The antibody developer, Creative Biolabs, Inc., identified six monoclonal antibodies to the 30 amino acid peptide antigen used, which corresponds to an extracellular loop of the Candidatus Liberibacter asiaticus outer membrane protein NodT. The materials were received by Dr. McNellis’ lab at Penn State University in September of 2012. Four of the antibodies appear to be useful for the project, based on molecular analyses of their binding efficiency to the epitope target and their structural integrity. This type of single-chain engineered monoclonal antibody is provided to us as a DNA clone, from which we express the antibody in bacteria. We are currently working on producing the antibodies in E. coli bacteria. This material will allow us to test whether the antibodies can be used to detect NodT protein in protein extracts from psyllids and citrus trees. The transformation construct for expressing the FLT-antiNodT fusion protein in citrus has been initiated and will be completed soon.
From the three FT constructs created (FT1, FT2, FT3), the FT3 construct has shown significant induction of flowering on transformed tobacco plants. FT3 shows promising results at shortening the juvenility period in both citrus and tobacco. Compared to a wildtype control of tobacco the flowering time of F2 generation plants transformed with the FT3 construct is over 3 months earlier. A multi-faceted approach to understand the activity of citrus FT is underway. One of the approaches involves measuring expression levels of FT1, FT2, and FT3 in different Citrus varieties using Real Time PCR to determine FT behavior at different stages of growth. Analysis for the first 3 months has been performed on Pummelo and pineapple sweet orange varieties and all three genes are actively transcribed at different levels depending on the time period. The biological effects of various phytohormones such as ethylene and gibberellic acid on FT3 expression and flowering will be monitored. This approach will allow us to devise a method to delay flowering induction at early stages in citrus due to the observed premature formation of flowers at tissue culture stages. The final approach is to isolate the FT3 mobile protein and introduce it into phloem of citrus and tobacco through various methods in order to induce early flowering. The protein will be synthesized and various trials of exogenous protein application will be performed. This approach will allows us to create a practical protocol for shortening juvenility periods. In both citrus and tobacco the FT3 genomic construct with the constitutive FMV promoter is highly effective, causing very early flowering. Unfortunately, in citrus, the flowering occurs on the plate, before the transformed material is useable. Some work has been done in an attempt to control the speed of flowering using day length, temperature, and gibberellic acid. In a further attempt to control the activity of the FT3 gene, a construct using an inducible promoter is being produced. This inducible promoter is based on the activity of an ecdysone receptor and is induced using the chemical methoxyfenozide. Before this construct is developed, the effectiveness of the FT3 cDNA is being compared to the FT3 genomic DNA using the original FMV promoter in the hopes that the smaller cDNA will be just as effective and can be used in the new construct without changing the flowering character.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in wild type (Col-0) background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers in the isolated transgenic plants. We have been in the process of testing disease resistance of these plants to Pseudomonas syringae infection. Besides ctNDR1, our recent test of Arabidopsis overexpressing ctEDS5 also showed a complementation of the eds5-3 mutant with the citrus gene. Transgenic plants with potential enhanced disease resistance will be further selected to obtain homozygotes for additional tests of disease resistance. Such constructs will be preferentially used to transform citrus for citrus disease resistance tests. We continue to characterize citrus transgenic plants transformed with ctNDR1. We confirmed with PCR that 29 independently transformed plants carry the transgene. In addition, we conducted second round of infection with Xanthomonas citri (Xac) and results showed again that citrus transgenic plants overexpressing ctNDR1 were more resistant than untransformed controls. We are growing the plants and prepare them for a HLB test in the future. In the meantime, we have made additional transgenic plants with other citrus SA genes. The transformation is generally conducted with two to four genotypes for each construct because there are significant variations in transformation efficiency and resistance to HLB and citrus canker diseases in different genotypes. Besides transgenic plants overexpressing ctNDR1, we have so far made transgenic plants expressing ctEDS5, ctPAD4, ctNPR1, and ctEDS1, which are in US-802, US-812, US-942, and/or Hamlin background. The presence of these transgenes was confirmed with PCR in some genotypes. More transgenic plants are to be obtained from these transformation events from different genotypes. Additional constructs will be placed in the pipeline of transformation once they are ready. The transgenic plants will be prepared for resistance tests for citrus canker and HLB diseases as having been planned for the ctNDR1 transgenic plants.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time, we have about 60 different antimicrobial peptides or RNAi constructs are under test against HLB. Plant infected with the CTV vector plus a peptide or RNAi sequence are being inoculated by HLB in a psyllid containment room.
The objective of this project is to examine existing sweet orange/poncirus hybrids for tolerance to HLB and acceptable juice quality. We found that a poncirus hybrid developed by Herb Barrett, USDA Orlando, many years ago is tolerant to HLB. However, it has too much poncirus to be commercially acceptable. However, this and sister hybrids were used in further crosses to sweet orange. We are testing some of those hybrids. At this point we have propagated selected hybrids and are beginning to test them for tolerance to HLB.
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
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