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 library is estimated to contain 2.1 x 10 7th primary, unique antibody clones. Because our antigen was individual psyllids from Florida infected with high concentrations of ‘Ca. Liberibacter asiaticus’, the library contains antibodies for both the pathogen and the vector insect. 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. These approaches are described in previous reports. We have now obtained a unique set of scFv antibodies including ‘B665, B705, B717, B734 and B743’ that bind to a portion of the major outer membrane protein, OmpA; B900, B905, B913, B932, B1037, B1071, B1072, B1075 and B1083 that bind to flagellar protein FlgL; B947, B968, B969, B982, B984, B1096, B1114, B and B1115, B1119 and B1128 that bind to flagellar protein FlhA; B435, B466, B467, B468, B475, B500, B547, B556, and B557 that bind to a pilus protein; B442, B443, B479, B494, B520, B1191, B1194, B1199, B1202 and B1212 that recognize a protein that polymerizes cell surface polysaccharides.We have also isolated a number of scFv that recognize the TolC protein of ‘Ca. Liberibacter asiaticus. We isolated these scFv by using genomic sequence data to clone and purify specific proteins to beused as capture antigens. Portions of these proteins predicted by software to be exposed on the cell the surface were cloned. Correct cloning was confirmed by DNA sequencing and these genes have been expressed in E. coli and the encoded proteins have been purified. Emphasis is on recovering the scFvproteins in native, soluble form, which was difficult, but we have now been able to accomplish it. Thus we have developed and demonstrated a protocol that will allow us to isolate scFv antibodies that, in principle, will recognize any proteins from “Ca. Liberibacter asiaticus” or the insect vector, Diaphorina citri. Several of these proteins have also been successfully used in Das-ELISA assays and in dot blot assays of plant and insect extracts containing ‘Ca. Liberibacter asiaticus’ from Florida. Premature termination of scFv proteins has been encountered in the soluble expression system. This is because ‘stop’ codons accumulate in the phage selection system because the host E. coli is an amber suppressing strain. But these ‘stop’ codons become a problem for production of soluble scFv. Therefore we are checking all clones by sequence analysis to be sure premature stop codons are not present. This approach allows production of soluble scFv. Several of these antibodies detect antigen in plant but not in insect extracts. scFv antibodies may be useful as labels for ultrastructural studies of infected plants and insects, advanced detection assays, and possible even for HLB control through a ‘plantibody-based’ approach.
The main objective of this project is to manipulate calcium signals by over-expressing calcium signal modifier genes (CSM) from citrus to develop broad spectrum disease resistance. During the fiscal year 2010-2011 we produced 17 transgenic C-22 rootstocks, three Valencia and six Hamlin oranges over-expressing the CSM-1 gene. Previous grapefruit plants produced with this gene Were tested for disease resistance and shown to be resistant to citrus canker, Phytophthora nicotianae and Alternaria alternate and to the toxin tentoxin. Furthermore we engineer a new Agrobacterium binary vector with a citrus lectin gene to be used for genetic transformation during the fiscal year 2011-2012. As part of the transformation procedure, we are trying to accomplish genetic transformation without the use of antibiotic, and using only citrus genes. We were able to find a substance that increase the regeneration capacity of citrus stem segments used for genetic transformation and at the same time have the potential to be used for selecting transgenic plants and replace the antibiotic selection system. We are currently optimizing the system. We performed several crosses of Rio Red X Hirado Buntan pummelo and Rio Red X Wilking tangor. Hybrids will be recovered in November 2011.
