The four most promising anti-NodT scFv antibodies have been selected for further development. Anti-NodT antibody #1 has been successfully expressed in E. coli. This means that we can generate as much of the antibody as needed. The antibodies are being augmented with two 6xHis epitope tags – one at the amino terminus, and one at the carboxy terminus. The protein can be detedcted with anti-His antibodies. The anti-NodT scFv antibody is soluble, and should be usable for protein immunoblotting and other applications. We experienced some delays in cloning the scFv antibody DNA into the appropriate citrus transformation vector. However, these difficulties have now been solved and we now expect to have the scFv citrus transformation construct completed within a few weeks, and we will commence transformation immediately.
McTeer trial – (3-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Continued evaluation of this trial shows significant differences in tree health among rootstocks, with Orange #19 showing the healthiest trees. However, HLB has significantly impacted the quality of fruit trees on all rootstocks, even from very healthy looking trees. We will look at these trees one more season to see if continued remediation will improve fruit quality next season. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 4.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Full data (yield, fruit quality and HLB infection rates) was presented in our Field Day handout. Big differences have shown up in HLB infection rates per rootstock. Control commercial rootstocks have the highest infection rates, with most >70% infected. The rate of infection on tetraploid rootstocks was half that of diploids, with tetrazyg Orange #15 showing the lowest rate (just 7%). Disease severity of infected trees is also being impacted by rootstock. Good candidate rootstocks for ACPS are emerging. Greenhouse Experiments – Rootstock liners have been grown off for the nutrition and rootstock comparison studies, and have been moved to the HLB house for graft inoculation, to begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Transgenic tetraploid lines containing an insecticidal Snowdrop lectin gene were regenerated from the tetrazyg selections Green #7 and Orange #4. 12 transgenic lines of Orange #4 and 3 of Green #7 have been successfully micrografted, acclimatized and transferred to the greenhouse. A construct containing the Snowdrop Lectin insecticidal gene combined with the antimicrobial gene CEMA was completed. Transformations are underway. We have codon optimized the Snowdrop Lectin insecticidal gene (GNA) for optimal expression in citrus. Two vectors containing this optimized gene have been produced; a) codon optimized GNA fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells; b) codon optimized GNA fused with a HDEL C-terminal extension for retention of the GNA protein in in the endoplasmic reticulum. Dual protection against psyllids and Liberibacter: A construct containing the native Snowdrop Lectin insecticidal gene with the antimicrobial gene CEMA have been constructed. Transformation using this vector are being carried out.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance.A construct containing CEMA gene stacked with the NPR1 gene has been constructed. Also, another vector containing a AttacinE gene stacked with the NPR1 gene is also under construction. Correlation of transgene expression with disease resistance response: More than 150 transgenic lines with different genes have been analyzed using ELISA by either C-myc or LIMA antibody (which also works for CEME) to measure transgene expression. As expected, significant differences were observed in our transgenic plants. Correlations between the data obtained from ELISA and other molecular data with HLB challenge response data are underway. Transgenic lines examined by ELISA include 40 lines with NPR-1, 50 lines with LIMA, and 9 lines with CEME. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): We have finished construction of several parts of the T-DNA region of a pCAMBIA0390 derived binary vector for cre-lox based marker-free selection. A fusion codA-hptII gene driven by the d35S promoter have been constructed and a cre gene driven by a glucocorticoid-responsive elements promoter have also been constructed and cloned into a pUC based vector. We are experiencing problems cloning the glucocorticoid receptor gene driven by a constitutive mirabilis mosaic virus promoter as all sequenced clones have mutations and/or deletions in them. Work is underway to rectify this.
