Obj. 1.DNA bar coding to establish the identity and diversity in south Florida (Stansly, Brown). new mtCOI primers were designed based on amplicons obtained using general homopteran primers, resulting in increased specificity. PCR primers were used to obtain a ~1200 bp product; sequence analysis is underway. Obj. 2. qPCR detection of Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different IAPs has been optimized and analysis is underway for the first two replicates. Obj 3. Define the gross association of Ca. Liberibacter in thick sections’develop a ‘gross anatomical road map’ of Ca. Liberibacter accumulation in key organs, tissues, and cells; Obj. 4 Using the resultant ‘thick section road map’, elucidate at the TEM level the specific organs, tissues and cells where Ca. Liberibacter accumulates in the vector. We have adopted the potato psyllid, Bactericera cockerelli, as a surrogate for the citrus psyllid because we can rear it on tomato easily, and set experimental parameters (‘acquisition access’, ‘inoculation access’ etc.) that have the tightest possible definition with regard to timespan of actual feeding and source infectivity at the locus of the stylet. Our rearing system is ramped up to broad, robust capacity, providing for multiple studies by several experimenters in the transmission context simultaneously. Guts of infective and uninfective specimens of all life cycle stages (1st thru 5th instars, metamorphosing adult, unpigmented emerging adult (‘tenerals’), and mature adult) have been processed, mounted on stubs, and are being studied under the scanning electron microscope (SEM). Graphic differences occur that correlate directly to host-vector relationships at the histological level. These comparisons assist us with interpretation of cross-sections, and demonstrate the abundance of bacteria in infected adults relative to indigenous flora, as well as the buildup of bacteria during each consecutive stage. The pre-oral organ complex, referred to as the ‘oral box’, is the 0.1mm3 chamber that houses the convergence of the salivary ducts and esophagus, and the transfer of their fluids to the stylets, which in turn, penetrate the plant for feeding. The potential for bacteria to access the stylets, through this box, during egestion events, is very high and cannot be overlooked. Along these lines, it is known that in Psyllidae, the unused length of the larval stylets is externally wound into a groove around the face, but the unused length of the adult stylets is held inside an internal sac (Pollard 1970, Ullman and McLean 1986). Our studies of the oral box have shown that the internalization process retains the cuticular barrier between stylets and blood. The basal sections inside the oral box, however, may undergo modification during metamorphosis that can admit bacteria, and must be given scrutiny as well. 10-15 infective and uninfective adults + source plant material were collocated to the same paraffin block face in four different runs. 4-micron thick sections were cut from these blocks and mounted in alternation on slides and stubs for, respectively, the light microscope and the SEM. Slides were treated with ‘in-situ hybridization’ techniques that attach a visible gold/silver label to bacteria, allowing them to be localized inside organs, and directly compared to sections magnified under the SEM. These methods give consistent label in the gut, as well as a more general distribution in the abdomen. Strong signal also occurs in the spaces between salivary gland cells and inside the oral box. These localizations are best pursued further under the transmission electron microscope (TEM). Strong signal occurs in new apical growth (‘flush’) of infected source plants, but in sharply delimited patches, or ‘compartments’.
