The goal of this project is to develop a method for rapid and efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System. We are evaluating several sets of plants that we have injected with the HLB bacterium-containing extracts using different conditions. Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. Craig Davis, a scientist from my lab who is involved in this project, visited Gonzalez’s lab and participated in injection system-based inoculations in order to learn the protocol that Dr. Gonzalez’s lab uses. Craig Davis brought back some new nozzles that Dr. Gonzalez uses with his injection device. We are testing those nozzles to see if they would increase the efficiency of our inoculation procedure. Now we are looking to see if we can improve our method by doing some modifications. We also try our injection system with Liberibacter crescens culture. We obtained a USDA-APHIS permit for working with this bacterium. Liberibacter crescens culture was received from Dr. Triplett’s lab and has been to inject several different plant hosts. We also have been assisting Dr. Dean Gabriel with injections of his L. crescens culture into citrus plants using our device
Copper continues to be used as antibacterial/antifungal agent for citrus crop protection due to its effectiveness and affordability. However, prolonged and aggressive use of copper compounds has raised concerns due to copper accumulation in agricultural soil and development of bacterial resistance. This demands a search for suitable Cu alternatives. The goal is to develop Quaternary ammonium compound (Quat) based bactericide as an alternative to Cu compounds. Quat exhibits strong antimicrobial properties. However, direct foliar spray of Quat to plant is not practical as it causes severe phytotoxicity. Moreover, Quat is highly soluble in water, therefore exhibits poor rainfastness. This project is focused on developing Fixed-Quat formulations which will overcome phytotoxicity and rainfastness issues of Quat material without compromising its strong antimicrobial properties. A series of Fixed-Quat materials are being developed using three different EPA approved Quat materials. In this reporting period, two most promising Fixed-Quat nanogel and a Fixed-Quat nanoparticle formulations have been synthesized which remained stable for over two months. Scanning Electron Microscopy (SEM) characterization of Fixed-Quat nanogel material has revealed the formation of highly polydispersed submicron to micron size silica-gel particles. Phytotoxicity studies have been carried out on Vinca sp. (known to be highly susceptible to phytotoxicity and often used in agricultural industry as model test plants) to observe any potential plant tissue damage from Fixed-Quat materials. Phytotoxicity results (observed for 72 hours) have shown zero plant tissue damage when applied at rates as high as 900 ppm. Antibacterial studies have been conducted using in-vitro microplate alamar blue assay, bacterial viability expressed as colony forming units (CFU/mL) and growth curves using gram-negative model bacterial system, E coli. Optimized versions of these formulations showed complete bactericidal efficacy at concentrations as low as ~ 2ppm over our previously reported ~8ppm concentration. One of the Fixed-Quat nanogel materials (containing all EPA approved ingredients) has been delivered for 2014 field trials. Field efficacy will be evaluated at two different foliar spray rates.
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 citrus and citrus relatives 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 using miSEQ and PacBio approaches.
The goal of this project is to generate antibodies of selected secreted proteins from Ca. Liberibacter and use them as detection markers for HLB. This method is more reliable than examining host (i.e. citrus) changes (including DNA, RNA and protein) in the infected plants, which may not be specific for HLB. The basic idea is to generate antibodies targeting selected proteins secreted from Ca. Liberibacter asiaticus (CLas), which are relatively independent on live pathogen cells and can spread out systemically in the infected trees through phloem flow. Using these secreted proteins as detection markers, we have a better chance to detect Ca. Liberibacter in a direct and highly specific manner by overcoming the large variability in titer and distribution of the pathogen within citrus trees, types of tissue tested and degrees of disease progression. We have made significant progress and obtained exciting results. We identified CLas secreted proteins that are highly expressed in citrus, generated antibodies against four unique CLas secreted proteins, and developed detection assays using direct tissue imprints. Our results suggested that these antibodies were able to specifically detect HLB from infected citrus trees. We are now in the process of extensively evaluating these four antibodies using HLB-infected field trees from California and Florida. We will also monitor the presence of movement of the secreted proteins using graft-inoculated citrus seedlings generated at the Contained Research Facility at the University of California Davis. Our results will be compared to other detection methods targeting plant metabolites and CLas DNA. This method holds promise for allowing early detection on newly infected trees and large-scale field surveys.
