FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted by an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. As reported previously, methyl accepting chemotaxis proteins (MCPs) from Xanthomonas strains were deduced based on whole genome sequence data and PCR analysis. MCPs from citrus pathogenic strains (Xcc) were closely related to one another and distinct from X. campestris pv. campestris (Xc). MCPs from Aw strains were more related to X. fuscans pv. aurantifolia (Xfa) and X.alfalfae subsp. citrumelonis (Xac) than to strain 306 of Xcc. Another analysis has been conducted to convert whole sequence data to binary form according to the absence or presence of each MCP. New specific PCR primers have been designed to detect the presence or absence of certain MCPs. Gene expression analysis confirmed the higher expression of fimA (XAC3241) the major subunit of Pilus type IV, at early stage of the biofilm formation in A and Aw when bacteria grown in LB or XVM2 minimal media; another fimA gene (XAC3240), however, showed a different expression pattern in Aw, showing the highest expression in mature biofilm. Expression of fimA gene (XAC3240) in Xcc A strain is higher in mature biofilm when this strain was grown in LB medium, while it is higher in early stage of the biofilm formation when grown in XVM2 medium. Differences in gene expression between Xcc A and Aw strains in the two nutrient environments are in accord with the structural changes observed during biofilm formation. Objective 2. To investigate biofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads. DNAse treatments were performed to confirm DNA content of aggregates and elucidate the role of DNA in biofilm formation. DNAse treatment of Xcc A, A*, Aw, Xac and Xc for different incubation times reduced biofilm formation for all citrus pathogenic strains but had little or no effect of biofilm formation by Xc and Xac. Biofilm formation by wide host range strains of Xcc was more affected at the beginning of the incubation period (0 to 24 h) while formation by restricted host range strains which were affected after a longer incubation of 72h. Xac biofilm was only affected at early stages of incubation. DNAse treatment of preformed biofilms reduced biofilm 40 to 50% confirming the high DNA content of mature aggregates. By comparison preformed biofilm of Xac and Xc was reduced only 20%. Differences in development of DNA structures of the biofilm between wide and narrow host range strains is related to the rate of biofilm formation by these strains analyzed by microscopic observations.
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 are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, but are eliminating many that are not effective and are focusing on about 20 that still are under test and about half of a dozen that have some 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. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
The goal of this project is to examine 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 gene (i.e. a gene of an effective antimicrobial peptide).The majority of trees in Florida are already infected with some CTV isolates. The main question for us is 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 polyprotein. 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.
The objective of this enhancement project is to characterize the chemical composition for different citrus cultivars that show different degrees of the susceptibility and tolerance. Previously we developed a centrifugation based method to collection the pure phloem sap in order to be able to quantify the compounds. The phloem sap of fourteen different citrus cultivars (listed in the end of report) was collected from one-year-old greenhouse plants. The collected phloem samples were derivatized either by methychloroformate (MCF) that specific for organic and amino acids or trimethylsilyl (TMS) that is specific for sugars. The derivatized samples were analyzed by GC-MS. Thirty-two compounds were identified in the phloem sap after MCF derivatization. These compounds were classified to three groups: amino acids, organic acids, and fatty acids. Over than fifty compounds were detected in the citrus phloem sap after TMS derivatization. The compounds detected after TMS derivatization included sugars, sugar alcohols, organic acids, fatty acids, and amino acids. Our MCF results showed a significant correlation between the concentration of phenylalanine, tryptophan, tyrosine, serine, leucine, valine, and histidine with citrus resistance to Candidatus Liberibacter asiaticus (CLas). Phenylalanine, tryptophan, and tyrosine are precursors for phenylpropanoid biosynthesis phenylpropanoid biosynthetic pathway which is induced in response to biotic and abiotic stress. At this point we are processing the GC-MS data obtained after TMS derivatization. Once we process all the data, we will analyze it to identify other potential phloem sap metabolites that are linked to citrus resistance to CLas. Citrus cultivars included: Valencia, Pineapple and Madam Vinous sweet orange (C. sinensis (L.) Osbeck), Duncan grapefruit (C. paradisi MacFadyen), Sour orange (C. aurantium L.), Volkamer lemon (C. limonia Osbeck ‘Volkameriana’), C. macrophylla Wester, Palestine Sweet lime (C. aurantifolia), Mexican lime (C. aurantifolia), Carrizo citrange (X Citroncirus webberi J. Ingram & H. E. Moore), Severinia buxifolia (Poiret) Ten, Poncirus trifoliata (L.) Raf. and Citrus latipes (Swingle),
