Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, April 2012. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding is for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for nearly four years, and as of mid-April, 2012, we have processed approximately 27,500 grower samples, with approximately 600 arriving since the last report, January 2012. Additionally, over 22,000 samples have been received for research, of which more than 4,000 were within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. We now lyophilize all plant samples prior to BeadBeating, which enables superior sample maceration when compared to use of liquid nitrogen. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology departments that also contribute funds from their research grants to support the labor and supply costs for research samples. The protocol established in 2010 for the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve is being used for research and extension samples. The basic diagnostic service remains available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR-processed-samples to include data from individual groves that consented to have their data used. In conjunction with an epidemiologist and computer mathematician, the spread of the disease will be modeled. These studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
We have cloned the potential effectors outlined in the proposal into binary vector based CTV vector at a position that maximizes the expression of the effectors.These clone have been infiltrated into Nicotiana benthamiana plants to purify CTV virions for inoculation of citrus. The following potential effectors have been already in citrus; CLIBASIA_05165 at position 1134356- 1133904, CLIBASIA_05605 at position 1216188- 1215856, CLIBASIA_01555 at position 334228- 333266, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. Additionally, we are also testing full-length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the Pathogen-associated Molecular Patterns (PAMP) activity associated with Flagellin domain of HLB. These plants have been tested by ELISA for CTV and consequently for the presence of the effectors. Initially for citrus inoculations we use Citrus macrophylla which is a preferred host for CTV. Subsequently a large batch of either sweet orange and /or grapefruit citrus varieties will be used for graft inoculation from CTV infected C. macrophylla, since we have to screen for HLB resistance with sweet orange and /or grapefruit. These plants are presently being tested at HLB resistance by Mike Irey at US Sugar Corporation, Clewiston, FL, and at the DR. Dawson green house facility at CREC, Lake Alfred, FL. We also want to screen for HLB resistance reciprocally by graft inoculation of HLB positive citrus plants with buds from citrus plants expressing effectors.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. solanacearum and published in PLoS ONE 6(4): e1913, 2011. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of GENOME REDUCTION. Based on the variations within the Las prophages, eight different populations (genotypes) have been identified. In addition, a major chromosomal deletion/insertion of Las was also identified, and their correlations with phenotypes and disease severity of HLB are under investigations. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, confirming only one operon responsible for zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 48 hrs based on qPCR estimation. The Las bacterial growth reached Stationary Phase and Death Phase in 48-72 hours in the liquid cultures. We are looking into factors affecting further growth. Fifty-one BAC clones with overlapped Wolbachia endosymbiont of Diaphorina citri (wDia) genome sequences were screened using wDia specific primers from BamHI BAC library and were sequenced. The average size for the 51 clones was 85.4kb with 95-100% coverage and the average GC content is 34.3%. Assembly results indicated that due to large amount of repeat elements, such as transposase, only 13 BAC clones were able assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.
Single color light emitting diodes (LEDs) were decided to be used in the leaf measurement system instead of regular light source and band-pass filters for cameras. By using the LED-illumination, the problem with low transmission efficiency of band-pass filters was resolved. LEDs in 591 nm (target wavelength) and 400 nm (reference wavelength) as well as a LED driver were purchased and their responses were evaluated and confirmed using a mobile spectrometer. An electronic circuit was designed for LED illumination system which is able to switch between two wavelengths and also control the amount of light in each wavelength separately. In order to use the maximum light of LEDs, the angle of LEDs’ light were narrowed down using proper lenses with a field of view of 24′. A box containing two cameras, LEDs, control circuit and also the imaging platform was design and created. As this box covers the leaf during the imaging and only the LED’s light is illuminated on the platform, the effects of other light sources in the grove can be eliminated so that imaging during the day is also possible. Also the distance between cameras and leaf can be kept fixed during the imaging. A visible linear polarizing laminated film was purchased and cut to be placed in front of the cameras and LEDs. The polarizing film in front of the LEDs and one of the two cameras was set to be in the same direction, however a polarizing film was installed in a perpendicular direction in front of the second camera. The frame grabber’s software was customized so that it can capture two snapshots from two channels at the same time. The preliminarily evaluation of leaf measurement system was performed in the Lab with some citrus leaves in different conditions. Four images were captured from each leaf, two images at 591 nm with parallel and perpendicular polarizing directions with light source and also two images at 400 nm with the same directions. The visual evaluation of images confirmed that there are differences between the images from the same scene when they were captured with different situations. The leaf measurement system is now ready for an experiment in a citrus grove.