Early detection using cost-effective surveillance techniques is crucial to successfully fighting the spread of HLB. The strategy of early detection of HLB focuses on the analysis of host VOC responses that are triggered early in the infection cycle as part of the plant innate immune responses. Based on previous CRB-funded effort, there is strong evidence that VOC analysis of citrus trees can lead to early detection of the HLB and other citrus diseases. VOC field testing is performed using EZKnowz’ instruments supplied by Applied Nanotech, Inc. (ANI). The EZKnowz’ trace chemical analyzer uses a gas chromatograph (GC) combined with a differential ion mobility spectrometer (DMS). Our effort in this program is the following: ‘ ANI delivered the 1st non-radioactive analyzer to UC Davis in Nov. 2013. They are optimizing the protocol for sampling and analysis before putting the tool into the CRF at UC Davis. Extensive dialog between ANI and UC Davis has lead to improvements in the next deliverable due Sept. 2014. ‘ ANI has improved the handheld sampler, adding battery power for over 8 hours of runtime, added airflow and air volume control. We are working to improve cleaning and conditioning of VOC traps. We have produced about 30 demountable traps to date. Initial data using the handheld sampler was collected in South Texas and compared to our standard instruments, indicating where the improvements are needed to bring the new instrument up to expected standards. ‘ Researchers at UC Davis are developing a graphical user interface for the Yuma that will be used for analysis at point of operation. Expected Outcomes and/or functional product/solution The potential value of this early-detection solution on the citrus industry is tremendous. By ‘flagging’ infected trees at the asymptomatic stage, eradication would be both more effective, and kept to the minimum necessary, since it would take place well before other trees become infected and enter the latent period. This would interrupt the deadly infestation cycle at the source, significantly reduce the heavy costs of losing trees and citrus produce for a period of three to five years and cut down the costs of planting new trees.
Early detection using cost-effective surveillance techniques is crucial to successfully fighting the spread of HLB. The strategy of early detection of HLB focuses on the analysis of host VOC responses that are triggered early in the infection cycle as part of the plant innate immune responses. Based on previous CRB-funded effort, there is strong evidence that VOC analysis of citrus trees can lead to early detection of the HLB and other citrus diseases. VOC field testing is performed using EZKnowz’ instruments supplied by Applied Nanotech, Inc. (ANI). The EZKnowz’ trace chemical analyzer uses a gas chromatograph (GC) combined with a differential ion mobility spectrometer (DMS). Our effort in this program is the following: ‘ ANI delivered the 1st non-radioactive analyzer to UC Davis in Nov. 2013. They are optimizing the protocol for sampling and analysis before putting the tool into the CRF at UC Davis. Extensive dialog between ANI and UC Davis has lead to improvements in the next deliverable due Sept. 2014. ‘ ANI has improved the handheld sampler, adding battery power for over 8 hours of runtime, added airflow and air volume control. We are working to improve cleaning and conditioning of VOC traps. We have produced about 30 demountable traps to date. Initial data using the handheld sampler was collected in South Texas and compared to our standard instruments, indicating where the improvements are needed to bring the new instrument up to expected standards. ‘ Researchers at UC Davis are developing a graphical user interface for the Yuma that will be used for analysis at point of operation. Expected Outcomes and/or functional product/solution The potential value of this early-detection solution on the citrus industry is tremendous. By ‘flagging’ infected trees at the asymptomatic stage, eradication would be both more effective, and kept to the minimum necessary, since it would take place well before other trees become infected and enter the latent period. This would interrupt the deadly infestation cycle at the source, significantly reduce the heavy costs of losing trees and citrus produce for a period of three to five years and cut down the costs of planting new trees.
Down regulation of phloem specific Callose synthase and phloem protein genes of citrus host of HLB by the demonstrated silencing ability of CTV are the objectives of this research. Citrus plants (Citrus macrophylla) potentially expressing he dsRNA for Callose synthase 7 and Phloem Proteins B8 and B14 are in the green house. We are going to analyze them for dsRNA expression soon by checking for positive ELISA for CTV. As soon as they are positive for CTV, we will multiply these Citrus macrophylla plants and inoculate with HLB by grafting and/or exposure to psyllids positive for Candidatus Liberibacter asiaticus. Previously we have shown that psyllids acquire dsRNA from citrus plants while feeding. We will use this strategy to simultaneously target both vector and host genes for silencing. Towards this end we have ligated in-tandem truncated genes for psyllid Awd and troponin, and citrus endogenous callose and phloem protein genes into CTV vector and will inoculate citrus. The goal is to silence the vector and the host genes simultaneously and mitigate the effects of HLB.