Work has been continuing on the development of a construct using the FT3 cDNA insert and an FMV promoter. This construct will eventually be used to test the efficacy of the FT3 cDNA as compared to the genomic DNA construct currently being used. Over the past several months, extensive testing was conducted to establish a more effective disinfestation technique for use on seeds and other explant tissue. This technique should allow for the continuous use of seed for transformation, even many months after their initial collection. Transformation of Carrizo has picked up following the most recent harvest of seed. These transformants will be used in the experiments examining the effects of GA and day length on FT phenotype. This is month 7 of the in vivo tracking of FT1, FT2, and FT3 and samples are continuing to be collected and processed. These data will be evaluated at the end of the year-long trial to compare month-to-month variations in gene expression. The FT3 protein that was commercially synthesized has finally arrived and experiments with direct application of the protein will be commencing shortly.
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 60 different peptides for activity against HLB. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids.
The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are beginning to be tested for tolerance and horticultural properties.
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 three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sunburst’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, Fl. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Preliminary results indicate that there are a range of host responses with ‘Temple’ in the most tolerant group. A second project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. In February 2013, budwood with various concentrations of CLas, derived from the antibiotic treated plants, will be evaluated for their potential to result in HLB symptoms in disease free material. The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants.. Development of periclinal chimera using resistant geneotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of Red Carrizo, Temple, Hamlin, and Valencia. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). After successfully generating the chimeras with HLB resistant vascular system and good fruit using the previously mentioned cultivars, additional cultivars such as ‘Sweet Orange’ and grapefruit will be added to this study. An additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle procedure is being investigated. If this screening method is successfully, it may be useful to quickly identify new sources of resistance varieties that may enhance and improve citrus production in Florida.
Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. New constructs have been used to transform citrus scions including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed and will be used in transformations next quarter. A series of transgenics scions produced in the last several years, continue to move forward in the testing pipeline.
Development continued on new rootstocks with outstanding attributes for Florida production, including tolerance to HLB. Yield, fruit quality, and tree size data were collected from ten rootstock trials with early ripening scions. Propagation continued to prepare trees for four more field trials to plant this year. Propagations of supersour selections were prepared for budding of trees for field trials, and another group was prepared for field planting. Cooperative work was continued with a commercial nursery to multiply 250 advanced supersour selections for placement of trees into cooperative field trials with growers at multiple locations. Work continued to assess supersour tolerance of CTV, Phytophthora, and Diaprepes using carefully controlled tests in the greenhouse and the field. Preparations were made for controlled testing of supersour selections for tolerance to high pH. Specialized testing of the supersour hybrids and concurrent field trials will effectively identify specific supersour selections that are equal or superior to sour orange in horticultural attributes and effects on fruit quality, as well as provide disease resistance or tolerance. Experiments were initiated to study the most important components of the tolerance to HLB exhibited by some citrus rootstocks. The final stage of a study of metabolic changes in HLB infected germplasm is being completed to supplement the gene expression study completed last year, including HLB susceptible and tolerant cultivars. Detailed evaluation of specific defense-related genes continued, including CtCDR1 and CtPDF2, identified by microarray as being responsive to HLB in tolerant rootstocks. Constructs are being built using this knowledge and that will allow the creation of new cultivars with increased HLB tolerance using only citrus origin genes. Knowledge gained about specific citrus resistance genes will also help guide crosses for the creation of