The first task in this proposal is to find citrus version of the two proteins that make up the functional design of a chimeric antimicrobial protein previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have completed the discovery of the replacement of the first component the human neutrophil elastase (HNE) the surface binding component. Since HNE is a serine protease with elastasin as a substrate and whose crystal structure has been determined, we used 3D shape criteria and electrostatic properties to search the PDB data base for a plant protein with the identical active site structure. Focusing on a set of 288 non-redundant plant derived proteins from the entire PDB database, we used CLASP to search for a match using the electrostatic and structural features of amino acids that make up the active site. This was successful and the tomato PR14a was identified as an exact match. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that is identical in the Citrus sinensis (Cs) and clementina (Cc) genomes. We are focusing on the 165 amino acid protein from Cs which we call CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains a 25 amino acid signal sequence. We are focusing on the 137 aa mature protein and begun the construction of two genes that encode this protein. The first is a CaMV35S expression cassette that will be used to express just the mature form of the CsP14a protein, however, we have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag is part of the secreted protein after cleavage of the signal peptide. The second version is like our CAP protein and has Cecropin B (CecB) at the C-terminal linked via a flexible linker as we have described earlier. This protein is designated CsP14a-CecB. These genes will be used for in planta expression of the proteins CsP14a and CsP14a-CecB that will be isolated and used to test their efficacy against the bacteria as we described in our last report.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project are being used to support the construction of a new greenhouse at the IRREC. UF-IFAS Facilities Planning & Operations (FP&O) conducted a competitive bidding process and identified a vendor capable of constructing FDACS-DPI-approved propagation structures. Funds were transferred to IFAS FP&O to initiate construction of a new greenhouse to support the goals of this project. Currently, site preparation at the IRREC is being performed to ready the location, and building will commence as soon as possible. – The initial cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation have been identified. They are currently emerging from the ‘HLB gauntlet’ and will soon be ready for en masse propagation.
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. A total of 3,805 transgenic plants have passed through inoculation process. A total of 76,160 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.
The Texas Citrus Budwood Certification Program has completely migrated into insect-resistant structures, as well as rebuilding the Foundation and Increase tree collection. The progress made will allow the Budwood program to continue supplying pathogen-free, high quality, true-to-type citrus budwood for the Texas citrus industry. PROJECTS – In October, 2012, construction was completed on the Phase I-II and III projects, enclosing the existing Increase tree collection inside an insect-resistant screen structure. The enclosed screen structures have new irrigation drip lines pumps and injector system. – Foundation Screenhouses 1 & 2:A new irrigation system was installed in Foundation Screenhouses 1 & 2. Foundation trees were moved from Screenhouse 5, the Stephenville greenhouse, and newly propagated trees from CCPP bud sources, and planted into SH-1 and 2. – A new irrigation system was installed in Screenhouse 5. New trees that were propagated from CCPP bud sources in October, 2012 were added to replace the Foundation trees moved to SH-1 and 2. There are currently 200 containerized trees in Screenhouse 5, and another 50 will be added this fall to bring it to capacity of 250. – The ‘TajMahal’ building that has served as a research and biological indexing greenhouse and screenhouse began renovation in May, 2013. The rooms will be made insect resistant, for housing additional Foundation trees. – All Foundation and Increase trees at the Citrus Center and Stephenville greenhouse were tested in the fall and spring for HLB and in the spring for CTV. All trees tested negative. – The Foundation collection began rebuilding in September, 2012. 34 new varieties were budded with buds from the California Citrus Clonal Protection Program (CCPP). In June, 2013 another 26 varieties were budded from buds from the CCPP. Currently there are 81 Foundation trees planted in-ground in Screenhouses 1 and 2. There are 200 Foundation trees located in Screenhouse 5. There are 83 Foundation trees located at the Stephenville greenhouse. Budwood from the Stephenville trees is cut 4 times per year and brought back to the Citrus Center to propagate new Increase trees for SH 3 and 4. – Establishment of new Increase trees began in Screenhouse 3 in September, 2012. There are over 1,000 active Increase trees in pots in Screenhouse 3, with another 1,200 trees budded to be transplanted. All Increase trees source budwood came from Foundation Screenhouse 5 and the Stephenville greenhouse. The first budwood cutting of trees in Screenhouse 3 was in April, 2013, about 4 months ahead of schedule. As soon as the capacity is reached in Screenhouse 3, then Increase trees will be started in Screenhouse 4.
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 have performed the designed microarray experiment to identify genes that are induced by NAD+ in citrus. The microarray data is currently under analysis. For objective 2, in last quarter we cloned two non-host resistance genes against citrus canker into the T-DNA vector pBI1.4T and mobilized the plasmids into Agrobacterium. In this quarter we have started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria. Several putative transformants have been identified.
