A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. Dr. Grosser removed the unsuccessful trees from the first planting and planted additional transgenics among the promising trees still under trial. Additional plantings are welcome from the research community.
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 with citGRP1 were transferred to soil. Nineteen of them were test by PCR and ten of them are transgenic plants with citGRP1 insertion. They will soon be ready for RNA isolation and RT-PCR to check gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. 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 kan resistant transformants were generated on the selective media. About twenty kan resistant shoot are rooted in rooting medium and one of Hamlin transformatn was planted in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. 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. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About forty resistant shoots were rooted in rooting medium and ten Hamlin transformats were plant in soil. 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.
Our recent progress towards proposed research goals: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A] Mutagenesis of EFR to produce elf18-CLas responsiveness: Our initial approach of random mutagenesis and screening in tomato was unsuccessful, indicating the necessity to generate multiple mutations for elf18-CLas recognition. Presently we are evaluating phage display for this purpose. To this end, we have defined suitable conditions for specific binding of ectodomain fragments of EFR to biotinylated elf24. Elf24 has been used for these experiments to allow linkage of the biotin group to Lysine 24; this peptide is fully functional in the elicitation of ROS. We are in the process of evaluating binding of biotinylated elf24-CLas to different regions of EFR. Once optimal regions are determined, mutagenesis will be performed on these regions and cloned into phage display vectors for screening. B] Screening for natural variants of EFR: A small selection of Brassicae has been screened for elf18-CLas response, however none of these were positive. Pending the outcome of mutagenic approaches, the screen for elf18-CLas response will be expanded to a large number of Brassicae. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. Assessing XA21 function in dicots: Transgenic XA21-EFR, XA21 and EFR lines have been generated in Arabidopsis and are now ready to assess their effectiveness in pathogen defence. We plan to test these lines against Xanthomonas, Pseudomonas and Argobacterium. In addition, we have generated transgenic tomato lines expressing XA21. These plants will be crossed with EFR tomato lines to determine the pathogen resistance conferred by these two genes in a heterologous system. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. We will initiate the construction of appropriate expression vectors of genes for citrus transformation and expression.
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 gene, the N-terminal region (LasA1-A), two version for the repeat region with different number of the repeat sequences (LasA1-B0 and LasA1-B1), the C-terminal region (LasA1-C), and the full LasA1 gene. We are analyzing these constructs for their transient expression in Nicotiana benthamana and stable expression in transgenic Arabidopsis thaliana. The transgenic lines were obtained by floral-dip transformation of Arabidopsis Col-0 plants and we are currently verifying the gene insertion and mRNA expression level on T2 Arabidopsis. Three transgenic T3 lines expressing the gene are selected for analyzing phenotypes and protein localization using GFP pGWB2 vector. We are testing the expression level of the gene constructs that were transiently expressed in N. benthamiana with 35S, PFLAG and GFP pGWB2, 6 and 12 vectors. Localization of different constructs of LasA1 and LasA2 proteins using GFP vectors and pull-downs for protein-protein interactions using the PFLAG vector are in progress.
Cytoplasmic (CiLV-C and CiLV-C2) and nuclear (CiLV-N) citrus leprosis virus cause citrus leprosis disease in North and South American. All types of the CiLV are reportedly transmitted by Brevipalpus mite species. We continued mite transmission experiments at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD utilizing endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida. We completed mite transmission experiments after receiving the citrus leprosis affected samples from Mexico (CiLV-N) & Colombia (CiLV-C2). Six to seven weeks after completion of the transmission experiments none of the citrus seedlings showed any leprosis symptoms. For confirmation of the negative test results asymptomatic leaf tissue from the experiments was analyzed by reverse transcription polymerase chain reaction (RT-PCR) using CiLV type-specific primers but all plant samples were negative. Recently, nuclear CiLV was reported from Mexico but no prior sequence information was available. In work on another funded project we have successfully determined the entire genome sequence of nuclear CiLV and deposited the sequence in the NCBI Genbank. A manuscript also has been accepted for publication in the journal Genome Announcement (‘Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus’. Contacts have been made with collaborators in Mexico, Colombia and Panama for further shipment of infected leprosis samples to continue the transmission experiments with all 3 types of CiLV. Using newly developed PCR primers we will determine the viruliferous status of the B. yothersii after acquisition from infected leprosis tissue. Based on these results we will determine if Florida endemic healthy Brevivalpus Florida mites are able to acquire the various citrus leprosis viruses. In addition the utilized mites will be sent to USDA cooperators to continue to compare their taxonomic status with those that do transmit in Colombia elsewhere. Mite transmission work in Colombia continued with work by our collaborator Guillermo Leon. In acquisition experiments it was found that B. yothersii (phoenicis) mites effectively acquired CiLV-C2 after 30 minutes feeding on leprosis symptomatic leaves of Valencia orange. In transmission experiments with mites that were allowed an acquisition period of 24 h, positive virus transmissions were accomplished after only 10 minutes of feeding. Transmission rates increased incrementally up to a maximum after 24 hours feeding. However in using PCR to detect CiLV-C2 in the mites variable results were found. It appears that a minimal level of virus must be present in the mite for positive detection however PCR negative mites may still acquire and transmit the virus.
