Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Previous results: We established a clone expressing the Liberibacter rpoH La rpoH) gene, and introduced it into Sinorhizobium meliloti strains that contain reporter fusions for 5 key genes we expect might be controlled by rpoH. As controls, we have the cloning vector along, and the vector expressing the native Sinorhizobium meliloti rpoH (Sm rpoH) gene. Results as of December 2014: 1. On each fusion strain, we performed GUS assays ‘ IPTG. We employed LB medium, with no stress conditions. Background GUS levels with vector alone are high, perhaps because strain background is WT for rpoH. ‘ The optimized La rpoH is able to regulate 4 of 5 fusions (groES, hslV, clpB, ibpA): each is induced (compared to empty vector) 2X to 4X, completely dependent on IPTG induction of rpoH transcription. ‘ We were surprised to find no induction of target genes with the Sm rpoH1 plasmid. This is a curious result. Why would optimized La rpoH be better at inducing expression of Sm rpoH target genes? We confirmed by PCR that we did not switch plasmids or strains. Here are possible explanation: (a) perhaps codon optimization of La rpoH allows for better expression in S. meliloti than the native rpoH1; (b) it might be that Sm rpoH1 levels are tightly regulated by proteolysis, but optimized La rpoH is not subject to this regulation; (c) For some unknown reason optimized La rpoH is better than Sm rpoH1 at initiating transcription at these target genes. 2. We plan to retest the La rpoH and Sm rpoH1 activities in RFF231 (.rpoH1H2; that is deleted for both native rpoH genes). We have introduced the 5 uidA fusions into that strain and will conjugate the rpoH constructs into each. 3. We are making a new optimized La rpoH construct with a slightly different insert than the one used above. The reason is to test whether using the RBS already present in the pSRK-Gm vector works better than introducing our own RBS (Mike Kahn ORFeome RBS+spacer) 4. Our next step will be to study more Ca. Liberibacter asiaticus genes for a regulatory role. We have designed optimized coding sequence for these genes: CLIBASIA_01180, 00835, 02900, 02905, 01545, 03950. In the next month, we will order the synthetic DNA segments corresponding to the optimized genes. 5. A note on technique: we anticipate that pSRK-Gm plasmid will work for our purposes. However, we will await the results of experiments in 2 and 3 before cloning the new genes into the vector. Summary of accomplishments: we have successfully expressed the Ca. Liberibacter asiaticus sigma factor RpoH in the heterologous Sinorhizobium meliloti, and we showed that it functions to control promoters for defined S. meliloti genes.
The overall goal of this project is to 1) determine the overall effects of ACPS/open hydroponics growing systems on drought susceptibility and 2) the efficacy of plant growth regulators on mitigating the effects of preharvest fruit drop resulting from HLB. The data are now being analyzed for a formal report/publication. Preharvest fruit drop data from the 4-acre plant growth regulator trial in Lake Alfred are in the final stages of analysis. The anatomical data from citrus trees in the different experimental blocks are also in the final stages of sectioning and analysis. As part of an extension of this project a greenhouse study has been initiated to test the efficacy of 2,4-D and other plant growth regulators on root health in small trees with and without HLB. The plants have been potted, graft inoculated, and their initial root mass measured prior to the first applications of the PGR treatments. These trees will be evaluated regularly over the next six months for changes in growth and physiology in response to the PGRs and HLB.
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.
Understanding the proliferation and movement of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (citrus greening), within the citrus tree remains a major obstacle in the efforts to undermine the pathogenicity and destructive nature of the disease. CLas still remains an unculturable organism, in part due to the lack of information on the phloem contents and internal environment. Once established in the phloem sieve elements, CLas movement within a tree has been assumed to follow the photoassimilate stream. Our current belief of the CLas transport mechanism is dependent on a presumed bidirectional flow of phloem sap, with basipetal movement from the site of infection, proliferation in the roots followed by systemic distribution through the tree. For CLas movement to occur under the current theory, CLas must be able to move laterally around the stems, trunks, and young roots, and then travel bidirectionally within the phloem sap. However, determinations based on general phloem physiology, our limited understanding of CLas, and on current anatomical studies have exposed serious inconsistencies with the accepted beliefs of CLas phloem transport. For example, based on general phloem anatomy and our current microscopy observations, lateral movement of CLas around a stem appears improbable given the size of cytoplasmic plasmodesmatal connections between adjacent sieve elements and the isolated nature of phloem cells. Furthermore, spreading of CLas from infected roots to healthy aerial tissues through the phloem conduits is difficult to reconcile without a reversal of the phloem flow, a condition not demonstrated for any evergreen species. The objective of this study is to define the biochemical properties and transport direction of citrus phloem elements that support CLas proliferation and allow movement within a citrus tree. The data will provide the basis for developing culturing conditions for the bacteria and for mitigating strategies based on movement of the phloem sap. This project is well under way, with all preliminary work completed within this period. To determine phloem movement, two separate devices have been constructed following existing literature. To follow phloem sap movement in young tissue, a device consisting of 2 arm probes has been successfully tested. In one arm, a UV light source and fluorescent sensor are mounted on the tip, whereas the second arm probe contains just fluorescent sensor. An externally applied fluorescent phloem-mobile substance is excited upon passage through the UV and its fluorescence detected by both probes as it moves down within the phloem sap. Time is recorded and velocity can be calculated. For this section of the project, data has been collected from both HLB and healthy greenhouse trees and data is being analyzed. For phloem movement in stem tissue, an electronic device was constructed. The device is based on heat transfer through the plant cortex. It contains 2 heat sensors that will determine time of heat transfer from a centrally located heat supply. The instrument has gone through a series of improving steps including solar energy power installation, miniaturization, test for different bark thicknesses, etc. We are finishing the construction of 10 instruments, 9 to be placed in field trees and one for future greenhouse experiments. Field installation is scheduled for December 10.
Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. Improving Consumer Acceptance: Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. Induction of early flowering to reduce juvenility (Carrizo transformed with the FT gene): After numerous attempts, we have finally produced transgenic Carrizo citrange plants expressing the clementine-derived CFT3 gene. Several of the plants have flowered once in the greenhouse in the juvenile state. These plants have been micrografted to standard rootstock and are in the greenhouse for further evaluation and observation. The new transgenic field site at the Southwest Research and Education Center (working with Dr. Phil Stansly) was successfully established, and approximately 320 transgenic citrus plants were planted as follows: Constructs: pCIT 107O (35s-CEMA) Line/Events: 15 Constructs: pCIT 109 (35s-SABP2) Line/Events: 24 (SABP2 is a SAR-inducing gene showing great promise) Constructs: pCIT 109A (AtSUC2-SABP2) Line/Events: 57 Constructs: pCIT105 (35s-CEME) Line/Events: 34 Constructs: pLC 216 (35s-LIMA) Line/Events: 190. Note: most of these are transgenic LIMA rootstocks (Carrizo/Orange 16) with non-transgenic Valencia scion. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015. We have achieved rapidly growing transgenic sweet orange trees through thorniness. We are also planning an outdoor RES type structure for transgenics.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, we have cloned all the components necessary for this study. The plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter have been constructed. Vectors containing the CpTI gene driven by a SLREO promoter that targets the transgene to the mature root cortex have also been produced. In addition, plant transformation vectors containing the gus gene driven by these root specific promoters have also been produced to demonstrate proof of functionality of the root specific promoters. Transformation experiments to produce genetically modified rootstocks with each of this promoters will be initiated in the next quarter as seeds become available.
“This project aims is to express molecules in plant that Disrupt the growth and ACP- transmission of CLas ” project narrative: 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 detect AHLs in the insect vector (psyllid) healthy or infected with CLas but not in citrus plant meaning that Insect is the source of AHL. We investigated the effect of expressing Lux-R in citrus plants on the level of many compounds, especially those presents in citrus. These compounds includes, but restricted to Indole-3-acetic acid (IAA), indole, g-amino butyric acid (GABA), salicylic acid (SA), Riboflavin and Lumichrome. These compounds can activate the Lux-R of many plant pathogens [1, 2, 3, and 4]. The level of these compounds were measured in healthy and CLas-infected CLas expressing Lux-R as well as healthy and CLas-infected plants without Lux-R. We expect that the levels of some of those compounds will be reduced as possible binding to the Lux-R protein and/or bacterial cells. “WE FOUND THAT IAA, GABA, and SA INCREASED IN THE PRESENSE OF Lux-R PROTEIN IN PHLOEM SAP” “Similarly, these compounds increased in the infected citrus plants indicating that CLas used them as signals binding to Lux-R” Identification of citrus compounds that mimic CLas N-Acyl homoserine lactone signal activities and affect CLas population density will highlight new strategies for the prevention of citrus greening disease. References 1) Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism-plant Signaling. FEMS Microbiol Rev 31: 425’448. 2) Lee JH, Lee J. Indole as an intercellular signal in microbial communities. FEMS Microbiol Rev: 34 (2010) 426’444. 3) Yuan ZC, Haudecoeur E, Faure D, Kerr KF, Nester EW. 2008. Comparative transcriptome analysis of Agrobacterium tumefaciens in response to plant signal salicylic acid, indole-3-acetic acid and g-amino butyric acid reveals signalling cross-talk and Agrobacterium’plant co-evolution. Cellular Microbiology 10(11): 2339’2354. 4) Rajamani S, Bauer WD, Robinson JB, Farrow JM, Pesci EC, Teplitski M, Gao M, Sayre RT, Phillips DA. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. Molecular Plant-Microbe Interactions: 21 (9): 1184’1192.
