Work has been continued on the experiments initiated last quarter. Fresh seed has been obtained to generate new citrus seedlings and, as expected, germination rates are much improved. Therefore transient expression assays are being reported with larger numbers of seedlings and our new peptides. This quarter, I have been dividing work among between all of the proposal objectives. Plasmid constructs for a silencing experiment using the citrus analog of NPR1 are being verified and it should be possible to test silencing soon.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are now underway. The transformations are being performed at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette is being introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Transformations were begun on November 5, 2013. The transformation construct includes a green fluorescent protein (GFP) marker. Within a few weeks of initiating the transformation experiment, clusters of green-fluorescing citrus cells were observed, indicating that the FLT-antiNodT fusion protein transformation vector was working. This was important, because we had encountered significant difficulties in the development of the FLT-antiNodT fusion protein transformation vector. All indications are that the transformation vector is working as expected, which is good news. Transgenic shoots were successfully regenerated that are green-fluorescing and presumably carry the FLT-antiNodT fusion protein expression cassette in their genome. As of December 13, 2013, micro-grafting of transformed shoots onto recipient plants was begun. Successful regeneration of shoots containing the FLT-antiNodT fusion protein expression cassette is critically important. Sometimes certain proteins and transgenes might interfere with the normal plant biology in such a way that regeneration of normal plant tissues cannot occur. We are pleased to report that this has not been a problem so far for this project. All indications are that we should be able to obtain transgenic plants suitable for testing for HLB resistance. This project has gone a bit more slowly than initially anticipated. All steps of the project took more time than initially predicted, from antibody development and cloning to the development of the transformation construct. It is likely that we will take at least until August, 2014, to obtain transformed plants that are suitable for HLB resistance testing. We will also need time to do laboratory experiments characterizing the expression of the FLT-antiNodT fusion protein in the transgenic plants. Therefore, we fully anticipate needing to request a minimum of one year no-cost extension to this project. A no-cost extension of the project will be necessary to achieve the goals of the project fully.
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. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. 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. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus of building new constructs to controlling psyllids until we have more conclusion from the peptides under screen. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
This is a joint project between CREC and USDA, Fort Pierce. 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 being screened for tolerance and horticultural properties. The hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. At this time, all 4 of 5 hybrids still have no symptoms. The inoculated plants are growing in the greenhouse as we wait to determine the degree of disease symptoms in each line.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are continuing at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette has been introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Plants resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Plants are developing and appear to be healthy and normal, without any deleterious effects of the transgene noticed in the plant regeneration process so far. We anticipate that 1-foot tall plants will be available to begin testing as early as August of 2014. We are requesting a no-cost extension to the end of 2014 to have enough time to test the HLB resistance status of the trangenic plants.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we tried citrus leaves with NAD+ or water (negative control) and collected the treated leaves at 0 hr, 4 hr, and 24 hr. Three biological replicates were collected for each time point/treatment. RNA samples were extracted and subjected microarray analysis. The microarray data have been analyzed and the results showed that a group of salicylic acid pathway genes were upregulated in NAD+-treated samples. We also repeated one more time of the NAD+ treatment experiment in citrus and are currently analyzing the results. We are testing NAD+ analogs in Arabidopsis and will then use test them in citrus if effective. For objective 2, in the last quarter we started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria carrying a T-DNA vector with non-host resistance genes. Several putative transformants were identified in the last quarter. In this quarter, about 20 independent lines have been generated for each construct. They transgenic are growing in the greenhouse. We expect to perform molecular characterization in the next quarter.
