In our work from April to July 2015, we moved forward to start molecular analysis of Liberibacter asiaticus rpoH effects on transcription. We also began the second major phase of work, in which we will study Liberibacter transcription regulators. We made use of new information about the effect of endogenous S. meliloti genes and the vector ribosome binding sequences (RBS) described in the March 2015 report. 1. Molecular analysis with the newly-characterized RpoH-expressing constructs: a. We have prepared RNA for S. meliloti strains containing the following plasmids: plasmid expressing Sm rpoH1; plasmid expressing optimized La rpoH; and the empty vector control, pSRK-Gm. These RNA preparations now will be processed for Affymetrix GeneChip analysis. 2. In our proposal, we aimed to study RpoH and also a set of putative transcription factors. Having completed construction of rpoH strains, we designed the optimized genes for the remaining six regulators: as before, we substituted codons of the native La rpoH gene for codons that are compatible with the GC-rich genome of S. meliloti. We ordered and received optimized DNA for these six genes. These were cloned into the pSRK-Gm vector, using the minimal background vector RBS. a. The codon-optimized La visNvisR genes were cloned as a single insert to maintain operon structure. We used the same intergenic spacer sequence between visN and visR that is present in S. meliloti. 3. We did not originally propose to create deletion strains lacking each orthologous S. meliloti regulator. However, because we found that the native rpoH genes interfered with our ability to observe the full effect of plasmid-based rpoH, we decided to construct strains with each corresponding regulator deleted. For example, we now plan to make a visNR deletion strain of S. meliloti in which to test regulation by the optimized La visNR gene; we will construct a S. meliloti ctrA deletion strain to host the La ctrA plasmid, etc. a. We are in the process of making these deletion strains using our customary sacB plasmid deletion method. In this method, a vector bearing a resistance marker and the sacB gene is used to clone DNA from regions flanking the gene to be deleted. After recombining into a wild-type S. meliloti host by a single cross-over event, the strain is plated on sucrose, which is lethal unless the sacB-containing vector has recombined a second time and been lost from the cell; among such double-crossover events, some restore the wild type gene and some have a clean deletion of the target sequence. Deletion strains are confirmed by performing PCR and sequencing. b. Results to date: We have single-crossover strains containing the sacB construct for all six chosen regulators. c. Of these six, two strains are complete and confirmed for deletion: the visNR deletion strain and ldtR deletion strain. d. We are presently screening for putative deletions of phrR1, phrR2, and lsr, and are planning a two-step construction of a ctrA deletion strain (needed since ctrA is required for basic viability).
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports 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 over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the sire in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall will be leading assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site in July in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested. Carrizo transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. RT-qPCR showed 50% of 16 chimera transgenic Hamlin have relatively high gene expression, with 100x difference between high and low expressers. These promising transgenic lines were replicated by grafting for HLB challenge. Twenty transgenic Hamlin lines were confirmed to contain thionin gene by PCR, and six have high gene expression by RT-qPCR. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptide from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. There is only slight canker resistance by infiltration, but considerably resistance to spray inoculation. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Primers were designed for two citrus FLS2 orthologues. They showed low gene expression in transgenic and nontransgenic citrus. Results on FLS2 transgenics against canker disease were summarized and a manuscript was written and submitted to MPMI. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
Objective 1: Using the Hamlin/Swingle trees available at the Gapway Grove ACPS site in Auburndale, FL we were able to determine the following. 1) The vascular anatomy of Hamlin trees dramatically adapts to the irrigation program. Trees growing under the traditional grower irrigation program with 1-2 pulse irrigation events per week were, generally, much more resistant to the effects of drought and better able to maintain water transport. The daily minimum water potential was significantly less negative (i.e. less water stress) in the grower irrigation program compared to ACPS trees. 2) The ACPS trees, with abundant and regularly applied irrigation water, appear to have maximized the efficiency in delivering water to the canopy. ACPS xylem specific conductivity was nearly two fold higher than trees in the grower irrigation program, meaning that water could flow through these trees to the canopy at significantly higher volumes. This increase in conductivity came at the cost of resistance to drought induced embolism formation that decreases conductivity. 3) The increases in ACPS conductivity were largely attributable to slightly wider conduit diameters, changes in vessel grouping, and decreased pit membrane thickness, which is one of the major points of resistance to water flow in the xylem. 