Microarray time course studies comparing tolerant rough lemon to susceptible sweet orange have been completed. More genes were differentially expressed early in HLB-affected rough lemon than sweet orange but the opposite was observed at later time points; many of these were associated with defense responses, carbohydrate metabolism, and disease resistance pathways. Microscopy showed callose deposition in phloem of infected plants of both species, but phloem transport was much greater in lemon leaves than orange. Proteomic analysis of infected orange revealed greater abundance of several stress related proteins, and microarray data indicated that their underlying genes were also upregulated; these results are leading to better understanding of the underlying causes of HLB disease. Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced; many of these are currently in greenhouse testing and field trial locations. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for a next round of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. An international project to produce a high density genetic linkage map to aid genome sequence assembly and validation has been completed, with >900 sequence based markers. Several new rootstock trials with more than 15,000 trees were planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one 22 months symptom-free). Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 are growing off. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valqarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release approval have been developed for 2 new sweet oranges.
Update for 6/30/10: Two microarray platforms (our Agilent chip containing disease resistant genes and the Affymetrix Citrus GeneChip) are being used to compare gene expression over time in response to canker and HLB. Comparisons are being made between resistant kumquat and susceptible grapefruit for canker, and HLB tolerant rough lemon and susceptible sweet oranges are being compared. Comparisons of carbohydrate metabolism in HLB-infected and healthy plants revealed that starch, sucrose, and glucose accumulated in infected leaves, maltose decreased and fructose levels were unchanged. Comparisons of cell-wall bound invertase, a critical enzyme involved in sucrose metabolism and plant defenses, have shown its activity to be induced 4-fold in both symptomatic and asymptomatic leaves of infected sweet orange. Further, the expression profiles of 2 starch breakdown genes are downregulated. Transgenic studies are proceeding, with several hundred plants containing various new combinations of natural or synthetic genes and promoters produced; these are currently being propagated for field trials or are already in greenhouse testing. New candidate genes have been identified from citrus and other plants for HLB and canker resistance, from gene expression studies and data mining of public EST databases; vectors are being produced for a next round of transgenic plant production. Phloem-specific promoters are being identified for incorporation in constructs to produce more consumer friendly transgenic plants, by limiting transgene expression to specific tissues. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed their roles, and these will be used in new transformation experiments to produce canker resistant citrus. Cybrid grapefruit plants showing tolerance to canker in greenhouse tests have been planted in the Indian River area to assess field tolerance to canker. New DNA samples from a genetic population are being genotyped with previously developed SSR markers to develop a high density genetic linkage map to aid genome sequence assembly and validation; this is part of the international genome sequencing collaboration. Several new rootstock trials with more than 15,000 trees have been or will be planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings are being identified routinely using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks are showing some suppression of HLB symptoms in greenhouse tests that are still underway. New crosses for rootstock improvement were made in 2010, using previously proven parental lines. New pummelo-grapefruit seedless hybrids selected the past 2-3 years, as well as breeding parents, are being assayed for fruit furanocoumarin content; several have been found FC-free. Genetic studies are underway using in silico expression approaches to EST data analysis to define the genetic controlling elements of this trait, which seems to be simply inherited based on preliminary results.
Update for 9/30/10: Data from microarray time course studies comparing tolerant rough lemon to susceptible sweet orange are being analyzed. Microscopic studies of anatomical features in HLB-infected rough lemon and sweet orange plants are underway, along with experiments to study carbohydrate loading into phloem tissues using fluorescence labeled sugars; the results from these experiments are being compared with the results of microarray time course experiments to elucidate the mechanisms underlying HLB-disease symptom development. Transgenic studies are proceeding, with several hundred plants containing various new combinations of natural or synthetic genes and promoters produced; these are currently being propagated for field trials or are already in greenhouse testing. New candidate genes have been identified from citrus and other plants for HLB and canker resistance, using results from gene expression studies and bioinformatic data mining of public EST databases; vectors are being produced for a next round of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and some of these will be incorporated into new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed roles of one kumquat R-gene, two kinases, and one transcription factor, and these will be prepared as new constructs for transformation experiments to produce canker resistant citrus. Cybrid grapefruit plants were produced with kumquat, and preliminary results show that some behave like kumquat in challenges. Previously tested cybrids showing some tolerance to canker in greenhouse tests have been planted in the Indian River area to assess their field tolerance to canker. New DNA samples from a genetic population are being genotyped with previously developed SSR markers to develop a high density genetic linkage map to aid genome sequence assembly and validation; this is part of the international genome sequencing collaboration. Several new rootstock trials with more than 15,000 trees have been or will be planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings are being identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks are showing some suppression of HLB symptoms in greenhouse tests that are still underway. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made in 2010 have been harvested. Seed from other candidate rootstock plants have been planted, and additional ones collected, to study their nursery performance attributes. Data collected from a field trial of various Valencia somaclones and seedless Midsweet selections across the past several years have pointed to candidate oranges likely to be released in 2011 have entered the DPI-Parent Tree Program.