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 have so far cloned six full-length cDNAs of citrus genes that potentially regulate SA, ET, and/or JA defense signaling. Agrobacterial strains containing these constructs were placed on the pipeline of citrus transformation in the co-PI Dr. Bowman’s laboratory. We also transformed Arabidopsis to overexpress these genes and to eventually test their defense function in Arabidopsis. We harvested T0 transformed seeds for some constructs and our initial screening of these constructs has yielded several transgenic plants for each construct. Arabidopsis transformation and screening have been continued during this past quarter. In addition, we are in process of cloning additional citrus genes. Six new full-length cDNAs of different citrus genes were cloned into the entry vector and will be further moved to the binary vector pBINplusARS for both Arabidopsis and citrus transformation and eventually for defense tests with the corresponding transgenic plants. 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.
In the past year, much attention has been drawn to the CRISPR/Cas9 system. In light of the system’s simplicity and efficiency, and since it has proven to work transiently in citrus (Hia and Wang, 2014), we have decided to use the CRISPR/Cas9 system in conjunction with our CPP transient expression experiments. It is our hope that we can use a modified version of CRISPR/Cas9, in order to activate and suppress target citrus genes depending upon their regulatory role. For example, we intend to suppress the expression of citrus terminal flowering protein (CiTFL), which is responsible for negatively regulating flowering during citrus adolescence, in order to reduce juvenility. The CRISPR/Cas9 system may allow us to perform this as well as other beneficial modifications, without the insertion of a foreign transgene. The focus of our work for this quarter has been primarily concerned with cloning of citrus optimized versions of a nuclease-free Cas9 (Cas9m4) with either an activator domain, an ecdysone receptor (EcR-B), or a suppression domain, Krueppel-associated Box (KRAB). These Cas9 vectors will be cloned into two different reporting vectors for transient expression assays with CPPs and, if necessary, agroinfiltration. The other aspect of our work has been concerned with designing proper sgRNAs, essential for the CRISPR/Cas9 system, for CiTFL and citrus phytoene desaturase (CiPDS), for use as a visual control vector. We intend to be done with most of the cloning by next quarter and intend to produce some transient expression data.
An experiment involving ciFT3 transgenic tobacco plants and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol(PBZ) is currently being monitored. The effects of the chemicals are evident due to the variation in phenotype observed. The transgenic line being used is a T2 that has shown to be homozygous for the ciFT3 transgene from previous GUS assay experiments. PBZ produced a short phenotype with succulent leaves and transgenic plants flowered earlier in comparison to the other treatments. Flower buds appeared 39 days after germination. Controls are being monitored for flowering and are expected to flower around the 150 day mark. The outcome of this experiment will be used to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages by perhaps using one of the compounds. RNA will be extracted at three different time points from each treatment to determine the relative expression of ciFT3 when treated with the compounds. New cDNA concentrations from the 12 month collection period of citrus tissue was used to run a new set of quantitative real time PCR amplification curves for the FT1, FT2, FT3 FLD, FLC, ELF5, and AP1 genes. Statistical analysis is being conducted to find any correlation in expression and flowering time. Work has proceeded designing a transcription activator-like effector (TALE) system inducible by methoxyfenozide that will chemically activate the naturally present FT3 gene in citrus. An 18 monomer TALE has been constructed based on the endogenous FT3 promoter region common to ‘Duncan’ Grapefruit, ‘Carrizo’ Citrange, Pummello, and Poncirus citrus. Progress is currently being made to assemble this FT3 TALE into a plasmid with a FMV promoter, a VP16 activation domain and the inducible ecdysone receptor-based expression system. The efficacy of this construct will be evaluated by co-transforming tobacco with the chemically inducible system and with a plasmid that contains the endogenous citrus FT3 promoter followed by a GUS reporter gene.
