In FL nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008 seed were collected from mature Pineapple sweet orange trees in Collier Co. and in 2009 from Murcott tangor trees in Hendry Co., FL. For Pineapple orange, 415, 723 and 439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on symptomatic branches of 2 Carrizo trees, and in 2009, 125 seedlings were recovered from 2 trees of Swingle citrumelo, 649 from 4 trees of ‘Kuharske’ Carrizo, 100 from 1 tree of Cleopatra mandarin and 100 from 1 tree of Sekwasha mandarin. In 2008, one suspect HLB+ Carrizo seedling was detected but HLB+ status was not confirmed after repeated testing. In 2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Subsequent detection occurred in only 75 and 33% of repeated 16S runs from two DNA extractions and the assay was negative using beta-operon primers. Despite the occasional HLB+ test results, no plants have ever developed HLB symptoms and repeated testing has never confirmed anything other than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees.
Preliminary work to explore optimal parameters for genetic transformation of Murraya has been carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, to attempt establishment of an optimal and standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots has occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures have revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth is being monitored, as well as the stability and uniformity of GFP expression over time, and further production of new transgenic events is underway.
Progress in the project was disrupted by the unexpected departure, with no advance notice, of the previous employee responsible for the research underway. Another employee was assigned responsibility and she has made excellent efforts to retain forward momentum and to make credible progress. Seeds of Murraya paniculata were procured from the USA-ARS National Clonal Germplasm Repository for Citrus and Dates, from a commercial company (Seeds & More), and from a local source in Florida. The limited seed availability and their relatively poor quality, along with consequent low germination rates and very poor growth, have been a hurdle in the process. Such seedling materials present a major challenge for Agrobacterium based transformation efforts. Therefore, a comprehensive experiment on screening optimal conditions for in vitro seed germination was conducted. Various culture media, using Murashige and Skoog’s (MS) basal salt, with or without GA3 (3 mg/l), with varying sugar (25, and 50 g/l) and agar concentrations (4, 6 and 8 g/l), have been examined, with a goal to achieve an acceptable seed germination rate. In order to achieve the high quality (with stronger stems) of seedlings necessary for the regeneration and transformation experiments, germinated weaker seedlings were subcultured twice on MS basal medium without any plant growth regulators for another month. Stronger seedlings obtained were used as explants for regeneration and transformation experiments.
Because of successful efforts to achieve acceptable seed germination rates and to produce more vigorous seedlings in vitro, higher quality seedling explant materials have been obtained. This accomplishment has enabled work to proceed to establish a suitable and efficient protocol for in vitro regeneration of Murraya, which can then be used for the various planned transformation experiments. Research was conducted to develop a suitable and robust organogenic shoot regeneration protocol, using various parts of germinated seedlings including hypocotyls (longitudinally cut vs. no cut), epicotyls (longitudinally cut vs. no cut), roots, leaves, and cotyledons. These explants were cultured on MS basal medium supplemented with various concentrations of BA, TDZ alone, or in combination with NAA. Two suitable and reasonably effective shoot regeneration protocols have been developed and optimized for transformation experiments (a manuscript is in preparation). Simultaneously, the capacity of shoot tips and lateral buds to undergo multiple shoot formation and multiplication in vitro has been examined. Shoot tips and lateral buds were excised directly from in vitro germinated seedlings and cultured on MS medium supplemented with BA at 0, 1, 3, and 6 mg/l. Newly formed shoots were subcultured on the same medium at 3 week intervals to determine optimal conditions for maximal shoot multiplication.
Quarterly report April 2011: Reserach to explore optimal various parameters for genetic transformation of Murraya was carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, and we established an optimal, standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events is being pursued.