Update for 6/30/10: This project will assess a wide range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. The largest seedlings of these accessions have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination. They are now being grown in a climate controlled, DPI-certified greenhouse. Additional seedlings not yet large enough for inoculation are retained in a separate, DPI certified propagation greenhouse, for future inoculations in the greenhouse and for field planting once an acceptable site has been secured. Further, we have explored planting out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, but we are waiting on DPI to grant permission for planting at the Picos Road Farm with USDA-ARS. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. In advance of DPI-approval, we have begun testing for HLB and CTV, to ensure DPI that the plant materials are safe for field planting. Numerous somatic hybrids of citrus with related genera are also being prepared for inclusion in the plantings. We are in the process of acquiring additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal. Finally, we made a number of crosses in spring 2010 to produce segregating families of several purported tolerant and susceptible types to begin searching for evidence of genetic control of HLB tolerance/resistance within the citrus gene pool. Our collaborators in China have sought domestic funding support but have not been successful, so no materials will be sent there. One company in Florida that initially offered land for the project has since withdrawn its offer. We are currently exploring other options within Florida, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and data on disease progression will be collected. Additional seedlings that have reached sufficient size have now also been inoculated with the same HLB source. The individuals of the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been propagated. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. Eleven crosses between 10 different susceptible and reputedly tolerant parents were made in spring 2010, and several thousand seeds were harvested. Populations of at least 150 from each cross have been planted and now are growing in a DPI-certified propagation house. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Update for 9/30/10: This project will assess a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. The largest seedlings of these accessions have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination. They are now being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development; to date, no symptoms have been observed. Additional seedlings now large enough for inoculation have been moved into our certified HLB-testing greenhouse, and inoculations are about to take place. Further, we have been granted DPI permission to plant out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, at the Picos Road Farm near Ft. Pierce, with USDA-ARS. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. Testing the material for HLB and CTV has been completed and only pathogen free selections safe for field planting have been identified and budwood samples sent to the USDA-ARS for propagation and subsequent field planting. Approximately 100 selections were chosen and trees have been budded in late August. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced; many of these are currently in greenhouse testing and field trial locations. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for a next round of transgenic plant production, already underway. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual and citrus-derived markers for genetic transformation; several candidates have been identified for further experiments. More than 875 transgenic plants have now been planted with a collaborator in Martin county, and these are being monitored regularly, along with a second site in Indian River county. The plant materials growing out include sweet oranges, grapefruit and mandarin hybrids. Several new rootstock trials with more than 15,000 trees were planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly, and data has been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We initiated a program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house (in collaboration with Dr. Stelinski) to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 were planted in the field. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved by the UF-IFAS Cultivar Release Committee for release and commercialization according to UF-IFAS policy.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
Researchers at the USDA Ft. Pierce: 1. Source of mature tissue. Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication. A manuscript was submitted to the journal Plant Cell, Tissue and Organ Culture that documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants. A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. Agrobacterium-mediated transformation of mature tissue explants. Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit (1 plant), US-942 (4 plants), and Etrog citron (4 plants) using the beta-glucuronidase reporter gene. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. New tissue culture method of Agrobacterium-mediated transformation of tissue explants. Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, research confirmed the work of others showing that the flush used to generate transformation explants was critical. In the Machado laboratory in Brazil, research on the regeneration ability of various sweet oranges continues. These differences are present in both mature and juvenile tissues. In the Moore laboratory in Gainesville, experiments still are focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. A transient transformation expression system has also been developed using citrus leaves.
Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. This objective is proposed as an alternative strategy in case constitutive expression of the mutant R proteins proves to be detrimental to the growth or vigor of the plant. By restricting expression of the R protein to the single cell that is pierced by the insect stylet, a defense can be mounted without endangering the overall health of the plant. In the long run, this may be the most effective strategy in fighting Liberibacter infection since the response can engineered to be quite robust. Results: To further insure more specific expression of the R proteins in the phloem, we identified a wound-inducible, phloem-specific promoter that triggers expression upon wounding resulting from aphid feeding. We substituted the AtSUC2-940 promoter (phloem-specific) with the AtPAD4-1002 promoter (PAD4) in SNC1, snc1, SSI4, and ssi4 R gene constructs in the pCAMBIA1305.1 vector. Additionally, we created a PAD4/GUSplus construct to verify phloem- and wound-specific activation. The PAD4 constructs were introduced to Arabidopsis plants, grown to seeds and harvested this week (Jan 2011). The transgenic seeds will be subjected to selective screening for R gene transformants. Due to the cloning limitations (restriction site configuration), all PAD4/R constructs had to be first assembled in the 1305.1 pCAMBIA vector prior to transfer into the 2301 pCAMBIA for transformation into citrus. We selected two constitutive R protein mutants and PAD4/GUSplus constructs, and transferred them to the Citrus Research and Education Facility at Lake Alfred (Dr. Vladimir Orbovic). Preliminary reports indicated that transformants are proving difficult to obtain, potentially due to the lethal nature of at least one R protein mutant. Conclusions: Our hypothesis is that phloem-restricted expression of the R protein constitutive mutants would limit the potential negative impacts on plant growth. Use of the PAD4 promoter to express the R protein constructs should further increase the stringency of transcriptional control over expression of the potentially harmful R proteins. Our goal is have only the single phloem cell that is penetrated by the stylet express the constitutively active R protein, hence limiting potentially detrimental effects and focusing a robust defense at the source of Liberibacter introduction. Additionally, these constructs with the PAD4 promoter may reduce spurious expression in callus cells during the transformation protocol into citrus.
Our presentations at the Second International Research Conference on HLB in Orlando documented recent progress in characterizing HLB resistance/tolerance as found in Poncirus trifoliata and hybrids of that species with citrus. Some trifoliate hybrid citrus rootstocks, including US-802 and US-897, were demonstrated to become infected by HLB more slowly, to develop smaller amounts of bacterial even after infection, and to exhibit dramatically less symptoms in response to HLB infection than other citrus cultivars. A greenhouse experiment was initiated to compare the utility of the trifoliata-type resistance in the rootstock and scion position in grafted trees. Evidence was presented that documents significantly higher levels of some antimicrobial metabolites in the HLB-resistant trifoliate hybrid germplasm. Gene expression and metabolomic studies are underway to further characterize the genes and metabolites responsible for the resistance that can be used in creation and selection of conventional and transgenic varieties with improved tolerance or resistance to HLB. In this quarter, fruit quality, yield, and/or tree size data were collected from fourteen rootstock and scion field trials. Data was collected from one large cooperative rootstock trial with Ray Ruby grapefruit scion to evaluate rootstock effect on grapefruit quality and sheepnosing. Scion growth was measured on a greenhouse experiment to develop a more rapid way to evaluate CTV resistance and to evaluate supersour rootstocks for tolerance to four different decline strains of CTV. Rooted cuttings of supersour rootstock hybrids were budded with scions to propagate trees for field trials. One hundred supersour-type hybrids were selected from among new progeny for propagation and additional testing. A field trial was planted to compare HLB reaction of standard rootstock varieties to that of new transgenic rootstocks. Budded greenhouse trees for field trials were grown to planting size. Experiments continued to assess the utility of different methods for testing germplasm for resistance or tolerance to Asian Citrus Psyllid (ACP) and HLB disease. Data continued to be collected from four field experiments to assess the HLB tolerance of sweet orange trees on 15 different rootstocks. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). Rootstocks were transformed with an early flowering gene to produce selections that will allow more rapid advancement through generations and thus, more rapid genetic improvement. Selected transgenic rootstocks were challenged with HLB to assess potential resistance, including constructs with two new bacterial resistance genes. Research is continuing to use HLB responsive genes and promoters identified in a previously published gene expression study for engineering resistance in citrus. A microarray analysis was completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. This data is being analyzed now for publication and to guide future breeding and transformation research. The new hybrid rootstock US-942 was officially released for commercial use, based on outstanding performance in several different field trials. Seed of US-942 was provided for distribution to Florida citrus nurseries.
