In the previous reports, we have reported the development of Soft Nanoparticles (SNPs) using two essential oils, EO A and EO B. The formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter Crescens (L.Crescens) and both the formulations and the controls showed > 90 % inhibition at 1, 5 and 10 % (v/v) dilution. Phytotoxicity of select formulations were performed at 1:1, 1:10 and 1:20 dilutions and all formulations showed low phytotoxicity when applied at 1:20 dilutions. The developed formulations were tested for their stability with adjuvants by a technique similar to the well-known ‘jar test’. Cohere and Cling are spreader-sticker type adjuvants that are frequently added to pesticide spray tank before spraying to enhance pesticide penetration. It was seen that most of the formulations were stable. Based on the efficacy, phytotoxicity and stability tests, formulations for EO A and EO B have been short-listed for possible field trial. SNPs have also been developed and characterized with Thyme Oil. The droplet size ranged from 3 to 18 nm and the oil loading ranged from 1 to 20% (w/w) using agriculturally approved surfactants. The surfactant loadings are similar with those in formulations with EO A and EO B. Addition of a co-surfactant greatly enhanced the oil loading to up to 20% (w/w). Selected formulations were also tested for stability with the adjuvants and most of the formulations were stable and the addition of adjuvants changed the particle size of the SNPs marginally but was still in the required range (5-16 nm). Future plans include testing of selected formulations of thyme oil for antibacterial activity / efficacy against the surrogate bacteria as well as phytotoxicity studies. To separate the efficacies of the essential oils from the surfactants, emulsions and microemulsions have been developed with low and ultra-low surfactant and oil loadings. Formulations have also been developed with different surfactants that are known to be safe and used in pharmaceutical formulations (e.g. Pluronic’ F127 and Tween’ 80). These formulations will be tested for their inhibition efficacy on the surrogate bacteria. These experiments will assist in understanding the efficacy of the oil in SNP as well as in determining the minimum amount required for L. crescens inhibition. Dye doped SNPs have also been developed to understand their foliar uptake. In addition, experiments are being planned to quantify the effectiveness of the adjuvants (Cling and Cohere) performance on citrus leaves.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the lasA1 and lasA2 genes. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana and citrus. We have obtained transgenic lines with these constructs. These transgenic Arabidopsis lines were verified by PCR and RT-PCR and their segregation in T2 and T3 were analyzed. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and/or overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. We also expressed LasA1 protein using the Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E. coli. Purified LasA1 protein are used for antibody production and crystallization study. Immunoprecipitation and elution of FLAG-tagged autotransporters from Agro-infiltration in Nicotiana benthamiana yielded several protein candidates, indicating LasA1/LasA2 interacted with mitochondria and chloroplast proteins. It is worth noting that we are able to detect LasA1 protein from Las-infected plant tissue using Western blot. The successful generation of this antibody will enhance our research in several aspects. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein has also been generated.
During the 3 years of funding, we identified and characterized a regulon from ‘Ca. L. asiaticus’ involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of ‘Ca. Liberibacter asiaticus’, using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. When bound to LdtR, the chemicals inactivate the protein, which disrupts a cell wall remodeling process that is critical for survival of the pathogen when exposed to osmotic stress (i.e. within the phloem of a citrus tree). Several model strains were used to confirm that the newly identified transcription factor (LdtR) and its regulated genes (ldtR and ldtP) confer tolerance to osmotic stress.These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease. The logical subsequent step of our research is the optimization of all necessary parameters to use the chemicals identified directly on field applications. The results were published in PlosPathogens Journal: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004101
Transgene Stacking for Long-Term Stable Resistance: PCR analysis of transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) resulted in identification of 7 lines that contain both transgenes. Agrobacterium mediated transformations to produce transgenic plants containing other combinations of stacked genes are in progress. Transgenic plants with the PCR positive stacked genes are being clonally propagated for further evaluation. Improving Consumer Acceptance: 1. In efforts to develop an intragenic citrus plant, we have in addition to the binary vector for an inducible cre-lox based marker free selection containing the cre gene driven by a Soybean heat shock gene promoter, a binary vector containing the cre gene driven by a citrus-derived heat shock promoter. Citrus rootstock Carrizo has been transformed with both of these constructs and numerous transgenic plants are being regenerated. 