Website creation and development The past year saw the addition of a genome browser to the CG-HLB genome resources website, allowing users unprecedented access to a wide range of bioinformatic predictions for the Liberibacter genomes. Initially set up for the Ca. L. asiaticus psy62 genome, the Ca. L. solanacearum CLs0-CZ1 genome and large subgenomic regions from Ca. L. asiatucus UF506 were added upon their public release in January 2011. In-house analyses and those generated at other sites are accessed through a series of hyperlinked tracks, with the linked data including functional domain predictions, functional role within the overall metabolic picture of the organism, predictions of protein location within the cell, locations of signal peptides, and gene operon structure. Links are also provided from individual genes to the secondary structure predictions generated by the Grishin lab. To aid in development of tools for discriminating between strains, locations of known and ab initio repeats in the three genome-scale sequences have been identified and added to the browser. Genes found to be unique between Las and Lso are also shown. The browser platform has been designed to readily accommodate addition of more genome sequences as they become available. Outreach In January, project personnel attended the 2nd IRCHLB research conference in Orlando FL to promote features of the site and invite collaboration. Subsequent work with Manjunath Keremane has concerned the assembly and analysis of sequence data for Ca. L. psyllaurous (LPS) which infects tomato and appears to be a system more amenable to biological characterization of Liberibacter-plant interactions. Collaboration with Wayne Hunter has focused on de-convolution of the Asian citrus psyllid metagenome sequence with a particular focus on assembly of a draft genome for the ACP endosymbiont, Wolbachia. Liberibacter is dependent on other organisms for its nutrients both in the plant and vector, so understanding the metabolic capabilities of endosymbionts in ACP may reveal sources of nutrients critical to the Liberibacter-vector association. Bioinformatic analysis A 32 GB server was recently purchase with grant funds and installed at the Cornell Center for Advanced Computing, significantly enhancing our group’s capability to analyze large data sets. This is particularly important for analysis of the high volume next generation sequencing data being generated for LPS and the Asian citrus psyllid metagenome. Analysis of the first quarter of the LPS genome to be assembled indicates high similarity with the Lso genome with a small number of clearly divergent loci. Analysis of the Asian citrus psyllid metagenome focused initially on assembly of reads derived from the Wolbachia endosymbiont. A draft genome has been produced, using prior Wolbachia sequences as a scaffold. Comparisons with other Wolbachia strains are ongoing. To better identify the full complement of endosymbionts in the Asian citrus psyllid and compare the resulting list to those previously identified by ribosomal RNA sequencing, different strategies are being employed for sorting reads and assemblies for the purpose of determining the source organism.
We have completed computational and manual analyses of Candidatus Liberibacter asiaticus (C. L. asiaticus) proteome providing the following results to the scientific community: 1. We built a pipeline for comprehensive protein sequence analysis and presented it as a public web-server at: http://prodata.swmed.edu/msas/msas.cgi. 2. We applied the pipeline to the C. L. asiaticus proteome and compiled an ‘encyclopedia’ for the bacterium at http://prodata.swmed.edu/congqian/, gathering prediction results from automatic methods for each protein. 3. We manually curated these predictions, analyzed sequence features and domain architectures, provided structure models and tried to predict exact function of each protein making the results available at: http://prodata.swmed.edu/congqian/curated From a proteome-wide view, we were able to generate confident structure models for about 80% of all proteins. While NCBI only annotated 68% of proteins, after careful analysis we predicted the function and sub cellular localization for 78% of all proteins and sub cellular localization for 9% more. Based on these results we further carried out three in-depth projects aimed at understanding the mechanism of the disease: 1. Identification of potential virulence factors. Effectors can be distinguished from housekeeping genes by many features, such as special sequence motifs, evolutionary history, predicted function, genome context and homology to known effectors. Combining all these criteria, we generated a list of potential virulence factors at: (http://prodata.swmed.edu/congqian/curated/effectors.html) One example is a predicted RTX toxin (NCBI gi: 254780384), which can be secreted by the type I secretion system and may affect host cells. 2. Analysis of ATP-Binding Cassette (ABC) Transporters. ABC transporters are essential for the survival of the bacterium as they mediate the communication between the bacterium and the host. We detected 14 ABC transporters in C. L. asiaticus. By clarifying their substrate specificity, evolutionary history and classifying their structure, we hope to promote experimental efforts on targeting these transporters and controlling the disease. 3. Metabolic reconstruction of C. L. asiaticus and identification of potential drug targets (in collaboration with Dr. Andrei Osterman, in progress). We reconstructed and verified the metabolic model of C. L. asiaticus and generated a list of essential genes. By comparing these essential genes with their eukaryotic counterparts we hope to identify proteins that are structurally different enough from eukaryotic proteins or without eukaryotic orthologs. These proteins might be good drug target as they are crucial for the C. L. asiaticus but different enough from the host proteins. We hope our effort on the analysis of the C. L. asiaticus proteome will advance understanding of this bacterium and will inspire further work to develop the strategies to control the disease.
