This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. We now are examining the possibility of treating infected plants with antimicrobial peptides to allow them to recover from an HLB infection. We are screening a large number of transgenic plants for other labs. We have promising transgenic plants that are being rescreened to ensure efficacy against HLB. We are beginning to work with a couple of teams of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms. Results from the budding material came back all positive, meaning that all trees had been exposed to HLB as intended. On July 2015, all trees from both treatments were sampled for HLB. In one set of trees, leaf samples were taken from both sides of the girdle to test for the potential transfer of CLas material across severed phloem cells. In the other set of trees, in which the girdle was placed in the main trunk, was also tested for HLB. In the tree with the girdle on one side branch, 23 out of 25 trees turned out HLB+. In the other treatment, 24 out of 24 trees turned out HLB positive. From the data, we conclude that genetic HLB signal is sub-cellular in nature and can be transferred between non-phloem living cells. This new finding is important in that it shows that HLB causing effector is not necessarily the entire CLas bacterium.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Six months after grafting, HLB- budded tissue continues to grow in all treatments, especially after spring flush. At this time (May 8, 2015), leaf samples were taken from the uninfected tree and sent for HLB analysis. This type of analysis will be conducted every three months. Meanwhile, all trees plus controls are being monitored for HLB symptoms.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner. Graft material was allowed sufficient time to connect and grow onto the tree, or to perish and be replaced. Budded material began to show signs of growth after 2 months of grafting with a high success rate. Once buds began to grow, trees were trimmed and kept under observation until sufficient growth takes place for HLB testing.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. To conduct tree girdling in specific patterns to severe direct phloem connections, several techniques were assessed in a preliminary experiment in the greenhouse. To start with, a technique to create spiral girdling was developed and chosen. Trees have been purchased and a total of 25 trees were girdled with this particular spiral pattern on the main stem. A similar girdling pattern is now being applied to a secondary branch on a second group of 25 trees. The trees are kept in a greenhouse and watered regularly. Once all treatments and controls are finalized, they all will be challenged with HLB-affected tissue and assessed for HLB by PCR regularly.
The overall objective of this project is to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two sets of 25 trees previously girdled in a spiral pattern were allowed to recover and monitored for possible secondary effects of the girdling process. After determining that the trees were in good health condition, they were all challenged with 4 grafts of HLB infected tissue. These grafts were placed in precise locations in reference to the girdled area and were of 3 different types to ensure HLB transmission. The trees are now in the greenhouse and allowed to grow prior to testing on a quarterly manner.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct have been completed at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette has been introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Fifteen (15) independent transformant lines resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Of these 15 lines, 10 are strong expresses of the green fluorescent protein (GFP) transgenic marker, indicating successful transformation and expression of the transgenic marker genes. The other 5 show spotty expression of the GFP in cells of all tissues examined. This could indicate gene silencing might be affecting GFP expression in these plants. The effect that this might have on FLT-antiNodT fusion protein expression are not known, but will be tested later. The plantlets range from 5 – 15 cm in height, with the smaller plants being younger than the larger plants. All of the 15 lines appear to be growing and developing normally, which is a good sign that the FLT-antiNodT fusion protein is not having any unexpected deleterious effects on the plants. Permits to move the plants from Florida to Pennsylvania have been obtained, in order to study the FLT-antiNodT fusion protein expression levels and phloem-localization of the FLT-antiNodT fusion protein. Plans are being made to test the HLB resistance of these transgenic lines in collaboration with Dr. Tim Gottwald at the USDA Horticultural Research Lab at Ft. Pierce, FL.
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 have been preparing citrus plants for root treatment with NAD+. We are testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, about 15 more transgenic lines have been generated. We are currently characterize the transgenic seedlings. For the 20 transgenic lines generated previously, we have confirmed 15 of them containing the transgene. Expression of the transgenes have also been tested. These plants are growing in the greenhouse and will be tested for canker resistance. We are cloning the citrus homologs and will confirm the sequences of the genes before transformation.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. From the screening of random mutants which had previously been performed, it became clear that we needed to screen a larger number of mutant clones to find recognition of elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas we have been developing a FACS based screen. To this end we have generated a number of reporter lines in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4 cell suspension lines for responsiveness to elf18 and both of these give clear induction of the reporter gene following treatment. We will also test the WRKY30 and FRK1 cell suspension lines once the liquid cultures are established. In addition we have generated transgenic plants with these reporter constructs and are currently waiting to collect seed from primary transformants. We plan to test the activity of the pPER4:GFP cell suspension lines following protoplast transformation to ensure that the reporter is not activated during. If these tests are clear then we will proceed to FACS screen of mutant EFR libraries. In addition to the mutagenesis approach we are currently setting up to screen the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas, in order to determine whether a natural variant of EFR capable of binding elf18-CLas can be isolated. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Constructs containing EFR alone or in combination with XA21 or XA21-EFR were provided to the Moore lab, and epicotyl transformation experiments are ongoing (May to July 2014). To date a total of 2,945 ‘Duncan’ grapefruit and 879 sweet orange epicotyl segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). Preliminary GUS histochemical assays were carried out on 6 sweet orange shoots regenerated from transformation experiments with EFR, EFR-XA21 and EFR-XA21-EFRchim. Results indicate that no GUS positive shoots were obtained, however 1 shoot from the EFR-XA21-EFRchim construct was chimeric for GUS.