2. Hamlin and W Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells. One of the T-DNA contain a grapevine myb gene under the control of a 35s promoter and the other contain T-DNA containing the selectable positive/negative fusion marker cassette. Plants have been regenerated that are purple in color from the anthocyanin production. Pending molecular analysis of the regenerated lines, we speculate one of two possible scenarios: a) The plants contains only the T-DNA of interest or 2) The plant contains both T-DNAs integrated into the genome. Somatic embryos are now being germinated and resulting transgenic plants will be evaluated. Induction of early flowering: The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 has also been produced for testing. Since previously generated FT and AP1 plants flower in vitro but not as young plants in the greenhouse, we are testing the possibility of a synergy when both are present together. Transgenic plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. Propagation of new transgenics for field testing: ‘ Propagation of LIMA-B (AMP) transgenic plants for further study; ‘ Propagation of Carrizo transgenic lines with LIMA gene to test a potential rootstock effect on non-GMO scion. Efforts to establish a new transgenic field site: Working with Dr. Phil Stansly, we have submitted an addendum to our transgenic field permit with APHIS to add an additional field site which would be located at the UF Immokalee Research and Education Center. We plan to plant 400 new transgenic trees at this site after approval. A few hundred trees have also been prepared for planting at the USDA Picos Farm site.
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. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107/CFU. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. 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 showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slightly canker resistance by infiltration test. A series of transgenic scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
This quarter we have made good progress on our transformation approaches: 1. Stable transformations in citrus using new vectors. Transformation experiments were carried out in Duncan grapefruit and carrizo with 5 constructs in a preferred vector backbone, including an empty vector control . 52 carrizo transformants were selected and analyzed by PCR. The genes of interest that were screened for included avrGf1, avrGf2, and NPTII. PCR results showed that 5 of 6 transformed with the avrGf1 gene were positive and 9 of 30 plants were positive for the avrGf2 gene. NPTII was detected in 22 of the 52 carrizo plants. 6 out of 7 Duncan plants were positive for NPTII. These results demonstrate improved transformation efficiency using this vector backbone. Further epicotyl transformation experiments were carried out with both ‘Duncan’ grapefruit (624 segments) and ‘Pineapple sweet orange (136 segments) with 6 constructs. These segments were placed on media without selection (kanamycin). This modification was done to see if transformation efficiency would improve. Rooting experiments and transplanting segments are ongoing. Of 1,507 transformants selected so far, 168 putative transgenic shoots of grapefruit, sweet orange and Carrizo were confirmed as transgenic by GUS assay (35S:GUS construct in vector) this period. To date, although no GUS positive or chimeric shoots have been observed for the sweet orange cultivar, Duncan grapefruit and carrizo citrange had totals of 4 and 134 GUS positive shoots, respectively. We will continue to screen and progress positively transformed plants for further analysis. Pathogenicity tests will be carried out as soon as plants reach adequate size. 2. Stable transformation in test systems Tobacco: N. tabacum lines transformed with the 14 EBE promoter fused to GUS, generated at the UC Davis transformation facility, were tested by GUS assay in the presence and absence of the X. citri effector PthA4, delivered as a 35S expression construct via Agrobacterium. Nine of 10 of the lines showed GUS expression only in the presence of PthA4. This system is now established as functioning and will provide a rapid means of testing the ability for individual and combinations of X. citri effectors to induce the promoter construct. We are in the process of harvesting seed for full scale screening. Tomato: A second test system was designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. Positive transformants of Bonny Best and large Red Cherry tomato cultivars were confirmed using DNA dot blot. T0 transgenic tomato were screened for pathogenicity reaction with X. euvesicatoria strains (8510 avrBs3 transconjugant & Race 9) and X. gardneri, and several Bonny Best and large Red Cherry were found to be resistant. This demonstrates that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance. We are harvesting seed from transgenic plants and are in the process of testing seedlings for resistance.