This project is a continuous support of the Texas Budwood Certification Program that was established in the late 1990’s, to ensure the Texas citrus industry receives true-to-type, pathogen free, certified budwood. The program currently has over 90 varieties that have been shoot-tip grafted and found free of all major graft-transmissible pathogens by either the California Citrus Clonal Protection Program or the Texas A&M-Kingsville Citrus Center. A portion of the funds were applied to salaries of the supervisor of the Stephenville greenhouse, Dr. Charles Simpson, and a technician that assisted with the maintenance and care of the trees in Stephenville. There were a grand total of 142,915 buds cut from 73 different citrus varieties from Sept. 1, 2010 to Aug. 31, 2011. The top variety was Rio Red grapefruit (53.5%) The testing for Citrus tristeza virus and Huanglongbing (Citrus greening) in the Foundation and Increase blocks was completed in January, 2011 and all samples were negative. CTV and HLB testing for the trees at Stephenville was completed in May, 2011, with all samples testing negative. Testing for viruses/viroids is currently in progress. Because of the CTV find in some Standard Valencia increase trees in 2009, all trees in the four orchards adjacent to the fenced budwood area were tested – no infected trees were found. 29 trees were added to the Foundation screenhouse, located in the Foundation/Increase Block area at the Citrus Center. The additional trees brings the total volume in the Foundation Screenhouse to 85. Budwood from 4 new varieties from the California Citrus Clonal Protection Program (CCPP) was received in October, 2010. In January, 2011 two new employees, a program coordinator and a lab technician, were hired utilizing funding from the National Clean Plant Network (NCPN).
The goal of the Texas Citrus Budwood Program is to have all foundation and increase trees under insect resistance screen. One screenhouse has been erected at the Citrus Center and currently has potted foundation trees of the main commercial varieties. A grant was obtained through Texas Department of Agriculture (specialty crop block grant) to renovate a greenhouse at the Texas A&M AgriLife Center in Stephenville, located well outside the commercial citrus area to house a back-up collection of foundation trees. Because AgriLife was unable to secure funding to renovate the whole structure, funds were requested from the Board to repair a temporary greenhouse, and to supplement the TDA grant for the renovation project. The renovation was completed in January 2011, and the 100 potted citrus plants were moved into it from the temporary housing. In May, the plants were transplanted into 20 gallon containers. The greenhouse has a double entry door with a wind fan, insect screens over the cooling pads and exit fans, and propane heating for winter. During the winter of 2010/11, the heating provided sufficient protection, and no plants were lost or damaged. An agreement is in preparation between AgriLife and TAMUK to allow the plants to be housed in Stephenville for an initial period of 5 years. A permanent secure foundation facility has to be built in the very near future, so the use of Stephenville beyond this time frame.
The influence of nutritional status on cold tolerance in Citrus has shown controversial results and the information on how nitrogen fertilization can affect cold tolerance is very scarce for grapefruit trees The objective of this study was to assess the cold tolerance of grapefruit trees with different levels of leaf N content. For this study, thirty one-year old Rio Red grapefruit trees were grown in a greenhouse between January and April. Two treatments were carried out: 1) trees receiving five applications of foliar urea (1.5%) at 7-day intervals; 2) trees that did not received any N application, although they all received the same fertigation treatment. At the end of April, when trees started showing growth differences, freeze tolerance was estimated from lethal freezing temperature (LT50) based on solute leakage from frozen leaf tissue. Tissues were subjected to decreasing temperatures between 0 and -10 ‘C (32 to 14 F). Results: Trees with low content of leaf N showed a lower late-freeze tolerance than well-nourished trees. Leaves from trees with low N content showed a freeze tolerance of -5 ‘C (23F) whereas well-nourished trees had a freeze tolerance of -6 ‘C (21.2 F). Since achieving excess nutrient concentration in leaves requires a long fertilization experiment and that the plant physiology lab at Citrus Center started its operation in winter 2011, it was not possible to reach excess nutrient levels before April (where late-freezes are supposed to take place) and only low vs. optimum concentrations were compared. Continuation Plans and Benefits to Citrus Industry: Further studies on the effect of fertilization on freeze tolerance of grapefruit trees are being carried out at Citrus Center, as part of the research works for a M.S. thesis, including the effect of nitrogen fertilization on early-freeze tolerance and how an excess of nitrogen fertilization can affect freeze tolerance. These works are integrated in a broader study on the interactions of freeze events with other environmental stresses as well as on the search of strategies based on the use of plant growth regulators to increase freeze tolerance. All these works are being possible after purchasing the refrigerated water bath for what the funds of this project were required.
