We have concluded the remaining activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). As mentioned in our last report we used two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. The CsISS15 peptide displayed no activity and this was an expected result based on the PAGAL predictions. Based on these results we have decided to include CsPPC20 as an additional construct for testing in planta. As mentioned in earlier reports we have CsP14a as a replacement for neutrophil elastase (NE). CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and currently being used to infect citrus plants. Binary vectors for expression of CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and have been used to generate transgenic tobacco and transgenic Carizo citrus plants. The construction of both CTV and binary vectors for the expression of CsP14a-CsPPC20 are currently under way. We have also used as a positive control NE-CB to develop plants with CTV based delivery and transgenic tobacco and transgenic Charrizo citrus that can be used to validate the efficacy of the citrus derived CAP proteins against HLB.
Report for period ending 6/30/14 During the period of 4/1/14 to 6/30/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. These daily activities included (but was not limited to) the following: Worked on standardization of data collection spreadsheets, set up water testing contracts, ordered supplies (soil probes, harvesting bags, etc…) for crop consultants (CC’s), talked with CC’s about trial progress, set up trial sites around the state including southwest, east coast and ridge locations, hounded CC’s for data, worked on details of new proposals, collection of picutres from field trials to add to data base whcih was setup, maintain the organization of the database, conducted rankings from trial photos received from CC’s, perfromed DI ratings, setup locations for thermal therapy trials, provided CC’s with protocols for leaf sampling for pcr, and assembled budgets for trials. Overall, Mr. Page was constantly working on a daily basis with different CC’s to ensure open lines of communication between the CC’s and the CPDC to ensure information was collected and delivered in a timely manner. Additionally, this required frequent travel to study sites around the state which during this period included a visit to trial site in Labelle (4/4/14), trip to trial site in Ft. Pierce (4/9/14), visited trial site in Balm (4/10/14), attended the CPDC meeting (4/17/14), field visit to thermal therapy trial sites with Reza Ehsani (6/18/14), photographed ridge grove site trial (6/19/14), and additional photographs of a ridge trial site (6/26/14).
Report for period ending 9/30/14 During the period of 7/1/14 to 9/30/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. These daily activities included (but was not limited to) the following: Attended routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintained constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sorted out results from testing labs, sorted and submitted invoices, contact product reps for material samples, continue to organize spreadsheets for data analysis, and reschedule submission dates for the CC’s filed trial data. Mr. Page also traveled to field sites and attended meetings to ensure projects were up to date. Travels durign this period included attending the CPDC meeting (7/1/14), went to ridge grove regarding a soil microbial trial (7/8/14), attended a thermal therapy field day at SWFREC (7/10/14), visited ridge grove sites and took photos (7/11/14), grove site visit in Labelle (7/15/14), setup new field trial at ridge grove location (7/22/14), conducted DI ratings at northern ridge grove site and photographed another field trial on ridge (7/25/14), was onsite for a thermal therapy treatment evaluation in a ridge grove trial (7/29/14), visited an east coast field trial site (7/30/14), sampled leaves for pcr testing from thermal therapy trial at a ridge grove trial site (8/7/14), took photographs of two thermal therapy trials at different ridge locations (8/15/14), photographed ridge grove trial site (9/4/14), field trial visit with company representative (9/8/14), field trial visit to Labelle (9/9/14), attended thermal therapy field day (9/10/14), attended CPDC meetings (9/18/14), and took photographs of thermal therapy trial at a ridge trial site (9/24/14).
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of December 12, 2014, a total of 6,402 transgenic plants have passed through inoculation process. A total of 124,795 bacteriliferous psyllids have been used in no-choice inoculations. Additionally, since our last report we have exposed 664 plants to a total of 14,060 infected psyllids in no-choice situations to answer questions about our inoculation procedures. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of March 31, 2015, a total of 7,066 plants have passed through inoculation process. A total of 138,855 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. As reported in December 2014, we initiated a series of experiments during fall 2014 specifically to evaluate inoculation success and to investigate different parameters related to the inoculation process. For example, does the presence of flush enhance transmission? In a colony of bacteriliferous psyllids, why are there sometimes large fluctuations over time in percentages of psyllids that test PCR-positive for the pathogen? Are lemon and citron equally suitable for maintaining colonies of infected psyllids? How effective is our inoculation program? Some of these questions are being answered based on transgenic material that has already been passed through the inoculation process. Recent feedback from inoculations of rootstock material gives some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The percentage of success was 62% for assays conducted 12 to 19 months after inoculations. There was no difference in the success rates for transformed and non-transformed seedlings.
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.
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