This project has been difficult from the beginning and when we were coming up on the third year, all of my Co-PIs decided they did not to continue on this project for various reasons. I did want to continue because my part of the project was going well. It took some time to sort this out but by May 9, 2012 I had e-mailed Dr. Browning for guidance. He indicated that I could submit by myself. I did this on June 19. I know it was received because Ms. Nowicki had me change something in the budget. And the executed contract was sent. Then I heard nothing. On September 28, Dr. Turpen called me about why I had not submitted paperwork. I told him I had and resent the third year paperwork to him. Again I heard nothing until an NOA was sent in the middle of November for $39,000. When someone in DSR asked Ms. Nowicki about a no-cost extension, she said there would not be one, I had until December 31 to spend the money because I had been so late in getting in the paperwork. I was not extremely late in getting in the paperwork! And of course I could not spend all of the funding in six weeks (during the holiday season) without doing things I am not comfortable doing because I have no desire to fail an audit or get in trouble with either CRDF or UF. A 6 month no-cost extension was finally executed. This allowed us to continue preliminary work on cell penetrating peptides, which led to the funding of Project 752. How to write the reports was problematic because of the gaps in funding and although I asked for guidance, I did not receive any.
There was no funding received during this quarter, so progress could not be made.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Significant progress has been made with research on the use of RNAi as a means of Asian citrus psyllid control. We have made use of a Citrus tristeza virus (CTV) dsRNA expression system which when inoculated into C.Marcrophylla results in leaf phloem containing dsRNAs which target essential ACP genes. When ACP feed on leaves from these “paratrangenic” (CTV transfected) citrus plants, mortality was double that observed when the ACP fed on artificial diets containing this synthetic dsRNA, greater than 80% mortality. Currently, new versions of paratransgenic citrus are being produced to determine the best sequence to use targeting this essential gene. The use of the Ion Torrent (by Life Technologies) next generation sequencing has provided rich insight into the transcription profile of “paratransgenic” fed “sick” ACP using comparative RNA Seq analysis. The data support the specific down-regulation of the dsRNA target gene as the cause of mortality as seen by the significant perturbation of genes in the molecular pathway in which this gene functions. RNA Seq data continues to be collected from ACP fed on a variety of gene specific dsRNA containing diets. Experimentation has also begun using artificial diets containing dsRNAs synthesized by a novel “mass-production” technique that would make it practical for the use of RNAi technology as a field application. The use of topical application in conjunction with “paratransgenic” plant varieties presents a strategy for effective delivery and a multiple gene targeting employment of the RNAi pest control technology.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Field day was held on December 12th, attendance was approximately 130. New HLB frequency of infection data was presented. Infection frequency of commercial rootstocks has now reached 89%. Overall, diploid rootstocks are now 67% infected, and tetraploid rootstocks are 57% infected. Four trifoliate orange x papeda hybrids from Sicily are still showing lower infections frequencies (31-59%). Additional seed of these selections has been acquired from Dr. Reforgiato-Recupero for additional trials. Most of the tetraploid rootstocks selected for release are still around 50% infected. New HLB severity assessment of all infected trees in the trial is underway. McTeer trial (ACPS trial of SugarBelle on 15 tree-size controlling rootstocks, nearly 100% HLB): every tree was scored for HLB severity. Rootstock White #4 (UFR-5) showed the overall best and most consistent tree health. Fruit quality was still poor in general, but there was a significant increase in harvestable fruit as compared to last year. Greenhouse Experiments – unsuccessful HLB-infected budstick grafts in the nutritional and rootstock studies were all regrafted (about 15% of the total trees). Most of the successfully grafted trees have flushed out, with minimal HLB symptoms in both experiments. Protecting Seed Source Trees: 1. The insecticidal Snowdrop Lectin gene is being employed in efforts to protect rootstock seed trees from psyllids (and thus Liberibacter inoculation), and for Diaprepes resistance. More than 100 ransgenic trees of Green #7, Orange #4, Orange #16 and Orange #19 containing the Snowdrop lectin gene were produced. Progress was also made stacking this gene with the CEME antimicrobial gene.
In collaboration with Mike Irey, we completed evaluation of a population of transgenic lines for their resistance to Clas. Transgenic trees were kept in a greenhouse containing CLas+ Asian Citrus psyllid (ACP) adults and trees were evaluated every 6 months for HLB for 2.5 years. In addition, psyllids were tested periodically for the presence of CLas. Although we had transgenic trees from four transgenes (LIMA, AttacinE, CEAD and NPR1) showing resistance to HLB, the results from NPR1 were the most promising. 27% of the trees containing the 35S-NPR1 construct and 57% of trees with the phloem specific AtSUC2-NPR1 construct survived and were PCR- after 30 months of inoculation. The HLB-free trees were moved to the SG field site under MTA and DPI petition (many in poor condition due to severe psyllid damage). Additional clones of the promising transgenic lines are currently being propagated for additional field testing in the Dunwoody Grove of Southern Gardens. In addition, Several newer NPR1 lines are already in the field test at the Picos Farms (USDA) and results there are promising as well. Our current goal is to combine transgenes that function by completely different mechanisms as to have a back-up to prevent the pathogen from overcoming single gene resistance in the field. We have produced 40 new transgenic plants of Hamlin cominging NPR1 with the successful AMP genes CEME or CEMA. We have successfully developed a heat inducible Cre/loxP site specific based recombination system for efficient excision of antibiotic resistance genes in citrus. In our construct, the nptII gene under the control of the NOS promoter and the Cre recombinase gene driven by either a soybean heat shock protein (hsp17.5E) promoter or a Arabidopsis thaliana small heat-shock protein (HSP20) gene promoter were flanked by two loxP recognition sites in direct orientation. An anthocyanin biosynthesis gene from Vitis vinifera (VvMYBA1) was placed outside the loxP sequence. Transformation efficiencies were similar using either soybean or the Arabidopsis promoter. Anthocyanin activity analysis on transformed Carrizo citrange (Citrus sinensis x Poncirus trifoliata) demonstrated that approximately 30-40% of transformation efficiency could be obtained following Agrobacterium mediated transformation and heat shock treatment. Molecular analyses have demonstrated that 100% selectable marker gene deletion occurred in all regenerated plants expressing anthocyanin. We have completed building a RES type structure in our greenhouse#7. Transgenic trees have reached the top of the structure and have been bent downwards. We did not observe any flowering this past year; however, downward growing branches on many of the trees have completely lost their thorns, indicating a rapid reduction in juvenility. Since this greenhouse is heated during the winter, flowering induction may be inhibited.
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.
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