Besides the physical defects due to the destructive Huanglongbing (HLB) citrus greening disease on oranges, the infected fruit and resulting juice have been perceived as being more sour, bitter and off-flavored. In the symptomatic juice, the off-flavor was correlated with lower sugars, and sometimes with higher acids. Nevertheless, differences in flavor between symptomatic and healthy fruit were also correlated with higher limonin levels, although they were below taste thresholds. Thresholds (concentration of a compound at which it can be detected) of limonin and nomilin have been previously reported in water but never in combination and or in a complex mixture, such as a sugar-acid matrix or orange juice. Therefore, as a preliminary step to understand and characterize what the metabolites are that are responsible for the bitter off-favor of HLB orange fruit, thresholds of limonin, nomilin, and their combination in a sugar and acid matrix, as well as in Valencia orange juice were determined by taste panels. Between 16 and 23 panelists, previously trained to differentiate bitter and sour tastes, were asked to participate in the taste panels. Food grade limonin and nomilin were added alone or in combination to a simple matrix solution composed of 6% sucrose and 0.15% citric acid in drinking water; or to a complex matrix solution containing 5.2% sucrose, 2.1% glucose, 2.5 % fructose, 0.75% citric acid and 0.25% malic acid, or added directly into Valencia and commercial orange juice. All stimuli were prepared by dissolving the compounds in solution and once the highest concentrations were prepared, the rest of the samples were diluted in a series by a factor of 2. Each series of stimuli were presented 3 times on different days. A detection threshold was determined using a forced-choice ascending concentration series method of limits described in ASTM E679-91. At each selected concentration, a triangle sample set consisting of one test and two blank samples was presented to subjects; subjects were asked to determine which the test sample was. The subjects were also asked to rate the difference from 0 to 10. The best-estimate threshold for each panelist was considered to be the geometric mean of the concentration at which the last miss occurred and the next higher concentration. In the simple matrix, the threshold of limonin was lower than nomilin, indicating that it would be detected at a lower concentration. Panelists perceived the nomilin bitterness as being more astringent, metallic and lingering than the limonin bitterness, as has been described for HLB juice. The synergetic effect of limonin and nomilin was significant in decreasing their individual thresholds in a sugar and acid matrix. Interestingly, the thresholds of limonin and nomilin were lower in Valencia and commercial orange juice compared to the thresholds measured in the complex matrix. Our current results show that the threshold concentrations of limonin and nomilin in healthy ‘Valencia’ orange juice are higher than the concentrations of both limonin and nomilin found in HLB ‘Valencia’ fruit, which was perceived bitter by a taste panel. Perhaps the lower sugar and higher acid content of HLB fruit decrease the threshold of those bitter compounds which will be explored in further studies. We mixed normal juice with symptomatic HLB juice and found that panelists could detect the symptomatic HLB juice at different levels depending on variety and more information will be forthcoming on this in the next report.