Five field studies are 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 trees in Maury Boyd’s grove in Felda. We have recently completed the third Hamlin harvest on these trees and will harvest Valencia as fruit reach maturity. 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. Recently, a summary of the past 10 years of yield for Maury Boyd’s grove and a nearby grove practicing tree removal was completed. This summary clearly showed that MB’s yield 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 will be harvested on 31 January 2011 and the yield data will be compared with the previous year’s. The third trial has been discontinued because, unknown to us, the grower-cooperator began applying a foliar nutrient program to this grove, including our treatment plots. 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 the trees (Valencia) will be harvested in spring 2011. 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. Samples have been collected from the Hamlin trees for quality analysis and total yields will be estimated when harvested. Both blocks continue to be scouted for HLB occurrence, but trees are not being removed. A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Trees are growing well in the system and have begun to show foliar symptoms of nutrient deficiencies for the different treatments. Trees are being monitored for the development of HLB symptoms and will be confirmed by PCR when suspects are detected. Once trees are known to be infected data collection of how the disease develops in trees under different nutrient deficiencies will be collected. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and development.
Abstract Citrus Huanglongbing (HLB) is caused by the phloem-restricted, Gram negative, and uncultured bacteria Candidatus (Ca.) Liberibacter (L.) africanus, Ca. L. asiaticus, and Ca. L. americanus, according with the continent in which they were first detected. Both Ca. L. asiaticus (CaLas) and Ca. L. americanus (CaLam) are transmitted by Diaphorina citrii, and are present in Brazil. Several studies about the transcriptional response of citrus plants during HLB symptoms have been reported, all of them are focused on the CaLas infection. This study proposed to evaluate the transcriptional reprogramming of sweet orange challenged with CaLam using a customized 385K microarray chip containing about 32,000 unigenes. A set of candidate genes from this study and from others was used to validate the expression profile in symptomatics and symptomless positive PCR tissue of sweet orange infected with CaLas or CaLam. We propose a global feature of the defense mechanisms of citrus in response to CaLas and CaLam. Methodology Challenge with Ca. Liberibacter spp ‘ Transcriptome experiments by microarrays were performed using plants of sweet orange (Citrus sinensis L. Osb. cv Pera) grafted onto Rangpur lime (C. limonia L. Osb.). The first challenge with Ca. Liberibacter americanus was carried out in 25 plants of four months old plants by grafting infected (PCR positive) budwoods. They were monitored bimonthly by end-point PCR for detection of the bacterium. PCR positive plants were challenged again by new grafting with infected budwoods, and pruned. Since Ca. Liberibacter americanus is heat sensitive, all plants were kept in growth chambers at 22 to 24 ‘C and 12/12 h light for six months. Full expanded leaves of three plants displaying initial symptoms of HLB and PCR positive for the bacterium, and leaves of healthy plants in the conditions were collected and stored at -80’ C. To validate specific gene expression, eight plants of sweet orange (cv Hamlin) grafted onto Rangpur lime were challenged with infected budwoods, either with Ca. Liberibacter americanus or Ca. Liberibacter asiaticus. Plants infected with Ca. L. asiaticus were kept in screen house at 28 to 30 ‘C, and plants infected with Ca. L. americanus in growth chambers at 22 to 24’C. Full expanded PCR positive leaves for Ca. L. americanus and Ca. L. asiaticus, respectively, were collected and stored at -80’ C. Genome wide transcription approach – Global transcriptional levels of diseased and healthy plants were evaluated with a Roche Nimblegen Systems customized 385K chip containing 32,000 unigenes of sweet orange. Three probes for each unigene with optimal predicted hybridization characteristics were designed to compose a probe set. Each probe set was represented onto the final array by four replicates. All probes were designed as perfect match oligonucleotides. Total RNA concentration and purity were determined from the ratio of absorbance readings at 260 and 280 nm using a Nanodrop ND8000 spectrophotometer (Nanodrop Technologies), and RNA integrity was tested in a denaturing agarose gel. Reverse transcription was performed with 1 ‘g of total RNA in a total volume of 