The main accomplishments during this quarter: We have confirmed the K gene overexpression-mediated improvement on transformation efficiency of a lemon cultivar we used. We have tested the effects of the K gene on genetic transformation efficiencies of 6 citrus cultivars and we observed 3-15 fold increases if compared to our control vector, and 3-11 fold increases if compared to the highest transformation efficiencies of the same cultivars previously reported by others. We have observed and confirmed the stimulatory effects of one non-conventional regulator of gene expression on shoot regeneration efficiencies of some citrus cultivars. We have been testing the effects of that factor and other factors in combinations of the K gene on transformation efficiencies of both mature and juvenile citrus explants and our preliminary results suggest that there are significant improvements in transformation efficiency for both juvenile and mature tissues. We are also repeating the effects of endogenous auxin and the auxin transport on efficiencies of shoot regeneration and Agrobacterium-mediated infection of mature tissues of citrus. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been accepted for publication in “Plant Cell, Tissue and Organ Culture”. The article is currently in the production stage and should be out in either February or March issue.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and five PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for identification of transformants. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Constructs are also being created in which Bs2 is co-expressed with other R genes that may serve as accessory factors for Bs2. Constructs with tagged Bs2 have been confirmed to function in transient assays, and have been transformed into Arabidopsis. Protein expression will be confirmed by immunoblot. GFP is currently being added to the constructs to facilitate selection of transformants in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. C1 and C2 have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. “C1” clearly cut the target sites of both varieties, causing 100-bp deletions. Sequence analysis confirms that “C2” cuts the target site in Carrizo. Considering this site is identical in both Duncan alleles, we expect it to cut Duncan as well. And, considering “C3” and “C4” are built from “C2,” we expect them to target the cut site as well. Sequence analysis is underway to confirm these expectations. In addition, to aid in the selection of positive transgenics, we are currently adding a GFP reporter into each CRISPR construct.
A 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 six 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 has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. 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 6 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, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. 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 site 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 led 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. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg 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.).
Background information The objective of this project is to quantify the relative effect of copper (Cu), windbreak (Wb) and leafminer control (Lc) on the spatial and temporal progress of Asiatic citrus canker (ACC) under conducive conditions for epidemics and disease loss. The experiment is set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Paran , Brazil. The different treatments are the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represents a plot. The presence or absence of copper sprays and leafminer control represents a subplot. Each subplot is composed of 112 trees. Each of the eight treatments has three replicates. Cu treated plots are being sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc is being performed with application of abamectin at 150 ml/ha every 21 days. Casuarina is used as a natural Wb around the plots. Disease evaluations started in December 2013 and include percentage of ACC symptomatic trees, proportion of the canopy affected by the disease, incidence of symptomatic fruits at harvest, and yield. CRDF funding will cover the period of November 2015 to October 2016. Pre-funding progress results Nineteen months since the onset of the epidemics (July 2015), the incidences of diseased trees in the plots with complete management (Cu, Wb, Lc) and no management reached 45.2 and 97.6%, respectively. ACC diseased trees under complete management showed a minimal citrus canker severity of 0.8% as opposed to 31.4% observed on trees without any control. At first harvest, the incidences of symptomatic fruits from trees treated with the tree measures and none were 3.8 and 58.5%, respectively. Finally, production of trees in the first harvest revealed the same trend observed for other assessments. Fruit yield of trees under complete management (40 kg/tree) was 186% higher than control trees (14 kg/tree). The combination of Cu and Wb is showing the greatest importance for disease control. Post-funding progress results Trees continued to be assessed monthly as previously described. By the end of this report, disease assessments of December 2015 had been performed but not processed. The assessments of January 2016 had not been concluded. Thus, up to November 2015, disease has not progressed since July 2015 as conducive conditions for the pathogen in the trial started in December. In November 2015, the incidences of diseased trees in the plots with complete management and no management reached 43.8 and 97.9%, respectively. Severity of citrus canker in the canopy dropped for all treatments and reached 0.6 and 2.5%. The same trend was observed in the previous year and it was mainly due to drop of ACC-affected leaves, production of spring flushes and lack of favorable weather conditions for disease outbreaks, which are expected to occur in the upcoming months.
A 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 six 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 has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. 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 6 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, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. 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 site 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 led 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. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg 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.).
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, all transgenic lines as described in the project proposal have been regenerated and most plants have been acclimatized and transferred into the greenhouse for further growth. A few putative transgenic lines have been transferred to fresh in vitro rooting medium in efforts to stimulate root production. Root samples from 21 lines have been analyzed for gene expression using qPCR. Of them, 12 were determined to be high expressers while the rest were medium to low in expression. We are in the process of evaluating the remaining greenhouse acclimated lines for gene expression. The high expresser lines will be propagated for subsequent evaluation with Diaprepes neonates.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol is being optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs were provided to the Lake Alfred transformation facility, but the transformation attempts have so far been unsuccessful, possibly due to negative effects of the constructs in Agrobacterium or in citrus. Troubleshooting of these transformations with a negative control construct is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting a site overlapping the two conserved leucines, and one targeting two flanking sites to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we are propagating a number of citrus transgenic lines overexpressing Arabidopsis defense genes. Our previous results indicated that these transgenic lines are likely resistant or highly tolerant to HLB. The progeny plants are growing in the greenhouse. For objective 2, we performed on round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results showed some of the lines may be chimeric. We are repeating the qPCR experiment. For objective 3, we are propagating the previously generated gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB. The progeny plants are growing in the greenhouse.
