The main objective of this proposal is to find ways to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. This is a new project and the funds for this project were released. We already have designated personal for conducting the proposed research. The research is in progress. Plant material that will be used in this project is being prepared in our greenhouse. Using plant material and inoculum sources that are already available, we are conducting initial experiments to examine the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. Young citrus trees are being inoculated with various isolates of CTV. Upon establishment of the initial infections the trees will be inoculated with the green fluorescent protein (GFP)-marked CTV vector. Levels of vector expression (monitored based on GFP expression) will be evaluated. The objective here is to investigate how previous infection of trees with the virus affects the ability of the vector to infect and multiply in those trees. The second objective is to examine the effect of various rootstock/scion combinations on the superinfecting ability of the vector in order to evaluate what combinations would support high levels of vector expression. Currently we are preparing plants that have different rootstock/scion combinations that will be used for the experiments proposed under this objective. Both first and second objectives represent our main focus for the first year of funding.
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
A number of experiments and observations at the USDA/ARS Ft. Pierce citrus scion improvement program demonstrate resistance or tolerance to HLB in some conventional citrus genotypes. In the funding cycle that began in 2012 our efforts in this area are markedly enhanced by funding for a postdoctoral researcher. Dr. Sharon Inch, who has extensive experience in plant pathology and histology, began working on this project 10/9/2012. She will move forward with established experiments comparing HLB development in specialty cultivars, which displayed resistance in a study of commercial groves, vs. susceptible standards. A set of replicated plants have been initiated for 50 genotypes representing advanced USDA selections, genotypes which display apparent resistance/tolerance in field observations, and susceptible standards: these will be exposed to HLB in a hot psyllid house and will be assessed for CLas levels, HLB symptoms, and growth. The remaining proposed experiments relating to assessing and characterizing HLB resistance/tolerance will be initiated in the next quarter.
The funding cycle that began in 2012 provides for a postdoctoral researcher in the USDA/ARS scion transgenic program. This will markedly enhance progress. Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. She will move forward with new constructs and resulting transgenics, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline.
In the initial funding of the current grant we have made progress on several of our objectives: Objective 1. Evaluate existing transformed lines: We have been maintaining a steady effort in the growing and testing out of candidate transformed lines from our large transformation efforts. Over 34,000 transformations of Duncan grapefruit have been carried out with 8 different reporter and resistance constructs, from which we have generated over 600 T0 plants that are being used for PCR analysis and pathogen testing. We are currently testing plants. The frequency of functional transformants may be lower than typical given that inappropriate leaky expression will be counter-selected. Objective 2. Expand stable transformations Our efforts to transform additional commercial citrus species has been focused on Ruby Red grapefruit and sweet orange. At present we have introduced eight constructs consisting of promoters with1,4, or 14 TAL effector/PthA binding sites with GUS or resistance gene coding regions in more than 1200 Ruby Red and over 600 sweet orange transformations, producing about 60 T0 plants for analysis. Objective 3. Refine constructs We have initiated additional resistance gene constructs that contain additional promoter elements and/or use another resistance gene known as AvrGf2. As each new construct is completed, we are testing these it in transient and stable transformation assays. Objective 4. Sequence more TAL effectors from additional canker accessions We have new sequences of TAL effectors from strains from Florida, Argentina, and Brazil, including the Miami “type” strain, as well as several strains with altered growth phenotypes. Last, we are expanding efforts at examining effects of resistance gene constructs on population growth of a range of Xanthomonas citri strains.
The current main priority is hiring the project postdoctoral fellow in the Zipfel laboratory. Whereas an initial candidate was identified, the hiring was not completed and additional candidates are currently being interviewed. The intention is to make an offer to a qualified postdoc by mid-November. Others in the lab are progressing the initial objectives: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. We have prepared constructs for EFR, XA21, EFR-XA21 and XA21-EFR chimera. We have carried out transient expression assays in N. benthamiana and found that the proteins express well. We have shown that the XA21-EFR chimera is functional, as exemplified by a gain of responsiveness to elf18 in a ROS burst assay. We are currently testing the functionality of XA21 and XA21-EFR by treatment with the axS17 peptide. This protein is unstable and difficult to synthesize, so we have obtained wild-type and ax21-minus strains of Xanthomonas euvesicatoria so that we can make extracts to test XA21-induced responses in N. benthamiana.