20 ‘L with oligo(dT) primer using Revertaid H-Minus reverse transcriptase (Fermentas). The final cDNA products were diluted 50-fold prior to use in RT-qPCR. Raw data were imported to NimbleScan 2.5v software, which employs three steps of preprocessing: convolution background correction, quantile normalization, and a summarization of expression measures in a probe-level with a robust multiarray model fit (RMA) using the median polish algorithm. Unigene transcripts with p-values ‘0.05, fold change |FC| ‘ 2.0 and odds probability ‘ 0.95 were considered as differentially expressed genes (DEG). To identify relevant molecular mechanisms potentially associated with sweet orange response against Ca.L.americanus infection gene set enrichment analysis (GSEA) method, that evaluates microarray data at level of gene sets was carried out. Gene set was defined as all predicted genes that sharing the same ontology based on GO database from A.thaliana, and can be classified as biological processes, molecular functions and cellular components over-represented in a set of DEG. Specific gene expression analysis – To obtain reliable of gene expression measurements, we performed a screening of candidate reference genes of our microarray analyses, adopting the following cutoff: absolute logFC ‘0.5, average expression (AveExp ‘7.0) and standard deviation (stdev ‘0.5). Unigene transcripts were then sorted by coefficient of variation (CV) standard deviation, and LogFC. Besides that, we decided to evaluate the expression stability of 18S ribosomal and GAPDH that were usually used to normalize transcript levels. Primer efficiencies, Cq values and normalized relative quantities (NRQ) were calculated as described. The most stable reference genes were identified using the geNorm 3.5v (medgen.ugent.be/~jvdesomp/geNorm/) algorithm. Stepwise exclusion of the reference genes with the lowest stability of expression (the highest M) allows ranking the reference genes according to their expression stability. The pairwise variation also determines the minimum number of reference genes required to a reliable normalization. Based on the predicted function, retrieved from TAIR, 24 genes were selected to be validated by real time quantitative PCR (RT-qPCR). To those eigth additional genes differentially modulated in response to Ca.L.asiaticus, were included in the validation. The gene-specific primers were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/) and IDT (Promega Corporation) software tools with melting temperatures of 60’C, amplicon length of 150 to 200 bp, and a GC content of 50 to 60%. Amplicon specificity was checked by 2% (w/v) agarose gel electrophoresis and by melting-curve analysis. Sequence identity was confirmed by direct sequencing of PCR products using an Applied Biosystems Model 3730 DNA sequencer. Relative quantification was carried out in a 96-well optical plate with an ABI PRISM 7500 FAST sequence detection system (Applied Biosystems) using the Fast SYBR green PCR master mix (Applied Biosystems). The standard thermal profile was used for all amplifications. All assays were performed using three technical replicates and a non-template control, as well as three biological replicates. To analyse dissociation curve profiles,program was run after the 40 cycles of PCR: 95’C for 15 sec followed by a constant increase in temperature between 60 and 95’C. Raw data of fluorescence accumulation for each individual assay were imported into R statistical package version 2.922 (R Development Core Team). Cq values were determined for each amplification by the maximum of the second derivative of the fitted sigmoid curve. The comparison of means of normalized expression values among groups were performed by a nonparametric one-way ANOVA with 1000 unrestricted permutations, followed by pair-wise comparisons with Bonferroni adjustment. Results and Discussion The complete manuscript is been submitted to an international journal for evaluation and, if accepted, to be published. Results and discussions are been focused on the following points: – Detection of the bacteria in the tissues of sweet orange. – Genome wide transcription approach. – Main affected processes or pathways in response to the infection o CaLam. – Specific gene expression by RT-qPCR. As soon as the manuscript is accepted I will send the final report of the project.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We now are focusing on when and how psyllids acquire Las from newly infected plants. This information is necessary of the epidemiology models for managing HLB. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. We have greatly optimized conditions to allow exposed plants to become rapidly inoculated by infected psyllids. We continue be a resource of healthy and infected psyllids and plants for other laboratories.
