The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them.
In this exploratory study soft nano-particle (SNP) formulations were developed for delivery of anti-bacterial essential oils to plant phloem where Liberibacter asiaticus resides. Two essential oils (EO-A and EO-B which are single component) as identified by InnoCentiveTM assay were selected for developing SNP formulations. Both oils are naturally occurring terpenoid essential oils, generally regarded as safe (GRAS) with a number of botanical sources, including oregano and thyme. SNPs with Thyme Oil have were also formed and characterized. Microemulsion formulations were developed using agriculturally approved surfactants with different hydrophilic – lipophilic balance and charge. Formulations containing EO-A loading up to 7% (w/w), 35% (w/w) for EO-B and 20% for Thyme Oil, having droplet size ranging from ca.3 to 30 nm were developed. The stability of the formulations was tested by dilution with water, dilution with phosphate buffer saline (PBS) and in the presence of commonly used spreaders sticker adjuvants such as Cohere and Cling. While most formulations were stable, some made with ionic surfactant displayed instability when diluted with PBS. The developed EO formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter crescens (Department of Microbiology and Cell Science, UF). The antibacterial efficacies of the SNPs were tested at three different dilutions 1, 5 and 10% (v/v). All SNP formulations showed > 90% inhibition at 1, 5 and 10 % (v/v) dilution. Some of the control solutions containing surfactants also showed high bacterial inhibition. High (> 90%) inhibition was observed with most formulations at concentrations as low as 100 ppm of EOs. While initially the SNPs were developed to achieve high EO loadings for maximum bacterial inhibition, it led to SNP formulations having relatively high surfactant concentration. The high concentration of the surfactants used is likely contributing to the antibacterial efficacy of the EO SNPs. Phytotoxicity of select formulations was tested at 1:1, 1:10 and 1:20 dilutions (formulations EO-A and EO-B) at Lake Alfred facilities (UF). All SNP formulations showed low phytotoxicity when applied at 1:20 dilutions. SNP formulations at these and lower dilutions showed high inhibition of L. crescens. Antibacterial and phytotoxicity results indicate that with suitable dilutions the SNP formulations can be used to for performing foliar applications tests on citrus crops. Future experiments are being planned for testing efficacy of SNP formulations in HLB infected plants.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications. This spring we detected a 20% in crease in root mass density of Swingle trees associated with a drop in root zone pH from 6.4 to 5.9 at 9 months after soil amendment with prilled sulfur (Tiger 90). Although the soil levels were high, the drop in pH did not result in evelvarion of leaf copper concentration into the toxicity range. Root mass density and well as copper levels in leaves in groves under acidification management will be monitored in 8 ridge and 4 flatwoods sites for changes in pH of irrigated root zone, root mass density and and nutrient levels in leaves.
We hypothesized that groves with high bicarbonate stress are suffering from HLB because they support lower fibrous root density compared to groves with lower bicarbonates (less than 100 ppm) in irrigation water and/or soil pH (less than 6.5). To confirm this relationship, we surveyed 37 grove locations in Highlands and Desoto counties with varying liming history and deep vs. shallow wells mostly on Swingle and Carrizo. Lower root density is significantly related to well water pH greater than 6.5 and to soil pH greater than 6.2. Yield records from these blocks reveal that groves under high bicarbonate stress production have declined 20 percent over the last three seasons (2009-2012) in contrast to Ridge groves with low bicarbonate stress which have increased 6 percent in production even though HLB incidence has accelerated. The yield losses are correlated with less fibrous root density, which reduces root system capacity for water and nutrient uptake. Evidence from research on other crops indicates that bicarbonate impairs the root’s ability to take up important nutritional cations including Ca, Mg and K, as well as micronutrients, especially Mn and Fe. In Florida, Bryan Belcher of Davis Citrus Management has acidified irrigation water with sulfuric or N-furic acid (a mixture of urea and sulfuric acid) by injection at the well in the same way as fertigation. N-furic has the advantages of being safer to handle and providing some additional N due to the urea component, but the disadvantage is higher cost of treatment compared to sulfuric acid. Since irrigation is not necessary when it rains, acidification treatment only occurs during the dry season when the bicarbonates are loading into the wetted area under the tree. When the rain begins, these bicarbonates are flushed from the rhizosphere. Our labs and grower cooperators are also evaluating acidification of soil by amendment with elemental sulfur applied in prilled or finely ground form. Sulfur (S) releases acid when it interacts with Thiobacillus bacteria in soil to form acid (H+) ions. This process of acidification with S is slower than treatment of the water but provides for longer lasting reduction in soil pH. Sulfur can be applied in prilled form with a fertilizer spreader or with a herbicide boom as a slurry. Sulfur can also be added to dry and fertigation formulations to lower the pH by as much as one unit after repeated ground applications. This spring we detected a 20% in crease in root mass density of Swingle trees associated with a drop in root zone pH from 6.4 to 5.9 at 9 months after soil amendment with prilled sulfur (Tiger 90). Although the soil levels were high, the drop in pH did not result in evelvarion of leaf copper concentration into the toxicity range. Root mass density and well as copper levels in leaves in groves under acidification management will be monitored in 8 ridge and 4 flatwoods sites for changes in pH of irrigated root zone, root mass density and and nutrient levels in leaves.
Stress intolerance of HLB trees is a direct consequence of more than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded an additional 20% by the interaction with bicarbonates in the rhizosphere as a result of irrigation with well water high in bicarbonates and/or over-liming with dolomite to avoid copper toxicity. HLB and bicarbonate stress also favor infection by root pathogens such as Phytophthora spp. In field surveys and greenhouse trials with trees with and without Liberibacter asiaticus (Las) inoculation, populations of Phytophthora spp. are elevated in the rhizosphere of HLB trees. When Las interacts with Phytophthora, fibrous root loss is greater than that caused by HLB alone depending on the stage of Las infection and time of year. Also, HLB damage of fibrous roots alters the concentration of soluble sugars by increasing leakage that attracts and accelerates infection by root pathogens such as P. nicotianae. Although HLB associated root loss has been attributed to carbohydrate depletion by HLB and Phytopththora this relationship may vary with rootstock. Rootstock tolerance to Phytophthora is broken by the interaction with Las such that resistance to biotic or abiotic stress is greatly reduced. This results in premature root loss and reduction in root system capacity for water and nutrient uptake. The normal annual cycle of root death and replacement of fibrous roots appears to be disrupted. To confirm this, bimonthly changes in fibrous root mass density and rhizosphere population Phytophthora are currently under study in 8 ridge and 4 flatwoods sites in south central Florida.
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.
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