DNA from a close cultured relative to Liberibacter asiaticus, strain BT-1, was given to our group by Dr. Michael Davis. In Dr. Davis’ studies strain BT-1 showed 92% 16S rRNA gene homology with Liberibacter americanus. From this DNA we obtained 16S sequences that matched most closely to Liberibacter solanacearum at 95% DNA sequence homology. Based on this close relationship to the Liberibacter genus we have begun sequencing the genome of strain BT-1 on the Illumina GaIIx platform. This genome being from a close cultured relative to Liberibacter asiaticus is anticipated to greatly improve metabolic models and aid in the development of a L. asiaticus growth medium.
Additional sequencing is needed to close the current Bt-1 draft genome. Two 454 mate-pair libraries were prepared with 3 and 8kb fragment length. PacBio and IonTorrent shotgun libraries were also sequenced. Using this data the draft genome has been reduced from 109 to 6 contigs as of June 1st. With the aid of the optical map we are able to order these remaining contigs and are attempting to close the remainder of the genome with both targeted Sanger sequencing and bioinformatic methods. All genomic and physicological data has lead us to believe that the babaco bacterium BT-1 is the closest cultured relative to Liberibacter asiaticus and Liberibacter solanacearum. We plan to propose the name Liberibacter crescens for strain BT-1 and will refer to it as such in all subsequent reports.
An analysis of the candidate pathogenesis-specific proteins was made to determine which ones would serve as good candidates for protein purification. The citrus genome was not yet publicly available so it was impossible to design primers that could amplify the host genes that appear to be preferentially expressed during pathogenesis. Primers are being designed for the 26 Liberibacter proteins that are expressed exclusively during pathogenesis.
Multiple medium formulations based on the metabolic reconstruction of Liberibacter asiaticus have yielded no positive results. More information on the intracellular environment in the plant and in the psyllid is needed. Additional Liberibacter genomes will also increase the specificity of these metabolic models. We are attempting to isolate the undescribed beta-proteobacterial endosymbiont of the psyllid gut. Correlations between Liberibacter asiaticus populations in the psyllid gut with this unknown beta-proteobacterium were found in our 16S study. If isolated this bacteria may serve as a partner for L. asiaticus in culture. Only three isolates have been obtained thus far and none proved to be the beta-proteobacterium based on 16S sequences.
An analysis of the pathogensis-specific Liberibacter proteins was made. Most are not obvious drug targets but one certainly is, topoisomerase IV subunit A. This enzyme is the target of quinolones. We also expect quinolones to be pholoem mobile. All that remains is whether quinolones such as can inihibit a Liberibacter infection in planta. In the meantime, we will determine whether quinolones inhibit the colosely related babaco bacterium.
A fatty acid profile of the babaco bacterium isolate BT-1 was generated at OpGen. This profile could not be compared to members of the Liberibacter genus as they are not currently cultured; therefore placement of BT-1 in the Liberibacter genus was inconclusive. A comparison of Liberibacter asiaticus genome and the draft BT-1 genome suggested several potential inadequecies in L. asiaticus. Based on these findings, additions to L. asiaticus media preparations were suggested to Dr. Michael Davis. The transcriptome of L. asiaticus in culture media may provide insight into metabolic insufficiencies and is anticipated to improve the metabolic model. We have begun working toward the sequencing of the L. asiaticus transcriptome across time in Dr. Davis’ static cultures as well as in removed psyllid midguts. Preliminary RNA extractions had insufficient yields for transcriptome sequencing.
Since the completion of the metabolic model of L. crescens we have been focusing on the development of a genetic system in this Liberibacter model. Specific genes involved in carbon metabolism, regulation, and cell wall recycling have been selected as targets for knockout in L. crescens. These genes are present in L. crescens but not L. asiaticus and through the development of knockouts in L. crescens we hope to better understand the constraints on L. asiaticus growth in culture. Electroporation using several plasmids / cosmids, such as p15TV-L, pLAFR1, pGS9, pHRGFPGUS, and pUFR071, were performed to test the transformation efficiency on L. crescens. It was found that only the vector pUFR071 was able to transform and replicate in L. crescens, which leads to an assumption that plasmids of smaller or similar size of pUFR071 should be able to electroporated into L. crescens. The gene targetted for knockout will be cloned into a suicide vector (such as p15TV-L) with a kanamycin cassette inserted in the middle of the gene and transformed into Escherichia coli DH5a. The construct will be confirmed by sequencing and the development of kanamycin resistance. The final construct will be electroporated into L. crescens. Chromosomal crossover will occur and mutants will be selected for kanamycin resistance. The knockout mutant will be confirmed by sequencing of the target gene.
Media development based on the Liberibacter asiaticus metabolic reconstruction continues. This process is slowed by an incomplete annotation and the lack of a closely related bacteria with an established growth medium. It was suggested that Liberibacter asiaticus may be dependent on other microbes in the psyllid gut and the citrus phloem. The bacterial diversity in citrus phloem was determined previously (Tyler et al 2009). The microbial communities of the psyllid will be elucidated through targeted 16S amplicon sequencing. If a relationship is discovered a co-culture methodology will be explored further. To this end DNA was obtained from psyllids and their Liberibacter asiaticus titer was evaluated with qPCR. A universal bacterial 16S rRNA gene primer set was then used to amplify this gene from any bacteria present in the psyllids.
Amplification of the pathogenesis-specific proteins in Liberibacter continues. The strategy for cloning these genes once amplified is being planned.