4) These data suggest a high degree of plasticity of Hamlin orange to irrigation programs, and the increased water transport capacity that developed in ACPS trees may be one of the main contributors to the faster growth rate and higher yield found in these blocks of trees. Objective 2: For this objective, a ~100% HLB-affected mature Hamlin grove with both Swingle and Carrizo rootstocks was used to determine whether plant growth regulators (PGRs) could be used to mitigate preharvest fruit drop. We applied 2,4-D (synthetic auxin) at a low (below labeled rate) 5 mg/L dose in 100 gal/acre foliar spray using an orchard airblast sprayer, starting from the time petal drop completed on 3/28/13, and repeating every six weeks. The 2,4-D sprays were timed to coincide with the four routine foliar nutrition sprays applied in the growing season to help correct nutrient deficiencies caused by HLB. Therefore the final treatment spray was applied in September. In this field experiment we also added EcoAgra , a commercially available plant-based surfactant based on nano-emulsion technology, with claims of improving plant health and vigor. The treatments were structured factorially, so that 2,4-D and EcoAgra were tested individually as well as in combination. We monitored fruit drop nine times with hand-raking of dropped fruit, every two weeks starting in September through harvest in December, and found no consistent significant differences between the different treatments. For example, statistical differences were found on only the third sampling date, where 2,4-D and EcoAgra both reduced fruit drop relative to the untreated control. At this point we do not recommend 2,4-D at the concentration and frequency applied in this study to be used to mitigate preharvest fruit drop, but these data should be considered within the scope of other PGR trials occurring simultaneously around the state, including those using 2,4-D at a single application, labeled rate. Part of the reason for lack of statistical significance in the total season-long fruit drop count is due to compensatory effects. A reduced fruit drop due to 2,4-D treatment early on like at the DROP[3] stage may have been compensated by a higher drop later on. This is a likely outcome since fruit retention represents cumulative stress to a tree. Only if everything works out perfectly with the remedy meaning total health improvement season-long will the tree likely retain its fruit in the long term as it would do if it was uninfected.
During the life of this project, 121 mature citrus transgenics were produced using genetic transformation with Agrobacterium. Sixty-six were transgenic for reporter genes and provided proof of concept that this protocol works in our hands. In the last 21 months, 55 transgenics with disease tolerance genes, most without reporters, were produced and micrografted onto rootstock. Agrobacterium transformation efficiencies were relatively low (3.47% positive shoots for constructs with reporters in scion, and 3.0% positive shoots for constructs with no reporters in scion and rootstock). Only 0.78% shoots/total explants plated were transgenic. Many more transgenics were probably produced, but were lost in the micrografting process. Attempts are being made to increase micrografting efficiencies. Although 9 vectors carrying disease tolerance genes were used in sizable transformation experiments, only 4 vectors yielded transgenics. The 55 mature citrus transgenics were produced with these 4 vectors to confer disease tolerance to HLB, canker, or both. These primary transgenics are being propagated into vegetative progeny to facilitate replicated field trials this fall. Numbers were low for Ray Ruby grapefruit (3 transgenics) and efforts are being made to optimize the tissue culture and transformation protocol for grapefruit. Experiments are underway to root mature scion because a larger scion could be easily micrografted onto rootstock with greater efficiencies. To increase our chance of success, we have been utilizing nurse cultures to supply additional nutrients to developing mature shoots. Additional vectors are being acquired from scientists around the country and worldwide. Budding is now done entirely in-house. A gene gun was purchased in July, 2014 to develop a high-throughput biolistics transformation system for mature citrus. Transient expression levels are relatively high, and a few stable transgenics have been produced. Optimizations for mature citrus have been hindered by the limited supply of mature scion in the growth room, which was primarily used for Agrobacterium transformations. In the future, we will purchase mature citrus from nurseries to continue optimizations. We have developed a high throughput screening system in which thousands of putative transgenics can be rapidly screened. A number of equipment expenditures were necessary to achieve this high level of efficiency. Equipment expenditures included a refrigerated centrifuge, a plate reader, a tissuelyser, a laminar flow bench and an incubator. A hybridization oven and dry baths were purchased for molecular analyses. Maintenance expenses for lighting, AC repair, sensors and expansion cards for RCWebview, and the water softener in the growth room are ongoing. This project depends upon a continual supply of healthy, viable rootstock seed and this was problematic last year. Rootstock of Swingle and Volkameriana had poor germination, and Macrophylla and Carrizo had disease/endophyte issues that negatively impacted budding of mature citrus and the tissue culture process.