While oxytetracycline has been used in agriculture for over 60 years, it is just one compound of a chemically diverse family of agents having variable activity across many different microbial species. Within this family, a select subgroup of compounds have discreet and targeted activity against alpha-proteobacteria, including CLas, the agent responsible for Huanglongbing (HLB), while devoid of activity against human bacterial pathogens. Such selectivity against HLB by these compounds can be used to treat plants while sparing the possibility of antibacterial resistance, creating microbial agents or biocides in their own class of compounds affecting HLB alone. Our efforts in tetracycline chemistry continue to produce the most highly active compounds found effective against the surrogate strain Liberibacter crescens (see http://citrusrdf.org/wp-content/uploads/2014/07/Antimicrobials.pdf), where most of the derivatives were 3 or more orders of magnitude more active than oxytetracycline. Two of the most potent compounds derived from these screening studies, designated EBI-601 and EBI-602, are being studied in models of phloem transport in citrus and were found to be transported throughout the plant by either foliar or bark application of the formulated compounds. In infected citrus trees both compounds showed the ability to suppress HLB (CLas) bacterial growth as determined by PCR levels as compared to infected control trees (studies ongoing), while leaves from HLB infected Valencia orange shoots showed significant repression of L10 and 16S mRNA levels, another indicator of HLB growth suppression and treatment. These results with both compounds show that chemically-modified tetracyclines are specifically active against alpha-proteobacteria and target HLB, decreasing the infection in whole citrus plants, without plant toxicity, and are considerably more active than currently used agents. Furthermore, these compounds have been described as some of the most potent compounds tested to date against HLB, and can be commercially produced in amounts needed to treat infected groves. Further compounds are being studied against the surrogate strain and in infected citrus, and as the chemical structure of the tetracycline is changed, major increases in activity against HLB are observed, describing the mechanisms by which tetracyclines can enter the plant and affect the bacterium while decreasing infectious disease symptoms. The ultimate goal of this study is to obtain the most suitable compound with highly potent anti-HLB activity, one with increased stability for field use, and to ensure its safety in the environment for registration through the EPA for use in agriculture. This study demonstrates for the first time that an effective and targeted microbicide can be chemically designed to combat agricultural diseases in commercially valuable crops.
Within the tetracycline family of compounds, a select subgroup of compounds have discreet and select activity against alpha-proteobacteria, including CLas, the agent responsible for Huanglongbing (HLB), while devoid of activity against human bacterial pathogens. Such selectivity against HLB by these compounds can be used to treat plants while sparing the possibility of antibacterial resistance, creating microbial agents or biocides in their own class of compounds affecting HLB alone. Our efforts in tetracycline chemistry continue to produce the most highly active compounds found effective against the surrogate strain Liberibacter crescens, where most of the derivatives were 3 or more orders of magnitude more active than oxytetracycline. Two of the most potent compounds derived from these screening studies, designated EBI-601 and EBI-602, are being studied in models of phloem transport in citrus and were found to be transported throughout the plant by either foliar or bark application of the formulated compounds. This quarter, we have engaged in the synthesis and characterization of new derivatives of EBI-601 and EBI-602, modifying physico-chemical parameters related to size, electronic character and lipophilicity at chosen positions within the tetracycline nucleus, using chemical techniques of spanning substituent space. By creating an array of new compounds that are chemically tractable with commercial methods of synthesis, we will provide new compounds effective against HLB, and with the ability to product commercial quantities. While other methods of control are currently being studied under funding mechanisms supported here and by other agencies, these methods, including siRNA, older compounds already used and compounds from commercial libraries, may be of limited use due to high cost, low effectiveness, toxicity or therapeutic activity and use as human therapeutics. Our compounds are currently effective against the surrogate Liberibacter strain, have no use in human medicine, have favorable biodistribution in the plant, are inexpensive, novel and patentable, and can be scale-up with starting materials in ready supply under contract agreement by prominent manufacturers. The compounds synthesized this quarter have been fully characterized chemically and by MIC testing against the Liberibacter surrogate, represent some of the potentially most potent compounds found to date against HLB, the causative agent of citrus greening. This study demonstrates for the first time that an effective and targeted microbicide can be chemically designed to combat agricultural diseases in commercially valuable crops.
