentially expressed genes: GSEA (Gene set enrichment analysis) which is Gene ontology based method and Mapman-Mapman pathway based method. Root samples were collected again from 12 trees in the selected citrus grove at St. Cloud in March 2014. Interestingly, further test in April indicated that two previous healthy trees became citrus blight positive. Further analysis of those trees are being conducted. All the sequencing data have been uploaded to public database. We analyzed the hormones in the blight diseased trees and healthy trees. Quantitative reverse transcription PCR was used to further compare the gene expression of selected genes of citrus. We sampled for the third time and further analysis of those trees are being conducted. Metagenomic analysis of the sequenced samples is being conducted. We are in the process of sampling fourth time for metagenomic analysis.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. A visiting scientist arrived and began work 6-15-2013. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. Inserts from the phage vector that encode the single chain antibodies have been amplified and recloned and some have been submitted for sequencing to confirm the expected sequences. The scFv selected for this purpose recognize a TolC-like epitope of CaLas and an InvA-like epitope of CaLas. Both of these protein targets are expected to be essential for the interaction between the pathogen and host and thus are vulnerable targets for impairment by scFv. These scFv sequences have been modified by the addition of a KDEL leader sequence. The KDEL leader sequence has been reported to stabilize expression of proteins in transgenic plants. The scFv sequences have also been modified to contain SmaI and and SpeI cloning sites to enable cloning into the pUSHL26 vector to be used for transformation of plants. Related research with the existing scFv is underway on project 551.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. Study of the literature showed that the media used to grow the E. coli and other details of the culture conditions greatly influence the yield of scFv obtained from culture lysates. We have found that a very rich and buffered medium, (2X yeast extract Tryptone broth with phosphate buffer) works best. The medium is supplemented with glycine, sucrose and IPTG at various stages of the expression protocol. With this protocol we have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Cross reactions with healthy plant tissue can be a problem, especially if the concentration of scFv is too high. However, color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. Previous work has used a secondary monoclonal antibody directed at the 6X His Tag of the scFv molecules conjugated with alkaline phosphatase and blocking of the membranes with skim milk. We have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. We have had unexpected results with the negative controls in these assays: When the secondary anti-FLAG monoclonal is used alone, without any scFv, it produces color tightly focused as a ring in the phloem cylinder of HLB but not healthy citrus. Thus the anti-FLAG monoclonal alone detects the presence of disease. As a further control, we have prepared polyclonal antibodies against the major outer membrane protein and detected them with alkaline phosphatase. The polyclonal antibodies produce distinct spots of color corresponding to individual phloem cells infected with ‘Ca. Liberibacter asiaticus’. This is a useful assay for ‘Ca. Liberibacter asiaticus’, and we are using it to describe the distribution of ‘Ca. Liberibacter asiaticus’ in infected citrus trees, fruit and seed. We have continued work with these antibodies applied to tissue printing of plant samples during the current reporting period. A manuscript on the original scFv antibodies has been prepared and two manuscripts on the tissue printing assays are in preparation.
As outlined in previous reports the potential effectors of HLB in Citrus Tristeza Virus (CTV) vector have been transferred into citrus. They are being screened for their response to HLB pressure. They include the following; CLIBASIA_05165, CLIBASIA_05605, CLIBASIA_01555, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. We are also testing full-length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the Pathogen-associated Molecular Patterns (PAMP) activity associated with Flagellin domain of HLB. We use Citrus macrophylla which is a preferred host for CTV for expression of putative effectors and subsequently transferred into sweet orange and /or grapefruit citrus varieties by graft inoculations. These plants are being challenged inoculated with HLB by exposing them to infected psyllids. Additionally, we have used a faster approach using Potato virus x (PVX) vector system to screen for virulence genes of HLB. Transient expression of prophage-related gene cluster of HLB in PVX vector did not induce obvious phenotype changes in tobacco plant. However, transient expression of HLB-flagellin, and flg22, HLB flagellin domain, induced cell death and callose deposition in N. benthamiana. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. However, the synthetic peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to HLB. These results have been published in PLoS One. Huasong Zou, Siddarame Gowda, Lijuan Zhou, Subhas Hajeri, Gongyou Chen and Yongping Duan. The Destructive Citrus Pathogen, ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Flagellin Characteristic of a Pathogen-Associated Molecular Pattern. 2012 7: (9) e46447