Samples for phloem plugging determination were collected from HLB infected mature field grown trees and from young greenhouse grown trees. A total of 29 trees and 4 cultivars were sampled; 22 trees were field grown. Callose plugs were 2.7 and 2.4 times more prevalent than phloem protein 2 (PP2) plugs in the field and greenhouse samples, respectively. Three Murcott field trees has a ratio of 13 callose plugs per each PP2 plug. For grapefruit, Valencia and Hamlin orange samples, the ratio range was 1.8 to 4.2 callose/PP2 plug. This data suggests that blocking callose production may be more productive than blocking PP2 production. Transformation experiments were initiated in efforts to produce plants that over-express the citrus ‘-1,3-glucanase gene, both with the constitutive 35S promoter (p35S ‘ BG-35T) and the Suc 2 phloem specific promoter (pSuc2-BG-35T). Using a modified Agrobacterium-mediated transformation protocol, 26 transgenic sweet orange shoots were regenerated containing p35S ‘ BG-35T (Valencia and Vernia sweet oranges) and 24 containing pSuc2-BG-35T (Valencia and Vernia). All have been micro-grafted to Carrizo citrange to expedite their greenhouse evaluation. Twenty transgenic Carrizo shoots were also recovered containing p35S ‘ BG-35T. Construction of plasmids for protoplast transformation has also been completed for the ‘-1,3-glucanase gene, and protoplast transformation experiments using new robust sweet orange suspension lines are being initiated. Protoplast transformation generally results in higher transgene copy numbers than Agrobacterium-mediated transformation. Twenty-eight genes containing a signal peptide (identified by SignalP) were cloned in TMV30b GFP viral vector. Thirteen constructs were successfully assayed in tobacco plants for symptom expression. Two (AS7 and AS13) out of the 13 showed interesting symptoms, and hence were selected for further characterization. AS 7 produced wilting and death of the whole tobacco plant within 2-3 weeks. AS13 transformed plants developed phyllody, stunting and very clear growth defects. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of these genes in planta was checked using RT-PCR. The leaf, petiole and roots of tobacco plants infected with AS7 were collected after ~3weeks for observation under the light microscope. AS7 caused very clear phloem collapse in the petiole, after 3 weeks of inoculation. These symptoms were not observed earlier, 10 days after infection.
A scFv library with activity against ‘Ca. Liberibacter asiaticus’ has been prepared at Beltsville. mRNA was purified from mouse spleens and converted into cDNA. The mice had been immunized with psyllid extracts confirmed to be carrying a high concentration of “Ca. Liberibacter asiaticus” A complete library of variable heavy chain (VH) and variable light chain (VL) genes were made by PCR amplification of the cDNA using a set of 44 primers. The (VH) and (VL) gene segments were then joined in a random combinatorial fashion by overlap extension PCR. The scFv genes were then ligated into the pKM19 phagemid vector which was used to infect Escherichia coli DH5. F’ cells with the aide of a helper phage. The resulting phage library is presently being screened to select phage clones expressing antibodies that bind to “Ca. Liberibacter asiaticus”. Our first attempts to select desired antibodies using extracts from HLB-infected rough lemon were not successful, probably because the concentration of the target bacteria in the rough lemon extracts was too low. We have therefore modified the screening procedure by incorporating magnetic microbeads. These microbeads bind to rabbit antibodies. To use them we raised standard polyclonal antisera in a rabbit against the outer membrane protein of “Ca. Liberibacter asiaticus”. These beads are added to plant and insect extracts to bind the “Ca. Liberibacter asiaticus” and then concentrated by magnetic separation. The phage libraries are then added to the “Ca. Liberibacter asiaticus” on the beads and screened in that manner. The outer membrane protein and antibody system for immunocapture of ‘Ca. Liberibacter asiaticus’ has however not proven to be successful. The polyclonal antibodies made in rabbits against purified outer membrane protein from ‘Ca. Liberibacter asiaticus’ work very well in detecting the purified outer membrane protein but are not effective in immunocapture. The most likely explanation is that the outer membrane protein is not well enough exposed on the surface of the ‘Ca. Liberibacter asiaticus’ cell. We have therefore cloned genes encoding the type IV pilus protein of ‘Ca. Liberibacter asiaticus’, since this protein should be well exposed on the cell surface and should be useful for immunocapture in place of the outer membrane protein. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins and have beenpurified. Emphasis is on recovering the proteins in native, soluble form, which is difficult, but we have now been able to accomplish it. These proteins will be used as antigen to prepare polyclonal rabbit antiserum for the immunocapture portion of the antibody screening process. In related research, we have also cloned and expressed a dinucleotide polyphosphate hydolase, and a polysialic acid capsule expression protein. These proteins, as well as the type IV pilus protein will be biotinylated, combined with strepavidin coated magnetic beads and used to bind phage expressing antibodies that recognize these targets. Thus we will finish with uncharacterized scFv antibodies binding “Ca. Liberibacter asiaticus” as well as a set of antibodies that bind to specific pathologically relevant proteins of the pathogen.