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. 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. 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. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, 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. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have had unexpected but very interesting 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. Interestingly, the anti-FLAG monoclonal will give similar results with tissue prints from trees infected with CTV and other plant viruses. Thus the anti-FLAG monoclonal alone detects the presence of disease in mature fully expanded leaves but not in young flush, and may be useful as a general diagnostic. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. 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. Two manuscripts on the original scFv antibodies have been submitted and two manuscripts on the tissue printing assays are in preparation. The latter manuscripts compare the scFv-based and polyclonal antibody based tissue prints with FLAG based detection and detailed high resolution anatomical and spatial distribution of CaLas within infected citrus plants.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. All Las bacteria examined to date have been found to carry prophages in their genomes. In periwinkles, but not in citrus, lytic phage particles are formed and can be visualized. We previously reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”), SC2-gp100 (“glutathione peroxidase”) and particularly SC1-gp110 (‘holin’) were much higher in periwinkle than in citrus. These results were confirmed and now extended, with identification and cloning of several late gene promoter region fragments. To functionally confirm lytic cycle genes, we cloned and expressed the annotated prophage holin (SC1_gp110) and endolysin (SC1_gp035), as well as a gene outside of the prophage region annotated as an endolysin, CLIBASIA_04790. Functional expression of SC1_gp110 in pET27B in Escherichia coli revealed bacteriostatic activity characteristic of holins. Expression of SC1_gp035, but not CLIBASIA_04790, in pET27B led to lysis of E. coli, confirming SC1_gp035 as an active endolysin. Four potential SC1_gp110 promoter regions were fused with a lacZ reporter; none exhibited LacZ activity in E. coli. One of these promoter regions was also fused with a uidA reporter and exhibited strong GUS activity in Liberibacter crescens, indicating strong promoter activity in L. crescens. Surprisingly, the GUS activity was suppressed by 1:100 diluted, crude, cell-free leaf extracts from citrus, eggplant, tobacco and periwinkle in a dose-dependent manner. Notably, inhibition of GUS activity was not observed following heat inactivation of the leaf extracts. Additional experiments are in progress to determine if a similar effect is noted using psyllid extracts, as well as attempts to identify the specific mechanism of transcriptional repression/activation. The fact that a promoter from a holin gene with demonstrated anti-bacterial activity is functionally active in L. crescens grown in culture and suppressible with plant cell extracts has implications for culturing L. asiaticus (Las). It is likely that this same holin gene is activated in Las when attempts are made to grow Las in culture. Such activation could be expected to halt Las bacterial growth, regardless of whether complete phage particles are formed or not. Further, the fact that plant leaf exacts can suppress this promoter activity indicates that the addition of plant cell extracts to the culture medium might allow additional growth of Las cells and increased overall titers in culture.
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. 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 scFv have been modified by the addition of a four amino acid leader sequence (KDEL) and both Sma I and Spe I cloning sites. The KDEL sequence is expected to stabilize the concentration of scFv in phloem cells by facilitating proper folding of the protein in the microtubules and thereby protecting the ScFv from proteolytic digestion. Eleven scFv inserts have been sequenced to be sure that the expected sequences are correct, and five ScFv sequences have been successfully cloned into the recombinant vector pUSHRL-26 for transformation of citrus rootstocks. These inserts include three different scFv that bind to the protein InvA and two that bind to the protein TolC. The protein InvA is produced by CaLas and secreted into the host to prevent the infected host cells from entering into apoptosis, and the protein TolC targeted by the scFv, is in the external membrane and is essential for the removal of antimicrobial substances produced by the plant. The vector is designed to direct expression of the scFv into the phloem cells of citrus, where CaLas grows, and the vector encoding the scFv genes is being introduced into rootstock varieties by Agrobacterium mediated transformation.
