A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
A number of experiments and observations at the USDA/ARS Ft. Pierce citrus scion improvement program demonstrate resistance or tolerance to HLB in some conventional citrus genotypes. In the funding cycle that began in 2012 our efforts in this area are markedly enhanced by funding for a postdoctoral researcher. Dr. Sharon Inch, who has extensive experience in plant pathology and histology, began working on this project 10/9/2012. She will move forward with established experiments comparing HLB development in specialty cultivars, which displayed resistance in a study of commercial groves, vs. susceptible standards. A set of replicated plants have been initiated for 50 genotypes representing advanced USDA selections, genotypes which display apparent resistance/tolerance in field observations, and susceptible standards: these will be exposed to HLB in a hot psyllid house and will be assessed for CLas levels, HLB symptoms, and growth. The remaining proposed experiments relating to assessing and characterizing HLB resistance/tolerance will be initiated in the next quarter.
The funding cycle that began in 2012 provides for a postdoctoral researcher in the USDA/ARS scion transgenic program. This will markedly enhance progress. Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. She will move forward with new constructs and resulting transgenics, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline.
In the initial funding of the current grant we have made progress on several of our objectives: Objective 1. Evaluate existing transformed lines: We have been maintaining a steady effort in the growing and testing out of candidate transformed lines from our large transformation efforts. Over 34,000 transformations of Duncan grapefruit have been carried out with 8 different reporter and resistance constructs, from which we have generated over 600 T0 plants that are being used for PCR analysis and pathogen testing. We are currently testing plants. The frequency of functional transformants may be lower than typical given that inappropriate leaky expression will be counter-selected. Objective 2. Expand stable transformations Our efforts to transform additional commercial citrus species has been focused on Ruby Red grapefruit and sweet orange. At present we have introduced eight constructs consisting of promoters with1,4, or 14 TAL effector/PthA binding sites with GUS or resistance gene coding regions in more than 1200 Ruby Red and over 600 sweet orange transformations, producing about 60 T0 plants for analysis. Objective 3. Refine constructs We have initiated additional resistance gene constructs that contain additional promoter elements and/or use another resistance gene known as AvrGf2. As each new construct is completed, we are testing these it in transient and stable transformation assays. Objective 4. Sequence more TAL effectors from additional canker accessions We have new sequences of TAL effectors from strains from Florida, Argentina, and Brazil, including the Miami “type” strain, as well as several strains with altered growth phenotypes. Last, we are expanding efforts at examining effects of resistance gene constructs on population growth of a range of Xanthomonas citri strains.
The current main priority is hiring the project postdoctoral fellow in the Zipfel laboratory. Whereas an initial candidate was identified, the hiring was not completed and additional candidates are currently being interviewed. The intention is to make an offer to a qualified postdoc by mid-November. Others in the lab are progressing the initial objectives: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. We have prepared constructs for EFR, XA21, EFR-XA21 and XA21-EFR chimera. We have carried out transient expression assays in N. benthamiana and found that the proteins express well. We have shown that the XA21-EFR chimera is functional, as exemplified by a gain of responsiveness to elf18 in a ROS burst assay. We are currently testing the functionality of XA21 and XA21-EFR by treatment with the axS17 peptide. This protein is unstable and difficult to synthesize, so we have obtained wild-type and ax21-minus strains of Xanthomonas euvesicatoria so that we can make extracts to test XA21-induced responses in N. benthamiana.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are under canker resistance test. These plants have been propagated and will be used for citrus greening test. As an extension of the project, we tested whether exogenous NAD+ could induce resistance to citrus canker. Exogenous NAD+ has recently been found in our lab to be a strong inducer of systemic acquired resistance (SAR). Since SAR has been shown to be effective against citrus canker, we expected exogenous NAD+ would induce resistance to canker. Indeed, our preliminary result showed that exogenous NAD+ activated strong resistance to citrus canker. We are confirming this promising result. For objective 2, we are continuing the direct genetic screen for citrus varieties with increased resistance to citrus greening. Seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds have been inoculated with psyllids carrying greening bacteria. Seedlings developing greening symptoms have been removed. The remaining seedlings will be re-inoculated with psyllids carrying greening bacteria.