Project narrative: We aim to understand the specific interactions between Candidatus Liberibacter asciaticus (CLas) and the insect vector Asian citrus psyllids (ACP) to block the transmission. ‘The transmission process of CLas depends on the success of specific interactions between CLas and the insect vector ACP. The bacterium passes through the intestinal barrier to reach the hemolymph where they multiply then they must invade the salivary glandes in order to be inoculated in a new plant host while insect feeding. Passing these biological barriers needs specific interactions between CLas cells and the epithelial cells in the guts and the salivary glands cells.’ Here we report our effort in investigating ACP protein expression response for the infection with CLas after fractioning the proteins to hydrophilic and hydrophobic proteins In order to get a better understanding of the transmission mechanism of citrus Huanglongbing (HLB) by the insect vector, Asian citrus psyllid (ACP), we have been investigating the protein-protein interactions (PPI) between ACP and the HLB associated bacterial agent, Candidatus Liberibacter asiaticus (CLas) by means of proteomics. During the transmission process, CLas bacteria systemically circulate inside ACP and series of molecular interactions must be involved; therefore, our aim is to find the proteins involved in the CLas-ACP interactions by studying the differentially expressed membrane proteins between CLas-free and CLas-infected ACP. So far, we have established a two-dimensional electrophoresis (2-DE) system and a blue native polyacrylamide gel electrophoresis (BN-PAGE) system in our lab. The 2-DE system used an optimized protein fractioning protocol for hydrophobic proteins and was designed to study membrane proteins, which can help identify potential receptor proteins of ACP. The BN-PAGE system was used to study all the protein components consist of the PPI complexes, which can provide further information of the CLas-ACP interactions especially from the pathogen side. By comparing the gel differences between CLas-free and CLas-infected ACP, a total of 13 hydrophobic proteins and 20 protein components have been located in the 2-DE and BN-PAGE gels, respectively, and all were sent for identification by mass spectrometry (MS). After protein identification and annotation, our next step is to use far-western blotting to show the specific protein-protein interactions between the identified protein(s) and CLas, which will further validate our results from gel work. Our work should contribute to our aim of disrupting the connections between CLas and ACP, thus help slowing HLB spread in the field.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants will need to be grown for some time to produce larger plants, which will be used to vegetative propagation of multiple individual plants for each independent transgenic line. Interestingly, at least one of the transgenic lines at University Park is blooming, despite being less than 8 inches tall. This is an expected phenotype of the transgene, since overexpression of the FLT domain of the engineered FLT-antiNodT fusion protein is expected to trigger precocious flowering due to the “florigen” blooming promotion activity of the Flowering Locus T (FLT) protein. This bodes well for the successful expression of the FLT-antiNodT fusion protein from the transgene in these plants. Plants at University Park will be used for analysis of the transgene expression, while plants at Fort Pierce will be tested for resistance to citrus greening, in collaboration with Dr. Gottwald.
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.
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 that are important for 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 significantly expanded 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 citrus defense control. We originally proposed a three-year research with three specific objectives: 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. However only one-year funding was provided to support our research. With this support, our effort has been focusing on Aim 1. We have cloned 15 citrus defense genes affecting SA, ET, and/or JA pathways from citrus cDNA libraries and made overexpression constructs with the full-length cDNA clones of these genes in the binary vector pBINplusARS. While our focus is on gene cloning with the one-year support, we also initiated Arabidopsis transformation and testing disease resistance of the transgenic plants for these cloned genes (Aim 2). So far we obtained T1 seeds for 10 gene overexpression constructs of the cloned citrus genes and preliminary testing of transgenic plants overexpressing CsJAR1 or CsACD1 revealed promising results for future studies. In addition, citrus transformation with 10 citrus gene overexpression constructs has been initiated (Aim 3). 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 was already terminated. Our recent analysis of transgenic citrus overexpressing CsNDR1 (five out of ten transgenic plants) showed that these plants had lower rates of HLB positive three months after inoculation psyllids carrying Liberibacter asiaticus, suggesting that CsNDR1 overexpression confers enhanced HLB resistance. This result is consistent with CsNDR1 overexpression in conferring enhanced disease resistance in Arabidopsis (Lu et al., 2013). We will follow up with these plants for a further test of HLB resistance in the next few months. Lu, H., Zhang, C., Albrecht, U., Shimizu, R., Wang, G., and Bowman, K.D. (2013). Overexpression of a citrus NDR1 orthodox increases disease resistance in Arabidopsis. Front Plant Sci 4, 157.