Sequence information of Candidatus Liberibacter asiaticus (CLas) revealed number of putative effectors unique to CLas, the causal agent of citrus Huanglongbing (HLB). The effectors identified to be part of prophage related gene clusters and/or pathogenicity islands believed to contain bacterial pathogenicity, multiplication and virulence determinants. We cloned the effectors into binary vector based citrus tristeza virus (CTV) vector, between CP and CP ORFs as a separate cistron under an heterologous beet yellows virus coat protein controller element. Additionally, based on the observation that engineering the foreign genes at the 3′ end of CTV vector resulted in optimal expression, the potential effectors were cloned into CTV and citrus plants were generated containing the following; CLIBASIA_05165, CLIBASIA_05605, CLIBASIA_01555, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. Citrus plants were tested by reverse-transcription-PCR (RT-PCR) for the presence of the effector sequence as part of CTV genome. However, the effectors did not elicit any unusual phenotype in citrus. We also tested full-length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the Pathogen-associated Molecular Patterns (PAMP) activity associated with Flagellin domain of HLB. We used a faster approach using Potato virus x (PVX) vector system to screen for virulence genes of HLB. Transient expression of prophage-related gene cluster of HLB in PVX vector did not induce obvious phenotype changes in tobacco plant. However, transient expression of HLB-flagellin, and flg22, HLB flagellin domain, induced cell death and callose deposition in N. benthamiana. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. However, the synthetic peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to HLB. These results have been published in the journal PLoS One. A useful outcome of this project is the observation that CTV-vector can be used as an Virus Induced Gene Silencing (VIGS) vector. We used this property of CTV to silence citrus endogenous Phytoene desaturase (PDS). It has also been used to potentially silence endogenous genes of psyllids (vecotr of HLB). In co-operation with scientists at CREC and USDA, Fort Pierce we have been successful in silencing of psyllid endogenous genes. This line of research potentially holds great promise in mitigating the spread of HLB by psyllid vector. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, upon replication in citrus generated RNA silencing triggers, which induced altered Awd expression when ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed wing phenotype in adults and increased adult mortality. This curtailed the ability of D. citri to fly, and potentially would limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. The work accomplished during this investigation have been presented in national and international meetings and another manuscript is submitted to Plant Pathology journal. Two post doctoral researchers, DR. Husong Zou and Dr. Subash Hajeri mailnly were involved in these investigations. Dr. Zou returned back to China to a new assignment in Fujian Agricult and Forestry University and Dr. Hajeri has since accepted a position with California Citrus Industry.
Liberibacter crescens BT-1 (Lcr, isolated from mountain papaya), has been cultured and sequenced (GenBank CP003789, Leonard et al., 2012) and has the potential to be developed into a model Liberibacter system for functional analyses of Las, Lam and Lso genes. To become a model system, BT-1 needs to be 1) reliably and stably transformed with a shuttle vector so that different genes can be added in; 2) gene knockouts in BT-1 must be reliably made, and 3) the strain needs to be introduced into a plant and be shown capable of growth in planta. The first two of these 3 goals have already been met. We previously reported the minimum inhibitory concentrations (MICs) of several antibiotics commonly used for plasmid selection, and reported that BT-1 was readily transformed at high frequencies with pUFR071 (RepW; Al-Saadi et al., 2007) and pUFJ05 (Bordatella replicon; Reddy et al., 2007), and that pUFR071 was quite stable in BT-1. In addition, we reported a site-specific gene knockout in BT-1 of the Type I restriction-modification system restriction subunit R (GenBank B488_07050). We now report that a EZ-Tn5-based random mutagenesis approach was developed and successfully used in BT-1. In a single representative mutagenesis experiment, 93 kanamycin resistant transformants carrying EZ-Tn5 insertions were recovered following electroporation into BT-1 cells. Southern blot analyses revealed that the EZ-Tn5 insertions were random. We are now attempting a series of “knock-in” mutations in BT-1; specifically, we are adding a series of phage and other genes suspected of being involved in pathogenicity of Ca. L. asiaticus (Las), Ca. L. americanus (Lam) and Ca. L. solanacearum (Lso) to BT-1. Our Lam genome sequencing paper has been accepted by Molecular Plant Microbe Interactions and will be available this week. The Lcr strain BT-1, isolated from mountain papaya (Babaco) is not known to be pathogenic, therefore its development as a true model Liberibacter system for functional genomics depends upon an as yet unknown capacity for infection and replication in a plant host. Several genes found in Las, Lam and Lso are missing in BT-1, and these are among the knock-in candidates being investigated. Attempts to artificially inoculate marked BT-1 strains into tobacco, citrus and periwinkle are currently in progress.
