In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
Expression in citrus of dsRNA targeting a psyllid gene, through use of the paratransgenic CTV expression vector, was further characterized. Our analysis showed that mortality of psyllids feeding on citrus producing target dsRNAs was directly correlated with accumulation of total psyllid gene RNA (ssRNA + dsRNA) produced within the leaf tissue. As much as 80 to 90% mortality of adult psyllids was observed after 6 days of feeding on leaves with the highest level of psyllid target gene RNA. Citrus leaves expressing RNA from the green fluorescent protein (GFP) cloned into the CTV expression vector induced no mortality in adult psyllids. These results support the hypothesis that mortality is associated with psyllid gene specific dsRNA ingestion. Currently experiments are being conducted on performance of all psyllid life cycle stages.
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): PCR analysis (run by the SG diagnostic laboratory c/o Mike Irey) validated the work of the CREC HLB scouts, as approximately 98% of the trees in this trial were confirmed as HLB positive. Complex tetraploid rootstocks Orange #19 and Orange #4 (Nova+HBP x Cleo+Arg.trifoliate orange), and the somatic hybrid Changsha + 50-7 trifoliate orange continued to show minimal disease symptoms, with a few symptomatic fruit beginning to show up on trees on Orange #4. Assessment of the rootstock effects on fruit drop and percentages of symptomatic fruit are underway. 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, approximately 5 acres of 3 year old trees,Harrell’s UF mix slow release fertilizer and daily irrigation). The entire trial was assessed for HLB by the CREC scouts, September 2012 data was compared to 2011 data, with very interesting results. Commercial rootstocks are showing extremely high HLB infection rates (Kuharske 87%, Swingle 70%, and Volk 80%), where as some of the complex tetraploid rootstocks showed much lower infection rates (tetrazyg Orange #15 just 7%). PCR analysis (run by SG) also showed that for a few rootstocks including Orange #15 and Orange #16, non-symptomatic areas of trees that have been PCR positive for more than one year were still PCR negative, suggesting that pruning maybe a management strategy for trees on these rootstocks. Assessment of the rootstock effects on fruit drop and percentages of symptomatic fruit are underway. 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 will begin during this quarter. Greenhouse study of HLB/nutrition interaction: 10 liners each from 15 different promising complex tetraploid rootstock candidates (and Carrizo as a control) were stepped up to 4×4 citripots for this study. Also, 150 liners of tetrazyg rootstock Orange #15 and were stepped up to 4×4 citripots for the individual nutrient/HLB interaction study.
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. 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. Correlating transgene expression with disease resistance response: We continued work to optimize an ELISA protocol to detect lytic peptide in transgenic Citrus plants using the LIMA antibody. This protocol has should be useful for evaluating transgenic plants containing either LIMA or CEME antimicrobials, using the same antibody. Since most of our constructs have the C-myc tag, ELISA and Western blot protocols have been optimized for large scale rapid screening of the transgenic plants to identify those with maximum transgene expression. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): A vector containing a dual T-DNA border has been constructed. To test the vector functionality and determine T-DNA segregation, we have incorporated a visual Anthocyanin expressing gene from Grape (VVMYB) into one of the T-DNA. This gene on expression turns cells purple. The other T-DNA contains a fusion negative-positive selectable marker gene for selection (codA/nptII; Vector 1). We are currently constructing another fusion negative-positive selectable marker gene, by replacing the nptII gene with a gene that encodes for resistance to the antibiotic hygromycin (hptII). This construct will be used for transformation of citrus cell suspension cultures (Vector 2).
Dr. Cecilia Zapata, PI, resigned August 2012. Before leaving, we planned for reduced effort until a replacement could be found. Dr. Vladimir Orbovic agreed to assist with oversight of the mature tissue transformation facility in the interim. Below is the quarterly report Dr. Orbovic submitted. In the first three months of the funding period, Mature Tissue Transformation Laboratory (MTTL) has undergone big changes. The person who supervised the Lab for the last three years has left that post in the beginning of September. In the anticipation of prolonged period without managerial supervision for MTTL, departing supervisor made a decision to discard high percentage of plants from the growth room to prevent accumulation of unused plants. The transformation experiments were scheduled at the rate of one per month. However, temporary supervisor revised the plan up to two experiments per month. To accommodate such change, certain batches of plants that were used a source of explants only once were not discarded as planned. Also, some of the smaller rootstock plants left for practice and as surplus were transplanted and will serve as an additional batch of rootstock plants. Throughout this period, nine co-incubation experiments were performed. Four of those experiments were done with Hamlin explants, four with Valencia explants, and one with Pineapple sweet orange explants. In the Hamlin experiments, 2390 explants were cut for treatment with Agrobacterium; 2520 explants were used in Valencia experiments, and 690 explants were used in Pineapple experiment. Here are the results of GUS assays: 270 shoots harvested from different experiments with Hamlin were tested and two were positive. Out of 210 tested shoots of Valencia harvested from different experiments, five were positive. And finally, out of 68 shoots of Pineapple orange harvested from two experiments, two were positive. One of two positive Valencia shoots died upon grafting. Other positive shoots appear healthy and will be moved to growth room soon. These results mark a milestone as all three commercially important cultivars of sweet orange were successfully transformed. There are four Ray Ruby plants completely cleaned from microorganisms and ready to become source of shoots for production of branches. These Ray Ruby plants were obtained from USDA as ‘clean’ although additional testing in MTTL has shown that they did harbor some microorganisms. Repeated micro-grafting of meristem regions to new and clean rootstock plants resulted in selection of plants that were purged of any pests.