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. One mechanism we investigated to see whether it contributed to this resistance was plant hormones. We sprayed salicylic acid, methyl jasmonate, and abscisic acid, which are all common plant hormones, on susceptible citrus plants to test the influence on host choice, oviposition, development, and survival of ACP. Abscisic acid cut the life span of adult ACP in half compared to untreated control plants. However, the plant hormones had no other effects on ACP. A chemist-collaborator also is continuing to analyze plant volatiles collected from resistant and susceptible plants in hopes of identifying differences that promote resistance. We hope all of the volatiles are identified within the next several months so we can begin testing ACP attraction and deterrence to these volatiles in the field and laboratory sometime next spring. We continued to screen citrus for resistance to ACP. However, mites have delayed our greenhouse experiments because they interfered with egg-laying and feeding by ACP. We performed a series of tests with six miticides to see which ones could be sprayed on plants and experimental ACP without killing them. Acramite, Kelthane, and petroleum oil do not kill ACP eggs and Acramite is also safe for nymphs and adults. This information is invaluable because we now have a management tool to reduce mite populations and enhance rapid screening of citrus plants for resistance to ACP. We have begun screening 20 genetic lines of citrus hybrids for resistance to ACP. We think these lines may have some resistance because they contain 1/4 to 1/16 of P. trifoliata in their genetic background. We evaluated resistance to oviposition and adults. Only one replication has been performed so far, but preliminary results indicate that as many of 15 of these genetic lines may express some resistance to ACP. We are initiating a second replication. Collaborators at the Fujian Academy of Agricultural Sciences conducted behavioral bioassays on ACP to determine how quickly it identifies host plants and the different structures of plants. They discovered that ACP quickly distinguishes between a host and non-host plant, but they take longer to distinguish between two host plants and structures within a host plant. They also conducted free-choice tests with all major groups of citrus and found differences among and within groups. Most groups of citrus were colonized by ACP, but lemons were the most preferred group and sour oranges and kumquats were the least preferred. The differences among citrus varieties within a group may be useful because volatile and phloem contents that differ between the least and most preferred species can be compared.
3rd Quarter (final funding period): This quarter was largely devoted to the development of experimental approaches to be employed in the assessment of resistance to Las infection in the transformed lines. These experiments were aimed at developing protocols that can detect and quantify the survival of Liberibacter in the early stages of infection in our transformed lines expressing various constructs of R proteins. 1-Test for possible feeding preference between transformed versus non-transformed citrus: Preliminary analyses were conducted to determine whether the introduction of inducible and constitutively expressed resistance R genes affected the feeding preferences of uninfected psyllids. The cuttings of all transgenic citrus plants were subjected to uninfected psyllids feeding. There was no observable difference in psyllid feeding behavior preference between transformants and control citrus plants, which is a condition that will facilitate the assessment of resistance in the transformants. 2-Development of a one-step DNA extraction protocol for PCR analysis of Las infection: Our strategy was to develop a facile and sensitive assay using heavily infected citrus leaves from nontransformed citrus initially, before subsequent application to transformed lines. A variety of genomic DNA extraction procedures were tested with an emphasis on limiting the quantities of plant material to the smallest possible, as well as assessing protocols that involved addition of plant material to extraction solutions followed by a brief heat treatment and then direct addition to PCR reactions. Detection of Las rDNA was reproducibly obtained using 1 mm and 0.5 mm midvein cores. Overall, extraction procedures that did not require prior genomic DNA purification (one-step approach) gave better results at lower extraction solution volumes; however, quantitative real time PCR was adversely affected to some extent by some of the extraction solutions utilized in the one-step approach. In order to precisely quantify the copy number of Las in infected citrus plants we established standard curves for Las using the plant mitochondrial cytochrome oxidase (Cox) gene as a control to measure the amount of plant material in the sample. Likewise, the wingless Wg gene served as a control in psyllid extractions. Standard curves were based on calibration curves constructed using purified PCR amplicons obtained from plant and psyllid genomic DNAs. Based on the assumption that cloned Las, Cox, and Wg amplicons may represent more accurate copy number reference, all three standards were cloned in pUC19 (Las and Wg) and pUC119 (Cox). In construction of a heat map of infection using a heavily infected leaf, Las copy number (16S rDNA) varied across midvein sections, with the secondary vein and a non-vein section of the blade showing the lowest amount of Las. The ability to detect Las 16S rDNA in 0.5 mm midvein cores suggests that fine-scale mapping of the early infection is feasible. 3-Netted single-leaf clip cages used to detect initial infection stages: Ten psyllids from an infected population (furnished by the Dawson laboratory) were placed in single-leaf clip cages and allowed to feed for a period of 7 days and then removed for PCR determination of Las infection. A total of 10 leaves were exposed to infected psyllids and were harvested at one week intervals for PCR analysis of Las copy number. Las/Wg copy number ratios varied from 2,238×10-5 to 23,575×10-5 in the psyllids. Early detection of Las from midveins was feasible; however, the sensitivity of the assay in its present form was still needs improvement. We continue to make adjustments to our testing conditions including the modification of psyllid-containment cages.