‘Sun Chu Sha’ and ‘Nagami’ kumquat plants were pre-treated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide 24 hours prior to inoculation with Xanthomonas citri pv citri (Xcc) in an attempt to determine whether CLas-flg22 was capable of inducing PAMP-triggered immunity (PTI). The Xcc population/concentration in the inoculated leaves was determine via colony forming units (cfu) by a standard procedure in a time course of up to 2 days post inoculation (dpi). Bacterial growth in the CLas-flg22 pretreated leaves was lower than in those treated with water in both Nagami and Sun Chu Sha, indicating that PTI was triggered. We used Xcc instead of CLas because of the impossibility of infiltrating the latter pathogen. Similar experiments using Xcc-flg22 also triggered PTI but to much higher levels than CLas-flg22. In other words, bacterial growth was slowed much more.
This is a new project that only just was funded, so there is not yet much to report. However, we do have some preliminary results. Cell penetrating peptides (CPPs) are small protein fragments that have been shown to be able to pass through the cell membrane that surrounds mammalian cells. More significantly, when the CPPs translocate in this manner they can also escort ‘cargoes’ across the membrane. Cargoes include proteins, plasmid or linear DNA, RNA, and antibodies that cannot enter the cell or blood-brain barrier without the presence of CPPs. CPPs have also been shown by others to work to introduce cargoes into plant cells. We have determined what CPPs work effectively in citrus for the import of proteins and nucleic acids. Imported DNA clones transiently express marker proteins; experiments on stable transformation have begun.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus is nearing completion. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA appeared to be unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA should not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We are now in the process of sequencing and verifying the pTLAB21-FLT-antiNodT transformation vector. Once this process is complete, we will commence citrus transformations. We anticipate that transformations will begin within the next reporting period.
In the period between April and July of 2013, Citrus Core Transformation Facility (CCTF) experienced significant changes in personnel. These changes reflected negatively on the productivity and as result, the goal of 100 transgenic plants per quarter was not reached. The experimental efforts were directed towards orders that were placed within previous six months. Produced transgenic plants were all Duncan grapefruit. They belong to following orders: MED16-11 plants; X20-four plants; X16-three plants; X19-one plant; X4-one plant; Bi121-six plants; N5-31 plants; N7-nine plants; HGJ3-one plant; and W14-one plant. As opposed to a few previous quarters when the number of placed orders was unusually high, facility received three orders during this time. The ‘genes’ of interest in those vectors were: gMOD1, ELP3-CIV, and ELP4-CIV. All three orders require transformation of Duncan grapefruit plants. Actually, two more orders were placed but after initial attempts to mobilize binary vectors into appropriate Agrobacterium strains the quality of cultures was reviewed and client decided to withdraw them. Within the first year (14 months) of funding of this project, CCTF received 35 orders for production of transgenic plants. Altogether, about 430 plants were produced. They belong to 23 orders received within this funding period and eight orders from previous period. Transgenic plants that were requested from clients in new orders were mostly Duncan grapefruit, but there were some requests for Valencia oranges, Carrizo citranges, and Mexican limes. Ratio of produced plants reflects well the ratio of requested plants in placed orders. CCTF remained a reliable producer of transgenic material. High influx of orders speaks of the important role that CCTF plays in efforts directed towards disease tolerance improvement of Citrus cultivars. Researchers that have well developed ideas or initial encouraging data about the beneficial activity of certain genes know that CCTF can help them test their hypotheses by producing citrus plants that carry those genes in them. While waiting for transgenic material from CCTF, those research groups can direct their efforts towards development of appropriate challenge tests that will be performed on transgenic plants or work on other aspects of protection against citrus diseases.