The antibody developer, Creative Biolabs, Inc., identified six monoclonal antibodies to the 30 amino acid peptide antigen used, which corresponds to an extracellular loop of the Candidatus Liberibacter asiaticus outer membrane protein NodT. The materials were received by Dr. McNellis’ lab at Penn State University in September of 2012. Four of the antibodies appear to be useful for the project, based on molecular analyses of their binding efficiency to the epitope target and their structural integrity. This type of single-chain engineered monoclonal antibody is provided to us as a DNA clone, from which we express the antibody in bacteria. We are currently working on producing the antibodies in E. coli bacteria. This material will allow us to test whether the antibodies can be used to detect NodT protein in protein extracts from psyllids and citrus trees. The transformation construct for expressing the FLT-antiNodT fusion protein in citrus has been initiated and will be completed soon.
From the three FT constructs created (FT1, FT2, FT3), the FT3 construct has shown significant induction of flowering on transformed tobacco plants. FT3 shows promising results at shortening the juvenility period in both citrus and tobacco. Compared to a wildtype control of tobacco the flowering time of F2 generation plants transformed with the FT3 construct is over 3 months earlier. A multi-faceted approach to understand the activity of citrus FT is underway. One of the approaches involves measuring expression levels of FT1, FT2, and FT3 in different Citrus varieties using Real Time PCR to determine FT behavior at different stages of growth. Analysis for the first 3 months has been performed on Pummelo and pineapple sweet orange varieties and all three genes are actively transcribed at different levels depending on the time period. The biological effects of various phytohormones such as ethylene and gibberellic acid on FT3 expression and flowering will be monitored. This approach will allow us to devise a method to delay flowering induction at early stages in citrus due to the observed premature formation of flowers at tissue culture stages. The final approach is to isolate the FT3 mobile protein and introduce it into phloem of citrus and tobacco through various methods in order to induce early flowering. The protein will be synthesized and various trials of exogenous protein application will be performed. This approach will allows us to create a practical protocol for shortening juvenility periods. In both citrus and tobacco the FT3 genomic construct with the constitutive FMV promoter is highly effective, causing very early flowering. Unfortunately, in citrus, the flowering occurs on the plate, before the transformed material is useable. Some work has been done in an attempt to control the speed of flowering using day length, temperature, and gibberellic acid. In a further attempt to control the activity of the FT3 gene, a construct using an inducible promoter is being produced. This inducible promoter is based on the activity of an ecdysone receptor and is induced using the chemical methoxyfenozide. Before this construct is developed, the effectiveness of the FT3 cDNA is being compared to the FT3 genomic DNA using the original FMV promoter in the hopes that the smaller cDNA will be just as effective and can be used in the new construct without changing the flowering character.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in wild type (Col-0) background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers in the isolated transgenic plants. We have been in the process of testing disease resistance of these plants to Pseudomonas syringae infection. Besides ctNDR1, our recent test of Arabidopsis overexpressing ctEDS5 also showed a complementation of the eds5-3 mutant with the citrus gene. Transgenic plants with potential enhanced disease resistance will be further selected to obtain homozygotes for additional tests of disease resistance. Such constructs will be preferentially used to transform citrus for citrus disease resistance tests. We continue to characterize citrus transgenic plants transformed with ctNDR1. We confirmed with PCR that 29 independently transformed plants carry the transgene. In addition, we conducted second round of infection with Xanthomonas citri (Xac) and results showed again that citrus transgenic plants overexpressing ctNDR1 were more resistant than untransformed controls. We are growing the plants and prepare them for a HLB test in the future. In the meantime, we have made additional transgenic plants with other citrus SA genes. The transformation is generally conducted with two to four genotypes for each construct because there are significant variations in transformation efficiency and resistance to HLB and citrus canker diseases in different genotypes. Besides transgenic plants overexpressing ctNDR1, we have so far made transgenic plants expressing ctEDS5, ctPAD4, ctNPR1, and ctEDS1, which are in US-802, US-812, US-942, and/or Hamlin background. The presence of these transgenes was confirmed with PCR in some genotypes. More transgenic plants are to be obtained from these transformation events from different genotypes. Additional constructs will be placed in the pipeline of transformation once they are ready. The transgenic plants will be prepared for resistance tests for citrus canker and HLB diseases as having been planned for the ctNDR1 transgenic plants.
