Huanglongbing (HLB) and Citrus Bacterial Canker present serious threats to citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters are used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. Use of this promoter may reduce the number of independent transformants needed. A new ubiquitin promoter from citrus (Belknap) is being tested. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and are being tested further for suppression of CLas in culture, before creating transgenes. ARS-Albany (Belknap) collaboration is providing genes from Carrizo citrange sequence generated using USDA and now CA-CRB funds, and other citrus genomic data, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Sequence data are being mined for citrus AMPs and defensins to test in-vitro and ultimately in-planta. Anthocyanin production genes,give bright red shoots (UGray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes have been developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 30 months and a repeated trial has been in the field for 18 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our library is estimated to contain 2.1 x 10 7th primary, unique antibody clones. Because our antigen was individual psyllids from Florida infected with high concentrations of ‘Ca. Liberibacter asiaticus’, the library contains antibodies for both the pathogen and the vector insect. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. These approaches are described in previous reports. We have now cloned two genes encoding type IV pilus proteins of ‘Ca. Liberibacter asiaticus’ (ACT57201,ACT57197) and the polysialic acid capsule protein (ACT57308), two genes from the flagellar operon (ACT56849, ACT56985), a TolC protein (ACT57403) and the major outer membrane protein (ACT57245) These proteins should be well exposed on the cell surface and should be useful for labeling ‘Ca. Liberibacter asiaticus’ in insect or plant tissues and for any serological detection assay. Portions of these proteins predicted by software to be exposed on the cell the surface were cloned. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins have been purified. Emphasis is on recovering the proteins in native, soluble form, which was difficult, but we have now been able to accomplish it. These proteins, except for TolC, have been used to select scFv particles specifically binding them. Thus we have developed and demonstrated a protocol that will allow us to isolate scFv antibodies that, in principle, will recognize any proteins from “Ca. Liberibacter asiaticus” or the insect vector, Diaphorina citri. Several of these proteins have also been successfully used in Das-ELISA assays and in dot blot assays of plant and insect extracts containing ‘Ca. Liberibacter asiaticus’ from Florida. Premature termination of antibody proteins has been encountered in the soluble expression system. We are checking all clones by sequence analysis to be sure premature stop codons do not appear. Several of these antibodies detect antigen in plant but not in insect extracts. After our presentation at the 2nd IHRC in Orlando, several researchers expressed interest in our offer to select scFv antibodies upon request for any protein of interest to their research programs. scFv antibodies may be useful as labels for ultrastructural studies of infected plants and insects, advanced detection assays, and possible even for HLB control through a ‘plantibody-based’ approach.
Four field studies are currently underway to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first trial is a survey-type trial to monitor the health and yield of mature trees in a commercial grove in Felda. We have recently completed the third consecutive harvestsof both ‘Hamlin’ and ‘Valencia’ trees. Statistical analyses of this data set across time are being completed and we anticipate having a manuscript draft of results from the first three years complete by late summer 2011. It is clear from this observational study that HLB infection does not kill well managed citrus trees, and that trees can be maintained with economically profitable yields of quality fruit for at least five years after known infection. Although absolute yield varies from season to season, the three years of data we now have suggest that this is largely due to environmental factors and not HLB. The effects of HLB on yield appear to have been suspended in this study grove as the percent of symptomatic fruit is remaining constant over time. Recently, a summary of the past 10 years of yield for this same grove and a nearby grove practicing tree removal was completed. This summary clearly showed that the yield of the nutritionally managed grove has remained constant since HLB was discovered whereas the nearby grove’s yields are declining as the tree population declines. The second of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received eight foliar applications of nine different treatments. Untreated trees serve as controls. The trees were harvested on 31 January 2011 and the yield data is being compared with the previous year’s. This trial has been terminated because of it utilized single tree replicates and there was not sufficient replication to overcome natural tree-to-tree variability. Two additional field studies were begun during 2010 in research blocks at the CREC. One of these trials involves the application of a commercially available foliar nutrient product with and without the application of SAR inducing compounds. Five treatment