Experiment described in the last report continue as the main focus of the project. In addition, we have formed a collaboration with researchers from UF’s Department of Chemistry and the IRREC (Mingsheng Chen, Brent Sumerlin, and Zhenli He) who work with nano-particles chemically assembled using amphiphilic polypeptides (polymers). Our groups seek to develop a method using a combination of encapsulating nano-particles and cell penetrating peptides (CPPs) to deliver cargos to selected tissues (such as the phloem). The first step, undertaken this quarter, is to elucidate the potential toxicity effects of different nano-particles using a combination of tissue culture techniques and fluorescence microscopy. Then, we will examine combinations of benign polymers and CPPs that we have found to be effective in transferring cargo to citrus.
During the course of this project we found that PAMP-triggered immunity (PTI) plays an important role in citrus resistance against canker and HLB. Furthermore, there was a correlation between the level of resistance observed in different genotypes (from most susceptible to most resistant: ‘Duncan’ grapefruit, ‘Navel’ sweet orange, ‘Sun Chu Sha’ mandarin, ‘Nagami’ kumquat) and the extent and intensity of the PTI in terms of transcriptional gene induction of defense genes. We also found that PTI induced by flg22 restricted growth of Xcc in planta only in the resistant genotype (‘Nagami’ kumquat). Flg22 from CLas also induced defense gene expression reprogramming. Given these exciting results we used genomic resources available online to identify the receptor gene for flg22 (named FLS2). We will test whether the ectopic expression of FLS2 from resistant kumquat is capable of increasing the resistance in a susceptible genotype (grapefruit). We will clone the FLS2 gene from kumquat into an expression vector (we have all the necessary plasmids available) and perform transient expression experiments to compare defense gene expression levels and bacterial growth that would indicate an increase of resistance in grapefruit.
In the previous reports, we have reported the development of Soft Nanoparticles (SNPs) using two essential oils, EO A and EO B. The formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter Crescens (L.Crescens) and both the formulations and the controls showed > 90 % inhibition at 1, 5 and 10 % (v/v) dilution. Phytotoxicity of select formulations were performed at 1:1, 1:10 and 1:20 dilutions and all formulations showed low phytotoxicity when applied at 1:20 dilutions. The developed formulations were tested for their stability with adjuvants by a technique similar to the well-known ‘jar test’. Cohere and Cling are spreader-sticker type adjuvants that are frequently added to pesticide spray tank before spraying to enhance pesticide penetration. It was seen that most of the formulations were stable. Based on the efficacy, phytotoxicity and stability tests, formulations for EO A and EO B have been short-listed for possible field trial. SNPs have also been developed and characterized with Thyme Oil. The droplet size ranged from 3 to 18 nm and the oil loading ranged from 1 to 20% (w/w) using agriculturally approved surfactants. The surfactant loadings are similar with those in formulations with EO A and EO B. Addition of a co-surfactant greatly enhanced the oil loading to up to 20% (w/w). Selected formulations were also tested for stability with the adjuvants and most of the formulations were stable and the addition of adjuvants changed the particle size of the SNPs marginally but was still in the required range (5-16 nm). Future plans include testing of selected formulations of thyme oil for antibacterial activity / efficacy against the surrogate bacteria as well as phytotoxicity studies. To separate the efficacies of the essential oils from the surfactants, emulsions and microemulsions have been developed with low and ultra-low surfactant and oil loadings. Formulations have also been developed with different surfactants that are known to be safe and used in pharmaceutical formulations (e.g. Pluronic’ F127 and Tween’ 80). These formulations will be tested for their inhibition efficacy on the surrogate bacteria. These experiments will assist in understanding the efficacy of the oil in SNP as well as in determining the minimum amount required for L. crescens inhibition. Dye doped SNPs have also been developed to understand their foliar uptake. In addition, experiments are being planned to quantify the effectiveness of the adjuvants (Cling and Cohere) performance on citrus leaves.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the lasA1 and lasA2 genes. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana and citrus. We have obtained transgenic lines with these constructs. These transgenic Arabidopsis lines were verified by PCR and RT-PCR and their segregation in T2 and T3 were analyzed. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and/or overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. We also expressed LasA1 protein using the Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E. coli. Purified LasA1 protein are used for antibody production and crystallization study. Immunoprecipitation and elution of FLAG-tagged autotransporters from Agro-infiltration in Nicotiana benthamiana yielded several protein candidates, indicating LasA1/LasA2 interacted with mitochondria and chloroplast proteins. It is worth noting that we are able to detect LasA1 protein from Las-infected plant tissue using Western blot. The successful generation of this antibody will enhance our research in several aspects. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein has also been generated.