For EDS1 cloning, we currently confirmed by sequencing the cloning of the full-length ctEDS1 and already moved the sequence from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we designed primes to clone the 3′ end sequence of ctSID2, which we were previously unable to obtain with several methods. We performed the RACE reaction and have now obtained this missing sequence. We will subsequently design primers to amplify the full-length sequence of ctSID2. In addition, we did bioinformatics analysis and identified additional 9 citrus homologs of Arabidopsis defense genes that have available sequences in the database. We designed primers for these citrus genes and have been conducting RACE in order to amplify the genes. So far we have obtained the 3′ end sequences for ctNHL1, ctSFD1, and ctFAD7. Further cloning of 5′ end and/or 3′ end sequences of these citrus defense genes are currently underway. We continue to characterize the transgenic plants expressing ctNDR1/pBINplusARS. We obtained 5 homozygous ndr1 + ctNDR1/pBINplusARS. Through HR test and disease resistance assay with infection of the avirulent strain P. syringae avrRpt2, we further confirmed that over-expression of ctNDR1 could complement Arabidopsis ndr1 mutant. We are going to further characterize the defense phenotypes of these transgenic plants.
Over the past quarter, we have continued to develop all aspects of our project. In particular we have progressed in the following areas: 1. Building and testing additional TAL effector and promoter constructs. We have synthetically assembled a number of TAL effector genes matching X. citri TAL effectors and showed that they transcriptionally activate our broad recognition or “super” promoter in a Nicotiana benthamiana system. We have also assembled promoters with individual TAL effector binding sites to test activity and specificity. 2. Testing activation of gene constructs against a diverse world wide collection of X. citri isolates. Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. Additionally, we are preparing and testing X. citri strains with single or multiple disruptions in their TAL effector complement to test the role of specific TAL effector proteins in the disease and resistance process. 3. Stable transformations. The transformed lines generated last Fall and Winter have progressed through selection, shoot formation and rooting, and are now growing in soil. These lines are tested by PCR as they reach adequate size, and positively scored lines have been subjected to pathogen testing by pin-prick assay with X. citri. We have identified several canker resistant transgenic lines. We are currently setting up additional transformations to generate more transgenic material for line testing and with new promoter constructs. 4. Manuscript preparation We are in the process of drafting a manuscript of our results.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but the identification and incorporation of ACP resistance traits from uncultivated Citrus spp. and Citrus relatives is seen as a potential disease management strategy. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field for resistance to natural South Florida populations of ACP. The majority of genotypes surveyed hosted all three life stages of ACP, however there were significant differences among genotypes in the mean ranks for ACP eggs (F = 3.13, df = 86, P < 0.000), nymphs (F = 9.01, df = 86, P < 0.000), and adults (F = 4.21, df = 86, P < 0.000). The only sampled genotype that was completely avoided by all life stages of ACP was Casimiroa edulis, commonly known as white sapote, which was one of the few plants included in the study belonging to the Rutaceae subfamily Toddalioideae. Although not completely avoided, very low levels of ACP were found on two surveyed genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The identification of partial resistance in this species to ACP may prove useful in future citrus breeding programs efforts aimed at controlling the damage caused by reducing the incidence and spread of HLB. In recent psyllid no-choice oviposition studies using six different genotypes of Poncirus trifoliata, with Citrus aurantium and C. macrophylla as susceptible controls, a greatly reduced number of ACP eggs were laid on the Poncirus trifoliata selections when compared with the controls. The post doc assigned to the project resigned in December to take a permanent job elsewhere. A new post doc has been hired and is set to start during late January. A major thrust of upcoming work will be on no-choice experiments with Poncirus trifoiata selections. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae : one with 31 citrus varieties and one with 40 citrus varieties. Both experiments are free-choice studies under greenhouse conditions. A delegation from FAAS will be visiting USDA-ARS during April to coordinate research.
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