In the first quarter, a greenhouse hydroponics experiment was established to investigate the relationships between boron nutrition and HLB pathogen viability and / or disease expression in citrus. Many of the symptoms of HLB, especially the earlier symptoms have remarkable similarity to boron deficiency. The most notable similarity is that of vein corking, which is associated with rapid unorganized replication of phloem tissue that results from plugging and collapse of the original phloem sieve tubes. It is uncertain why the phloem is especially sensitive to boron deficiency. Boron deficiency also has root symptoms similar to HLB. In the early stages of boron deficiency, the roots stop developing and the tips become disorganized. After extended boron deficiency the roots begin to die back. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or antipsyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another large experiment is underway. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, so far do not look like they have resistance to HLB.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
1- The first objective of the second year was to build a plant growth room at the Citrus Research and Education Center in Florida (CREC). The physical construction/renovation of the growth room has not been started so far. The construction is expected to start at the end of September. The rootstocks that are currently growing in the lab are big and we will need to start growing a new batch until the growth room is ready. They cannot be transferred to another greenhouse because it will defeat the purpose of growing under controlled conditions. 2- Training of the manager Dr. Zapata has been completed at the IVIA under the supervision of Dr. Pena. 3- Initial material to establish the mother plants are being produced at the Department of Agriculture with Dr. Peggy Sieburth. She will start releasing the in vitro plants in September. Ideally these plants should be used immediately for grafting on the rootstocks; plants will be kept in vitro until the growth room is ready. The plants cannot be kept for more than 4-5 months on in vitro conditions. 4- A search for a full growth room technician started. The final hiring process will be completed once the growth room construction starts. We expect this technician to go for 2 weeks of training in Spain. At this moment, construction/renovation of the growth room is a major bottleneck for the progress of the project. Clean materials for the most important scion varieties of Florida have been obtained with the help of Dr. Peggy Siebuth through shoot-tip grafting. Clean rootstocks (stored at the lab at this moment-the only clean area we count with) are already 6-month-old and will be ready to be grafted with the clean scions for next October. However, we do not know yet when the growth room will be finalized. Only after this facility is fully operative, we will be able to perform the grafts with the clean materials. If the construction of the growth room is delayed further (more than 3 months), we have the risk of losing both the scions and the rootstocks, and consequently lose months of work (that could not be repeated until next year due to Dr. Sieburth’s agenda) and a lot of money. These delays (no growth room after 1.5 years of project) are making impossible to fulfil our objective for the end of this 3-year project. This situation is out of my understanding- Leandro Pe’a
Concerning the Nested PCR one step we are working on improvement of sensitive of reactions but with few progress in the sensibility of NPCR one step comparing against double step nested PCR, as discussed in the annual report. On the other hand, we did great progress on development of antibodies against Ca. Liberibacter asiaticus. All the 9 peptides mentioned on the third report were injected in rabbits for the polyclonal antiserum (PA) production. Female New Zealand white rabbits weighing 2 kg were endovenously inoculated with 200 ug of each antigenic peptides. Afterwards, this procedure was repeated two times at 14 days intervals, using 300 ug and 400 ug of antigenic peptides, respectively. Seven days after the last injection, the blood was collected through cardiac puncture and the serum titer and specificity were determined by indirect enzyme-linked immunosorbent assay (ELISA) according to Clark et al. (Clark, M.F., R.M. Lister & M. Bar-Joseph. 