Obj 1A: All components of the user-friendly web versions of the single (sTCW) and multi-(mTCW) psyllid transcriptome databases have been finalized in preparation for the public release which will allow researchers to quickly identify targets, not only based on expressional differences but in combination with predicted functions. Recent data obtained from the sTCW databases containing salivary glands and midgut tissue transcripts show that the salivary glands contain more transcripts that are differentially expressed in response to Liberibacter than midgut tissues. Also there are drastically more ‘biological process’ Gene Ontology categories that contain a significant number of differentially expressed transcripts in the salivary glands compared to the midgut. These data highlight the potential importance of tissue specificity, specifically the role of the salivary glands in Liberibacter transmission. Previously, it was reported that members from 3 OrthoMCL-defined clusters (mTCW) were validated in both POP and ACP. To date, the expression of transcript members from an additional cluster predicted to be important for bacterial nutrition has been validated in both ACP and POP by RT-PCR using primers designed from conserved regions. Obj 1B: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions. Previously it was reported that a total of 9 CLas candidate genes from a list of 19, had been moved into the Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut and salivary gland libraries. To date 12 gut library matings and 12 salivary gland library matings have been performed. Data analysis has been completed for 20 of those experiments with the remaining being in various stages (PCR, cloning, sequencing, etc.) moving towards completion. Through those 20 experiments we have thus far discovered roughly 44 ACP gene products that have high levels of interest making them good candidates for RNAi and will be moved into that phase of the project if they fit the criteria specific for RNAi. Previously we reported the findings from the first 3 mating projects (1 midgut and 2 salivary glands). Since the last update, we have discovered more interesting candidate effectors that will be moved into the RNAi phase of the project many of which are predicted to be important in Liberibacter adherence to host tissues. One interesting candidate is an endopeptidase putatively involved in the dissolution of extracellular matrices. The overexpression of this gene could greatly interfere with CLas transmission. An ACP gene product similar to enzymes involved in signaling cascades important for bacterial communication and subsequent biofilm formation was also identified. A CLas ‘bait’ with protease function, identified an ACP gene important for bacterial nutrition and is made available to the bacteria after processing. An interesting finding came from using the CLas ‘bait’ FlgL protein. No interactors were discovered from gut matings, only from salivary gland mating experiments which suggest the motility of Liberibacter is most important in this tissue. To date, one candidate ACP gene putatively involved in bacterial adhesion has been mated against the CLas prey library interacting gene products (‘prey’ inserts) identified are currently being analyzed. Obj 2: RNA interference (RNAi) studies are underway to functionally validate candidate effectors. To date, good quality dsRNA has been synthesized for five psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport, or transcytosis. Previously we reported on the impact of the knockdown of one of the cytoskeleton-related gene on Liberibacter transmission using the oral delivery method. Recently, both microinjection and feeding studies have been conducted for actin, the other cytoskeleton-related gene whose knockdown is expected to result in high psyllid mortality and is used as a control to optimize our studies. Mortality results from oral delivery of dsActin show that 37% more psyllids died after feeding on dsActin-treated diet in comparison to the control. Mortality results from microinjection delivery of dsActin show that 15% more psyllids died from dsActin injections in comparison to control injections. These preliminary data suggest that the method of dsRNA delivery is crucial, and should be optimized accordingly.
The target for therapeutics that we focused on this quarter of funding is CLIBASIA_01810 (renamed LdtR). Over the last year we have found that this protein is a novel transcriptional regulator that modulates the expression of proteins involved in cell wall synthesis. Since we have proved that inhibitory molecules bind specifically to LdtR to reduce the transcriptional activity of ‘Ca. L. asiaticus’ in infected plants, our goal was to identify where and how small molecules affect the protein stability and activity. Using structural modeling we selected residues that may be involved in the binding of small molecules and performed site directed mutagenesis. The consequences of mutations in the LdtR protein was tested on the binding to its cognate DNA binding sequence as well as on the ability to bind small molecules. We were able to identify three residues involved in ligand binding. Currently, molecular dinamics experiments are being performed in collaboration with IBM with the end goal of optimizing chemicals for treatment of infected trees. A manuscript with the results obtained in the LdtR studies has been submitted for publication to a peer reviewed high impact journal.
Processing of the agreement for this project was delayed and funds were only recently received by the PI. Nevertheless Hamlin orange fruit samples were harvested from neighboring groves with normal cultivation and fertilization treatment and those receiving one or two different nutritional foliar sprays from healthy and infected trees (PCR tested), in December, 2013 and January, 2013. Similarly, Valencia orange samples were harvested in April and May of 2013. Size and color measurements were conducted on the fruit prior to processing. Fruit were then washed and extracted using JBT extractors, finished, pasteurized and frozen for future analyses. Sensory tests include difference from control tests (DFC) and some trained panel analysis, while chemical analyses include measurement of sugars, acids, aroma compounds, limonoids and flavonoids which are ongoing. Fruit from HLB treatments were smaller and more green than fruit harvested from healthy trees.
Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA was completed, and as expected, data shows a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology: 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene driven by either an embryo specific Carrot DC3 promoter or an embryo specific Arabidopsis At2S2 promoter. Transgenic plants expressed anthocyanin only in the embryogenic cells. Cotyledonary cells did not express anthocyanin indicating tissue specific activity of the promoter. Plants germinated from these embryos have been observed to lack anthocyanin production and have been micro grafted for growth. RT-PCR will be carried out on these plants to confirm inactivity of the visual selectable marker. 2. A binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cell transformation and selection have been constructed. Hamlin and W Murcott cells have been transformed with this construct. Preliminary data indicates that the transformation vector is functional and able to incorporate both T-DNAs into the plant genome. Somatic embryos containing this dual T-DNA cassette have been isolated and have been placed in embryo maturation medium for growth and development. Plants will be tested by once the plants are large enough. Subsequent experiments will confirm if negative selection pressure can differentiate between cells that contain the marker free T-DNA from the T-DNA containing the selectable positive/negative fusion marker cassette and if it can be removed from the citrus genome. 3. The binary vector for an inducible cre-lox based marker free selection has been constructed containing a heat inducible excision system containing the cre gene driven by a Soybean heat shock gene promoter. Tobacco tissue has been transformed with the construct and we are waiting for the plants to grow to a suitable size for heat shock treatment and excision. Renewal of transgenic field permit with APHIS: following completion and review of paperwork, our permit was renewed for 3 years; we now have permission to allow transgenic trees to flower and fruit; the Southern Gardens transgenic site was added to our permit. Transgenic testing: Nearly 200 new transgenic citrus trees were planted at the Picos USDA Farm site (resets and new tree positions). Paperwork is nearly complete to move HLB resistant transgenic trees from the SG hot psyllid house to field sites (petition approved by DPI). Two large sets of transgenic plants were propagated for testing in the SG hot psyllid house; they include new transgenes LIMA-B and SABP2 (the latter being a plant gene showing promise). We expect to move these plants to the SG psyllid challenge house within the next few weeks.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus to controlling psyllids until we have more conclusion from the peptides under screen.
The potential for intellectual property protection for the proposed prototype and technology was discussed. Protection will reconsidered upon collecting more field data. PI Ehsani visited with research team at the USDA-ARS in Ft. Pierce who are doing similar research to explore potential collaboration and effort leveraging. Expanding the research scope through this collaboration is being discussed. The results of experiments where trees where treated in the summer of 2012 and fruit harvested and analyzed in the spring of 2013 were presented at the 2013 Annual Meeting of the American Society of Agricultural and Biological Engineering. Experiments with the new prototype were initiated in May to treat the canopies at three different temperatures with varied heating periods ranged from 20 to 180 min. The 2012 field-trials confirmed the limitation of QPCR technique in quantifying HLB kill due to varied live and dead bacterial load in the leaves from same branch and within branches of the untreated tree. Therefore, the tree physiological measurements such as number of leaves are being conducted to quantify the effect of treatment on tree health. It was found that despite supplemental heating, reaching temperature above ___ was difficult. It was hypothesized that ground water evaporation is responsible for producing evaporative cooling and delaying the rate of heating. An additional heating element will be added to the system and the ground will be covered with a tarp to prevent evaporation.
The potential for intellectual property protection for the proposed prototype and technology was discussed. Protection will reconsidered upon collecting more field data. PI Ehsani visited with research team at the USDA-ARS in Ft. Pierce who are doing similar research to explore potential collaboration and effort leveraging. Expanding the research scope through this collaboration is being discussed. The results of experiments where trees where treated in the summer of 2012 and fruit harvested and analyzed in the spring of 2013 were presented at the 2013 Annual Meeting of the American Society of Agricultural and Biological Engineering. Experiments with the new prototype were initiated in May to treat the canopies at three different temperatures with varied heating periods ranged from 20 to 180 min. The 2012 field-trials confirmed the limitation of QPCR technique in quantifying HLB kill due to varied live and dead bacterial load in the leaves from same branch and within branches of the untreated tree. Therefore, the tree physiological measurements such as number of leaves are being conducted to quantify the effect of treatment on tree health. It was found that despite supplemental heating, reaching temperature above ___ was difficult. It was hypothesized that ground water evaporation is responsible for producing evaporative cooling and delaying the rate of heating. An additional heating element will be added to the system and the ground will be covered with a tarp to prevent evaporation.