The main focus is the selection of antibody fragments and peptides against Huonglongbing (HLB), Citrus Variegated Chlorosis (CVC) and the development of immunoassays that incorporate these protein products. We have been able to make significant progress with the identification of new markers and evaluating their application in conventional ELISA immunoassays. We have continued our selection process of scFv antibodies directly against three pathogens, this time we incorporated a subtractive selection through phage display. Last year we were successful obtaining several highly reactive antibody fragments that recognized surface proteins some of them were common between the gamma proteobacteria like Xylella and Xanthomonas both of which express a common though not identical outer membrane protein, MopB. This year we have incorporated a negative selection where the antibody library was bound to the other bacteria first, so if we are interested in CVC we first expose the library to E.coli and processed the phage that did not bind to E,coli. This was then repeated with Agrobacterium and Xanthomonas and then the remaining phage were exposed to Xylella. Those that bound after 2 rounds of selection were then processed to obtain individual clones that can be screened for specificity. Several highly reactive antibodies have been found and new ELISA tests have been performed, and validations are still under biological testing with promising results. Antibody fragments of different sizes were selected and reactivity was highly specific for X. fastidiosa for 13 clones that will expressed, amplified and purified for additional validation assays against infected samples from the field and greenhouse. We have also utilized a new strategy in Brazil, which consisted of a biopanning (selection) of 12-mer random peptides (2 billion clones expressed in M13 vectors) against sap extracts of infected HLB citrus and non-infected samples (controls). The controls were used for subtraction and then for positive selection against infected samples as shown in. We have identified 46 peptide sequences, which were further validated against the infected sap. After identification of sequences, we are getting custom synthesized five biotinylated random peptides with 8 additional residues of the pIII protein of the phage capsid, and with amidation of the carboxy-end. This specific request has not arrived yet, and they will be tested as a preliminary assay in Brazil in August/2012, in order to verify if the peptide design will work.We have also performed a highly sophisticated preparative fractionation Xylella in order to identify additional biomarker proteins. We have isolated proteins present in various subcellular locations in the Xylella bacteria and sequenced the proteins using high resolution proteomics procedures. We used the enzyme trypsin to shave off proteins present on the outermost surface of the cell the outer membrane and then we confirmed the presence and identity of various proteins by high resolution proteomics. We then isolated the membrane fractions to identify all of the proteins present in the outer and inner membranes. We fractionated the secreted proteins present in the periplasmic space as well as outside the cell. A major protein secreted was designated PD1 and we raised antibodies against this protein and found that this protein is present in all fractions and predominantly that are secreted. We compare this distribution of PD1 with that of MopB a protein that has previously shown been shown to be the major outer membrane protein. We are in the process of generating a high titer antibody against PD1. This hypothetical protein is highly conserved 95% between the PD and CVC strains. This antibody will be very useful for detecting the bacteria and possibly the disease at very early stages.
Thus far, 686 media formulations have been tested to support planktonic growth of Candidatus Liberibacter asiaticus (Las). No substantial planktonic growth was observed in any of the media. Various medium additives, such as a lipid mixture, individual amino acids such as asparagine, mineral salts, and peptone preparations appeared to promote biofilm formation of the surface of alimentary canals of Diaphorina citri used as a source of inoculum. Deposition of Las onto the bottom of culture plates by centrifugation in an effort to simulate formation of biofilms appeared to promote grow of Las in some medium formulations. This line of research is being further pursued.