Work continued to develop new rootstocks with outstanding attributes for Florida production, including tolerance to HLB. Tree infection and health information were collected from several established field plantings. Fruit quality evaluation began on one large rootstock trial with grapefruit scion. Grapefruit quality results from this trial last year yielded surprises in comparisons between sour orange and several other rootstocks, with US-812, US-852, and US-897 producing fruit of high quality early in the season. One new collaborative field trial with supersour rootstocks was established. Tree propagation continued for five more field trials. More than 9000 cuttings were made from supersour selections in preparation for disease testing and producing budded trees for field trials. Cooperative work was continued with a commercial nursery to multiply 250 advanced supersour selections for placement of trees into cooperative field trials with growers at multiple locations. Work continued to assess supersour tolerance of CTV, Phytophthora, Diaprepes, and high pH soils, using carefully controlled tests in the greenhouse and the field. Specialized testing of the supersour hybrids and concurrent field trials will effectively identify specific supersour selections that are equal or superior to sour orange in horticultural attributes and effects on fruit quality, as well as provide disease resistance or tolerance. Work continued to understand the genetic and physiological basis of tolerance to HLB exhibited by some citrus rootstocks. A study of metabolic changes in HLB infected germplasm is being completed to supplement the gene expression study completed last year, including HLB susceptible and tolerant cultivars. Detailed evaluation of specific defense-related genes continued, including CDR1 and PDF2, identified by microarray as being responsive to HLB in tolerant rootstocks. The results of these studies will provide target defense gene and regulatory sequences, as well as insights, to help design transgenic citrus with resistance to HLB infection. Manipulating expression of citrus genes will allow the creation of cultivars with increased HLB tolerance using only citrus origin genes. Knowledge gained will also help guide crosses for the creation of conventional hybrids with improved HLB tolerance. A detailed study comparing tolerance of rootstocks to HLB was accepted for publication. The second half of a study is underway to define the interaction of rootstock tolerance with scion tolerance/susceptibility, and the completed study is expected to be published by early next year. Trees were propagated for an expanded study to assess the additional benefit of expanding the amount of a tree that is the HLB-tolerant rootstock to include the trunk and scaffold branches. Selected anti-microbial and citrus plant resistance genes were inserted into outstanding rootstock and scion cultivars to develop new varieties with increased resistance to HLB. A manuscript was published comparing the gene expression with five different promoters transformed into US-802 citrus rootstock. More than 200 new transgenic plants were produced, including the genes CtEDS5, CtPAD4, CtNDR1, and CtACD1. Twenty new transgenic rootstocks with selected antimicrobial genes were propagated and entered into controlled greenhouse tests to assess tolerance to HLB. Eighty more transgenic rootstock selections were propagated in preparation for additional greenhouse testing with HLB. A field trial continued with selected transgenic rootstocks to evaluate performance under natural field infection with HLB. Collaborative work continued to assess rootstock interaction with scion, nutrition, and management factors in determining tree tolerance to HLB. Collaborative work continued to assess small RNA associated with HLB infection and tolerance. A presentation was made to Immokalee citrus growers on new citrus rootstocks and the USDA citrus rootstock development program.
After a long history of battle with pathogens, plants have evolved a complex immune system that consists of multiple layers of immune receptors and signaling regulators. One layer of this system is meditated by resistance (R) genes capable of recognizing pathogen effectors and subsequently inducing defense responses through a number of downstream regulators. R gene-mediated immunity is often associated the hypersensitive response (HR), characterized by localized, rapid cell death at the infection sites. It has been well documented that overexpression of a number of R genes and/or defense regulators from diverse plant species can trigger pathogen-independent HR in the model species Nicotiana benthamiana. This phenomenon has also been used to identify defense regulators. We recently identified two closely-related citrus genes, temporarily named CtHRT1 and CtHRT2, capable of inducing HR-like cell death when overexpressed in Nicotiana benthamiana by using Agrobacterium infiltration-mediated transient expression. Database searching and sequence analyses revealed that other plant species, including rice, Arabidopsis, tomato, Nicotiana benthamiana etc., also contain CtHRT orthologs and this group of genes shares high levels of sequence identity at the amino acid level, suggesting an invariant role in plant defense. To test this idea, we overexpressed the rice and Arabidopsis orthologs of CtHRTs in Nicotiana benthamiana and found that they all induced a similar HR. Therefore, CtHRTs represent a family of evolutionarily conserved defense regulators and could be used to heighten defense against citrus diseases including greening and canker
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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. 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 one week, 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. This report marks the end of the first quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project is being hired, two small greenhouses for rearing psyllids are almost completed, and general supplies including insect cages have been procured. USHRL dedicated an existing conventional greenhouse for the project, erected two new hoop houses for the project, and assigned a support scientist to the screening project. Additional ARS funds were used to increase the bio-security of the existing greenhouse to guard against invasion of parasitoids of the psyllid. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