Quarterly report 4-1-11 to 6-30-11 The major program conducted during the quarter was the Florida Citrus Growers’ Institute in Avon Park on April 6, 2011. This program is organized by the citrus extension agents to inform growers about selected citrus topics. Attendance exceeded 300 growers from throughout the state as well as a few international growers. Twelve presentations highlighted psyllid and HLB management, foliar applications of nutritionals and insecticides as well as topics on genomics and cultural management. All presentations (video and viewable) are posted to the citrus agents’ website at: http://citrusagents.ifas.ufl.edu/events/GrowersInstitute2011/GrowersInstitute2011.htm . Additionally, presentations from the 2008, 2009 and 2010 Institute programs are also available to provide reference information from earlier programs. Over the 4-yr period, psyllid and management of greening have been a major focus of the Institute program. Program support for meals is provided by sponsors with the rental fees and video recordings being supported from funding from the CRDF. The Mid Florida Citrus Foundation field day in May near Avalon, FL was the site of an Extension outreach effort highlighting various citrus management issues (HLB, psyllid, weeds and environmental factors). This field day had more than 150 growers participating in the educational program. Citrus extension agents collectively conducted more than 110 field visits during the 3-month period to discuss citrus production management issues with growers. Additionally, meetings and newsletters in each of the agent’s local area continue to provide supporting information and educational opportunities to strengthen citrus management practices and to address local issues and problems. Citrus HLB (greening) photo series continues to post monthly photos of the same trees at selected locations around the state. This web-based series allows growers to view the condition of specific HLB infected trees over time. Each agent collects photos from 4-6 sites within their respective area. All trees have been infected for at least 2-3 years and have tested HLB positive. Efforts are also devoted to ensuring growers have ample opportunity to obtain continuing education units (CEUs) by attending area meetings and by reading and completing tests in Citrus Industry magazine and on the web (Citrus Agents’ web site) to allow renewal of restricted use pesticide licenses. The licenses are essential to maintaining properly trained and licensed employees who must supervise or use insecticides in the battle to suppress psyllid populations. Agents are also actively involved in related areas to ensure urban groups also are aware of HLB and its impact on the Florida citrus industry.
Nutritional spray programs applied directly on the foliage of orange trees for management of the Huanglongbing (HLB) disease have been investigated in the last few years to maintain tree health and yield. The impact of these programs on HLB infected fruit flavor and aroma is unknown. This study looked at the effects of 3 different foliage nutritional spray programs (Maury Boyd, Keyplex and Wettable Powder) applied to healthy and HLB infected trees on the flavor and composition of their fruit, and compared them with the fruit treated with a conventional spray program. Three types of fruit samples were selected: healthy orange fruit from non-infected trees (healthy), asymptomatic fruit from HLB infected trees (HLB), and symptomatic fruit from HLB infected trees (HLBs).Four harvest times and two cultivars (‘Hamlin’ harvested in December 2009, December 2010 and January 2011, and ‘Valencia’ harvested in April 2011) were investigated. For all three harvests of ‘Hamlin’ fruit, HLB and HLBs juice samples for all treatments were significantly different by smell and by taste from the conventionally treated healthy juice, with the exceptions of conventionally treated HLB (harvested in December 2009) and the HLB Maury Boyd treated juice (Harvested in January 2011) which were not significantly different by smell from the healthy control. Generally, the panelists described the differences as being bitter, grapefruit like, more sour, astringent, and metallic. The limonin content was usually higher in the HLB and HLBs juice, regardless of the nutritional programs, while the nomilin content was found higher or at similar levels compared to the conventionally treated healthy juice. For the December 2009 harvest, the sugar content in HLB and HLBs juice samples of all treatments were the same as the levels as those found in conventionally treated healthy juice, while lower sugar content in all HLB and HLBs juice samples (regardless of the treatment) compared with healthy fruit were measured in the December 2010 and January 2011 harvests. The Valencia harvest in April, 2011, exhibited differences between healthy, HLB and HLBs for the conventional spray group, but less and inconsistent differences were observed between healthy, HLB and HLBs for Valencia juice from the nutritional spray groups. In the ‘Valencia’harvest of April 2011, conventionally treated HLBs juice was judged significantly different by taste compared with the healthy control. The HLBs juice was described as being sweeter, more sour, more tart, more bitter, metallic, off-flavored, and having more of a fruity-non-citrus flavor than the control. No significant difference by smell or by taste was observed between the Keyplex treated juice samples (healthy, HLB and HLBs) and the conventionally treated healthy control. Healthy Maury Boyd treated juice did not differ either by taste or smell from the conventionally treated healthy control, but the Maury Boyd HLB and HLBs juice sample were both significantly different by smell and taste. No clear conclusions can be drawn at this point whether the nutritional programs really have a beneficial impact and alleviate the negative effects of HLB disease on fruit flavor and composition. In most cases, the panelists could distinguish between a conventional treated healthy control and a sample that received a nutritional spray. Although, no significant difference in taste or smell were observed in the ‘Valencia’ harvest for the Keyplex nutritional treatment, there was either a difference between the HLB and HLBs samples from the conventional treatment compared with the healthy control. More harvests would be necessary to draw a conclusion. Also the effect of volatiles on juice aroma should also be investigated.