It has been established that three different viruses can individually cause citrus leprosis. Two different cytoplasmic viruses (CiLV-C and CiLV-C2) and one nuclear virus (CiLV-N) have now been sequenced and detection methods are available. In all types of the leprosis the viruses are apparently transmitted by Brevipalpus mite species. The Brevivalpus yothersii (syn. phoenicis) mites from Florida and from Colombia were found to be the same by R. Ochoa and G. Bauchan using low temperature scanning electron microscopy. We attempted mite transmission experiments at the USDA, ARS, Ft. Detrick, MD with endemic healthy B. yothersii from Florida using citrus leprosis affected citrus leaves from Mexico (CiLV-N) & Colombia (CiLV-C2) were attempted multiple times however no positive transmissions were found. However as previously reported mite survival in transportation from Florida was poor. Leaf tissue from the experiments were analyzed by RT-PCR using CiLV type-specific primers and were negative. However using newly developed PCR primers we were able to prove the viruliferous status of Brevipalpus mites taken from infected lesions, killed in ethanol and shipped from Mexico (CiLV-N) and Colombia (CiLV-C2). Virus transmission experiments were performed successfully by our collaborator, Guillermo Leon, in Colombia with Brevivalpus yothersii (syn B. phoenicis) mites. In acquisition access period (AAP) experiments it was found that the mites efficiently acquired CiLV-C2 after 30 min feeding on symptomatic Valencia orange leaves with healthy leaves producing typical leprosis symptoms. However positive RT-PCR detection of the virus in the mites was only possible after an AAP of 6 h. Positive virus transmission was accomplished with a 10 minute inoculation access period (IAP) with an AAP of 48 h. For periods of 10 and 20 minutes IAP 25% of the Valencia orange receptor leaves produced disease symptoms. At 30 and 60 min. IAP 50% of leaves produced symptoms and with 2 and 6 hours IAP 56.25% and 68.75% of the leaves respectively, produced symptoms of leprosis. After a five day AAP on symptomatic Valencia orange leaves 40% of B. yothersii mites populations sampled were RT-PCR positive for CiLV-C2The transmission of CiLV-C2 by the mite vector, B. yothersii was studied after virus acquisition from citrus, then feeding on non-citrus plants and finally feeding on healthy citrus plants. According to the appearance of visual symptoms of the disease, the mites were able to transmit the virus to Valencia orange plants at a rate of over 80%, after feeding from 2-20 days Dieffenbachia sp., Hibiscus rosa-cinensis, Codiaeum variegatum, Swinglea glutinosa, Sida acuta or Stachytarpheta cayennensis. The appearance of the first leprosis symptoms on the Valencia orange leaves was from 14-37 days after the mites had previously fed on S. glutinosa, Dieffenbachia sp. and C. variegatum. After feeding on S. acuta, leprosis symptoms occurred in Valencia between 18 and 46 days and after the mites fed on H. rosa-cinensis and S. cayennensis the symptoms appear between 26-51 days after transmission. This work suggests that CiLV-C2 may be persistently transmitted by B. yothersii. As mentioned above the mites in Florida appear to be the same taxonomically with those in Colombia. We have sequenced herbarium specimens from Florida and they have been shown to be CiLV-N. Because of the funding for this project and a leprosis project on diagnositics for leprosis we have assembled an international cooperative team of researchers who will continue research on this important citrus disease.
The goal of this project is to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins secreted by the causative agent Candidatus Liberibacter asiaticus (CLas) into the phloem. One of the most important virulence mechanisms of bacterial pathogens is their protein secretion systems, which secrete proteins (called ‘effectors’) to manipulate the hosts. CLas possesses the general Sec secretion system that secretes effectors carrying a specific N-terminal secretion signal. Sec effectors have been demonstrated to be important for pathogenicity of insect-transmitted, phloem-limited bacteria. Therefore, it is likely that Sec effectors of CLas also contribute to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are using these effectors as molecular probes to identify key components in HLB. We aim to find the citrus targets of these CLas effectors, which may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In this report period, we have completed the Y2H screening. For each effector (i.e. bait), more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the baits. This practice eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with each bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. We are still doing in-depth analysis of these data, but have already seen interesting citrus proteins that are potentially targeted by the CLas effectors. For example, one effector targets several transcription factors in citrus. These transcription factors may directly regulate the expression of genes that contribute to HLB susceptibility and/or symptom development. Indeed, one transcription factor targeted by this effector contains a “WRKY” domain, which is well-known for its regulatory role in plant defense. Interestingly, our microscopy analysis also shows that this effector is located in the nucleus of plant cells. These data suggest that CLas effectors may directly manipulate gene expression in citrus.
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