This project aims to understand important components during HLB pathogenesis. Our previous research identified four secreted proteins, called effectors, from the HLB-associated bacterium Candidatus Liberibacter asiaticus (Las). Effectors are known to play important virulence function in microbial pathogens. Our goal is to characterize the targets of these effectors in citrus. This project will advance our understanding on the basic biology of HLB pathogenesis and facilitate the development of control strategies. Our research has been focusing on four Las effectors that are highly expressed in HLB-infected citrus trees. We hypothesized that their targets in citrus contribute to Las infection and/or symptom development. The main approach we are using to identify the effector targets is yeast two hybrid (Y2H) screening. In the past two years of this project, we accomplished the following experiments: 1) expression analysis of the Las effectors; 2) cloning and expression of the effector genes in yeast; 3) construction of a normalized citrus cDNA library with more than 3 millions of primary clones using HLB-infected RNA samples; 4) Illumina sequencing-based Y2H screening using each of the four Las effectors as the bait and sequencing data analysis; 5) subcellular localization analysis of the Las effectors. At the beginning of the third year of this project, we focused on experimental confirmation of effector targets. From Y2H screening, multiple potential targets were identified for each effector. We performed extensive literature search and prioritized our effort on candidates with potential roles in plant defense or HLB symptom development. These candidates were re-cloned from citrus cDNA and were subsequently examined for their interaction with the corresponding effector by targeted Y2H. To this end, we have confirmed the interaction partner of two Las effectors. These two effectors are expressed more than 10 folds higher in citrus than in psyllids, suggesting that they may play a role in HLB pathogenesis. One of these two effectors specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability and are also well-known virulence targets of other bacterial and fungal pathogens. The other effector targets ion-binding proteins that are important for ion uptake and photosynthesis. Our on-going effort is to finish the in vitro and in vivo co-immunoprecipitation assays to further confirm the effector-target interaction. We will also determine the co-localization of effectors with their targets using confocal microscopy analysis.
The driving force for this project was the need to evaluate citrus transformed to express proteins that might mitigate HLB, which required citrus be inoculated with CLas. Although citrus can be manually inoculated by grafting with infected budwood, a program using CLas-infected ACP was preferred primarily because this is what occurs in nature. Nine colonies of infected ACP for inoculations were initially established in a walk-in chamber. Additional colonies of infected ACP were later established in two small greenhouses and in a second walk-in chamber. For each individual colony, a potted lemon or citron tree graft-inoculated with CLas (testing PCR-positive and showing HLB symptoms) was placed into a cage and trimmed to stimulate flush; ACP were introduced and allowed to reproduce; and the colonies were maintained over time by regularly trimming plants and introducing additional ACP as needed. The source of CLas for these lemon and citron trees was the original HLB-infected tree found during 2005 in south Florida. Germplasm to be challenged for HLB resistance was supplied by USDA-ARS citrus breeders. To inoculate plants, individual plants were caged for 2 wk with 20 psyllids from one of the infected colonies, after which the plants were held together for six months in a greenhouse with an open infestation of infected psyllids. After this two-step inoculation procedure, the plants were returned to the breeders. Challenges associated with the program have included variability in percentages of ACP testing CLas-positive in a single colony over time and among different colonies at any given time. Consequently for the first inoculation step we always used ACP from colonies with the highest percentages of infected ACP, and we increased the number of colonies to increase numbers having high percentages of infected ACP. Lots of qPCR assays were required to monitor infected ACP colonies and infected source plants. Growth chamber colonies generally have had greater percentages of infected ACP than greenhouse colonies, probably due to more stable environmental conditions in chambers. Another challenge has been that, among ACP that test PCR-positive for CLas, relatively low transmission rates have been reported (e.g., 12% or less). Therefore, caging an individual plant with 20 psyllids from an infected colony may not always result in inoculation. Success of the first inoculation step would likely be increased by increasing the number of ACP per plant. Subsequently holding the plants for six months in the greenhouse with free-roaming infected psyllids increases the probability of inoculation. With respect to the second inoculation step, population levels of ACP in the greenhouse were sometimes severely reduced as a consequence of damage to oviposition sites by western flower thrips or spider mites. Also, sometimes population levels of ACP eggs and nymphs were severely reduced by western flower thrips, and further reductions in populations of nymphs were sometimes caused by Tamarixia parasitoids that invaded the open infestation of ACP. Effectiveness of the inoculation program has been slow to gauge. A series of experiments was initiated during 2014 specifically to evaluate inoculation success. Meanwhile, recent feedback from inoculations of rootstock material gave some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The average percentage of infected ACP used in step one was 52% (range 24 to 80%). qPCR 12 to 19 months after the two-step inoculation process indicated an average of 62% success in inoculating citrus. Plants that escaped inoculation had another opportunity for inoculation after being transplanted to the field.