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of ‘Ca. Liberibacter asiaticus’ that are likely to be related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, a pilus protein, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. During work reported previously we have found that these scFv bind to their targets in extracts of infected plants and can be easily detected in a dot blot format. However, there is often a weaker reaction with uninfected plant material. One of the powerful aspects of scFv technology is that the binding affinity of any scFv for its target can be improved by mutagenesis. During this time period we have prepared mutated scFv libraries for several of our antigen targets. We have followed the procedures described by Pavoni et al., (Gene 391 (2007) 120’129). In summary, the antigenic diversity of the scFv is encoded in defined hypervariable regions designated ‘CDR3’ in both the Vh and Vl segments. The hypervariable regions were targeted by PCR mutagenesis using degenerate primers KM144’KM143 and KM148’KM145 to introduce random mutations in CDR3 regions of both the heavy or light chains. These mutated fragments were then assembled with the remaining portions of the scFv genes by combination with the products of amplification with primers KM148’KM157 and KM158’KM143 primers for HC and LC, respectively. The result is secondary mutated libraries that contain large numbers of phage with related sequences likely to recognize antigens potentially relevant to virulence. We have created five such mutated libraries, based on scFv1289 (TolC; 1.3 x 10_6th TU); scFv734 (Omp6f; 4.2 x 10_6th TU); scFv968 (FlhA; 4.0 10_6th TU); scFv932 (FlgL; 1.4 x 10_6th TU); scFv1202 (kpsA; 1.5 10_6h TU). Future work will be performed to isolate scFv with the greatest binding specificity (low cross reaction to uninfected plants) and affinity. These scFv will then be available for labeling proteins for in vivo visualization by various microscopic techniques, for testing as diagnostic reagents in various applications, and for introduction into plants to determine if they can confer resistance to HLB.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are under canker resistance test. These plants have been propagated and will be used for citrus greening test. As an extension of the project, we tested whether exogenous NAD+ could induce resistance to citrus canker. Exogenous NAD+ has recently been found in our lab to be a strong inducer of systemic acquired resistance (SAR). Since SAR has been shown to be effective against citrus canker, we expected exogenous NAD+ would induce resistance to canker. Indeed, our preliminary result showed that exogenous NAD+ activated strong resistance to citrus canker. We are confirming this promising result. For objective 2, we are continuing the direct genetic screen for citrus varieties with increased resistance to citrus greening. Seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds have been inoculated with psyllids carrying greening bacteria. Seedlings developing greening symptoms have been removed. The remaining seedlings will be re-inoculated with psyllids carrying greening bacteria.
Our objective is to determine how Asian Citrus Psyllid (ACP) behavior is affected by Las-infection in healthy and diseased citrus. In previous experiments we have determined that ACP adults initially settle on Las-infected plants. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding the psyllid experiences imbalanced nutrition and choose to seek a better host. To examine this hypothesis we planned to determine how ACP settles on plants with known nutrient deficiencies. In previous experiments we used asymptomatic Las-infected Valencia seedlings to show ACP movement and host acceptance. However, in this quarter we chose 4-year old Valencia trees for our settling experiments with (1) late-stage symptomatic Las-infections, (2) nutrient deficiencies, or (3) healthy trees as control. To ensure that we still had the same settling behavior that we found in previous work, we began our experiment with control vs Las-infected tree choice tests. Surprisingly, the older Las-infected trees were no longer initially attractive, and nearly all of the ACP settled and remained on healthy trees. We attributed this to the poor condition of the Las-infected trees that were no longer suitable hosts. We conducted the same experiment using Las-infected pineapple sweet orange trees that were highly symptomatic yet still actively growing and in relatively good condition. In this experiment, ACP settled evenly over both Las-infected and healthy trees. Over the course of the experiment, ACP moved; however, there was no clear pattern of movement to or from healthy or Las-infected trees. We hypothesized that as the disease progresses, the attractiveness of the Las-infected tree changes. To continue this line of questioning, we have obtained sweet orange trees that are (1) healthy, (2) asymptomatic, newly Las-infected, and highly symptomatic, Las-infected trees. We will conduct three way choice tests between these trees to determine ACP settling preference over time. We are also establishing nutrient deficiencies in seedling Valencia trees for use in future settling experiments.
Data analysis of the 2011 LAS experiments is currently being completed. A publication on this data is being written concurrently and should be ready for submission by the end of the year. For this publication, we are attempting to obtain more information about the apparent biofilm that was formed at the air-liquid interface of cultures supplemented with citrus juice in 2011 experiments. From one of the experiments, biofilm was harvested from all of the flasks and DNA extracted from these samples. PCR was used to amplify the V4 region of the 16S rDNA gene from these samples as well as from the initial seed coat-based inoculum originally used to inoculate the culture flasks. Amplification was verified by gel electrophoresis, and there are now confirmed PCR products (with concentrations determined by flurospectrophotometer) from the inoculum and biofilm samples. These PCR products will soon be sequenced via next-generation sequencing to determine the bacterial composition of the biofilm. We anticipate that the biofilm could be composed of LAS, a “helper” bacterium that provides necessary nutrients to the culture, or a combination of both. Exhaustive sequencing of these samples will answer this question. New experiments for the fall 2012 season are underway. Since the concentrations of LAS in citrus fruit seed coats are the highest from ~September-November, the experiments are being conducted during this time. The experiments are of a similar design to the 2011 experiments, but with only two media types: 100% King’s B media and 100% commercial grapefruit juice. Viability will be measured over time with EMA-qPCR as before but with lower frequency, as the viability pattern has already been established in previous experiments. The focus this time will be on examining biofilm formation, nutrient utilization as measured by ICP-OES, and attachment via microfluidic chamber observations. We are also trying to determine the best method for long-term storage of LAS cells by storing the same inoculum under different conditions and subsequently reviving the cells and comparing cell viability between the different storage methods. This would hopefully allow us to determine the best way to store the cells so that experiments with LAS can be conducted during times of the year when cell concentrations are not high enough in the fruit seed coats.