This is a project to continue one of the most fruitful leads that accidentally resulted from our previously funded work. We have found that citrus becomes a source of Huanglongbing (HLB) inoculum for spreading the disease to other plants much earlier than previously thought. The working hypothesis is that the female psyllid finds an area of new flush to lay her eggs. As she is laying eggs, she probes the phloem to feed and transfers Candidatus Liberibacter asiaticus (Las) to the tree. As the eggs develop into nymphs, Las begins to multiply in that localized area of the plant, where the new nymphs then feed and acquire Las. Thus, infection of only a micro area of flush tissue where the nymphs develop is sufficient for the first generation of psyllids to become infected and to be vectors to spread the disease to other trees. Thus, the time-period after a tree becomes infested by infected psyllids until it is a donor for other trees could be as short as 15-30 days or less. The limitation is actually the time for the second generation of psyllids to develop. We are working with a group in the Math Department of UF to develop a model of spread of HLB in new planting of citrus. A manuscript is being prepared reporting these results. We are using this system to screen RNAi constructs and different peptides against psyllids. Preliminary results are encouraging. We also are attempting to adapt the system into a method to screen peptides against HLB more quickly. We are using this rapid screen to determine whether a peptide can inhibit Las multiplication within 60 days instead of approximately one year. However, this rapid screen measures resistance, but not tolerance. We are still screening using infected psyllids to inoculate plants, but this information allows us to know which plants are inoculated with Las and is greatly improving those assays also.
This proposal concerns the down regulation of genes of phloem specific Callose synthase and phloem proteins involved in phloem plugging in citrus infected with Huanglongbing (HLB) pathogen. Potential down-regulation of these over-expressed genes responsible for phloem-plugging would negate the disease manifestation and severity. We have been able to isolate and clone Calloase-7 and Phloem proteins B-8 and B-14 from sweet orange total RNA. A truncated version of these genes have been cloned into the CTV silencing vector. We have also engineered these genes individually, in tandem and individually at different location in the CTV vector to evaluate their potential silencing abilility of endogenous citrus Callose Synthase and Phloem proteins. We have agro-inoculated some of these constructs into Nicotiana benthamiana in an effort to produce CTV virions to infect citrus.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. Study of the literature showed that the media used to grow the E. coli and other details of the culture conditions greatly influence the yield of scFv obtained from culture lysates. We have found that a very rich and buffered medium, (2X yeast extract Tryptone broth with phosphate buffer) works best. The medium is supplemented with glycine, sucrose and IPTG at various stages of the expression protocol. With this protocol we have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Cross reactions with healthy plant tissue can be a problem, especially if the concentration of scFv is too high. However, color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. Previous work has used a secondary monoclonal antibody directed at the 6X His Tag of the scFv molecules conjugated with alkaline phosphatase and blocking of the membranes with skim milk. We have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. We have had unexpected results with the negative controls in these assays: When the secondary anti-FLAG monoclonal is used alone, without any scFv, it produces color tightly focused as a ring in the phloem cylinder of HLB but not healthy citrus. Thus the anti-FLAG monoclonal alone detects the presence of disease. As a further control, we have prepared polyclonal antibodies against the major outer membrane protein and detected them with alkaline phosphatase. The polyclonal antibodies produce distinct spots of color corresponding to individual phloem cells infected with ‘Ca. Liberibacter asiaticus’. This is a useful assay for ‘Ca. Liberibacter asiaticus’, and we are using it to describe the distribution of ‘Ca. Liberibacter asiaticus’ in infected citrus. We are also interested in the protein recognized by the anti-FLAG antibody in ‘Ca. Liberibacter asiaticus’ infected citrus.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. A visiting scientist arrived and began work 6-15-2013. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. The primers have been synthesized and used to cclone the scFv gene fragments from the scFv phage display library. The fragments have been purified from agarose gels and another set of primers has been designed to incorporate a KDEL leader sequence on the scFV as well as adapters specific for the vector. Final cloning experiments are underway. Related research with the existing scFv is underway on project 551.
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