In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have now closed the circular genome of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. A circular genomic DNA sequence of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle (Vinca) in Brazil has been completed. The genome size is 1,195,199. The circular genome has been extensively confirmed by PCR. Particular attention has been paid to confirmation of truncated or incomplete proteins and pseudogenes. The average GC content of Lam is 31.12%. So far, 1,071 genes have been called, with 1,003 encoding predicted proteins, and 9 encoding rRNA genes and 45 encoding tRNAs. There is a surprising lack of overall nucleotide similarity among the Liberibacter asiaticus (Las) and L. solanacearum (Lso) sequenced strains. To see large scale conservation of gene organization, the PROmer version of MUMer had to be used. This analysis revealed that the overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 851 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 8 genes common to Lam and Las but not found in Lso. The latter included a DNA uptake competence gene. As with both Liberibacter asiaticus (Las) and L. solanacearum (Lso), two prophage were found, and with somewhat similar organization. As with Las, two circular forms were confirmed in Lam, with Lam-SC1 being 39,941 bp and Lam-SC2 being 16,398 bp in size. SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. This stable plasmid lysogen can be useful for lysogenic conversion (host range and pathogenicity enhancing), and indeed lysogenic conversion genes are found on these plasmid prophage—specificially peroxidases and adhesins. SignalP was used to identify 21 predicted Lam proteins with Type II secretion signals. Since Liberibacters occupy an intracellular niche, Type II secreted proteins would be immediately introduced into the phloem cell cytoplasm. These are currently being further analyzed as potential effectors of disease.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ (CaLas) and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of CaLas that are likely to be on the surface of the CaLas cell and/or related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, two pilus proteins, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. The vector and expression system that we have used for this project allows selection of the scFv antibody expressed from a phagemid genome but packaged on the surface of an M13 particle. This facilitates selection, but large scale expression of the scFv requires cloning of the scFv gene into a cognate plasmid expression vector. We have had problems with many of our scFv at this step, because ‘stop’ codons can accumulate in the phagemid without affecting the selection process, but prevent expression from the plasmid vector. During this reporting period we have repaired the improper ‘stop’ codons for several of our scFv to allow full expression of the scFv. These scFv were selected because they showed the desired specificity when selected in the phagemid format, but failed to produce scFv when used in the expression vector. The repairs were done by sequencing the defective scFv and identifying the stop codons, designing primers for a series of PCR that allowed amplification of the scFv while replacing the incorrect codons with corrected sequence, and transforming the corrected plasmid with the scFv into E. coli for expression. We have corrected the sequences for scFv that bind FlhA (scFv B947, B1096, B1072); KpsA (B520, B1199, B1202); the major outer membrane protein OmpA (B743); Pilus protein (B556, B557). These scFv are now available in pKM16, our expression vector for testing. The next steps will require expression of the scFv, purification of the scFv and testing it for yield and specificity against purified antigens. These scFv will be added to our inventory of multiple scFv for each target of interest to improve our chances of finding scFv that will be extremely useful for detection assays and for labeling cells for scientific studies. The visiting scientist who was performing this research returned to China during this reporting period. Due to the short time period remaining on this grant a replacement scientist was not found.
A comparative field trial involving two copper loaded silica nanogel (CuSiNG) formulations (Cankicide-pH7 and Cankicide-pH4) and a series of commercially available Cu biocides (Kocide 2000, Kocide 3000, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop WP, Nordox 75G and Magna-Bon) was conducted in a Ray Ruby grapefruit grove in Vero Beach FL. The disease incidence in the untreated checks was 60-65%. Overall, all copper based treatments were effective despite 45 inches of rain from August to October due to the presence of windbreaks surrounding the trial block. The Cankicide pH7 treatment set at the equivalent metallic copper to Kocide 3000 and Cankicide pH4 adjusted to a similar metallic copper rate as Magna-Bon controlled canker on fruit at the same level as the standards. Detailed materials characterization data using High Resolution Transmission Electron Microscopy (HRTEM), X-Ray Photoelectron Spectroscopy (XPS) and X-Ray Electron Diffraction revealed that copper is embedded in silica matrix in both particulate (<10 nm size; crystalline) form and in complexed (i.e. chelated Cu; amorphous) form. The XPS study indicated the presence of Cu in more than one oxidation states. The HRTEM image of CuSiNG material at low magnification showed micron-size structures. However, a careful investigation of the image at high magnification HRTEM revealed that the CuSiNG material consisted of a gel-like network of copper embedded nanoparticles. The XRD data showed that the material is overall amorphous. These unique features of CuSiNG material are not observed in traditional copper compounds.
A graduate student was hired for developing the proposed HLB detection system using polarized light and conducting experiments for this project. A suitable camera and a frame grabber for this project was identified and purchased. Proper bandpass filters and polarized filters are currently being searched. A relationship between two cameras, their field of view (FOV) angle, and distance between the cameras and leaf sample were investigated, and an overlapped area for the two cameras can be calculated. The overlapped area is where we acquire images of leaf, which will be analyzed for HLB infection status. With a distance of 26.5 cm between the camera and leaf, an overlapped area becomes 4 cm . 6.17 cm, which is similar to leaf size. The actual distance will be determined based on the actual size of an imaging area needed. A leaf measurement system was designed for detecting HLB infection status. The next step will be to acquire necessary filters, and to test the cameras in a laboratory setting.