applications were made in 2010 and two have been made thus far in 2011. The trees (Valencia) are currently being harvested. Leaf samples have been collected and analyzed for nutrient content and a photographic record of each tree is being kept. The second of these trials involves standard and high application rates of foliar nutrients in combination with standard and elevated ACP control. Trees in this 10 acre block are Hamlin and Grapefruit. Leaf and fruit (at harvest)samples have been collected from the Hamlin trees for nutrient and quality analysis, respectively, and total yield per plot was estimated at harvest. Both blocks continue to be scouted for HLB occurrence, but trees are not being removed. Due to severe canker in the grapefruit portion of this block we have determined that it is no longer viable to continue managing the grapefruit trees. They will be removed this summer and a new, intensively managed block of sweet orange will be planted to test whether new trees can be brought into production in close proximity to mature HLB-infected trees. A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Trees are growing well in the system and are now deficient in Mg, Ca, B and Mn. These deficient trees were inoculated with HLB and are being monitored for the development of HLB symptoms and are being sampled periodically for PCR confirmation of HLB. Once trees are known to be infected data collection on how the disease develops in trees under these nutrient deficiencies will be collected. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
In the second year of funding, the CCTF continued to maintain and improve the quality of service it offers, proving itself as a reliable partner and integral part of the wider research community engaged in fighting HLB and canker. CCTF has become truly known and recognized beyond the community of Citrus Research and Education Center (CREC) and that is reflected in the increasing percentage of orders coming in from main campus of University of Florida in Gainesville. Within the last year, CCTF received orders to produce transgenic plants by using following vectors: p33; p7; p10; pMOG800; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; pSucNPR1; pWG19-5; pWG20-7; pWG21-1; pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. This is the largest number of orders received during one year since the facility opened and it clearly describes high demand for transgenic Citrus plants. At the time this report is being written, the facility has already been informed of additional five orders (from UF researchers) and another two orders for which binary vectors for insertion of customer’s genes of interest were sent to Yale University. The initial goals of this project that were reached in the first year of funding were being met throughout the second year. Despite high flux of people, the number of employees was kept constant. That allowed the number of explants processed per week to stay at about 2500. This amount of processed material per week is sufficient for production of high numbers of shoots that are being screened for presence of transgene by using different methods. And this in turn creates situation where CCTF is capable of servicing multiple orders at the same time. Application of the new PCR-based screening method that we started using last year is proving to be extremely useful. By using PCR on small shoots and detecting those that are putatively transgenic before they get micro-grafted on the rootstock plants improved CCTF productivity. Considering the fact that many orders include the use of binary vectors with no reporter gene, introduction of PCR as screening tool has brought the efficiency of the facility to a new level that permits its present output. As a result, the production stayed on high level of above 400 plants per year. More importantly, the time needed for completion of order and delivery of transgenic plants has fallen to about 10 months for many vectors. Within the last quarter, additional plants were produced for the old orders: pHK (12) and pSuperNPR1 (2). However, most of the plants were produced for newer orders: pNAC1 (26), p33 (12), pMKK7 (16), pMOD1 (5), pAS7 (7), pAS13* (4), pSucNPR1 (3), pMOG800 (1), p7+p10 (15). Similar to last year, the plants that CCTF produced in the second year of funding belong to five cultivars: sweet oranges-Hamlin and Valencia, grapefruits-Duncan and Flame, and Mexican lime. All of the new orders received in this funding period had a goal of improving tolerance and/or resistance to Citrus pathogens. The production of plants for old orders listed here also continued: pCL1; p6; pN1*; pC5*; pNPR1; pSuperNPR1; pPiTA; pCIT108p; pCIT108p3; and pHK. This decisively confirms the relevance of this project for the overall effort to produce and challenge transgenic plants as soon as possible and present them to Citrus industry as prospective candidates for tolerance and/or resistance against huanglongbing (HLB), canker, and Citrus Tristeza Virus (CTV). All current orders are for faculty presently involved in research projects funded by CRDF to battle HLB, canker or CTV. Funding for the CCTF furthers the efforts of these research groups and brings some of their results into life by producing transgenic plants that carry genes with predicted protective roles against pathogen attack. Continued funding to CCTF will allow for this situation to continue by keeping production of transgenic material at high levels and uninterrupted.