During the last quarter of 2015 Core Citrus Transformation Facility (CCTF) continued to operate as planned and produced transgenic Citrus plants. The number of orders received for the last three months jumped to nine. Two of those orders were for transgenic Duncan grapefruit and the rest are the set of seven orders for production of genetically modified Pineapple orange. CCTF still has not received two groups of orders announced by prospective clients about 5-6 months ago. Also, one set of orders that included the use of three different vectors was withdrawn and the client is working on re-cloning of all vectors. CCTF produced 51 plants within last quarter. These plant belong to multitude of orders, altogether nine of them, some of which are old and some newer. Six of the produced plants were Valencia oranges, 11 were Duncan grapefruit, and the rest were Carrizo citrange. As I have previously reported to Dr. H. Browning, the analysis of transgenic plants carrying NPR1 gene was successfully completed by Yosvanis Acanda from Mature tissue transformation facility. Out of 67 tested transgenic rootstock plants produced by the CCTF, 23 were shown to be high expressers of NPR1. These plants are being kept in the CCTF s greenhouse until they reach the size at which they can be propagated so that they can serve as rootstocks for both transgenic and WT scion plants.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
The overall objective of this project was to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two girdling patterns were designed differing in the distance photoassimilates had to travel apoplastically (around the girdle) to re-enter the unobstructed phloem. We began experimentation with the girdling design (spiral) that presents the longest distance for apoplastic movement and the longest distance for CLas (or its signal) to travel. To test the hypothesis, spiral girdles were placed on the main stem or on a lateral stem, and trees budded with HLB material above the girdle. In both instances, and within 8 months, approximately 90% of the trees became HLB+ opposite to the girdle. The results are clear indication that CLas (or its signal) is capable of movement laterally across the phloem tube side walls. Based on the convincing results using the girdle pattern, the number of trees to be tested with the checkerboard pattern was reduced. As with the spiral pattern, HLB was transmisible through the severed phloem elements. The data collected in this project offer new insights into the nature of the HLB signal, as it seems to move through physical barriers smaller then the actual size of CLas. We conclude that genetic HLB signal is sub-cellular in nature and can be transferred between non-phloem living cells. This new finding is important in that it shows that HLB causing effector is not necessarily the entire CLas bacterium. The infected HLB trees were kept to be used in current CRDF-sponsored research projects.
The main accomplishments during this quarter: We continued testing the effect of the K gene on transformation efficiency of a lemon cultivar. In general, lemon cutilvars are difficult to be genetically transformed. We have observed the K gene can drastically improve the transformation efficiency of the lemon cultivar used, similar to the effects of the K gene on the other orange cultivars tested. We continued to repeat the effects of K and I genes on transformation efficiency of mature citrus explants of mature Pineapple orange. The effects of the K and I genes have been confirmed. We have also tested effects of a non-conventional regulator of gene expression on regeneration efficiency of Washington navel orange and Valencia orange. We observed several fold increases in shoot regeneration efficiencies of both cultivars. Our goal is to use this regulator and also its combination with the K gene to improve transformation efficiencies of of both juvenile and mature citrus tissues One manuscript reporting the drastically improvement of several citrus cultivars has been submitted in the end of August. We have started to write the second manuscript from the project.
Effectors are essential virulence factors in microbial pathogens. The HLB-associated bacterium Candidatus Liberibacter asiaticus (Las) is known to encode the Sec secretion system, which is predicted to deliver effector proteins into plant phloem. Our previous research identified four Sec-secreted proteins from Las. The goal of this project is to characterize the targets of these effectors in citrus. This research will provide important knowledge on the basic biology of HLB pathogenesis and facilitate the development of control strategies. Our research has been focusing on four Las effectors whose expression can be consistently detected from various citrus varieties that are infected by Las. We hypothesized that their targets in citrus contribute to HLB 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. During this report period, 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 individually and examined for their interaction with the corresponding effector by targeted Y2H. Due to the large amount of work, we have further focused on two effectors, which exhibit significantly higher expression (10-40 folds) in HLB-infected citrus tissues than in psyllids. So far, we examined a total of over 20 potential interacting proteins of these two effectors. Our results strongly suggest that each of these effectors specifically interacts with a class of citrus proteins in yeast. Importantly, these two citrus plant families have been reported to play a role in plant defense and they are also well-known virulence targets of other bacterial and fungal pathogens. Furthermore, one of the protein family has been reported to be present in the vascular system. We have made exciting progress in the project. Our on-going effort includes: 1) further confirm the physical interaction of the effectors and their targets using in vitro and in vivo co-immunoprecipitation assays; 2) characterize the target proteins as a family to understand how they interact with the effectors.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
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