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Random mutagenesis of EFR ectodomain to gain elf18-CLas responsiveness did not produce gain-of-function mutants. Using chimeric elf18 peptides of wild-type and CLas sequences, we could determine that both the N- and C-terminal regions of elf18-CLas were responsible for the lack of recognition by EFR suggesting that elf18-CLas is impaired in both binding and activation. In order to identify complex EFR mutants that respond to elf18-CLas, we are developing a FACS-based screen. We fused promoters that are expressed at a low basal level and PAMP inducible to nuclear-localized GFP and tested by Agrobacterium-mediated transient expression in N. benthamiana and in Arabidopsis protoplasts. All constructs gave significant basal expression, which is presumably due to unrestricted expression in these transient systems. Therefore, it is necessary to produce transgenic lines of these reporter construct. Modelling of the EFR/elf18/BAK1 complex has been performed, based on the available FLS2/flg22/BAK1 structure. Sites have been selected for targeted mutagenesis to improve recognition of elf18-CLas. We have also examined the interaction with BAK1 and identified two regions which may be involved in elf18 binding. Mutagenesis constructs of these regions is currently being screened for elf18-CLas response. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Reciprocal XA21 and EFR chimeric receptors have been produced and transformed into Arabidopsis for functional testing. Since it is now known that axYS22-Xac is not the ligand of XA21, we tested the response of the EFR-XA21 chimera to elf18, in order to determine the function of the XA21 cytoplasmic domain in dicots. These constructs performed in a similar manner to wild-type EFR. In addition we tested XA21, EFR, XA21-EFR and EFR-XA21 for pathogen resistance to Pseudomonas syringae pv. tomato DC3000 COR-. Interestingly, all constructs provided an elevated level of resistance, indicating that the chimera of XA21-EFR was functional and that XA21 was capable of perceiving a potential ligand Pseudomonas. A manuscript describing this work is currently being prepared for submission to PNAS. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs including EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore Lab for transformation. These were transformed into E.coli DH5. cells and Agrobacterium tumefaciens strain Agl1. Confirmation of clones was done using PCR (EFR and XA21 designed primers) and restriction analysis (NcoI and SpeI restriction enzymes). Initial transformation experiments have been carried out with a total of 100 ‘Pineapple’ sweet orange segments. Germination rate for sweet orange is 5%, which is extremely low. ‘Pineapple’ sweet orange and Duncan grapefruit seedlings are on germination media and transformation experiments will be initiated in May 2014.
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 performed another NAD+ treatment experiment, in which NAD+ was either infiltrated into the citrus leaves or sprayed on the surface. After 24 hours, the NAD+-treated leaves were inoculated with the canker bacterial pathogen Xanthomonas citri subsp. citri. Canker symptoms were analyzed two weeks later. Compared with the Actigard treatment, the NAD+ treatment did not provide strong protection to the plants in this experiment. It is possible that time between NAD+ treatment and canker bacteria inoculation was too short. We are design another experiment in which the NAD+ induction time will be longer. We are also preparing citrus plants for root treatment with NAD+. For objective 2, in last quarter, about 20 independent lines have been generated for each construct. The transgenic seedlings have been growing in the greenhouse. We have performed molecular characterization of the transgenic plants. DNA was extracted from each individual plants and PCR was performed to confirm the presence of the transgene. Only a fraction of the putative transgenic seedlings contain the transgene. We are currently testing the expression levels of the transgene in these plants. We will also generate more transgenic lines using the constructs.
The goal of this project is to understand the biology of HLB by identifying key host components and processes involved in disease development. We use secreted proteins (also called effectors) from the causative agent, Candidatus Liberibacter asiaticus (CLas), as molecular probes because they have been considered key virulence proteins of bacterial pathogens. Our previous research using bioinformatic and experimental approaches identified four CLas effectors that are highly expressed in infected trees. We will isolate the direct citrus targets of these effectors, which will reveal important information of HLB pathogenesis. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first two quarters of this project, we cloned the four CLas effector genes into the Y2H bait vector, transformed them into the yeast strain AH109, and confirmed that the effectors are highly expressed in the yeast without self activation activities. Therefore, these constructs are appropriate for Y2H screens. In the third quarter (Year 1) of this project, our main efforts include: 1) Construct citrus cDNA libraries that will be used for Y2H screening. We collected RNA samples from asymptomatic and symptomatic tissues of HLB-infected sweet orange leaves. These RNA samples were mixed with RNA extracted from healthy tissues to ensure that we would be able to cover as many genes as possible. The RNA has been sent out for cDNA library construction by a company. We have been working with the company to normalize the library in order to minimize the influence from the over-representative transcripts of highly expressed, housekeeping genes, which may bias the screening later on. 2) Make gene expression constructs that produce fusion proteins with each CLas effector gene tagged to a gene encoding the yellow fluorescence protein (YFP). These fusion proteins have been transformed to plant cells to determine the localizations of these effectors in plants using microscope.