Our group continued to make excellent progress in the second year of our project. Over the last twelve months we have accomplished the following: Testing TAL effector specificity: We have synthetically assembled six TAL effector genes from X. citri strains and have establishing a system to test their activity on our broad recognition or “super” promoter in transgenic Nicotiana benthamiana plants. We have also prepared constructs with promoters containing individual TAL effector binding sites to test their activity and specificity. Testing broadness of resistance: Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves with Bs3 promoter constructs. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. These studies concur with the preliminary results showing that the constructs limit X. citri growth and produce HR against a number of strains. Additionally, we have generated different versions of the constructs that are designed to detect TAL effectors in all known X. citri strains. We are currently testing these, as well as X. citri strains with single TAL effectors to isolate the role of specific TAL effector proteins in the disease and resistance process. We have isolated multiple new TAL effector genes from important X. citri strains, which we will sequence and analyze in the next project year. Production and analysis of transgenic grapefruit lines: Sixteen independent transgenic lines generated in the first project year have progressed through selection, shoot formation and rooting, and are now well established in soil. These lines were verified by PCR and have been used in several experiments to examine response to pin-prick assays with X. citri and controls. We have thus far tested nine of the lines, and all but one demonstrates some degree of canker resistance, with two showing strong resistance. We have also set up additional transformations on both cotyledons and epicotyls. In total, we have initiated transformations of 6,857 explants using seven promoter constructs, and the explants are moving through tissue culture and selection, with more than 200 additional plantlets in soil. Finally, we have consulted with industry personnel to identify the most industry-relevant commercial germplasm to transform, and as a consequence of this we have obtained seed of red grapefruit and mid-season sweet orange to test in transformation assays. Other outcomes: We filed a patent application in January 2011 on canker-resistant transgenic citrus, and we have drafted an initial manuscript of our results, to be completed following the outcome of pending experiments.
The overall objective of this proposal is to develop and use a high throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps). Most work during the second quarter of the project was focused on analysis of a potential model system in which potato psyllids inoculate the model plant Arabidopsis with CLps. We previously found significant differences among 19 diverse Arabidopsis lines in the percentage of plants that became infected, and in the amount of bacteria present in tissue samples from each plant as judged from the Ct value of qPCR detection. The most resistant and susceptible lines are being tested again with larger numbers of plants to confirm differences. In previous experiments we did not detect any visible disease symptoms on leaves or stems of infected Arabidopsis plants, despite relatively low Ct values. The current experiments also evaluate effects of CLPs on seed production, seed germination and root growth. If Arabidopsis is not affected by CLPs when bacteria are present at levels that induce symptoms in tomato and citrus, then we may investigate the basis for this difference in response. We are growing plants for experiments that will compare gene expression patterns in plants exposed to CLps-free psyllids vs CLps containing psyllids. These experiments should be informative about similarities and differences between the responses of citrus and Arabidopsis to closely related bacteria. During this quarter, we did not make any advances in development of a system for high-throughput chemical screening. This may depend on selection of the appropriate Arabidopsis ecotype, a step that should be completed within the next few weeks.
USDA Ft. Pierce (Neidz) Agrobacterium-mediated transformation of mature tissue explants: Transformation of mature internode explants from greenhouse trees has been demonstrated in four citrus types including Valencia sweet orange (1 plant), Ruby Red grapefruit (1 plant), US-942 (8 plants), and Etrog citron (8 plants). Current efforts are directed toward characterizing this system for routine transgenic plant production. Source of mature tissue: Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication: A manuscript entitled, ‘Bud emergence and shoot growth from mature citrus nodal stem segments’ was accepted for publication by the journal Plant Cell, Tissue and Organ Culture. The paper documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants: A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. New tissue culture method of Agrobacterium-mediated transformation of tissue explants: Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. Mineral effect on shoots regeneration: Preliminary results suggest that mineral nutrition significantly affects in vitro culture response. The effects on transformation are currently being studied. University of Florida (Moore, Grosser, Gmitter) Efforts continue with greenhouse grown tissue (CREC) Rootstock effect on mature tissue transformation: the experiment conducted to determine if vigorous allotetraploid rootstocks could increase transformation efficiency was compromised by endogenous fungal contamination. We are now testing coconut fiber, sterile liquid nutrition, and low humidity in a clean environment for growing mature tissue explants in efforts to minimize problems with fungal contamination. Characterization of mature-tissue transgenic ‘Hamlin’ plants: recovered mature-tissue derived transgenic ‘Hamlin’ plants from previous experiments were propagated via micro-grafting for further characterization. Research continues on using cell penetrating peptides (CPPs) to deliver cargo (proteins, chemicals, plasmid) to existing citrus cells (Gainesville). Using the easily visualized GUS enzyme, we have found that we can efficiently get protein imported into a number of citrus tissues, using several different CPPs. Currently we are testing import of plasmid DNA, which should let us test clones and constructs before we do stable transformation. Based on a recent report on woody plants, we are also investigating whether we can produce cultures of rapidly proliferating cambial cells from citrus (Lee et al. 2010. Nature Biotechnology 28:1213).