Core Citrus Transformation Facility (CCTF) continued to operate at the high level it did in the previous period and produced transgenic material in a timely and dependable manner. CCTF maintained its standing as reputable partner that delivers transgenic material in reasonable amount of time and continued to be sought for services. This quarter marks the end of three year period that saw significant but expected increase in number of orders. With six new orders received most recently, CCTF has just surpassed its 200th order and 96 of those were placed within the last three years. Clients requested transgenic Duncan grapefruit and Valencia orange in the newest orders. The number of plants produced in this quarter is 85 bringing a total to 730 plants for the duration of the funding period. Most of the produced plants were of Duncan grapefruit cultivar, followed by Carrizo rootstock, Valencia orange, some Citrus macrophylla, Swingle citrumelo, and Mexican lime. This strong bias towards Duncan grapefruit points towards need of researchers to get fast answers regarding candidate gene that could potentially render Citrus plants tolerant/resistant to diseases. Since Duncan grapefruit is highly susceptible to both Huanglongbing (HLB) and citrus canker, challenging transgenic plants expressing gene of interest with bacteria causing these two diseases will quickly reveal if that gene holds any promise or not. Throughout the duration of this project, CCTF processed about 400000 explants. Approximately 5% of explants were lost to contamination which brings the total number of explants producing data to 380000. Overall transformation efficiency expressed as the percentage of positive shoots out of all of those tested was about 3% which is low. One of the reasons for this is the nature of orders received. Amongst 96 received orders, there were 34 for which total of just a few transgenic plants were produced. This was a result of withdrawal of 10 orders, presence of sequence detrimental for shoot development in binary vector used for transformation in six orders, and for 18 orders transformation was either not possible or achieved at the very low rate. The second reason for low transformation rate is the quality of seeds/seedlings resulting from the effect HLB infection has on fruit growing on trees. In the immediate future, CCTF will dedicate increasing efforts to acquire fruit/seeds of higher quality than what it was used recently. The labor force and the income of CCTF remained relatively stable during the last three years and changes in facility s staff never compromised the level of operation. Number of orders presently serviced by the CCTF and those that were either announced or anticipated in the near future is clear indication of sustaining interest of researchers in transgenic citrus plants. They also assure CCTF will stay busy for the years to come.
The original and initial intent of this one-year-funded project was to supplement CRDF-CATP #707 by developing greenhouse and expanded field trials exploring the potential efficacy of 2,4-D applications to reduce citrus fruit drop and its effects on overall tree health. Potted Valencia trees were purchased from a commercial citrus nursery and half were bud-inoculated with CLas to determine the effects of 2,4-D applications on CLas+ trees in a controlled greenhouse environment. The project duration has since expired and funding no cost extension was not granted. It is therefore not presently clear as to the potential efficacy of 2,4-D in reducing citrus fruit drop.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and several plantlets have been regenerated. These constructs were also incorporated into Carrizo citrange in the previous quarter, but we have had a severe Mould mite infestation in our tissue culture room which destroyed most the promoter-gus citrus plantlets. A repeat experiment to produce fresh carrizo citrange plants expressing the root specific promoters were carried out in this quarter and plantlets are being regenerated. The Carrizo citrange plants expressing a root specific promoter – insecticidal gene construct (GNA, APA or ASAL genes individually or stacked with the CpTI gene) were not affected by the mite infestation and several plants have been hardened and transferred to the greenhouse for growth.