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. As of April 9, 2014, a total of 5,314 transgenic plants have passed through inoculation process. A total of 106,250 bacteriliferous 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. 60: 66-69. A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
Three replicated experiments were undertaken to evaluate the effectiveness of reflective mulch to repel ACP, reduce incidence of HLB, and improve growth and yield of newly planted citrus compared to standard practices. The first was planted 3-4 July 2012 in a 10-acre block on 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County. Plants were provided with 2 punch-in drip emitters for irrigation and fertigation. Experimental design is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots 5 rows wide and 13 trees long, mulch or no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film 3 mils thick and covered with a clear protective polyethylene coat. Monitoring ACP with flush inspection and sticky cards replaced weekly commenced 13 August. Of 1974 ACP found on sticky cards, more than 70% came from no-mulch plots, 11% from plots receiving insecticides and only 2% from plots with both mulch and insecticides. Of more than 59,000 flush shoots inspected, 7,368 were infested with ACP and 1565 were infested with Aphids. More than 65% of ACP and aphid infested flush were found in no-mulch plots, 477 ACP and 518 aphid infested flush in insecticide treated plots and 93 ACP and 166 aphid infested flush in plots provided with both mulch and receiving insecticide applications. Twenty positive HLB leaf samples were found from a sample collected mid-January 2014, of which none came from the mulched treatments, 12 from the no mulch + foliar nutrition but no insecticide with no other significant differences. Trunk diameter measurements made 21 January 2014 indicated better growth from trees planted in metalized mulch compared to non-mulched subplots. Greatest in growth difference among treatments between mulched and unmulched was seen without insecticides (18%) with only a 3% increase with insecticides. Foliar nutrition increased growth by 3% in all treatments when compared to no foliar nutrition. Leaf samples for nutrient analysis were collected before the March foliar nutrition spray. Normal grove care operations continued which included one gylphosate application in March for weed control, Kocide copper prayed monthly to control canker, and one application of Danitol as a dormant season spray to reduce ACP population to more realistic levels in all treatments. A 6-week insecticide drench rotation plan for designated plots was implemented in February starting with thiamethoxam and followed up mid-March with clothianidin. A second trial planted May 2013 at SWFREC consists of 24, 250 ft rows of ‘Ray Ruby’ grapefruit divided into 8 main plots, half with organic amendments of compost. Each 3-row plot is divided into 2 subplots: whiteface or metalized mulch. One of 160 leaf samples collected 28 Jan 2014 tested positive; it was located in a white mulch compost plot. Of 55 ACP found on sticky cards, 29 were from white mulch+compost, 16 white mulch no compost, 6 in metallized mulch compost and 4 in metalized mulch no compost. No infested shoots were found in trees on metalized mulch compared to 5 infested on white mulch no compost and 9 on white mulch with compost (N=7013). Trees in in compost plots had 15% more flush shoots than in plots without compost with no effect of mulch type on number of flush. The 3rd trial was planted on 17Mar14 at the Florida Sustainable Research Farm in Vero Beach to evaluate plantings in metallized reflective mulch and urban plant debris compared to a conventional cropping system. ‘Ray Ruby’ on ‘sour orange’ was planted 12′ x 25′ in a RCB with 4 reps. Irrigation and fertigation is supplied by 2 Bowsmith 2 gph drip emitters per tree. Trees were pruned after planting, trunks wrapped in 10′ aluminum foil and trees treated with imidacloprid again after planting. These last 2 trials only received funding for one year which ends 30 June 2014.