During the 3 years of funding, we identified and characterized a regulon from ‘Ca. L. asiaticus’ involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of ‘Ca. Liberibacter asiaticus’, using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. When bound to LdtR, the chemicals inactivate the protein, which disrupts a cell wall remodeling process that is critical for survival of the pathogen when exposed to osmotic stress (i.e. within the phloem of a citrus tree). Several model strains were used to confirm that the newly identified transcription factor (LdtR) and its regulated genes (ldtR and ldtP) confer tolerance to osmotic stress.These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease. The logical subsequent step of our research is the optimization of all necessary parameters to use the chemicals identified directly on field applications. The results were published in PlosPathogens Journal: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004101
Transgene Stacking for Long-Term Stable Resistance: PCR analysis of transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) resulted in identification of 7 lines that contain both transgenes. Agrobacterium mediated transformations to produce transgenic plants containing other combinations of stacked genes are in progress. Transgenic plants with the PCR positive stacked genes are being clonally propagated for further evaluation. Improving Consumer Acceptance: 1. In efforts to develop an intragenic citrus plant, we have in addition to the binary vector for an inducible cre-lox based marker free selection containing the cre gene driven by a Soybean heat shock gene promoter, a binary vector containing the cre gene driven by a citrus-derived heat shock promoter. Citrus rootstock Carrizo has been transformed with both of these constructs and numerous transgenic plants are being regenerated. 2. Hamlin and W Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells. One of the T-DNA contain a grapevine myb gene under the control of a 35s promoter and the other contain T-DNA containing the selectable positive/negative fusion marker cassette. Plants have been regenerated that are purple in color from the anthocyanin production. Pending molecular analysis of the regenerated lines, we speculate one of two possible scenarios: a) The plants contains only the T-DNA of interest or 2) The plant contains both T-DNAs integrated into the genome. Somatic embryos are now being germinated and resulting transgenic plants will be evaluated. Induction of early flowering: The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 has also been produced for testing. Since previously generated FT and AP1 plants flower in vitro but not as young plants in the greenhouse, we are testing the possibility of a synergy when both are present together. Transgenic plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. Propagation of new transgenics for field testing: ‘ Propagation of LIMA-B (AMP) transgenic plants for further study; ‘ Propagation of Carrizo transgenic lines with LIMA gene to test a potential rootstock effect on non-GMO scion. Efforts to establish a new transgenic field site: Working with Dr. Phil Stansly, we have submitted an addendum to our transgenic field permit with APHIS to add an additional field site which would be located at the UF Immokalee Research and Education Center. We plan to plant 400 new transgenic trees at this site after approval. A few hundred trees have also been prepared for planting at the USDA Picos Farm site.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants transformed with citGRP1 were test by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107/CFU. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slightly canker resistance by infiltration test. A series of transgenic scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
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