The project was extended with no cost for an additional nine months in order to conduct the final destructive sampling during the dismantling of the hydroponics greenhouse experiment, and to collect the final year of 2012/13 fruit yields from the ‘Hamlin’ and ‘Valencia’ field trials at Orange Hammock.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Twenty “Hamlin” trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were harvested in December 2012 for the fifth consecutive year. Half of the trees (10) were symptomatic for HLB (PCR+) prior to the first harvest five years before and the other half were non-symptomatic (PCR undetermined; healthy). The five consecutive harvests show conclusively that HLB, when managed properly, does not kill trees in 2 to 3 years as commonly reported in the literature. Also, the yield of the trees has remained statistically unchanged for the five years of the project, demonstrating conclusively that HLB can be managed and the profitable yields of trees infected with CLas can be sustained. For the 2012-13 harvest season, HLB-affected trees yielded an average 179 lbs of fruit per tree and healthy trees yielded an average 533 lbs of fruit per tree. Furthermore, the juice from all of the fruit (healthy, symptomatic and asymptomatic) has been and continues to be of superior quality, meeting or exceeding USDA standards for Grade A pasteurized not- from-concentrate juice. We state again, although HLB-affected trees have lower per tree yields than healthy trees, their yields are not declining and are statistically similar today as they were five years ago. It is important to note that the added nutrition has not produced a rampant, uncontrolled spread of HLB as evidenced by the healthy trees still producing very high yields. This is because psyllid control in the grove is excellent and the psyllid is the vector of the disease, not nutrition. The grove also shows no evidence of other serious pests and diseases impacting the root systems of trees, such as sting nematodes, Phytophthora and Diaprepes root weevils. HLB is most successfully managed with intensive horticultural practices in the absence of other pests and diseases, many of which don’t have a viable long-term solution. Multiple pest and disease problems superimposed onto HLB-affected trees are likely to result in their rapid decline and death.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Twenty “Valencia” trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were harvested in April 2013 for the fifth consecutive year. Half of the trees (10) were symptomatic for HLB (PCR+) prior to the first harvest five years ago and the other half were non-symptomatic (PCR undetermined; healthy). The five consecutive harvests show conclusively that HLB, when managed properly, does not kill trees in 2 to 3 years as commonly reported in the literature. Also, the yield of the trees has remained statistically unchanged for the five years of the project, demonstrating conclusively that HLB can be managed and the profitable yields of trees infected with CLas can be sustained. For the 2012-13 harvest season, HLB-affected trees yielded 1.9 boxes of fruit per tree and healthy trees yielded 3.9 boxes of fruit per tree. The juice from the fruit is awaiting analysis; however, for the previous three years the juice from all fruit (symptomatic, asymptomatic, healthy) has met or exceeded USDA standards for Grade A not-from-concentrate juice and based on samples tasted during juicing we do not anticipate a different result this season. We state again, although HLB-affected trees have lower per tree yields than healthy trees, their yields are not declining and are statistically similar today as they were five years ago. It’s also important to note that the added nutrition has not lead to a rampant, uncontrolled spread of HLB as evidenced by the healthy trees still producing above-average yields. This is because psyllid control in the grove is excellent and the psyllid is the vector of the disease, not nutrition.
This project was initiated May 1 2013 and we have focused our efforts on Activity 1. In this activity we are using a rational design strategy to develop and rapidly test individually the two components of a chimeric antimicrobial protein (CAP) based therapeutic that could provide broad spectrum resistance to young citrus plantings against a range of biotrophic bacterial pathogens (HLB and CBCD). Gene mining bioinformatics tools are being used to search existing citrus genomic DNA sequence information available in Phytosome (http://www.phytozome.net) to identify structurally relevant candidate citrus components that can be fashioned into a CAP molecule. The current and highly functional CAP design described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725) is being used as a scaffold/guide to search for the citrus components. Working with the developer of a recently described bioinformatic tool CLASP we have successfully searched using the active site architecture (3D shape) of the forst component HNE and have found a citrus PR14a sequence whose shape is similar. We have determined the similarity of the citrus protein shape to that of the tomato PR14 and grapevine PR14a proteins. We used the tomato sequence as the protein 3D structure has been determined and could thus be used to thread the citrus and grapevine protein sequences whose structure is yet to be determined. We have also been working on the finding the citrus DNA sequence to replace CecropinB component. We have identified several candidates and are evalusting the similarity in their structure with respect to the known structure of Cecropin B. We have begun the process of designing the vector system to express the citrus PR14a sequence that we have identified. This vector system will be used for the plant based expression system described in our Activity 2. Once designed and constructed will be expressed in Nicotiana benthamiana to produce active protein that can be tested for activity. We have also submitted an application to APHIS-PPQ for the movement of the recently cultured Leberibacter crescens (Lcre). Once we get permission then later this year we can test these proteins for their efficacy using the Lcre to evaluate growth and mortality.