Pathogenicity islands observed in the CLas genome potentially harbor bacterial effectors required for pathogen virulence, multiplication etc., and probably insect transmission determinants for the horizontal gene transfer. Many of the hypothetical proteins in CLas which do not have any homolog’s in other phytopathogenic prokaryotes potentially may function as effectors of pathogenicity. To functionally identify and express these putative effectors in citrus and study their role in pathogenicity we are using the CTV-vector system which expresses foreign genes in the citrus phloem, where the CLas bacteria are located in the infected plant. We have cloned most of these potential effectors outlined in the proposal into binary vector based CTV vector and have infiltrated Nicotiana benthamiana plants to purify CTV virions carrying putative effector for inoculation of citrus. The following potential effectors have been already in citrus; CLIBASIA_05165 at position 1134356- 1133904, CLIBASIA_05605 at position 1216188- 1215856, CLIBASIA_01555 at position 334228- 333266 and wild type CTV as control. They have been tested for CTV accumulation (consequent presence of effector) by ELISA and are presently being tested at HLB resistance by Mike Irey at US Sugar Corporation, Clewiston, FL. Other CLas putative effectors in citrus and need to test by ELISA before challenge inoculation include the following; CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. Additionally, we are also testing full- length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the PAMP activity associated with Flagellin domain of HLB.
During two years of this project, we made substantial progress in the detection of Candidatus Liberibacter asiaticus (CLas) in citrus tissues and psyllid vector using non radioactive digexigenin labelled probes. We developed and used both digoxigenin-labeled Plabeled PCR probes as well as RNA probes. We produced digoxigenin-labeled PCR products using specific primers for the OMP, RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA intergenic regions.Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the tissue for the blots and to isolate total nucleic acids template necessary for the amplification of CLas specific DNA. Primers were designed based on the CLas sequence information and amplified specific amplicons from the DNA isolated from HLB infected plants, and amplification was not observed from the healthy control plants grown under similar conditions. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes failed to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription and the probes gebnerated substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region and whole leaf and branch containing multiple leaves from healthy and infected citrus on nylon membranes. The hybridization observed with the midrib imprints and whole leaves showed clear signals compared to stem imprints. In other experiments we used imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprinted on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. This procedure, was observed to be to be very useful to the determine the distribution of CLas in infected tissue. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. The procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify CLas specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of CLas has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. . Success of this work would benefit the citrus industry by providing simple, sensitive and rapid detection method for large-scale detection of HLB under field conditions. Tissue imprints, once on the membrane could be stored for later use, can be shipped easily and generally are stable. The blots can be made on site and does not involve live tissue storage and transportation. The modified form of tissue blot, the ‘squash-blot’, for psyllids will be useful to understand the epidemiology of HLB. Two manuscripts have been prepared for publication (one for Phytopathology and the other is for Molecular Probes) and are being reviewed by CO-PI’s.
ZnuA was successfully purified. Over the past year, great effort has been put into the expression and purification of enzymatically active ZnuA, due to the low solubility of the protein. Using eleven plasmids and five variants of the ZnuA sequence, over 250 genetic constructs were developed to test expression of ZnuA, in a wide range of hosts (E. coli, Bacillus subtilis, Pichia pastoris, Sinorhizobium meliloti). To increase protein solubility, a variety of N-terminal and C-terminal fusion proteins (CBD, GST, MBP, 6x-His) were also constructed. From these, we found that the N-terminal fusion to the maltose binding protein (MBP) successfully increased the solubility of the protein. With the construction of the ZnuA-MBP fusion protein in vector pMAL-c2x (New England BioLabs), we were able to successfully purify the ZnuA protein. We are actually working on the biochemical characterization of the enzyme and the library screening. We expect to communicate the results during the first months of the second granting year. During year 1 we also focused on the cloning, expression, purification and screening of five transcription factors selected from the genome of Candidatus Liberibacter asiaticus. We were able to clone and express all of them as His-tagged fusions. The proteins, CLIBASIA_01510, CLIBASIA_01180 and CLIBASIA_02325, were highly expressed in soluble form and purified. Alternative fusion tags and hosts are being tested (as described for ZnuA) to purify CLIBASIA_00835 and CLIBASIA_03370. Next, the purified proteins were tested for thermal denaturation as a preliminary test for the high throughput small molecules screening assay. CLIBASIA_01510 and CLIBASIA_01180 were screened against the Prestwick library of small molecules and chemicals that increased the melting temperature by more than 2 degrees were considered hits. The screening of CLIBASIA_01510 resulted in 17 positive hits. The dose dependency was tested by thermal denaturation with increasing concentrations (1 nmol to 1 umol) of each chemical. Since CLIBASIA_01510 modulate transcription by direct interactions with the RNA polymerase the results obtained in vitro are being validated in vivo using a two hybrid system. The screening of CLIBASIA_01180 resulted in the identification of 9 chemicals. The results were validated by determining the ability of each of these compounds to modulate the binding of the transcription factor to its target DNA sequence. The binding region of CLIBASIA_01180 was located upstream of CLIBASIA_01180 using an electrophoretic mobility shift assay (EMSA). Using this assay, it was found that the small molecules disrupted the formation of the DNA/ CLIBASIA_01180 at concentration between 5 and 50 uM. The results obtained on the small molecule screens will be validated in vivo using Sinorhizobium meliloti as a surrogate strain. To this end we have obtained 8 mutants in CLIBASIA_03370, CLIBASIA_00835, CLIBASIA_01180 and znuA. Their phenotypes have been tested in planta and under laboratory conditions. There were subtle differences in the nodulation potential of the strains, but none was incapable of forming nodules. Interestingly, CLIBASIA_01180 was expressed only in younger nodules (anti-sense constructs were not expressed, as expected), suggesting that it has a function during the early stages of the infection/interaction with the plant host. All CLIBASIA_01180 mutants affect biofilm formation on abiotic surfaces. We are currently constructing expression vectors carrying CLIBASIA_01180 to reconstitute phenotype and test the effect of the chemicals identified on the screens. During the second year of funding we plan to construct and test mutants and reporters in CLIBASIA_01510 to validate the results from the screens in Sinorhizobium.