1986. ELISA techinques, p.742-766. In H. Weissbach & A. Weissbach (eds.), Methods in enzymology, v.118. New York, Academic Press, 820p). Preimmune serum was used as a negative control. Antibody titres of nine PAs were checked by an enzyme- linked colorimetric assay (ELISA) using the Anti-Peptide Antibodies methodology (Table 1). Just one (T3699-3) of the nine PA resulted in low reactivity against the antigen (10’g/mL). For the nest steps we will test the eight PA against the Liberibacter asiatic infected and or symptomatic plants. Table 1. ELISA assays of Polyclonal antibodies against the antigens. Peptides name T3699-1 T3699-2 T3699-3 T3699-4 T3699-5 T3699-6 T3699-7 T3699-8 T3699-9 Dilution Antibody O.D. (405 nm) 1:500 2.100 1.936 0.875 1.455 2.032 2.117 2.153 1.819 2.042 1:1.000 2.037 1.695 0.500 1.311 2.001 2.026 2.132 1.548 1.928 1:2.000 1.904 1.286 0.264 1.129 1.917 1.793 1.993 1.332 1.625 1:4.000 1.601 1.408 0.126 0.758 1.884 1.569 1.825 1.109 1.560 1:8.000 1.336 1.077 0.038 0.573 1.706 1.297 1.584 0.750 1.346 1:16.000 1.110 0.761 0.000 0.284 1.495 1.112 1.388 0.509 1.220 1:32.000 0.832 0.550 0.000 0.164 1.252 0.998 1.060 0.287 1.013
Continued efforts to improve transformation efficiency: ‘ The effect of antioxidants lipoic acid, glycine betaine and glutathione are being evaluated for increased transformation efficiency. Some treatments are showing a significant increase in transformation efficiency across a range of citrus genotypes.’ We have successfully developed an efficient transformation system for embryogenic citrus callus, (publication in Plant Cell Reports in press). This system works well for polyembryonic mandarin types (i.e. W. Murcott, Ponkan) that are seedless or more recalcitrant using the common Agrobacterium-mediated citrus method. The method should also work well for lemons. Horticultural manipulations to reduce juvenility in commercial citrus: The RES (Rapid Evaluation System). ‘ Commercial sweet orange, grapefruit, and specialty mandarin cultivars were propagated and planted in the RES. Several juvenile hybrids from our breeding program flowered and set fruit after only one year – our goal is to force flowering and fruit set in juvenile sweet oranges and grapefruits in 1-2 years. If successful, the same approach could be applied to transgenics. A two-year old field trial using a juvenile Valencia budline on more than 70 rootstocks is showing significant rootstock affects on precocious bearing. Transformation of precocious but commercially important sweet orange clones: transgenic plants of precocious ‘Vernia’ sweet orange somaclones were regenerated and micrografted for further study of early flowering. Transgenic approaches to reduce juvenility: ‘ Whole plants generated from ciFT and empty vector control transformation experiments of Carrizo are being evaluated by both PCR amplification assays and a repeat of the screening histochemical GUS assay. No obvious phenotypes have yet been observed among the whole plants, however, flowers have occasionally been observed to occur on in vitro shoots. ‘ Putative transformed Duncan grapefruit whole plants in soil and shoots being rooted in vitro have been generated. We will be doing additional transformation experiments as soon as fresh seed becomes available. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. ‘Through a project being conducted by an HHMI-sponsored undergraduate student, Melanie Pajon, we will also be cloning the tomato FT ortholog and using it to obtain citrus transformed by a heterologous FT gene.Transformation of Samsun tobacco with the ciFT genes has resulted in a number of T0 plants of each of the 3 ciFT constructs, some of which have produced T1 seeds. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. Phenotypes of the T0 plants have ranged from early flowering, multi-branching, dwarfs to ones very similar in architectures in the wild type parent.
Weather towers are in place in a ridge grove location and collecting data for determining weather conditions to optimize low volume spray applications. Towers will be placed in a flatwoods grove location during September. As noted in earlier reports the ability to measure spray droplet size from low volume spray machines is not possible. Funding for that part of the research project has been returned to the granting agency.