The goal of this project was to create canker-resistant citrus through a strategy of an engineered cell death response to Xanthomonas citri pv. citri TAL effectors. During the project, we defined sequences of 14 distinct effector binding elements (EBE) recognized by Xcc TAL effectors. Constructs were created that incorporated these EBEs into the Bs3 promoter driving a pathogen effector gene (either avrGf1 or avrGf2) that triggers a hypersensitive cell death reaction in several citrus cultivars. Two key constructs were one with all 14 binding sites (14EBE) and one with four binding sites (4EBE) that was expected to be bound by at least two TAL effectors from each X. citri strain characterized. These constructs were shown to function as expected in a transient assay in citrus leaves: they produced a hypersensitive response and reduced bacterial growth when co-inoculated with a Xanthomonas strain carrying a TAL effector recognizing one of the EBEs. In the absence of pathogen inoculation, the promoters are tightly “off” in the transient assay. However, despite the tight regulation of these constructs in a transient assay and multiple attempts to produce stable transformants in Duncan grapefruit or sweet orange, we have been unable to recover transgenic lines. We also tested Carrizo citrange, a citrus variety that does not exhibit a hypersensitive response to AvrGf1 or AvrGf2, and we were able to recover transformants. These results suggest cryptic induction of the construct at some point during the transformation process, causing transformants to be selected against. A second promoter derived from the citrus Lateral Organ Boundaries (LOB) gene also failed to produce transformants when driving AvrGf2 expression. Therefore, despite the theoretical promise of the approach, more work would be needed to define a promoter and executor gene combination that could be efficacious in citrus.
Seasonal root sampling continues in two field sites for root density and root growth. We are collecting a second year of root growth data from Hamlin/Swingle and have 1 year of root growth data on Valencia/Swingle. Results so far emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. Preliminary tests of root tubes are almost complete. This will allow for more rapid quantification of root growth and death with less damage to observed trees. Field site selection for the first set of tubes is underway. Sampling at a rootstock trial site continues. Only one rootstock tested to date has shown a significant difference in response to HLB we will be installing root tubes to compare root growth and root death data to rhizotron experiments. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. The first experiment is nearing completion and data analysis of root growth and root longevity in response to Las is currently underway. The analysis is taking longer than expected because of some variations introduced by the original setup that need to be corrected for. Fine tuning is being done to the setup to reduce the data analysis steps in future experiments. Additional rootstocks are being considered for the second round of rhizotron experiments and should be planted and inoculated shortly. Method development to characterize the mechanism by which Liberibacter causes root death is underway and the experimental samples will be collected shortly.
To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we surveyed 8 ridge groves in Highlands county and 4 flatwoods groves in Hardee county with high bicarbonate stress as detected in our 2013 survey. All the blocks are less than 10 year old Valencia trees on bicarbonate sensitive rootstocks, Swingle and Carrizo. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2014 season block yields 1.0 to 1.5 years after acid treatments began. From May to November root density fluctuated but was maintained at about the same level throughout the season. Root density was 5-10X higher in the ridge than in the flatwoods groves. Phytophthora populations were low or non-detectable in ridge groves but were always present and about 2-3x higher in the flatwoods groves. Soil pH ranged from 5.0-6.0 in the ridge after 1-1.5 years of acidification. pH in the flatwoods groves was initially in this range but rose above 6.5 and remained at that level. Flatwoods groves with higher bicarbonates in irrigation wells and soil require higher rates of acidification treatment to reduce below 6.5 as recommended for Swingle and Carrizo rootstock groves. Block yields will be evaluated for response in production compared to previous seasons. Surveys are continuing in 2015 with the addition of 4 groves without acidification in Highlands and Hardee counties to serve for comparison with an un-managed check. Recommendations based on these results were published in the May 2015 issue of Citrus Industry magazine.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