The present project investigates timeline of the HLB infection in citrus, as reflected by emission of volatile organic compounds (VOCs) for advanced disease diagnostics. We have completed the preliminary arrangements at the Containment Research Facility (CRF) for the longitudinal study. We have developed experimental plans for plants testing that include testing schedule, randomization of plant position in green house, and work flow (sequence of testing steps with different methods). Supplies and parts necessary for the study have been arranged. An overhaul of instrumentation has been conducted for the study. We have conducted all the necessary personnel hire and training and established schedule tables for the rotation at the facility to ensure complete use of available time at the facility. We have conducted multiple sets of preliminary experiments to develop optimized protocols for the VOC testing using gas chromatography/differential mobility spectrometry (GC/DMS). The current protocol utilizes PTFE bags to envelop the tested branch and purge with dry air. The first sets of data using the developed protocols have been collected. 200 samples for GC/DMS have been collected so far. The data analysis revealed satisfactory data quality for the first sample collected, but poor data quality for consecutive replicate samples. Further improvements to the protocol are currently being considered based on the collected body of data. The improvements include changes in purge with the makeup air and different sample intake time. In parallel, we have conducted development of experimental protocols for mass spectrometry analysis. Specifically, we have conducted sampling using solid-phase microextraction (SPME) sorbents and stir bar sorptive extraction (SBSE). We have conducted sampling of trees in the laboratory conditions, in the greenhouse and at the CRF facility for the gas chromatography/mass spectrometry (GC/MS) analysis. Both the sampling protocols and the GC/MS protocols have been optimized. The collection of volatiles was conducted using active sampling device developed under previous CRF-funded effort. We have discovered that SBSE sampling resulted in large variability of the data. Currently we are investigating the ways to mitigate such variability by modifying sampling procedure. We have received a future-generation GC/DMS device for in-field use from the industry partner, Applied Nanotech Inc. and have been adopting the unit for the greenhouse testing. We have conducted, along with ANI, troubleshooting and modifications of the device. We have also compiled the set of guidelines/recommendations from the end user perspective for further improvements of the design and interface. Glenn Sellar from NASA JPL has visited UC Davis. We have installed the rig for the hyperspectral measurements of citrus canopy at the CRF facility and conducted training of all UC Davis personnel involved in sample collection to assure that we will be able to collect hyperspectral measurements independently as a part of multi-method rotation. Current data analysis is focused on ensuring consistent data are being collected and therefore is focused on unsupervised analysis to observe variations within the collected data. We have presented the VOC study aspect of the collaborative project of early HLB detection at the 2014 Citrus Showcase in Visalia, CA.
The present project investigates timeline of the HLB infection in citrus, as reflected by emission of volatile organic compounds (VOCs) for advanced disease diagnostics. We have completed the preliminary arrangements at the Containment Research Facility (CRF) for the longitudinal study. We have developed experimental plans for plants testing that include testing schedule, randomization of plant position in green house, and work flow (sequence of testing steps with different methods). Supplies and parts necessary for the study have been arranged. An overhaul of instrumentation has been conducted for the study. We have conducted all the necessary personnel hire and training and established schedule tables for the rotation at the facility to ensure complete use of available time at the facility. We have conducted multiple sets of preliminary experiments to develop optimized protocols for the VOC testing using gas chromatography/differential mobility spectrometry (GC/DMS). The current protocol utilizes PTFE bags to envelop the tested branch and purge with dry air. The first sets of data using the developed protocols have been collected. 200 samples for GC/DMS have been collected so far. The data analysis revealed satisfactory data quality for the first sample collected, but poor data quality for consecutive replicate samples. Further improvements to the protocol are currently being considered based on the collected body of data. The improvements include changes in purge with the makeup air and different sample intake time. In parallel, we have conducted development of experimental protocols for mass spectrometry analysis. Specifically, we have conducted sampling using solid-phase microextraction (SPME) sorbents and stir bar sorptive extraction (SBSE). We have conducted sampling of trees in the laboratory conditions, in the greenhouse and at the CRF facility for the gas chromatography/mass spectrometry (GC/MS) analysis. Both the sampling protocols and the GC/MS protocols have been optimized. The collection of volatiles was conducted using active sampling device developed under previous CRF-funded effort. We have discovered that SBSE sampling resulted in large variability of the data. Currently we are investigating the ways to mitigate such variability by modifying sampling procedure. We have received a future-generation GC/DMS device for in-field use from the industry partner, Applied Nanotech Inc. and have been adopting the unit for the greenhouse testing. We have conducted, along with ANI, troubleshooting and modifications of the device. We have also compiled the set of guidelines/recommendations from the end user perspective for further improvements of the design and interface. Glenn Sellar from NASA JPL has visited UC Davis. We have installed the rig for the hyperspectral measurements of citrus canopy at the CRF facility and conducted training of all UC Davis personnel involved in sample collection to assure that we will be able to collect hyperspectral measurements independently as a part of multi-method rotation. Current data analysis is focused on ensuring consistent data are being collected and therefore is focused on unsupervised analysis to observe variations within the collected data. We have presented the VOC study aspect of the collaborative project of early HLB detection at the 2014 Citrus Showcase in Visalia, CA.
