Without the ability to culture Candidatus Liberibacter asiaticus (CLas) in vitro, the pathogen can only be studied within the Asian citrus psyllid vector or in the citrus or other host plants. CLas DNA in citrus tissue can be detected with various highly sensitive and robust PCR protocols, however, these methods do not reveal if the DNA target is from living, and pathogenic cells, from dead cells, of from extracellular CLas DNA that may be excreted by the pathogen. Treatment of bacterial cells with DNA intercalating dyes prior to qPCR has promise for distinguishing between live and dead CLas cells in citrus tissues; however, because CLas resides in citrus phloem there are obstacles to this approach. The overall goal of this project is to extend previous findings regarding the use of DNA intercalating dyes and optimize them for quantification of live CLas cells in citrus. During months 1-4 of the project our objectives were to: 1) Determine suitability of PMA-qPCR for distinguishing between living and dead CLas cells in citrus; and 2) validate and compare results of PMA-qPCR with EMA-qPCR. We have made significant progress towards meeting each of these objectives. Specificity and efficacy of EMA- and PMA-qPCR were determined using both purified plasmid DNA containing the CLas DNA target sequence and E. coli cells transformed with the same plasmid. Results with this model system confirm that both EMA and PMA treatments are specific for the CLas target sequence. Amplification of plasmid DNA in qPCR was inhibited 100% by both EMA and PMA. Estimates of live cells using E. coli with EMA or PMA gave similar results of ca. 10% live cells. If cells are heat killed prior to dye treatment, amplification is inhibited 100% . In the course of these experiments we also optimized variables in the protocol to give greatest sensitivity in the assay and the widest working range. We have conducted preliminary tests of EMA- and PMA-qPCR for distinguishing between live and dead cells in citrus seed coat vascular bundles, a tissue known to contain high titers of CLas, and in citrus leaves, both from CLas-inoculated trees in the greenhouse and from HLB symptomatic trees in the field. DNA extracted from seed coat vascular bundles that had been treated with EMA prior qPCR protocol showed about 25% of the CLas copy number of that in DNA from non-treated seed coat vascular bundles. We compared results of EMA- and PMA-qPCR with citrus leaf samples. We used leaves that expressed a range of HLB symptoms for these experiments. Samples were collected both from the greenhouse and from the field. Estimates of the number of live CLas cells in leaves treated with EMA were typically less than those obtained using PMA. Over a range of total CLas titers, estimates of live cells averaged 15% based on EMA-qPCR and 50% based on PMA-qPCR. Experiments during the remainder of this project will focus on validating preliminary results.
The L. crescens BT-1 genome was shared with Chris Henry at Argonne National Laboratory. He and his group are using this information along with the culturing medium formulation provided by Dr. Michael Davis to optimize the metabolic reconstruction of Liberibacter asiaticus. This has been hindered slightly by the absence of a defined growth medium for L. crescens. We have begun development of a defined version of the standard L. crescens culture medium developed by Dr. Davis. Manual curation of the L. crescens genome annotation and comparison of this with the genomes of L. asiaticus and L. solanacearum continues. The RAST annotation is the starting point for this comparison. In this system the gene functions are divided into 28 different metabolic subsystems. Of these subsystems the greatest differences between L. crescens and L. asiaticus were observed in: ‘amino acids and derivatives’ with 154 genes in L. crescens vs. 69 in L. asiaticus; ‘membrane transport’ with 26 in L.c. and 18 in Las; ‘motility and chemotaxis’ with 39 in Lc vs. 14 in Las; ‘stress response’ with 40 in Lc vs. 28 in Las; and ‘virulence, disease, and defense’ with 28 genes in Lc and only 19 in Las. These differences have implications in both culturing and disease development.
Primers to amplify the pathogenesis-specific genes are in hand and being used to amplify those genes in infected citrus. The search for the full length genes that are pathogenesis-specific in citrus was hampered by the lack of availability of citrus genome sequences. We hope that these genomes will be made available soon.
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.
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