Testing the functionality of reporting – quarterly, annual and final. 4/19 received email that ‘annual’ report was not a choice – checking it out. “Annual” report can be indicated at the top of the pdf file. It was not a choice at the bottom of the researcher page completed when submitting report to the web site. We will look into this.
Samples collected and fixed from HLB affected and healthy trees for a complete evaluation of phloem development and disfunction from just above the fiberous roots to one-year-old shoots will be evaluated by light and electron microscopy with revised staining technology previously described. The trees selected are 22 years-old and will allow phloem age evaluations and natural and HLB elicited changes with aging. The molecular basis of the interaction of Las with its hosts is not well understood. We hypothesized that during infection, Ca. L. asiaticus differentially expresses the genes critical for survival and pathogenicity in either host. To test this hypothesis, .quantitative reverse transcription PCR was utilized to compare the gene expression of C Las in planta and in psyllid. Overall, 362 genes were analyzed for their gene expression in planta and in psyllid. Among them, 263 genes were up-regulated in planta compared with in psyllid, 18 genes were overexpressed in the psyllid, and 81 genes showed similar levels of expression in plant and psyllid. Our study indicates that Las adjusts its expression of genes involved in transport systems, secretion system, flagella, LPS, heme biosynthesis, stress resistance, hemolysin and serralysin in a host specific manner to adapt to the distinct environment of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Las genes in a plant host and its insect vector. Two of the hypothetical genes that were overexpressed in planta were screened on Nicotiana benthamiana and showed symptoms in planta. The genes encode for unique hypothetical proteins, and the products are 80 aa and 51 aa respectively, and both have associated signal peptide and transmembrane regions. The expression of respective genes in planta was confirmed by one step Reverse-Transcriptase PCR on total RNA isolated from younger leaves. Transgenic citrus plants (Duncan) expressing these two genes were constructed at the Citrus Transformation Facility. Another potential virulence factor overexpressed in planta when compared to the psyllid was hemolysin. The first step in the characterization of hemolysin produced by C. Liberibacter asiaticus is to confirm that the protein is active or functional in the organism. For this, the gene was cloned and expressed in E. coli, and grown on sheep blood agar plates (5%) to evaluate the hemolysis on the plates. Hemolysin shows three types of lytic activity, alpha, beta and gamma. Alpha hemolysis is characterized by incomplete hemolysis, where the area surrounding the coloines is greenish /yellowish or brownish. Beta hemolysis is complete hemolysis, characterized by clearing around the colonies. There is no hemolysis associated with the Gamma hemolysis, although the plate appears brownish. In this experiment, the use of a negative control of E. coli containing an empty vector is critical, because E. coli is also known to produce hemolysin proteins. Hence, it is necessary to confirm that the hemolysis is due to the C. Liberibacter asiaticus hemolysin. Our result indicates that C. Liberibacter encodes a functional hemolysin protein which might be involved in pathogen and host interaction. Citrus plants transformed for phloem limited callose production are being introduced into a greenhouse to challenged them with HLB infected citrus psyllids.
We reported last quarter the cloning of the ctEDS1 gene in the binary vector pBINplusARS. We have already transformed the Arabidopsis eds1-2 mutant with this construct. The T0 seeds were harvested and will be selected for the transgenic plants in the next few weeks. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. The cDNA fragments of these genes are now in the pGEM T-easy vector and were confirmed with sequencing. The next step will be to clone these cDNA fragments to the binary vector pBINplusARS for plant transformation. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes for the next round of cloning and complementation analysis.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: In addition to the SSI4 obtained from Arabidopsis thaliana var. Columbia genomic DNA, we created two new constructs (5-4) AtSUC2/SSI4 and AtSUC2/ssi4 mutant derived from Nossen genomic DNA (lines obtained from Dr. Klessig). Out of 49 ssi4 transgenics, 18 showed a reduced stature phenotype. However, none of the ssi4 expressing transgenics displayed a severe dwarf phenotype seen when expressed using their native promoter. We visited Dr. Orbovic’s laboratory at the UF CREC at Lake Alfred and resolved a technical problem regarding the PCR-based screening of citrus transformants. Dr. Orbovic has subsequently identified transgenic citrus for our two clones: AtSUC2/snc1 and AtSUC2/ssi4 mutants. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to the single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. The PAD4/ssi4 transgenics were difficult to obtain. We screened three times as many To seeds to obtain 18 plants. From these, 50% developed the “smaller stature” phenotype. Conversely, almost all transgenic plants containing SSI4 (wt) developed normally. Of the 33 transgenics with the PAD4/SNC1 construct, the majority showed no detrimental effect on growth. However, from the 37 PAD4/snc1 mutant transgenics, 10 were smaller than normal. We obtained 27 transgenics with the PAD4-reporter and analyzed GUS expression. Originally, the PAD4 promoter was selected based on literature reports of its phloem-specific expression and wound-inducibility upon the insect feeding. Our results indicated four basic groups of PAD4/GUS reporter expression in transgenic Arabidopsis leaves as follows: 1) phloem-specific expression with no wound induction; 2) universal expression with strong wound induction; 3) restricted expression with no wound induction; and 4) strong wound expression only. Conclusions: Our GUS analyses of two phloem-specific promoters (AtSUC2 and AtPAD4) revealed that transgenic plants do not maintain strict phloem-specific expression. Moreover, we found no PAD4 plants with the expected phenotype of wound-specific expression limited to phloem cells. However, substitution of the native promoters for ssi4 and snc1 genes with AtSUC2 and AtPAD4 did result in a significant reduction in the severity of the growth retardation phenotypes. Evaluation of more transformants will give us a better picture of the feasibility of utilizing these two promoters to obtain the desired expression patterns in citrus.