Initial funding for this project was finally obtained on July 03,2012. McTeer trial – (3-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): 450 trees in this trial were visually scored for HLB symptom severity, and leaf samples from each tree were collected for PCR analysis (being run by the SG diagnostic laboratory c/o Mike Irey). Complex tetraploid rootstock Orange #19 (Nova+HBP x Cleo+Arg.trifoliate orange) showed minimal disease symptoms, whereas Swingle was in the middle of the pack, and the worst average symptoms were observed on Flying Dragon and Rich trifoliate orange. There are no Phytophthora issues in this trial at present (as determined by JH Graham). St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 4.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). 200 trees identified by the CREC scouts as being HLB positive were visually scored for HLB symptom severity. As with the McTeer trial, complex tetraploid rootstock Orange #19 showed the least disease symptom severity. Swingle again placed in the middle, whereas Kuharske and Volk controls scored very high disease symptoms. Complex tetraploid Orange #4, a sibling of Orange #19 also showed minimal HLB symptoms at both sites. Leaf samples from around each HLB positive tree, and from symptomatic areas of each tree were collected and sent to the SG diagnostic laboratory for PCR analysis (again c/o Mike Irey). Percentages of trees diagnosed with HLB differed significantly as well, with Kuharske citrange showing a very high infection rate. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Initial experiments to transform promising complex tetraploid ‘tetrazyg’ rootstocks Orange #4, Green #7 and Orange #19 with protecting constructs are underway, and 20 transgenic lines of Orange 4 and 7 lines of green 7 have been recovered to date. Progress was also made building two-transgene constructs that contain an antimicrobial gene for HLB resistance combined with an insecticidal gene to protect against psyllids.
Initial funding for this project was finally obtained on June 20, 2012. Construction of the rapid flowering system (pvc pipe scaffolding system) in the greenhouse has been completed. Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks and growing in airpots have been entered into this RES system. The plants have been single stemmed, and some are already approaching 6 feet in height. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. The first experiment combines our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene). The two-transgene Gateway based cloning system was employed to build the 2-gene construct. The NPR1 gene is under control of the rolD promoter while the CEME gene is under control of the d35S promoter. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance. Experiments to combine the NPR-1 gene with other lytic peptide transgenes including CEMA and AttacinE are underway, also using the new Gateway technology.
More than two thousand transgenic lines we have produced thus far, and are planted in field trials (2 locations under permit) or are in greenhouse tests at the CREC and with grower-collaborators; we continue to monitor the HLB and canker resistance or tolerance of these lines over time. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified by genome mining as well as from other disease resistance plant research. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are being continued examining interactions of rootstock and nutrients in severity of HLB symptom expression. Hot psyllid greenhouse facilities are now being used routinely to assess performance of transgenic citrus (representing our most advanced constructs with phloem-limited promoters and previously proven genes), as well as hybrids between citrus and Poncirus, for responses to psyllid feeding and HLB development. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Additional seedless pummelo-grapefruit hybrids have been identified during the 2011-12 season, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that address canker and marketing issues of ordinary grapefruit. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, and planted in a new field trial. A second demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, was established to assess to demonstrate their performance and utility in commercial processing, in collaboration with a major juice processor; these trials allow comparisons to be made between different production regions with the same sweet orange candidate selections. A well-attended field day was held in mid-November at the large CREC field experiment at the St. Helena block in Dundee; this featured Vernia and Valquarius orange trees grown on a number advanced rootstock selections, and highlighted early performance (yield and HLB effects).
The Core Citrus Transformation Facility (CCTF) continued to produce transgenic plants at expected rate of about a 100 per quarter. Plants for the following orders were produced: eight Duncan plants (ELP3 gene); ten Duncan plants (ELP4 gene); 15 Duncan plants (p7 gene); three Duncan plants (p10 gene); three Duncan plants (pWG19-5 vector); three Duncan plants (pWG20-7 vector); five Duncan plants (pWG21-1 vector); 15 Duncan plants (pWG22-1 vector); five Duncan plants (pWG24-13 vector); 12 Duncan plants (pWG25-13 vector); five Duncan plants (pWG27-3 vector); four Hamlin plants (pLC220 vector); eight Duncan plants (p35 gene), 11 Duncan plants (35S-TRX vector), and two Duncan plants (SUC-TRX vector). CCTF received three new orders in this period. The decision was made to replace the soil to which plants get transferred to following successful grafting. Use of soil that would get contaminated occasionally resulted in a loss of plants in previous periods. A special, commercially available, foam product is being used instead of commercial potting soil. The problem of loss of plants due to soil contamination has now been remedied.
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