We completed prediction of local sequence features, function, and spatial structure for each Diaphorina citri psyllid protein by using MESSA server (MEta Sever for Sequence Analysis) developed in our lab. Total of 18,142 proteins were analyzed. Among these proteins, 94% proteins display similarity to known proteins database, and the closet homologous organisms are pea aphid and red flour beetle. 14,185 (78%) proteins have homologs with known of predicted functions. We also predicted Gene Ontology terms. 8,980 (50%) proteins have associated GO terms. We identified the homologous protein families by using HHsearch and RPSblast to search against the COG and Pfam database. 9,847 (54%) proteins were assigned to 1,191 COG clusters and 14,572 (80%) proteins were assigned to 3,191 Pfam families. We predicted spatial structure by using HHsearch and RPSblast to search against PDB and SCOP database. 83% psyllid proteins can find structure templates. Overall, the templates cover 70% of all residues in psyllid genome. All the results are available at
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant genes. A total of 25 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants were sequenced and analyzed. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of five samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and an FDR threshold of 0.01. Among them, 306 genes are up-regulated and 515 are down-regulated in resistant citrus. Using the program iAssembler, a total of 53,981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, compared with the citrus reference genome. We further identified 3073 DE genes by comparing the gene expression of another group of resistant and sensitive citrus samples using DESeq2 with adjusted P-value (padj) < 0.1. Among these DE genes, 1413 genes were up-regulated in resistant citrus significantly and 1660 genes were down-regulated in resistant citrus. Due to our comparing strategy, the DE genes should most come from the difference between Marsh and Jackson. As all of the 3 resistant citrus came from the offsprings of Jackson and all of the sensitive citrus trees came from that of Marsh. The resistance genes should come from Jackson. A total of 86 DE genes were identified in the tolerant and intolerant citrus group using DESeq2 with adjusted p-value less than 0.01. Among these DE genes, 69 genes were up-regulated and 17 genes were down-regulated in the tolerant samples. We tried to combine the DE genes of GROUP1, resistant and sensitive group, with that of group of tolerant and intolerant. There were only 2 down-regulated genes, XLOC_024967 and XLOC_028218, conserved in both groups. Gene XLOC_024967 is a pathogenesis-related thaumatin protein. The PR genes usually were transcribed when interacted with pathogens and were believed to be the response of host with pathogens. The other gene down-regulated gene in resistant and tolerant samples was XLOC_028218, a transducin family protein. A number of significant up-regulated and resistance-related genes are selected for further characterization.
Evaluation of existing standard cultivars (‘Temple’, Fallglo’, Sugar Belle’, ‘Hamlin’ and ‘Ruby’) for HLB resistance/tolerance was established in 2010 at Picos Farm, Ft. Pierce Fl. Leaves from each cultivar have been and continue to be sampled monthly. Growth (diameter and height) and disease development are rated at each of the sample times. Levels of Candidatus liberibacter asiaticus (CLas) in symptomatic leaves are being assessed using real time PCR. All cultivars showed symptoms of HLB and have a range of titer levels. ‘Fallglo’ and ‘Temple’ had the lowest levels of CLas. ‘Tango’ had the highest titer levels. A second project involves assessment of HLB tolerance measured as growth in presence of CLas at various titers. Infected bud-wood of nine varieties, 3 putatively HLB-resistant (‘Temple’, Gnarly Glo’, and ‘Nova’) 3 HLB-tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 HLB-susceptible (‘Flame’, Valencia’, and ‘Murcott’) was treated with antibiotics to generate a range of CLas titers. Treatments included; 1) 100 .g/ml penicillin and 10 .g/ml streptomycin, 2) 50 .g/ml penicillin and 5 .g/ml streptomycin, 3) 25 .g/ml penicillin and 2.5 .g/ml streptomycin, and 4) water only. In December 2012, treated buds were grafted on to sour orange rootstock. They will be rated for HLB symptoms on a monthly basis for a 2-year period. CLas colonization will be quantified on a quarterly basis using real time PCR. Due to bud-wood mortality, several treatments have to be repeated. Development of chimeras is currently underway. Seedlings of GUS transformed ‘Carrizo’ x ‘Hamlin’ and ‘Red Carrizo’ x ‘Sweet Pineapple’ have been successfully approach grafted together. A horizontal cut was made at the grafting union and the area was treated with plant growth regulators to promote formation of chimeral plants. Growth of chimeras is general slow and very few adventitious buds have formed. A high throughput evaluation method for HLB resistant biomarkers in Citrus and Citrus relatives is being assessed. This methodology may help identify potential candidates that can be used in the USDA citrus breeding program. Crude leaf extracts from several citrus varieties, Poncirus (trifoliates), and hybrids (sweet orange x Poncirus), were prepared. The compounds are separated and identified using liquid column chromatography coupled with mass spectrometry (LC/MS). The resulting spectrographs and retention times were compared and differences were observed. Citrus varieties and the hybrids had similar chemical profiles. Both of these groups’ chemical profiles were different from that of the trifoliates. The trifoliates also showed high levels of poncirin and naringin. Whereas the citrus varieties and hybrids screened in this study had no detectable levels of poncirin. Unique compounds will be identified and their role in disease resistant will be determined. A method for the rapid identification of potential sources of HLB resistance is also being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage that are exposed to HLB infect ACP feeding trials. CLas titer levels, using real time PCR, will be evaluated at 2, 4, and 8 weeks.