Obj 2 Employ qPCR to detect Liberibacter in experimental adult psyllid cohorts given a range of acquisition-access periods as immatures or adults. Pineapple citrus seedlings at 4-5 leaf stage will be used to study the transmission of HLB using (1) ACP reared on HLB positive citrus plants (hot colony), (2) field collected ACP from HLB positive citrus and (3) ACP reared on orange jasmine (cold colony). Experimental seedlings will be grown in plastic cones filled with Fafard 4 P soil media. Three seeds will be sown in each cone for a total of 60 cones. Upon germination, their number will be reduced to one per cone. Ten seedlings (replicates) will be used for each of the above three groups of psyllids and ten seedlings will be kept without psyllids. Each seedling will be isolated with 10 adults from the respective group within a ventilated plastic cylinder. Adults will be allowed to feed for 4 weeks and removed at that time or earlier when their progeny is in fifth instar and ready to become adult in order to separate the adults used to inoculate the seedlings from their progeny that developed on the seedling. At that time samples of initial cohort of 10 adults will be released on each seedling. Nymphal progeny (4-5 instars) and seedlings will be collected and processed using PCR for the presence of Liberibacter. The experiment will be conducted in an air-conditioned glass house maintained at 26 ‘C and 60-80% RH. Hopefully, seedlings will be able to withstand psyllid feeding pressure during the course of the experiment. Obj 3 Define the gross association of Liberibacter in thick sections. Develop a gross anatomical road map of Liberibacter accumulation in key organs, tissues, and cells. Obj 4 Using the resultant road map, elucidate, at the TEM level, where Liberibacter accumulates. The oral box (see Sept 2010), has become the center of attention because it is the terminal portal of the transmission cycle. Our exploration of the proliferation patterns of the bacteria in the deshelled abdomen still continues with FISH as our primary technique, but we must know if the bacterial titers in the abdomen are of secondary importance to the titers that can develop inside the oral box irrespective of them. We have formative ideas on the construction of a new model for transmission in this kind of insect vector/pathogen relationship, which is in strong contrast to models based on virus, from which scientists draw their initial methodical approaches. Our first Z-section library consists of 211 100nm thick plastic sections of an oral box of an infected adult under the transmission electron microscope. We have compiled most of them as needed into an animation (see Jan 2011). We’ve started on our second Z-section library. It uses the oral box of an uninfected adult from which much of the cellular material has been leached. This will allow us to accurately map, in three dimensions, the configuration of the salivary ducts and esophagus as they merge into the stylets for secretion and ingestion, respectively.