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 have about 60 different antimicrobial peptides or RNAi constructs are under test against HLB. Plant infected with the CTV vector plus a peptide or RNAi sequence are being inoculated by HLB in a psyllid containment room.
The objective of this project is to examine existing sweet orange/poncirus hybrids for tolerance to HLB and acceptable juice quality. We found that a poncirus hybrid developed by Herb Barrett, USDA Orlando, many years ago is tolerant to HLB. However, it has too much poncirus to be commercially acceptable. However, this and sister hybrids were used in further crosses to sweet orange. We are testing some of those hybrids. At this point we have propagated selected hybrids and are beginning to test them for tolerance to HLB.
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
A number of experiments and observations at the USDA/ARS Ft. Pierce citrus scion improvement program demonstrate resistance or tolerance to HLB in some conventional citrus genotypes. In the funding cycle that began in 2012 our efforts in this area are markedly enhanced by funding for a postdoctoral researcher. Dr. Sharon Inch, who has extensive experience in plant pathology and histology, began working on this project 10/9/2012. She will move forward with established experiments comparing HLB development in specialty cultivars, which displayed resistance in a study of commercial groves, vs. susceptible standards. A set of replicated plants have been initiated for 50 genotypes representing advanced USDA selections, genotypes which display apparent resistance/tolerance in field observations, and susceptible standards: these will be exposed to HLB in a hot psyllid house and will be assessed for CLas levels, HLB symptoms, and growth. The remaining proposed experiments relating to assessing and characterizing HLB resistance/tolerance will be initiated in the next quarter.
The funding cycle that began in 2012 provides for a postdoctoral researcher in the USDA/ARS scion transgenic program. This will markedly enhance progress. Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. She will move forward with new constructs and resulting transgenics, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline.
In the initial funding of the current grant we have made progress on several of our objectives: Objective 1. Evaluate existing transformed lines: We have been maintaining a steady effort in the growing and testing out of candidate transformed lines from our large transformation efforts. Over 34,000 transformations of Duncan grapefruit have been carried out with 8 different reporter and resistance constructs, from which we have generated over 600 T0 plants that are being used for PCR analysis and pathogen testing. We are currently testing plants. The frequency of functional transformants may be lower than typical given that inappropriate leaky expression will be counter-selected. Objective 2. Expand stable transformations Our efforts to transform additional commercial citrus species has been focused on Ruby Red grapefruit and sweet orange. At present we have introduced eight constructs consisting of promoters with1,4, or 14 TAL effector/PthA binding sites with GUS or resistance gene coding regions in more than 1200 Ruby Red and over 600 sweet orange transformations, producing about 60 T0 plants for analysis. Objective 3. Refine constructs We have initiated additional resistance gene constructs that contain additional promoter elements and/or use another resistance gene known as AvrGf2. As each new construct is completed, we are testing these it in transient and stable transformation assays. Objective 4. Sequence more TAL effectors from additional canker accessions We have new sequences of TAL effectors from strains from Florida, Argentina, and Brazil, including the Miami “type” strain, as well as several strains with altered growth phenotypes. Last, we are expanding efforts at examining effects of resistance gene constructs on population growth of a range of Xanthomonas citri strains.
The current main priority is hiring the project postdoctoral fellow in the Zipfel laboratory. Whereas an initial candidate was identified, the hiring was not completed and additional candidates are currently being interviewed. The intention is to make an offer to a qualified postdoc by mid-November. Others in the lab are progressing the initial objectives: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. We have prepared constructs for EFR, XA21, EFR-XA21 and XA21-EFR chimera. We have carried out transient expression assays in N. benthamiana and found that the proteins express well. We have shown that the XA21-EFR chimera is functional, as exemplified by a gain of responsiveness to elf18 in a ROS burst assay. We are currently testing the functionality of XA21 and XA21-EFR by treatment with the axS17 peptide. This protein is unstable and difficult to synthesize, so we have obtained wild-type and ax21-minus strains of Xanthomonas euvesicatoria so that we can make extracts to test XA21-induced responses in N. benthamiana.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