109,913 I did not receive the fund (and hence could not start the project) by this time because of the prolonged negotiation between UCR and CRDF on the intellectual property terms in the contract of this project.
Obj 1A: To increase database mining capabilities the single (sTCW) [C. Soderlund, W. Nelson, M. Willer and D. Gang. (2013) TCW: Transcriptome Computational Workbench; PLOS ONE: accepted for publication] and Multi-TCW (mTCW) psyllid transcriptome databases have been updated and finalized prior to the public release of the database. Now, the Gene Ontology categories (sTCW) can be organized by the number of differentially expressed transcripts (GOSeq, EdgeR) they contain using pairwise comparisons of the different libraries. The addition of statistical software (EdgeR, Pearson’s Correlation Coefficient) to the mTCW_ht_WN2 database allows for key word searches and differential expressional analyses amongst the clusters which will allow for better identification of candidate effectors. To date, the expression of transcript members from 3 clusters shown to be significantly differentially expressed in response to Liberibacter have been validated in both ACP and POP by RT-PCR using primers designed from conserved regions. Obj 1B: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions to find key players involved in the circulative, propagative pathway. Previously it was reported that 6 CLas candidate genes had been identified and were in various stages (PCR, cloning, sequencing, etc.) moving towards Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut library and the ACP salivary gland library. That list now contains 19 CLas candidate genes all ready for mating experiments except for; 2 dropped out as unclonable (without a lot of extraordinary measures), 2 cloned and waiting on sequence verification. To date 9 gut library matings and 6 salivary gland library matings have been performed. Data analysis has been completed for 5 of those experiments with the remaining being in various stages moving towards completion (each mating experiment from mating to complete data analysis takes about 18-21 days minimum so stages of completeness are staggered). Through those 5 experiments we have thus far discovered several ACP gene products (‘prey’) that have various levels of interest for making them good candidates for RNAi knock down. The first good RNAi candidate emerged from the mating experiments using both the gut library and the salivary gland prey libraries. The ‘bait’ in this case was CLas-OmpA (Outer membrane protein A). Both ‘prey’ ACP gene fragments that interacted with the ‘bait’ show similarity to integral membrane proteins and could possibly play a role in adhesion or invasion. The next two good candidates came out of the mating experiment again with CLas-OmpA as ‘bait’ and the salivary gland library. The first ‘prey’ insert showed similarity to extracellular matrix-related protein possibly indicating some role with either adhesion or biofilm formation. The second ‘prey’ candidate has domain similarity to a novel component involved in activating signaling cascades in the innate immune response. The fourth and final good candidate to date comes out of a mating experiment between the gut library and CLas-MviN as ‘bait’. MviN has been identified in a variety of bacteria including pathogens and non-pathogens and plant-symbionts. It has been shown to be a virulence factor in Salmonella and is require for motility in Rhizobium. The ‘prey’ candidate pulled out has domain similarity both a protease inhibitor protein (invasion defense) and a putative collagen-binding domain of a collagenase. These four candidates will be moved into the RNAi phase of the project if they fit all criteria specific to RNAi. Obj 2: To date, good quality dsRNA has been made for four psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport or transcytosis. Feeding studies have been conducted for the gene putatively involved in cytoskeleton formation. Quantitative PCR analysis of psyllids fed on either dsRNA of target gene or buffer showed that there was roughly 20-30% down-regulation of target gene. Results from the transmission experiments performed with this first gene showed subtle differences in the transmission efficiency between plants fed on by dsRNA-treated and control psyllids at 82% and 100% respectively.
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