The bacterium Candidatus Liberibacter asiaticus (CLas) is closely associated with the development of HLB and is transmitted into the citrus phloem via the psyllid insect. Using the fully sequenced CLas genome, we have identified 27 proteins that are predicted to be secreted outside the bacterial cell. These bacterial proteins are called effectors. In other bacteria, effectors are required for pathogen virulence enabling nutrient acquisition, insect feeding, and suppression of defense responses. The goal of the funded research will be to investigate the expression patterns and citrus targets of four different CLas effectors that are expressed in HLB-infected citrus trees. We hypothesize that these effectors are important for bacterial survival or HLB symptom development by targeting important citrus proteins to manipulate their host. A detailed understanding of these effectors and their citrus targets will facilitate HLB detection strategies as well as provide pathogen targets that can be manipulated to enhance plant tolerance and resistance. We have made significant progress in developing tools to assess CLas effector expression. When a gene is expressed, it is first transcribed into RNA and then translated into protein. In order to assess effector expression at the protein level, the Ma lab has previously generated antibodies that can recognize each of the four CLas effectors. During the funding period, we have purified each effector and used this to affinity purify each antibody. This has resulted in significantly enhanced detection of effector proteins with no interfering background detected in navel, mandarin, or Lisbon lemon. These purified antibodies can now be used for effector detection as well as effector targets in the funded work. In the Contained Research Facility at UC Davis, we have started a time-course experiment to investigate the expression of each effector in navel and mandarin orange over time after graft inoculation with CLas. We have validated that the infected material is PCR positive for CLas, contains the four effectors, and expresses the effectors at the RNA level. Leaf tissue from plants at time zero (before inoculation) and one month after inoculation has been collected and is currently being processed to extract DNA, RNA, and proteins. We anticipate that samples will be taken for at least six months, or until the plants exhibit severe disease symptoms. In collaboration with Siddarame Gowda at the University of Florida, citrus expressing each effector is being generated using the CTV-based expression system to determine if effector expression results in any obvious morphological changes, enhances susceptibility to CLas, or promotes psyllid feeding.
The productivity of the Core Citrus Transformation Facility (CCTF) in the time between January and April of 2014 was higher than it was in the previous quarter. We have continued to produce transgenic plants for different research groups from the state of Florida and beyond. The work done within this quarter concentrated mostly on old orders. In communication with one research group, which is the biggest client at this time, the decision was made to prioritize the work on some of the orders they previously placed so CCTF had to shift the efforts towards specific orders and disregard time when the orders were placed. Also, through the communication with the CREC Director, CCTF received direct order from CRDF to produce some rootstock plants transformed with the NPR1 gene. These efforts are being coordinated with the Mature Tissue Transformation Lab (MTTL). Considering the importance of this project, CCTF started working on it immediately. Initial co-incubation experiments were done just a few days upon receipt of this order with the plant material obtained from MTTL. However, the quality of seedlings obtained from the MTTL was not good and results from that series of experiments had to be discarded. Rootstock cultivars of Carrizo, Swingle, and Citrus macrophylla are being used in this project. C. macrophylla seeds used for production of seedlings, that are the source of explants for co-incubation experiments, seem to be carrying some endophyte as most of the material obtained from these seeds ended up being contaminated. Because of this observation, the decision was made to put the emphasis on the Carrizo and Swingle cultivars. The work on this order is continuing at high pace. Plants produced by the CCTF within this three months period belong to the following orders: pNah-10 plants, pN9-seven plants, pN18-six plants, pX11- eight plants, pW14- three plants, pX20- one plant, pX28- one plant, pN7- one plant, pHGJ4- one plant, pMed16- one plant, pMed14- two plants, pELP3-G-nine plants, pELP4-G- three plants, pTMN1-five plants, and pMG105- two plants. Altogether 60 plants were produced and they were all Duncan grapefruit.