conventional hybrids with improved HLB tolerance or resistance. A study to define the interaction of rootstock tolerance with scion tolerance/susceptibility is nearly complete, and is expected to be published by mid-year. Additional trees were propagated to examine the effect of rootstock tolerance to HLB in trees with the scaffold composed of the tolerant variety. Trees were also propagated for a field planting that will examine the same high grafting technique. Collaborative work continued to assess rootstock interaction with scion, nutrition, and management factors in determining tree tolerance to HLB. A manuscript was published on collaborative work demonstrating the association of particular small RNAs and nutrition, with HLB infection. Collaborative work continued on the relationship of small RNA to the HLB tolerance of selected citrus genotypes. Collaborative work began to compare the early response of trees infected by HLB to those infected by CTV, both phloem-limited pathogens. Selected citrus plant resistance genes were inserted by genetic transformation into outstanding rootstock and scion cultivars to develop new varieties with increased resistance to HLB. More than 300 new transgenic rootstock selections with potential resistance to HLB were produced, targeting increased expression of the citrus resistance genes CtNPR1, CtEDS1, CtMOD1, CtEDS5, CtPAD4, CtNDR1, or CtACD1. Twenty-five new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. A field trial continued with selected transgenic rootstocks to evaluate performance under natural field infection with HLB. The field trees are nearly 100% infected with HLB, but differ widely in the severity of symptoms and the effect of HLB on plant growth. Three presentations were made at the International Citrus Congress on research progress in the USDA rootstock program, including descriptions of new USDA citrus rootstocks, rootstocks for tree size control, and methods to test citrus selections for HLB resistance.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. One mechanism we investigated to see whether it contributed to this resistance was plant hormones. We sprayed salicylic acid, methyl jasmonate, and abscisic acid, which are all common plant hormones, on susceptible citrus plants to test the influence on host choice, oviposition, development, and survival of ACP. Abscisic acid cut the life span of adult ACP in half compared to untreated control plants. The plant hormones had no other strong effects on ACP, but abscisic acid may play a minor role in preventing oviposition by ACP. We are currently doing a third replication to verify effects of plant hormones on oviposition. In addition, we are preparing the data for publication in a peer-reviewed journal. Another mechanism that may confer resistance to ACP is the structure of the scelerenchyma (a fibrous ring) surrounding the phloem. The scelerenchyma in non-citrus plant species was found to prevent feeding by herbivorous insects. We are collaborating with a Research Entomologist at our facility to test whether ACP choose different feeding positions on leaves of resistant P. trifoliate versus suceptible xCitroncirus sp. We completed one replication and found differences in feeding position. We will soon initiate a second replication to verify our results. If we verify differences in feeding position on resistant and susceptible leaves, our collaborator will then evaluate the salivary sheaths from ACP and the scelerenchyma in leaves using scanning electron microscopy. Some of the research that we discussed in earlier progress reports is now in review or already published in peer-reviewed journals, including a paper on host plant resistance in HortScience, one in the journal Crop Science, a review paper in Entomologia Experimentalis et Applicata that focuses on control strategies of ACP, and a paper on miticides (investigating which miticides could be sprayed on plants and experimental ACP without killing them). Collaborators at the Fujian Academy of Agricultural Sciences conducted free-choice tests with all major groups of citrus and found differences among and within groups. Most groups of citrus were colonized by ACP, but lemons were the most preferred group and sour oranges and kumquats were the least preferred. The differences among citrus varieties within a group may be useful because volatile and phloem contents that differ between the least and most preferred species can be compared. FAAS continues their screening of germplasm including CRC accessions. To date, there is obvious resistance to ACP in Poncirus and among some distant relatives of Citrus. There is also at least a little ACP resistance within most of the Citrus groups but, in general, there is no evidence that any Citrus germplasm would offer as much genetic resistance to ACP as Poncirus. Among trifoliate hybrids, there is some material that appears as resistant as Poncirus itself, indicating that traits of interest (greatly reduced oviposition, reduced longevity) were genetically inherited.