Sensory impacts and flavor and aroma changes in HLB fruit: Hamlin fruit was harvested, juiced using a commercial extractor and pasteurized in late December, 2010. Symptomatic greening fruit was blended into healthy fruit at 0, 2.5, 5, 10, 20, 50, and 100% by fruit weight prior to juicing. These juices are being stored and will be pasteurized. Future taste panels are planned. Physiological changes in HLB fruit: We compared the visual appearance and characteristics of HLB-impacted fruit with girdled fruit to determine if restriction of carbohydrate movement caused symptoms characteristic of HLB. Girdling was performed on ‘Hamlin’ branches in groves in July and August 2010 on healthy trees. A ring (full girdled) or half ring (half girdled) of bark (‘8 mm width) around the twig located 10-cm above a fruit was removed. Leaves between the girdled region and fruit were removed. Four biological replications composed of eight trees (4 fruit/rep) each were used for girdling experiments; fruit were harvested on December 8th 2010. Relative gene expression in flavedo was analyzed using quantitative real-time PCR. Citrus glyceraldehyde-3-phosphate-dehydrogenase was used as the constitutively expressed internal calibrator. Visual analysis indicated that full-girdled (FG) fruit were similar in size to HLB symptomatic (SY) fruit, but girdled fruit were greener and not misshaped. No visual differences were seen between healthy (H) and asymptomatic (AS) fruit or ungirdled (UG) and half-girdled (HG) fruit. SY and FG fruit were lower in starch and sugar and had similar changes in juice quality than H or UG fruit, respectively. Generally, the impact on 5FG (5 months girdling) was greater than on 4FG (4 months girdling). Carotenoid content was reduced in SY when compared with H, but increased in FG flavedo when compared to UG. Expression of several genes was significantly changed in SY but not FG flavedo. Included in this list were a sulfate transporter, starch synthase, alpha-amylase, four phospholipases and two ethylene synthesis genes. Yield, peel color, fruit size and seed abortion in HLB fruit: FG fruit had significantly lower fruit diameter, fruit weight, flavedo starch and sugar content compared to HG or UG fruit. Such a pattern was similar when SY fruit were compared with AS or H fruit. Extension and education: The processor HLB posters were displayed at the following meetings: International Citrus and Beverage Conference, the Deputy Under Secretary of Agriculture visit, Florida Television visit, Florida Citrus Mutual Board Meeting, and a CRDF meeting. A total of 452 processors brochures were handed out in the fall that describe our results.
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 first quarter of the project was focused on development of a chemical screen using potato psyllid/CLps/Arabidopsis as the model. As a first step toward this objective, we tested 19 diverse Arabidopsis lines for susceptibility to infection by psyllids. We found significant differences among 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. No lines were completely resistant, the most resistant having only 1 of 5 plants infected. The most resistant and susceptible lines will be tested again to confirm differences. We did not detect any visible disease symptoms on leaves or stems of infected plants, but have not yet evaluated seed production or root growth. We also found that about 92% of psyllids from our colony test positive for bacteria, a level adequate to obtain a high frequency of infected plants from inoculation with 3 psyllids per plant. Methods to grow Aradidopsis seedlings in 24-well plates were developed. However, when psyllid nymphs were transferred to the seedlings, they appeared to feed but we did not obtain any infected plants. The reason for this is being investigated but it may relate to the composition of the growth medium. We have planted seedlings to measure gene expression responses to CLps. We have not yet initiated chemical testing because the system to produce suitable plants for screening has not yet been successful.
Researchers at the USDA Ft. Pierce: 1. 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 was submitted to the journal Plant Cell, Tissue and Organ Culture that 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. Agrobacterium-mediated transformation of mature tissue explants. Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit (1 plant), US-942 (4 plants), and Etrog citron (4 plants) using the beta-glucuronidase reporter gene. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. 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. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, research confirmed the work of others showing that the flush used to generate transformation explants was critical. In the Machado laboratory in Brazil, research on the regeneration ability of various sweet oranges continues. These differences are present in both mature and juvenile tissues. In the Moore laboratory in Gainesville, experiments still are focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. A transient transformation expression system has also been developed using citrus leaves.
Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. This objective is proposed as an alternative strategy in case constitutive expression of the mutant R proteins proves to be detrimental to the growth or vigor of the plant. By restricting expression of the R protein to the single cell that is pierced by the insect stylet, a defense can be mounted without endangering the overall health of the plant. In the long run, this may be the most effective strategy in fighting Liberibacter infection since the response can engineered to be quite robust. Results: To further insure more specific expression of the R proteins in the phloem, we identified a wound-inducible, phloem-specific promoter that triggers expression upon wounding resulting from aphid feeding. We substituted the AtSUC2-940 promoter (phloem-specific) with the AtPAD4-1002 promoter (PAD4) in SNC1, snc1, SSI4, and ssi4 R gene constructs in the pCAMBIA1305.1 vector. Additionally, we created a PAD4/GUSplus construct to verify phloem- and wound-specific activation. The PAD4 constructs were introduced to Arabidopsis plants, grown to seeds and harvested this week (Jan 2011). The transgenic seeds will be subjected to selective screening for R gene transformants. Due to the cloning limitations (restriction site configuration), all PAD4/R constructs had to be first assembled in the 1305.1 pCAMBIA vector prior to transfer into the 2301 pCAMBIA for transformation into citrus. We selected two constitutive R protein mutants and PAD4/GUSplus constructs, and transferred them to the Citrus Research and Education Facility at Lake Alfred (Dr. Vladimir Orbovic). Preliminary reports indicated that transformants are proving difficult to obtain, potentially due to the lethal nature of at least one R protein mutant. Conclusions: Our hypothesis is that phloem-restricted expression of the R protein constitutive mutants would limit the potential negative impacts on plant growth. Use of the PAD4 promoter to express the R protein constructs should further increase the stringency of transcriptional control over expression of the potentially harmful R proteins. Our goal is have only the single phloem cell that is penetrated by the stylet express the constitutively active R protein, hence limiting potentially detrimental effects and focusing a robust defense at the source of Liberibacter introduction. Additionally, these constructs with the PAD4 promoter may reduce spurious expression in callus cells during the transformation protocol into citrus.
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. We therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. The outer membrane protein and antibody system for immunocapture of ‘Ca. Liberibacter asiaticus’ has however not proven to be successful. The polyclonal antibodies made in rabbits against purified outer membrane protein from ‘Ca. Liberibacter asiaticus’ work very well in detecting the purified outer membrane protein but are not effective in immunocapture. The most likely explanation is that the outer membrane protein is not well enough exposed on the surface of the ‘Ca. Liberibacter asiaticus’ cell. We have therefore cloned genes encoding the type IV pilus protein of ‘Ca. Liberibacter asiaticus’, and the polysialic acid capsule protein since these proteins should be well exposed on the cell surface and should be useful for labeling ‘Ca. Liberibacter asiaticus in insect or plant tissues. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins and 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. The Type 4 pilus proteins and the polysialic acid capsule protein have been biotinylated and combined with strepavidin coated magnetic beads and used successfully to bind recombinant phage expressing antibodies that recognize these targets. 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. We will select scFv antibodies next against purified Outer Membrane Protein (OMP), using protein already purified. 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.
For EDS1 cloning, we currently confirmed by sequencing the cloning of the full-length ctEDS1 and already moved the sequence from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we designed primes to clone the 3′ end sequence of ctSID2, which we were previously unable to obtain with several methods. We performed the RACE reaction and have now obtained this missing sequence. We will subsequently design primers to amplify the full-length sequence of ctSID2. In addition, we did bioinformatics analysis and identified additional 9 citrus homologs of Arabidopsis defense genes that have available sequences in the database. We designed primers for these citrus genes and have been conducting RACE in order to amplify the genes. So far we have obtained the 3′ end sequences for ctNHL1, ctSFD1, and ctFAD7. Further cloning of 5′ end and/or 3′ end sequences of these citrus defense genes are currently underway. We continue to characterize the transgenic plants expressing ctNDR1/pBINplusARS. We obtained 5 homozygous ndr1 + ctNDR1/pBINplusARS. Through HR test and disease resistance assay with infection of the avirulent strain P. syringae avrRpt2, we further confirmed that over-expression of ctNDR1 could complement Arabidopsis ndr1 mutant. We are going to further characterize the defense phenotypes of these transgenic plants.
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