Data was prepared and a poster developed for the American Society for Horticultural Sciences Annual Meeting in Miami, FL in August (Albrigo). Data included field and greenhouse comparisons of plugging types and new photomicrographs of phloem plugging and necrosis in different size scaffold limbs. All tissues in diseased plants showed similar phloem disruption symptoms and more phloem cells were laid down in HLB affected plants, young and old. Material for an oral presentation was prepared for delivery at the American Society for Horticultural Sciences Annual Meeting in Miami, FL in August concerning the transformation of citrus with the beta 1, 3-glucanase gene, their propagation and eventual testing by challenging with HLB (Ahmad Omar).
Sampling of field trees for further evaluation of phloem plugging continued. Trees of citrus relatives were evaluated for HLB and psyllid activity in field rootstock trees. No HLB was detected in Poncirus, but HLB did occur in Carrizo. Further, no Poncirus was ever found to have any psyllid stages on the leaves in two locations examined 3 times. Plants were transformed with virulent genes from the Liberibacter bacteria. Preparations were continued for transformation of citrus with then glucanase gene. Sept-Oct 2010 report
Locations and HLB affected trees were identified, confirmed by PCR and sampled for phloem plugging evaluations. Some of the needed samples were taken and fixed for EM work. Continued transforming plants for virulence factor evaluations and also plants for glucanase additions for evaluations. Different types of citrus and transformation techniques are now being used. Arrangements for testing transformed plants being made. Sept-Oct 2011 report
3rd Quarter (final funding period): This quarter was largely devoted to the development of experimental approaches to be employed in the assessment of resistance to Las infection in the transformed lines. These experiments were aimed at developing protocols that can detect and quantify the survival of Liberibacter in the early stages of infection in our transformed lines expressing various constructs of R proteins. 1-Test for possible feeding preference between transformed versus non-transformed citrus: Preliminary analyses were conducted to determine whether the introduction of inducible and constitutively expressed resistance R genes affected the feeding preferences of uninfected psyllids. The cuttings of all transgenic citrus plants were subjected to uninfected psyllids feeding. There was no observable difference in psyllid feeding behavior preference between transformants and control citrus plants, which is a condition that will facilitate the assessment of resistance in the transformants. 2-Development of a one-step DNA extraction protocol for PCR analysis of Las infection: Our strategy was to develop a facile and sensitive assay using heavily infected citrus leaves from nontransformed citrus initially, before subsequent application to transformed lines. A variety of genomic DNA extraction procedures were tested with an emphasis on limiting the quantities of plant material to the smallest possible, as well as assessing protocols that involved addition of plant material to extraction solutions followed by a brief heat treatment and then direct addition to PCR reactions. Detection of Las rDNA was reproducibly obtained using 1 mm and 0.5 mm midvein cores. Overall, extraction procedures that did not require prior genomic DNA purification (one-step approach) gave better results at lower extraction solution volumes; however, quantitative real time PCR was adversely affected to some extent by some of the extraction solutions utilized in the one-step approach. In order to precisely quantify the copy number of Las in infected citrus plants we established standard curves for Las using the plant mitochondrial cytochrome oxidase (Cox) gene as a control to measure the amount of plant material in the sample. Likewise, the wingless Wg gene served as a control in psyllid extractions. Standard curves were based on calibration curves constructed using purified PCR amplicons obtained from plant and psyllid genomic DNAs. Based on the assumption that cloned Las, Cox, and Wg amplicons may represent more accurate copy number reference, all three standards were cloned in pUC19 (Las and Wg) and pUC119 (Cox). In construction of a heat map of infection using a heavily infected leaf, Las copy number (16S rDNA) varied across midvein sections, with the secondary vein and a non-vein section of the blade showing the lowest amount of Las. The ability to detect Las 16S rDNA in 0.5 mm midvein cores suggests that fine-scale mapping of the early infection is feasible. 3-Netted single-leaf clip cages used to detect initial infection stages: Ten psyllids from an infected population (furnished by the Dawson laboratory) were placed in single-leaf clip cages and allowed to feed for a period of 7 days and then removed for PCR determination of Las infection. A total of 10 leaves were exposed to infected psyllids and were harvested at one week intervals for PCR analysis of Las copy number. Las/Wg copy number ratios varied from 2,238×10-5 to 23,575×10-5 in the psyllids. Early detection of Las from midveins was feasible; however, the sensitivity of the assay in its present form was still needs improvement. We continue to make adjustments to our testing conditions including the modification of psyllid-containment cages.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. 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 one week, 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. This report marks the end of the first quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project is being hired, two small greenhouses for rearing psyllids are almost completed, and general supplies including insect cages have been procured. USHRL dedicated an existing conventional greenhouse for the project, erected two new hoop houses for the project, and assigned a support scientist to the screening project. Additional ARS funds were used to increase the bio-security of the existing greenhouse to guard against invasion of parasitoids of the psyllid. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
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