Expression of ZnuA was examined using two recombinant Pichia pastoris strains (GS115 and X-33). The expression level of three clones (WT, ‘SP, ’21) was analyzed during intracellular and secreted expression. Low levels of soluble ZnuA were obtained during intracellular expression of the protein in strain X-33, but purification of the His tagged protein by nickel affinity chromatography was unsuccessful due to the Pichia mitochondrial alcohol dehydrogenase isozyme III competitively binding to the nickel resin. As a result, we have designed multiple new ZnuA fusion proteins that will be used for purification by affinity chromatography on resins other than nickel. The new fusion proteins were prepared using all previously tested lengths of ZnuA (WT, ‘SP, ’21, ’28, and ’40), fused to three separate binding domains (GST, maltose, and cellulose). After sequencing results have confirmed proper gene orientation of the fusion proteins, transformation of Pichia will be carried out by electroporation. The construct expressing the optimal level of active protein will be selected for large scale purification of ZnuA. Assays done using Escherichia coli: ZnuA was expressed in the inclusion body of E. coli. Co-expression of ZnuA with E. coli Spy chaperone protein didn’t improve the solubility of ZnuA. Expression of ZnuA-GST fusion protein was detected in E. coli inclusion body but being process after 2 hours of induction. The HIS6x tag may interfere the folding of ZnuA. Thus, ZnuA genes (WT, .sp, .21, .28, .40) were cloned into other vectors with different tags (pET34b(+): cellulose binding domain. pMAL-c2X: maltose binding protein) for further analysis. Transcription factors: CLIBASIA_02325 (RirA, Rrf2 family, iron responsive) was purified as pure recombinant proteins (0.26 mg/mL, 6mL) in E. coli after adjusting the purification step. Thermal kinetics pre-screening was attempted immediately after the protein was purified, which indicated that this screening method is not suitable for RirA. In order to evaluate the effect of the positive hits from the thermal melting screening on the DNA binding activity of the MarR, CLIBASIA_01180, electrophoretic mobility shift assays (EMSAs) were performed. First, we selected an appropriate DNA fragment containing the putative promoters of both CLIBASIA_01180 and the divergently transcribed CLIBASIA_01185, coding for a delta aminolevulinic acid dehydratase. CLIBASIA_01180 was able bind this fragment as well as the optimal in vitro conditions for complex formation. The next step was to use the three most significant hits from the thermal melting screening, some less significant hits, and the substrate of the delta aminolevulinic acid dehydratase, aminolevulinic acid. This substrate was chosen because the substrate of an enzyme may be involved in regulating the enzyme’s expression. One of the small molecules disrupted complex formation at very low concentrations, whereas the other hits disrupted the complex only at higher concentrations. Aminolevulinic acid did not appear to stabilize or disrupt the complex, and this led us to hypothesize that CLIBASIA_01180 may not be regulating delta aminolevulinic acid dehydratase. Thus, we further examined the genomic context of CLIBASIA_01180 and found that the region upstream of the MarR is also well conserved in diverse species. This region contains genes involved in flavin biosynthesis (ribE and ribA) and amino acid metabolism (glyA), and if CLIBASIA_01180 is regulating these genes, disruption of this regulator would have deleterious effects on Ca. Liberibacter asiaticus.
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 recently identified a suitable candidate to be a visiting scientist and the necessary arrangements are underway. We have also contacted a laboratory in Germany about the availability of a promising vector developed there. Related research with the existing scFv is underway on project 551.
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