Our efforts over the last quarter have focused further on our transformation bottleneck using several approaches: 1. Stable transformation of citrus at UC Davis transformation facility. Previously we reported that we sent two of our constructs to the contract transformation laboratory at UC Davis. Carrizo transformants were obtained, albeit at lower efficiency than the transformation standard, but no transformants were recovered from Duncan grapefruit. These results are consistent with our own experiences, reflecting lower efficiencies of Duncan transformation over carrizo overall and in particular with our constructs. We will test the carrizo transformants and we have changed the vector backbones of our constructs to pCAMBIA 2201, which is routinely used in the Moore lab 2. Stable transformations in citrus using new vectors. New transformation experiments have now been carried out in Duncan grapefruit and carrizo now with 6 constructs in the pCAMBIA backbone. Plantlets are being transferred to soil, and initial PCR and GUS testing is in progress. Most plantlets are still too small for analysis, but this should be well underway next quarter. 3. Stable transformation of test systems – tobacco. UC Davis also made several lines of tobacco transformed with marker gene and resistance gene constructs. These have just been received in Gainesville and will be tested by PCR and pathogen assays. 4. Stable transformation of test systems – tomato We are testing a tomato system as a proof of concept. We used the same PIP14 promoter to drive expression of the avrBs4 gene. Expression of AvrBs4, a Xanthomonas effector, in tomato results in a hypersensitive reaction, this this construct will induce localized cell death when induced by pathogen effectors in Xanthomonas strains, as in our other systems. Explants of Bonny Best and large Red Cherry tomato cultivars were transformed to test the PIP14 construct efficiency via Agrobacterium mediated transformation. Transformants were recovered and PCR was conducted to assess transgene presence. With Bonny Best, several positive transformants were isolated. In addition, a few transformants showed a moderate resistance to Xanthomonas gardneri XV444, a strain that produces a TAL effector that can trigger our promoter construct. With Red Cherry, positive transformants were selected, and the resulting transgenics are being screened using PCR. We have some potential candidates that we are currently screening. 5. Transformation of mature citrus explants We will explore mature citrus transformation with the Lake Alfred group working on this effort.
Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
In the previous three months Core Citrus Transformation Facility (CCTF) has continued to provide services for production of transgenic material to interested clients. In comparison with the third quarter of 2013, productivity was higher in the last quarter. Within this quarter, CCTF has received eleven orders. For five of those orders together with the other two from previous quarter, limited number of co-incubation experiments was performed as they required Valencia seedlings which are out of season. Other orders required the work with Duncan seedlings and experiments were performed at full capacity. Considerable amount of work was put into few orders placed in the first half of the 2013. In coordination with the client who ordered these plants, efforts are continuing to find out if the genes requested to be introduced into citrus have negative developmental effect that is hindering production. Additional six vectors and/or Agrobacterium strains received from the same client fall into the group were problems with the (common) PCR primers developed and are not solved yet. Since for all except two current orders, screen for putatively transgenic shoots is done based on PCR reaction, the work on the orders with questionable PCR primers is stalled. Plants that were produced within these three months are from the following orders: pN4-one plant, pN5-one plant, pN7-two plants, pX28- six plants, pX19- 23 plants, pTMN1- seven plants, and pMG105- two plants. Altogether 47 plants were produced and they were all Duncan grapefruit. Despite problematic nature of some recent orders, they are being placed at high volume which shows continued need for the CCTF services.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. 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. This screening program supports two USHRL projects funded by CRDF for transforming citrus. 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 two weeks, 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. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. A total of 4,472 transgenic plants have passed through inoculation process. A total of 89,810 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. As an offshoot of the research, damage by western flower thrips was so severe that research was conducted to validate damage by this pest to developing flush and facultative predation on ACP, which led to the following publication: Hall, D. G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection. (in press). A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
Construction is completed. Parent plants will be moved into the structure over the next quarter.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, 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 (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants transformed with citGRP1 were test by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. They will be used for canker resistance screening by detached leaf assay and infiltration. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kanamycin resistant transformants were generated on the selective media. About twenty kanamycin resistant shoots are rooted in-vitro and three Hamlin transformants are in soil. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay and canker resistance test will be performed soon. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