The citrus Greening Bibliographical Database http://swfrec.ifas.ufl.edu/programs/entomology/hlb_db.php was created in 2009 by the entomology group at the Southwest Florida research and Education Center (SWFREC/IFAS) in collaboration with the Florida Center for library Automation at the University of Florida. The objective of the greening database is to make available in a single location, the literature and all the information related to HLB, the vectors of Candidatus Liberibacter species, Diaphorina citri and Trioza erytrea, the effects of the disease on these insects and on plants, and also information related with the management techniques of the vectors and the disease. This information can thus serve as a useful tool for the growers, consultants, researchers and students. The database is continually updated and includes refereed and non-refereed publications, proceedings, presentations, reports, extension publications, periodicals, dissertations, book chapters and abstracts. Since October 2013, 249 new references were added to the database giving at the present 3,378 references with more than 92.5% of them linked to the original resources. The database has open information from around the world and some of this information is in different languages such as English, Spanish, Portuguese, French, Japanese and Chinese. Last year, the Citrus Greening Bibliographical Database was presented in two nationals entomological meetings: the 96th Florida Entomological Research meeting (July 14-17 2013, Naples, Florida) and the 61st Entomological society of America meeting (November 10-13 2013, Austin Texas). Our goal in this project is to continue uploading the most current HLB related information.
Obj 1A: All components of the user-friendly web versions of the single (sTCW) and multi-(mTCW) psyllid transcriptome databases have been finalized in preparation for the public release, which will allow for rapid target identification based on differences in expression, and in combination with predicted functions and tissue tropisms in the midgut and salivary glands. Twenty-two transcripts with predicted importance in nutrition, adhesion, immunity, and defense have been validated (mRNA) for ACP adults by RT-PCR. Two manuscripts reporting these efforts are ready for immediate submission. Obj 1B: Yeast-2 hybrid studies to capitalize on in vitro protein-protein interactions important in psyllid-Liberibacter interactions that support CLas infection, multiplication, and circulation in the vector that facilitate CLas infection of citrus plants. Previously, reported 15 CLas candidate genes from a list of 25 putative candidates (identified in silco using the db, obj. 1), were cloned into the Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut and salivary gland libraries. To date, 17 gut library matings and 16 salivary gland library matings have been performed. Data analysis has been completed for 33 of those experiments with the remaining being in various stages (PCR, cloning, sequencing, etc.), and moving towards completion. From the 33 experiments, 50 putative ACP gene products of high interest were identified as candidates for RNA interference (RNAi) testing. Previously we reported results of the pilot test hits, optimization of RNAi feeding/transmission assays, and qPCR analysis of CLas detection in psyllid- inoculated tomato plants. Revised efforts are directed at promising candidate effectors that will be moved into the RNAi testing, many which are predicted to be important in Liberibacter adherence to host tissues. We last reported results from 9 ACP candidates mated against the CLas prey library, showing two ACP candidates with putative functions in invasion/adhesion and defense, interacting with three different CLas proteins, including a CLas pilus-associated protein. At present, 14 candidate ACP genes putatively involved in bacterial adhesion, endocytosis, nutrition, and defense were mated against the CLas prey library. Several interacting gene products (‘prey’ inserts) have been identified and additional candidates are under analysis, including a putative virulence factor identified using an ACP clathrin-like protein as bait. The identification of a potential CLas virulence factor from a mating using this ACP gene highlights the importance of our bi-directional Y2H analysis (‘ACP bait’ against ‘CLas prey’ and ‘CLas bait’ against ‘ACP prey’). This Y2H result suggests that the knockdown of this ACP candidate may inhibit CLas transmission. The identification of ACP/CLas interacting partners and subsequent knockdown of the ACP interactors is ongoing. The confirmation of Y2H interacting proteins and identification of additional interactors using immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are underway. Two CLasY2H candidates with putative involvement in adhesion and quorum sensing were selected to optimize IP and Co-IP assays among the high number of ACP ‘interactors’ identified thus far by Y2H. Outputs from this approach will be native interacting proteins for mass spectrometry identification following confirmation by western blot analysis. Obj 2: RNAi studies are underway to functionally validate candidate effectors that pass all validation steps. To this end, good quality dsRNA has been synthesized for eleven psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport or transcytosis, and nutrition. Previously we reported on the knockdown of 6 of these genes on Liberibacter transmission using oral, topical, or and microinjection delivery. To date, dsRNA for 10 genes have been tested for adult PoP psyllids, and knockdowns have been assessed by qPCR analysis, with a range of 40-94% knockdown per target transcript. They also have been tested in our transmission bioassay, with five showing reduced transmission frequency. Confirmation of knockdowns and transmission experiments are ongoing for additional candidates.
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