Construct optimization: We have engineered several new constructs for transformation in citrus genotypes. These include a ProBs314TBB-avrBs3: avrGf2 and a ProBs34TBB-avrBs3: avrGf2. In transient assays, these constructs elicited an earlier and stronger HR than constructs carrying avrGf1. We have also designed a construct which utilizes a portion of the promoter region of a citrus gene that demonstrates strong binding activity by PthA4 homologues. Transient assays demonstrate higher levels of activation than other constructs. Six new constructs are being used for stable transformation The constructs vary based upon the promoters used, the number of copies of the avrGf2 gene (single or multiple), the presence or absence of a terminator – nopaline synthase terminator (NOS T) upstream of the promoter and the plasmid used. Transformation summary In vitro germination experiments are ongoing with citrus varieties ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit and ‘Pineapple’ sweet orange for future epicotyl experiments. To date both epicotyl and cotyledon transformation experiments have been carried out with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange segments and the 6 new constructs designed with the avrGf2 gene. Sweet orange epicotyl and cotyledon transformation experiments have been carried out and a total of 1, 005 and 554 segments, respectively transformed with 5 of the 6 constructs. On the other hand 6 constructs have been used to transform altogether 3,623 grapefruit epicotyl segments and 5 constructs to transform 527 cotyledon segments in the transformation experiments. Shoots regenerated from transformed segments of sweet orange (53) and grapefruit (19) have been placed on rooting media. Putative sweet orange and grapefruit transgenic plants, 44 and 19 respectively originated from the regenerated shoots placed on rooting media have been placed in soil for acclimatization and will be tested via PCR for confirmation of integration of the transgene.
In our last progress report (9/15/2012), we reported the identification two citrus genes, temporarily named CtHRT1 and CtHRT2, capable of inducing hypersensitive response (HR)-like cell death when overexpressed in Nicotiana benthamiana. We also found that CtHRT1 and CtHRT2 belongs to a highly conserved family, in which other members from rice and Arabidopsis all can induce similar HR-like cell death in Nicotiana benthamiana. CtHRT1 and CtHRT2 are now renamed XBCT31 and XBCT32. Over the past three months, we carried out experiments to confirm the observed cell death response. An Arabidopsis protein with similar structure, but lower sequence identity, to XBCT31 and XBCT32 was expressed in Nicotiana benthamiana. No cell death was observed, indicating that the citrus XBCT31 and XBCT32-triggered tissue collapses are specific. HR cell death is often associated with electrolyte leakage caused by membrane damage. Ion leakage assays were performed to quantify the cell death. We found that significant amounts of ion leakage were detected 48 and 72 hours after infiltration with the XBCT31 or XBCT32 harboring Agrobacterium. In the infiltrated leaf discs, the development of visible tissue collapse kinetically correlated with the time course of ion leakage. Ion leakage was not induced by agroinfiltration of the empty vector. To confirm XBCT31 and XBCT32 accumulation, we carried out protein blot analyses. Leaf samples were harvested 40 hours after infiltration, at which time the necrotic phenotype was not visible. The XBCT31 and XBCT32 proteins were readily detectable in the infiltrated leaves. Interestingly, XBCT31 accumulated to a significantly lower level as compared with XBCT32. In contrast, a much more severe tissue death was induced by XBCT31. Therefore, XBCT31 appears to be a stronger cell death inducer, whereas XBCT32 seemed to have a weaker capability. Despite this difference, our data confirm the cell death activities of XBCT31 and XBCT32, and may be indicative of an activation of defense mechanisms. A manuscript with the data reported above and previously has been submitted to an international journal. We are currently constructing plasmids harboring XBCT31 and XBCT32, respectively, for stable citrus transformation to eventually test resistance to citrus canker and greening.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for one week, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. This report marks the end of the second quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project has been hired, two small greenhouses for rearing psyllids have been completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. Psyllids will be available for challenging test plants in January. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. Overexpression of AtNPR1 or its orthologs is often associated with a constitutive expression of some PR genes. We therefore carried out RNA blot analyses to detect levels of chitinase 1 (Chi1). High levels of Chi1 were found in all three CtNH1 overexpression lines, which is in contrast with an undetectable basal level of this gene in wild-type plants. These results indicate that Chi1 is constitutively expressed in the CtNH1 overexpression plants. Because induction of Chi1 has been correlated with a resistance response in citrus (Porat et al. J. Plant Physiol. 158: 1585), our observations suggest that overexpression of CtNH1 leads to a constitutive activation of defense in citrus.
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