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). Our qPCR study on dissected organs and tissues of ACP (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104:526-533) strongly suggested that the salivary glands constitute an important infection and/or transmission barrier to Las in the psyllid vector. This may partly explain why the proportions of Las-positive psyllids (by PCR) are normally much higher than those that can actually inoculate Las into citrus plants. A second study on the distribution of Las in various organs and tissues of ACP using fluorescent in situ hybridization (FISH) was completed and accepted for publication (Ammar et al., 2011, J. of Phytopathology, in press). Specific DNA probes with FISH and confocal laser scanning microscopy detected Las in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of Las-infected ACP. Our results showed the near-systemic infection of ACP by Las, indicating that Las may replicate and/or translocate into all these organs and tissues, and confirmed our PCR results suggesting that the salivary glands constitute a barrier to Las infection and/or transmission by ACP. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We developed a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity assays on ACP from the current 6-12 months (when using whole plants) to only 2-3 weeks (when using detached leaves). Las-infected ACP adults were tested for inoculativity by caging them singly or in small groups (5 or 10 per leaf) on detached healthy citrus leaves for 1-2 weeks (as described by Ammar & Hall, 2011, Florida Entomologist 94:340-342). These leaves were then assayed by qPCR 1-2 weeks later using two Las primers (Li and LJ900). The results of four trials on detached leaves using the more conventional Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings were used for inoculation. Our results, a summary of which appears in “Proceedings of the 2nd. Inter. Res. Confer. on HLB held in Orlando, FL, Jan. 10-14, 2011”, indicate that detached citrus leaves can be used to test the inoculativity of ACP and that this new method can speed up inoculativity tests on citrus psyllids considerably, which should enhance various studies on HLB epidemiology and vector interactions. [B) We conducted two large experiments to study the effects of various acquisition access periods on Las-infected citrus plants (between 1 day and 2 wks) on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. Preliminary results indicate significant differences between nymphs and adults as well as between various AAP’s in the acquisition and transmission of Las by ACP. But we are still analyzing the results on the replication of Las in both nymphs and adults in these experiments that will be repeated for verification. [C] A study on the ultrastructure and morphometrics of ACP stylets and the food and salivary canals (inside the maxillary stylets) is underway using both transmission and scanning electron microscopy. This study is expected to reveal the role of these canals in Las transmission (or lack of) by ACP nymphs and adults. [D] In a field study on ACP adults collected from HLB-diseased citrus trees in Fort Pierce, FL since Feb. 2009, there have been no strong trends observed over time with respect to percentages of Las-infected ACP or titers of the pathogen. However, during late summer 2010, there were eight consecutive weeks in which percentages of ACP testing PCR-positive for the Las bacterium averaged below 10%. Statistical analyses indicated that increased rainfall and excessively high air temperatures during these weeks were significantly correlated with the lower percentages of Las-infected ACP.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). [A] Our qPCR study on dissected ACP organs and tissues provided the first PCR confirmation of Las in the alimentary canal and salivary glands of ACP and strongly suggested that the salivary glands constitute an important infection and/or transmission barrier to Las in the psyllid vector. This partly explains why the proportions of PCR-positive psyllids are normally much higher than those that can actually inoculate Las into citrus plants. Our results (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104: 526-533) also suggested that Las may replicate or accumulate in both the alimentary canal and salivary glands of ACP. [B] We were finally able to study the distribution of Las in various organs and tissues of ACP by fluorescent in situ hybridization (FISH). Only one of the six protocols tested succeeded in the localization of Las in hemolymph smears and dissected organs of ACP adults that fed on Las-infected plants and in sections from infected leaves. Using specific DNA probes with FISH and confocal laser scanning microscopy, Las was detected in the filter chamber, midgut, Malpighian tubules, hemolymph, salivary glands, ovaries and in muscle and fat tissues of Las-infected ACP, as well as in the phloem of Las-infected leaves. Our results showed the near-systemic infection of ACP, which suggests that Las may replicate in various tissues of ACP, and confirmed our PCR results suggesting that the salivary glands constitute a barrier to Las infection and/or transmission. This study was recently accepted for publication (Ammar et al., 2011, J. of Phytopathology, in press). Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We have developed a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity assays on ACP from the current 6-12 months, when using whole plants, to only 2-3 weeks when using detached leaves. Las-infected ACP adults were tested for inoculativity by caging them singly or in small groups (5 or 10 per leaf) in 50-ml polypropylene tubes on detached healthy citrus leaves for one week (as described by Ammar & Hall, 2011, Florida Entomologist 94:340-342). These leaves were then assayed by qPCR, 1-2 weeks later, using two Las primers. So far, the results of three trials on Las-inoculativity tests on detached leaves using the more conventional Li primers are largely comparable to those recently reported for Las transmission when whole citrus seedlings were used for inoculation. Our results indicate that detached citrus leaves can be used to test the inoculativity of single or small groups of ACP and that this new method can speed up inoculativity tests on citrus psyllids considerably, which should enhance various studies on HLB epidemiology and vector interactions. An extended abstract on this new method is published in “Proceedings of the 2nd. Inter. Res. Confer. on HLB held in Orlando, FL, Jan. 10-14, 2011”. [B) We conducted two large experiments to study the effects of various acquisition access periods on Las-infected citrus plants (between 1 day and 2 wks) on both acquisition and transmission of Las by ACP nymphs and adults, as well as on Las replication in both life stages. We are still analyzing hundreds of individual psyllids from these two experiments with qPCR using primers to Las and to one of the psyllid genes for comparison. [C] In a field study on ACP adults collected from HLB-diseased citrus trees in Fort Pierce, FL since Feb. 2009, there have been no strong trends observed over time with respect to percentages of Las-infected ACP or titers of the pathogen. However, during late summer 2010, there were eight consecutive weeks in which percentages of ACP testing PCR-positive for the Las bacterium averaged below 10%. Statistical analyses indicated that increased rainfall and excessively high air temperatures during these weeks were significantly correlated with the lower percentages of Las-infected ACP.
The overall goal of this project is to identify a Bacillus thuringiensis (Bt) crystal toxin with toxicity to Asian Citrus Psyllid (ACP) and to further enhance toxicity by genetic modification. The long term goal of the proposed research is an effective management strategy for the psyllid and associated HLB disease that is more sustainable, less costly and more environmentally benign than the repeated application of broad spectrum insecticides. Our focus for the current reporting period has been on trypsin activation of the partially purified Bt toxins from the selected Bt strains. Trypsin activation of Bt toxins was carried out Iowa State University. Briefly, Partially purified Bt toxins were dialyzed against 50 mM Tris-Cl pH 8.5. Small aliquots of the dialyzed Bt toxins were incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Proteolysis was stopped by adding 1 mM phenylmethylsulfonyl fluoride. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. The SDS-PAGE profile of trypsin-treated Bt toxins exhibited multiple high intensity protein bands ranging from ~22 kDa to ~75 kDa . Based on the similarity of activated toxins bands, the toxin profiles of the nine Bt strains are categorized into the following five groups. Group one: Four Bt strains, each with five activated toxin bands of ~22, ~30, ~32, ~50 and ~70 kDa. Group two: Two Bt strains, each with three activated toxin bands of ~65, ~77, and ~85 kDa. Group three: One Bt strain with one activated toxin band of ~65 kDa. Group four: One Bt strain with one activated toxin band of ~85 kDa. Group five: One Bt strain with activated toxin bands of ~22, ~30, ~32, and ~70 kDa. Trypsin activated Bt toxins will be sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
Management of citrus Huanglongbing (HLB), which is associated with Candidatus Liberibacter asiaticus, could be achieved by application of antimicrobials and stopping the spread of HLB pathogen. Curing Ca. L. asiaticus infected citrus trees is one attractive goal due to the high value of citrus trees and the high cost of citrus tree removal and replanting. Wide spread of HLB throughout Florida renders curing Ca. L. asiaticus infected citrus trees necessary. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. First we filtered the structures based on their physico-chemical properties, e.g. Molecular Weight, H-Bond Donor, H-Bond Acceptor & Rotatable bonds and structurally similar to adenine moiety. Approximately 5000 structures were retrieved from ~5 million structures of commercially available databases. The identified data set was used for virtual screening by molecular docking method. Based on the dock scores we eliminated about 4500 low scored structures and selected ~500 (10%) for further molecular docking & minimization to evaluate the scoring functions (Dock glide scores, Hydrophilic, Hydrophobic e.t.c). Based on scoring functions, structural diversity, and our chemical intuition we have chosen forty structures for biological activity studies against purified SecA protein. For that purpose, SecA of Ca. L. asiaticus was expressed in E.coli using the pE-SUMO vector and purified. The inhibition assays of the 40 compounds against SecA was done as described previously by Denis et al. 1978. The IC50 ranged from 0.3’M to 100’M. Those findings were published in article entitled: “Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design” on Bioorganic & Medicinal Chemistry Letters Volume 21, Issue 14, 15 July 2011, Pages 4183-4188. Currently, the second screening was evaluated with twenty compounds identified and five compounds were determined to have nano molar activity against SecA by In-vitro studies. The most active five compounds will be used to apply on HLB infected trees for preliminary study in greenhouse and to identify more active compounds to develop an agrochemical/pesticide for HLB bacterial infections.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida.The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in X. axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. axonopodis. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida. Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker.Currently, five Xac related strains are being sequenced with FL-1195 completed, XacAw close in completion (chromosome completed, plasmids in progress) and annotation is on going, while three more genomes are in the gap closing stage. For XacAw, manual curation is currently in progress. The genome sequence has been compared to the reference strain XAC A 306 using MAUVE and global rearrangements were observed. Analysis of T3SS effectors revealed presence of unique XopF1 and avrGf1 effector genes which are absent from Xac A 306 strain. Mutant of xopF1 and double mutant of xopF1/avrGf1 are being constructed to determine their roles in host specificity. Illumina, 454 sequencing and optical mapping were used to obtain a complete genome sequence of Xacm strain FL1195, 4.9Mb in size. The strain lacks plasmids as compared to other citrus pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen Xcv 85-10 with a completely different host range. We also compared Xacm to the genome of citrus canker pathogen Xac 306. Comparative genomic analysis showed differences in several gene clusters like Type 3 effectors, Type 4 secretion system, lipopolysaccharide synthesis and others. In addition to pthA, effectors such as xopE3, xopAI and hrpW were absent in Xacm while present in Xac. These effectors might be responsible for survival and reduced virulence of this pathogen on citrus compared to Xac. We also identified unique effectors in Xacm that may be related to the different host range as compared to Xac. Xacm also lacks various genes such as syrE1, syrE2 and RTX toxin family genes, which were present in Xac. These may be associated with distinct virulence of Xacm and Xac. Comparison of the complete genome sequence of Xacm to Xac and Xcv provides valuable insights into evolution of xanthomonads and emergence of new strains with different host range and distinct virulence.
Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The citrus canker lesions will not rupture, thus do not release bacteria as normal lesions caused by the wild-type strain Xac. Our preliminary analyses indicate the 12 mutants released onto the citrus leaf surface from water-soaked spots were significantly less compared to the wild-type strain Xac A. Identification of the mutated genes in those mutants is underway.
We converted our experience gained from the computational analysis of Candidatus Liberibacter asiaticus proteome into a fully automated web-server for researchers to use. Computational sequence analysis, that is, prediction of local sequence properties, homologs, spatial structure and function from the sequence of a protein, offers an efficient way to obtain needed information about proteins under study. Since reliable prediction is usually based on the consensus of many computer programs, meta-severs have been developed to fit such needs. Most meta-servers focus on one aspect of sequence analysis, while others incorporate more information, such as PredictProtein for local sequence feature predictions, SMART for domain architecture and sequence motif annotation, and GeneSilico for secondary and spatial structure prediction. However, as predictions of local sequence properties, three-dimensional structure and function are usually intertwined, it is beneficial to address them together. We released a MEta-Server for protein Sequence Analysis (MESSA) to facilitate comprehensive protein sequence analysis and gather structural and functional predictions for a protein of interest. For an input sequence, the server exploits a number of select tools to predict local sequence properties, such as secondary structure, structurally disordered regions, coiled coils, signal peptides and transmembrane helices; detect homologous proteins and assign the query to a protein family; identify three-dimensional structure templates and generate structure models; and provide predictive statements about the protein’s function, including functional annotations, Gene Ontology terms, enzyme classification and possible functionally associated proteins. We tested MESSA on the proteome of Candidatus Liberibacter asiaticus. Manual curation shows that three-dimensional structure models generated by MESSA covered around 75% of all the residues in this proteome and the function of 80% of all proteins could be predicted. MESSA is free for non-commercial use at http://prodata.swmed.edu/MESSA/
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