Citrus Huanglongbing (HLB), also known as citrus greening has been threatening the citrus industry since the early of 1900’s and up to this date there is no effective cure for this disease. Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components cell-to-cell communication systems. But the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We confirmed the functionality of LuxR by expressing in E. coli and the acquisition of different AHLs We detected AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. In fact the plant has another signals that bind to LuxR and then, the complex control CLas gene expression to be pathogenic (in susceptible varieties) or less pathogenic (tolerant variates). “Enhancement funding” In order to identify the molecules that influence CLas signals for movement and aggregation in plant, we compared the phloem sap composition of many varieties of citrus and citrus relatives. Phloem sap samples were collected from 14 varieties of healthy (not HLB) infected citrus and 4 varieties of non-citrus, which were derivatized using silylation reagents (TMS) as well as methylchloroformate (MCF) and then injected into the GC-MS. MCF is most sensitive toward free amino acids and some organic acids, whereas TMS is most sensitive toward organic acids and especially sensitive for mono- and disaccharide sugars. Therefore, all samples were analyzed with both derivatization methods. Phloem saps from citrus were found to have compounds such as malic, citric and quinic acids, the sugars glucose, fructose and sucrose, and 17 of the 22 amino acids found in nature. Sugar alcohols such as xylitol, mannitol and several forms of inositol were also found. Altogether, more than 85 different compounds were identified and about 50 have been confirmed using analytical standards purchased from Sigma Aldrich. The distribution of organic compounds in healthy citrus phloem sap in this study was between 22% and 62% sugars, 10-35% organic acids, 2-10% amino acids and 20-35% minor and trace compounds such as fatty acids, amino sugars, sugar acids, and sugar alcohols. in general, varieties can be well separated using principal component analysis to tolerant and suspectable groups. The data is in the final stages of analysis and the manuscript in is the writing stage.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. We reported cloning six new full-length cDNAs of different citrus genes into the entry vector last quarter. Now we have moved five of these clones into the binary vector pBINplusARS. Citrus transformation with these five clones shall be initiated shortly. In addition, a seventh new full-length cDNA was obtained in the entry vector for further DNA construction followed by plant transformation. In addition, we continue to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
This project aims to characterize conditions for optimal deployment of a superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from the development of the HLB disease and to treat trees that already established the disease. In order to provide protection against HLB, the superinfecting CTV vector will be carrying an anti-HLB insert: a gene of an effective antimicrobial peptide or an RNAi-based insert that would target a psyllid gene. The majority of trees in Florida are already infected with a CTV isolate. The goal of this project to understand how these pre-existing isolates would affect the establishment of infection with the superinfecting vector and, thus, expression and the production level of an anti-HLB insert. I the experiments, we are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees. We are assaying the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 and/or T68 isolate of CTV, singly or in mixtures (these isolates were propagated in our greenhouse) as well as with CTV-infected material obtained from the field trees (FS series isolates). In addition to wild type isolates, we also included several CTV constructs that could be used as vectors for expression of genes of interest in trees to see how they compete with wild type isolates. Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Real time PCR quantification is also being employed to these tests. In these experiments we are using different citrus rootstock/scion combinations in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the anti-HLB protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla. Evaluation of results is ongoing. As a result of this activities, we mapped several genomic regions within the CTV genome that determine the ability of an isolate of CTV to block the secondary infection with another virus isolate. We also discovered how a CTV virus variant that is already established in a tree could be removed from a tree (replaced by a wild type naturally occurring CTV virus). This discovery could be important in the situation when a recombinant CTV vector has to be removed from field trees if its presence is no longer needed or desired.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
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