The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Ca. L. asiaticus with the advice and permission from program manager of FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. In addition to previous report, the following progress has been made recently. To understand how Ca. L. asiaticus make a living including nutrients acquisition and overcoming of plant defense in citrus, we first analyzed its metabolic capability and nutrient requirement by conducting curated metabolic reconstruction. Our data indicated that Ca. L. asiaticus could rely only on aerobic respiration rather than on anaerobic respiration. Ca. L. asiaticus could not synthesize 12 amino acids and needs external folate, quinines, biotin, thiamine, pantothenate, and pyridoxine for its growth. By analyzing the metabolic pathway, we also identified 5 genes that were missed in current genome sequence. The work is in progress. The transcriptome study is in progress.
Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. Characterization of the beneficial microorganisms associated with citrus in the local environment might identify beneficial bacteria for practical use to control HLB. Major achievements: This research has resulted in four publications (three published, one in preparation). 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida (Sagaram et al. 2009). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following publication ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ (Trivedi et al. 2010) which has been published on AEM. 3.Development of a proper in vitro screening system that provides repeatable and reliable results in shorter periods of time is an important step for isolation of efficient bacterial antagonists. We have developed a method to screen antagonistic bacteria against uncultured HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. 4.Isolation of plant growth promoting bacteria from potential escape citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. We specifically focused on delineation of the cultivated endophytic bacterial isolates and characterization of their salient metabolic features. A total of 227 and 159 morphologically distinct colonies were isolated individually from uninfected and infected samples, respectively and were taken for further analysis. We used a detailed approach for screening novel plant growth promoting (PGP) isolates by conducting qualitative, quantitative and PCR based assays for traits related to mineral nutrition [Phosphate (P) solubilization, siderophore production, Nitrogen (N) fixation], development [Indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relieve [production of 1-amino-cyclopropane-1-carboxylate (ACC) deaminase] and production of quorum sensing [N-Acyl Homoserine Lactones (AHL)] signals. A total of 39 bacterial isolates showing at-least 5 beneficial traits were further taken up for quantitative estimation of PGP activities. The selected isolates belonged to 12 different genera mostly belonging to species of genus Serratia, Pantoea, Pseudomonas, Bacillus Burkholderia. Several promising strains in our culture collection represent the first isolates of bacterial groups that have only been detected based on culture-independent methods. From the culture collection six bacteria isolates were found to reduce the number of viable Ca. L asiaticus cells by EMA-qPCR based method.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. We are currently assessing citrus genes modulated by Las infection in 1) citrus stems and roots, 2) citrus grove, 3) citrus varieties that show tolerance, and 4) at different infection stages. Using Affymetrix microarray analysis, we detected a total of 2,795 and 1142 probe sets with significantly (p< 0.05) altered expression levels in stems and roots of Valencia sweet orange (Citrus sinensis Osbeck), respectively. At a cutoff point of 1x log fold change (logFC), a total of 580 transcripts were significantly up-regulated and 350 down-regulated in stems. A relatively lower number was up-regulated (58) and down-regulated (58) in roots. Different sets of plant genes including those related to response to biotic or abiotic stress, transcriptional factors, transport, cell wall re-modeling and biogenesis were represented in both sets. Highly up-regulated genes (>3x logFC) in stems included 2OG-Fe(II) oxygenase family protein, WAK-like kinase, Lectin-related protein precursor, Cu-Zn superoxide dismutase, Zinc transporter protein ZIP1, many of which are involved in oxidative stress that produce highly toxic reactive oxygen species (ROS). Homologs of nucleotide binding and Leucine-rich repeat (NB-LRR) domain containing proteins involved in gene-for-gene resistance were down regulated in both tissues with some such as TIR-NBS-LRR proteins being re-pressed in stems only. Suppression subtractive hybridization analysis of RNA from Las-infected Mandarin line (Citrus x limonia Osbeck) showed up-regulation of pathogenesis/resistance, biotic stress related, cell wall re-modeling gene groups and transcriptional factors and down regulation of NB-LRR domain containing proteins. Zinc is a cofactor in many redox reactions and zinc deficiency-like in plants have been attributed to damage by ROS. This suggests that the zinc-pattern-deficiency symptoms associated with HLB is caused by ROS generated by citrus plants in response to Candidatus Liberibacter infection.