Function of individual X. citri transcription activator like effectors (TALEs): We previously reported the development of an assay using stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct to be tested for activation by a number of PthA homologs we have cloned from various X. citri strains. The PthA homolog-EBE specificity was were tested by creating transconjugants of X. campestris pv campestris 8004 transconjugants carrying the various PthAs and infiltrating into the stable transgenic Nicotiana benthiamiana plants. X. campestris pv campestris was used in lieu of X. citri for screening in Nicotiana because X. citri doesn’t infect Nicotiana. We observed strong gus activity in transconjugants containing pthA1(21.5 repeats), pthA2 (15.5 repeats) and pthA4 (17.5 repeats) from strain A44; pthA1- 17.5 repeats from Etrog; but not PthA4 homolog from Miami strain. Transformation and production of stable citrus lines: Because we have had difficulty recovering intact and functioning stable transgenic citrus lines, we have undertaken a number of efforts to overcome this bottleneck. Changes included testing a different transformation vector, different promoter and construct components, adjusting the transformation methodology, and adding Carrizo citrange, given it’s greater transformation efficiency, for comparison with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange. So far we have regenerated 312, 22 and 261 putative transgenic shoots from grapefruit, sweet orange and Carrizo, respectively and transferred to rooting media. Ongoing transformation experiments with previously used constructs have resulted in a number of putative transgenic shoots that have been rooted and transferred to soil in trays or 4′ pots. PCR screening of putative transgenic grapefruit and sweet orange plants regenerated from segments transformed with various constructs were analysed for 3 genes, 2 of which are present in the transgene of the constructs used (avrGF2 and nptII). A total of 54 plants were tested, but none of the plants tested contained the avrGF2 gene while 36 of the plants contained the nptII gene. We also tested for the presence of the virC gene which indicates bacterial contamination. One plant was positive for virC. Histochemical GUS screening was also carried out on putative transgenic grapefruit, sweet orange and Carrizo. Segments were scored based on the extent of the blue staining observed on the segments. GUS positive shoots were considered those segments that stained entirely blue while chimeric GUS shoots were those segments that stained less than 80%. No GUS positive shoots were observed for grapefruit or sweet orange cultivars for any of the constructs analyzed. Some grapefruit shoots were observed to be chimeric. Carrizo citrange showed the best results with several plants being GUS positive and a large number of plants chimeric for GUS. Another effort we have undertaken was to have an external contract transformation lab test our constructs along side their standard transformation control. In these experiments we provided a 14 EBE promoter construct in the original vector driving either AvrGf1 or GUS, for stable transformation of tobacco and Carrizo. The results so far indicate that our construct was considerable less successful in their hands for transformation compared to their control. These results suggest that our difficulties arise from the original vector used. Now that we have made new constructs we expect to have greater success in the production of stable transgenic lines
CLas colonizes its insect vector and is transmitted in circulative propagative manner. The bacteria multiply within the insect vector hemolymph. We noticed that the bacteria also form biofilm on the gut surface. In general bacteria need a cell-to-cell signaling system (Quorum sensing) in order to form a biofilm. Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. Main findings: 1-Using different bacterial biosensor, we partly identify these AHLs (number of Carbon). CLas biofilm formation on the surface of insect Gut confirms the presence of cell-to-cell communication in insect while the planktonic state of CLas in plant indicate the absence of this communication. 2- In plant, we found molecules that bind to LuxR but inactive its function (plant defense). We try now to characterize these molecule and study their effect on biofilm formation inside insect. We use purified molecule to feed infected insect through artificial diet system. 3-We produced citrus plants that express LuxR protein in the phloem sap in order to test I- If the acquired LuxR proteins in insect interfere with the biofilm formation in insect (cure the insect from CLas) II- if the expression of LuxR in plant induce biofilm formation (localize the infection in plant) We found that feeding infected ACP with CLas on the LUXR expressing plants reduce the bacterial populations in insect and reduced the infection rate significantly. This result strongly indicates that we can target this system to interfere with the insect transmission and the spread of Disease. In last few months we focused on identifying the AHL- like molecules in plant Phloem sap. for that were analyzed the phloem sap chemical composition from suspectable and resistent varities of citrus. We found some candidates and we start testing them. The main aim of this project is to express molecules in plant that interfere the growth of CLas in insect by feeding.
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 focussed in the vascular cylinder of HLB infected, but not healthy petioles. We will carry out further experiments with the scFv using the FLAG epitope for detection of the scFv. The scFv will also be used to label sections for examination by fluorescence and electron microscopy. We have provided several scFv to cooperating researchers at USDA ARS Ft. Pierce.
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