Once we have been able to establish at IVIA the procedures and conditions to transform mature Hamlin, Pineapple and Valencia sweet oranges, we are in conditions to transfer the basic protocols to Florida. We believe they can be reproduced with little or no modification at the new laboratory that is being set up at the CREC. As we have also developed a genetic transformation system for mature Carrizo citrange, we are now incorporating a construct of interest into this genotype. The second objective of our project was to develop genetic engineering strategies to improve citrus tree management. In this sense, we proposed to reduce endogenous gibberellin levels in transgenic rootstocks to make them dwarf or semidwarf. Such rootstocks could provide reduced size to non-transgenic scion varieties grafted into them. With this aim, we have incorporated a hairpin construct into Carrizo citrange to silence an endogenous GA20-oxidase gene and them reducing bioactive gibberellin levels in growing shoots. After Agrobacterium-mediated transformation, the explants regenerated abundant callus and showed prolific shoots formation. Around 70% of the explants regenerated shoots in the light step. At this moment there are several transgenic (PCR-positive) shoots micrografted in vitro. They are still pending of grafting on vigorous rootstocks in the greenhouse and Southern blot verification in coming weeks/months. New experiments will be run with this construct and fresh starting plant material within the next couple of months. For improving citrus tree management, we also proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. This objective was initiated one year ago or so, and we have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. The PI and his greenhouse/growth room manager, Josep Peris, travelled to Florida last March 20-27th to visit the CREC and supervise the last steps of the growth room construction before been finalized (by the end of April, we guess). We suggested to revise minor details to make the facility more reliable and helpful for operators. We also short-trained the tissue culture technicians in horticultural practices. We checked substrate, seed stock and plant nutrition issues with the manager Dr. Zapata.
Preparation of potato psyllid libraries for sequencing. During this time, we have collected 6 potato psyllid samples: uninfected guts, uninfected adults, uninfected larvae, infected guts, infected adults, and infected larvae. Based on the Trizol/RNeasy hybrid RNA extraction protocol we developed, we can extract 1 ug total RNA for Illumina sequencing library construction from: 80 Asian citrus psyllid larvae/100 Asian citrus psyllid adults or guts/250 Asian psyllid salivary glands or 20 potato psyllid adults/50 potato psyllid larvae/100 potato psyllid guts. mRNA isolation, cDNA synthesize and paired end library production. We have constructed 8 Illumina Paired end libraries for whole transcriptome sequencing from 6 potato psyllid samples (uninfected guts, uninfected adults, uninfected larvae, infected guts, infected adults, and infected larvae) and 2 Asian citrus psyllid samples (uninfected guts and infected adults). Illumina sequencing and assembly. We have received from NCGR the Illumina assembled sequences from 6 potato psyllid libraries and 2 Asian citrus psyllid libraries. Each library resulted in over 24M sequences and the total set of ~160M sequences for 6 potato psyllid libraries and of ~65M sequences for 2 Asian citrus psyllid libraries. The last two Asian citrus psyllid libraries are to improve coverage over initial 454 runs (429995 ESTs for uninfected guts and 233891 ESTs for infected adults). Taking together with ~100M sequences from other 4 Asian citrus psyllid libraries (infected guts, uninfected adults, uninfected larvae and infected larvae), we would be able to get a deep coverage of ACP whole transcriptome. Characterization of the psyllid transcriptome and Identification of midgut-specific genes. Annotation, qPCR analysis of ACP transcripts and biological validation of novel/tissue-specific ESTs/genes were carried out. Several psyllid genes have been selected for full length cloning, expression profiling, enzyme assay and/or metabolism analysis as potential targets for RNAi constructions, plant transformation and function identification. ACP sequence data from prior sequencing. The 454 reads from whole body and gut libraries of the Asian Citrus Psyllid have been submitted to Genbank and are available for download from http://www.sohomotera.org. This site also has a query interface to the annotated 454 unitrans and the annotated Hunter Sanger unitrans (unique transcripts). Both assemblies and annotatin were performed with the PAVE software, where the 454 annotation used the Aug2010 Invertebrate and full SwissProt and TrEMBL databases and the Hunter used the corresponding Feb2011 UniProt databases. We are currently updating both annotations to use the Mar2011 UniProt databases, where we are searching against the invertebrate, virus and bacteria databases separately so that the composition of the functional annotation can be easily deciphered. For example, the results from the Hunter annotation shows the following number of unique hits to each database (up to 25 hits are stored for each Unitrans for each database): 4294 SP-Invertebrate, 31112 TR-Invertebrate, 125 SP-Virus, 599 TR-Virus, 6122 SP-Bacteria, 13309 TR-Bacteria, 5 SP-All, 7 TR-All (the All hits do not include any of the species database hits).
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (in collaboration with D. Hall) is being conducted on a replicated set (8 plants of each) of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. It is anticipated that statistical analysis of CLas levels and symptoms will permit identification of material with significant resistance, for further testing. A series of promoters has been tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. Use of this promoter may reduce the number of independent transformants needed. CLas sequence data were used to target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. Radiolabelled ATP is being used assess effect of identified peptides in E. coli expressing the CLas translocase. Collaboration with a USDA team in Albany, CA is: providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns; providing genes from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Anthocyanin production genes,give bright red shoots (Gray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 27 months and a repeated trial has been in the field for 15 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
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