The babaco bacterium (BT-1) genome Illumina sequence data has been assembled into 109 contigs. These contigs cover approximately 1.43 Mb which gives BT-1 a slightly larger genome than those of L. asiaticus and L. solanacearum. Inital annotation was done in RAST and metabolic systems comparisons are underway. Initial analysis shows that BT-1 is able to synthesize more amino acids than Liberibacter asiaticus. This difference is not anticipated to be the key to culturing Liberibacter asiaticus since a full complement of amino acids was included in all media formulations to date. A culture of BT-1 was given to us by Dr. Michael Davis. This culture will provide DNA for additional genome sequencing which is needed to close the genome.
We were able to close the L. crescens BT-1 genome without the use of pcr. The final genome is 1,504,659 bp long. Genome alignments between BT-1 and Liberibacter asiaticus and Liberibacter solanacearum showed low overall macrosynteny. There was a high degree of gene-sequence conservation but gene order was very divergent. Further genome comparisons of the Liberibacter species have yielded primarily regulatory differences. Attempts to compensate for biosynthetic inadequecies in L. asiaticus through media additives have been unsuccessful. It does not seem that any single metabolic deficiency is responsible for unculturability in L. asiaticus. Two prophage regions were identified in L. crescens. These regions appear to be unrelated to those in L. asiaticus and L. solanacearum.
We have done initial studies on purifying Liberibacter cells from psyllid nymphs and adult psyllids. Our initial attempts to purify Liberibacter cells from nymphs were not productive. Based on qPCR data, the calculated population of Liberibacter cells in a representative portion of the nymphs was much lower than for an equivalent number of adults. Fractionation of Percoll gradients loaded with extracts from nymphs and analysis of the Liberibacter content of the fractions indicated lower than desired recovery of Liberibacter cells from nymphs. Our initial results using adult psyllids were more promising. A sampling of the adults showed a much higher level of Liberibacter than in the nymphs. The Liberibacter numbers from Percoll gradient fractions indicated that Liberibacter cells purified from adult psyllids band much higher up in the gradient than when extractions are made from plant tissue. Fluorescence In Situ Hybridization (FISH) microscopy performed on these fractions indicate that Liberibacter cells are embedded in a matrix which likely is remnant insect tissue. This suggests that the current buffer extraction does not effectively dislodge Liberibacter cells from insect membranes. We will continue working with adult psyllids and will modify the extraction buffers to include different non-ionic detergents as a means of dislodging bacteria from insect tissues and increasing the recovery of “free” bacterial cells. If the detergents act as desired, we expect to see a greater banding of Liberibacter cells further down in the Percoll gradient, indicating an increased number of “free” bacterial cells.
Data generated by Proteomics-based Change Mediated Antigen Technology were finalized to identify the pathogenesis-associated proteins. A total of 155 biomarkers were identified exclusively in the infected citrus phloem. 74 biomarkers were found within the green plant database. 55 biomarkers were found in the bacterial database. More importantly, 26 biomarkers were found in the Candidatus Liberibacter asiaticus database. The discovery of these citrus greening-specific biomarkers will be useful to design future experiments on citrus greening diagnosis, such as generating antibodies to detect citrus greening disease.
The visiting scientist who began work with the scFv library made with the earlier grant from CRDF has been making excellent progress. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. Study of the literature showed that the media used to grow the E. coli and other details of the culture conditions greatly influence the yield of scFv obtained from culture lysates. We have found that a very rich and buffered medium, (2X yeast extract Tryptone broth with phosphate buffer) works best. The medium is supplemented with glycine, sucrose and IPTG at various stages of the expression protocol. With this protocol we can produce purified scFv at concentrations in the mg/ml range. The tissue print assays are being done on nitrocellulose membranes. A range of parameters have been optimized, including the concentration of non fat milk used as blocking protein, duration of blocking and wash steps, the dilution and concentration of the scFv antibodies used for the detection of the ‘Ca. Liberibacter asiaticus’ in the tissue print, and the concentration of the detection antibody, which is a commercially available monoclonal antibody that recognizes the 6X His Tag and is labeled with alkaline phosphatase. Cross reactions with healthy plant tissue can be a problem, especially if the concentration of scFv is too high. Best results to date are with petioles. We have established tissue printing protocols for 18 scFv, that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. We have systematically sampled leaves from chronically infected rough lemon trees grown in our greenhouse. Petioles are sliced by hand for tissue prints and alternate slices are set aside for DNA extraction and qPCR to estimate the quantity of ‘Ca. Liberibacter asiaticus’ in the sections used for tissue printing. Interestingly, petioles from leaves with blotchy mottle and low qPCR values (= high concentrations of ‘Ca. Liberibacter asiaticus’ DNA) give poor results by tissue printing, but sections of petioles from asymptomatic leaves from the same branch produce higher qPCR values (fewer bacteria) but better tissue printing results. This is consistent with the concept that the Liberibacter cells present in phloem of severely symptomatic leaves are dead, and that therefore the proteins on their surface have been degraded and are no longer recognized by the scFv. Tissue prints typically show strong color development in the phloem ring. Tissue prints from healthy control plants are clear. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. We will carry out further experiments with the scFv using the FLAG epitope for detection of the scFv. The scFv will also be used to label sections for examination by fluorescence and electron microscopy. We have provided several scFv to cooperating researchers at USDA ARS Ft. Pierce.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. Efforts have been underway during this quarter to recruit a visiting scientist to work on this project. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have identified a suitable candidate for the visiting scientist position and the visa process is moving along. Related research with the existing scFv is underway on project 551.
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