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 Xanthomonas 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. a. 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. Major achievements: Currently, five Xac related strains are being sequenced, which includes Xac Aw and A* strains which have restricted host range compared to the A type strain, X. axonopodis pv. citrumelo strains (copper resistant and non-copper resistant), and Argentinian strain (copper resistant). Both 454 Titanium and Illumina (solexa) methods were used. The genome sequences are completed for Xac Aw strain and Xanthomonas axonopodis pv. citremelo. The papers are in preparation. Aw strain: Complete De novo genome sequencing of Xanthomonas axonopodis pv. citri strain Aw was done. 454 Titanium paired end reads were used for making contigs and scaffolds using Newbler with 24X coverage. It was then assembled and closed by using contigs made from Illumina 75bp paired end reads using CLC Bio with about 300X coverage. Finishing was done using Opgen MapSolver. Plasmids for the strain were obtained by reference sequencing using only the Illumina 75bp paired end reads. The size for genome was 5.34 Mb and for plasmids pAw1 and pAw2 was 29 and 60 Kb approximately. The plasmid sequences need further confirmation due to the repeat regions. The genome sequence has been compared to the reference strain 306 using Mavue and global rearrangements were observed. In detail analysis of T3SS revealed presence of AvrGf1 and absence of HpaA gene. Further analysis of other secretion systems, LPS, EPS, motility clusters and other regions different form reference strain is currently in progress. Currently, we are also working on closing the gaps of the other three strains XAC A*270, XAC A44 and XACM 1381.
This project has been revised extensively to take into consideration of suggestions from reviewers and CRDF council. The title has been revised as: Control of citrus Huanglongbing by screening small molecules which are antimicrobial against Candidatus Liberibacter asiaticus. Specifically, the target has been suggested to be SecA for the first year. Protein secretion in bacteria is a critical and complex process. SecA is the protein translocase ATPase subunit and a superfamily 2 RNA helicase, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. Identification of small molecule inhibitors that intrude the function of SecA could lead to potential antimicrobial agent. In order to find the novel inhibitory structures we followed the below steps in the current design. 1) Identification/Build the 3D protein structure & optimization 2) Pharmacophore design based on ATP binding site 3) Virtual screening of commercial databases 4) Generation of a small subset of structures 5) Molecular docking studies & filtration of the structures 6) Selection of best candidates by scoring functions & chemical intuition 7) Biological activity studies against SecA Several 3D structures of SecA protein have been reported in Protein Data Bank (PDB), nevertheless we used the best resolution (<2.0Ao) and ATP bound model 2FSG.pdb in our study. Maestro module of Schrodinger software was used to add hydrogen's, electro static potential charges to the protein and optimized the structure with molecular minimization by using AMBER force field. Then identified the pharmacophores at ATP binding site and subjected these to virtual screening with the Lead Like structures from commercially available ZINC databases. Further with the best hits from database structures we build a small set of ~5000 structures as subset and all these structures were docked at ATP binding site to evaluate the docking scores and their molecular poses at the active region. Based on the dock scores we filtered ~4500 structures and selected ~500 (10%) for further docking & minimization studies and evaluated the scoring functions. Based on scoring functions, physicochemical properties and our chemical intuition we have chosen 50 structures (10%) for biological activity studies. The activity studies against SecA are in progress and will be presented within couple of months. All the molecular modeling studies have been performed on Linux system by using Schrodinger suite programs.
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