The ultimate utility of airborne detection and mapping of HLB infection depends to some degree on the time lag between HLB infection and the development of a detectable reflectance signature. In Year 2 (FY 2014) JPL will participate in the longitudinal study proposed to CRB by UC Davis. UC Davis will provide citrus specimens, some of which will be inoculated with HLB in the UC Davis contained research facility. JPL will lead an effort to measure the reflectance spectral of these specimens with weekly frequency as the disease progresses. The following milestones have been achieved: * Design of a test station incorporating a reflectance spectrometer with spectral performance similar to AVIRIS-ng, an appropriate light source (simulating solar illumination), and calibration standards. * Fabrication of the test station. * Development of the experiment procedure. * Testing of the apparatus at JPL. * Delivery and installation of the test station at UC Davis. * Training of UC Davis personnel in the experiment procedure.
This project provides the infrastructure to support eight CRB-funded projects investigating detection of the putative causal agent of huanglongbing (HLB), Candidatus Liberibacter asiaticus (CLas) in citrus prior to symptom development. The 8 projects are using a systems approach that includes transcriptomics, proteomics, and metabolomics to develop early detection methodology. The researchers are all using the same cohort of plants, so that results of the various methods are comparable. The plants used to initiate the studies were grown and grafted using plant material certified to be free of pathogens by the Citrus Clonal Protection Program. The scions Washington navel, Tango mandarin, and Lisbon lemon were grafted onto Carrizo rootstock to produce 48 Washington navel plants, 42 Tango mandarin plants, and 54 Lisbon lemon plants that were delivered to the Contained Research Facility (CRF) on July 31, 2013. These scion/rootstock combinations are common in California citrus production. The plants were placed in a greenhouse at the CRF to allow the newly grafted buds to grow. Of these plants, 113 plants were available for the first round of experiments and the remaining plants were used to establish plants from which scion material could be harvested to continue to produce more experimental plants. Carrizo seed was obtained and the rootstock for the next cohort of experimental plants (approximately 175 plants ) is being grown in the CRF. The CLas (California Hacienda Heights strain) was obtained from USDA, ARS ‘ Beltsville as infected plant material on August 23, 2013, and grafted into Volkameriana citrus to initiate a culture at the CRF on August 27, 2013, using leaf-patch grafts and T-bud grafts. Once these plants became infected, buds from these plants were used to infect two Washington navels, two Tango mandarins, and two Lisbon lemons as CLas source plants for the experiments that began March 3, 2013. Samples of leaf tissue were taken from the source plants and sent to the Citrus Research Board Laboratory in Riverside for CLas testing. The source plants were PCR-positive for CLas and had CT values between 24.0 ‘ 30.17; and the control (not infected) source plants had CT values greater than 40. Establishment of a CLas-positive Asian citrus psyllid (ACP) colony began in January 2014, and ACP adults and nymphs from this colony will be tested for the presence of CLas in early April. This colony will be used to infect citrus plants in experiments in the future. This project provides addition support by providing the conference call line, and compiling minutes of the research group monthly meeting. The minutes and other pertinent materials for the group are archived on a secure website.
Present proposal involves the down regulation of phloem specific Callose synthase and phloem protein genes involved in phloem plugging in citrus infected with Huanglongbing (HLB) pathogen. There is substantial increase in the accumulation of the transcripts and proteins corresponding to these in infected plants. Down-regulation of these over-expressed genes responsible for phloem-plugging potentially would negate the disease severity. Towards this goal we have isolated and cloned Calloase-7 (CsCal-7) and Phloem proteins B-8 and B-14 (Cs PP2) 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 in the CTV vector to evaluate their potential silencing ability of endogenous genes of CsCal-7 and CsPP2. We agro-inoculated these constructs into Nicotiana benthamiana and isolated CTV virions carrying truncated genes of CsCal-7 and CsPP2 and have inoculated citrus plants for subsequent evaluation.
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