In our initial schedule, the mature transformation facility (lab and greenhouse) at the CREC in Lake Alfred had to be implemented during the first year of the project. An existing laboratory was modified to fulfill the requirements of a tissue culture facility. The laboratory is now fully operative. Regarding the greenhouse, it became impossible to accommodate the budget to our plans for constructing the outstanding facility we requested. Alternatively, a growth room was designed profiting an existing structure at the CREC. The growth room construction was initiated in October 22nd 2010. The projected date of completion was February 11th 2011. Technically it was finalized by mid-April, however there were several technical/operational problems that came out during the following 4-5 months, especially regarding the refrigeration system (environmental conditions were not estable; some air handlers were not producing the air the manufacturer claims, in some cases the thermal expansion valve was changed because it was defective, air filters were not the ones we requested and they were changed, the humidifier in the small room is not located in the appropriate place for working properly), computer program (growth room technician still doesn’t have access to the program though this is currently being solved), water elimination after irrigation is defective, soil sterilizer (it needs a special accommodation to work ‘safely’). A generator should be purchased; without it, any prolonged electricity cut could jeopardize the whole project. The manager from Florida (Dr. Cecilia Zapata) completed her training in Spain during the first year, moved to Florida and has been working hard to set up the mature transformation facility at the CREC during the whole second year. Two part time OP technicians were hired to work on tissue culture and on plant preparation and a third OP technician was hired to work care at the growth room, under the supervision of the manager (and the PI at the initial stage). Another technician has been recently hired to help in the growth room and the lab because one of the OP technicians is leaving soon due to personal reasons. The Spanish lab has been monitoring the progress of the Florida facility. The PI and his manager at the IVIA greenhouses traveled to Florida last March 2011 to supervise the growth room construction and to set up healthy citrus germplasm bank establishment. The PI and his greenhouse manager will travel again to Florida next October 2011. An IVIA scientist with experience in mature citrus transformation will travel to Florida to help setting up the facility tentatively next November 2011. It is programmed that the IVIA scientist will spend three months in Florida (November 2011-February 2012). In Spain, mature tissues from the three sweet orange types (Hamlin, Pineapple and Valencia) plus Carrizo citrange were readily transformed. For our second objective, improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either CsFT or CsAP1 flowering-time genes already established in the greenhouse. We have characterized these transformants at the molecular level and continue characterizing them phenotypically in detail in the greenhouse. Moreover, for generating a dwarf-dwarfing rootstock, we have incorporated a construct aimed to induce RNA interference to downregulate the expression of a crucial gene in gibberellin biosynthesis, CcGA20ox1, in mature Carrizo citrange.
Objective 1: Comparative methodology was initiated to compare phloem plugging and collapse with different staining techniques. Phloem samples were collected and prepared from healthy and HLB affected trees. Preliminary results suggest that a rapid survey of plugging and starch accumulation is possible for general surveys of functional and non-functional phloem. Objective 2: Transformation experiments to produce plants that over-express the citrus ‘-1,3-glucanase gene: Experiments to validate the stability of the ‘-1,3-glucanase gene in transgenic greenhouse plants were initiated. PCR analysis on 9 of 10 plants showed the ‘-1,3-glucanase gene band, indicating stable transformation. All recovered transgenic clones (mostly Valencia, Duncan and Carrizo) were micropropagated, producing 4-5 replicates of each clone for further testing, including HLB challenge. Embryogenic callus transformation with the ‘-1,3-glucanase gene: More than 200 GFP+ somatic embryos were recovered from sweet orange OLL#20, and > 100 transgenic shoots were transferred to rooting medium. About 50 GFP+ embryos and shoots from Jin Cheng sweet orange and 20 GFP+ embryos and shoots from Valencia were also recovered ‘ which should result in stable transgenic plants in these important cultivars. Objective 3:To identify the putative genes involved in HLB disease development and psyllid transmission, quantitative reverse-transcriptional PCR (QRT-PCR) assays using total RNA isolated from infected plants and psyllids were conducted. Gene specific primers were used to check the expression of 506 genes in Ca. L. asiaticus. The genes showing a differential expression of two fold or more in either the plant or psyllid were categorized into Clusters of Orthologous Groups of proteins functional categories. Potential virulence related genes including hypothetical genes, which were overexpressed in planta, were selected. Differential expression of these selected genes were also evaluated in susceptible and tolerant varieties of greening infected citrus. Several ABC transporter genes and genes involved in protein export, along with many genes involved in porphyrin and chlorophyll metabolism, were upregulated in the plant. Most of the genes that were overexpressed in the psyllid included those involved in cell motility. The genes overexpressed in planta identified in this study will also be screened by transient assays on Nicotiana benthamiana plants. The results from this study will be useful in identifying the potential virulence genes involved in symptom expression of this pathogen in planta.
For the construct containing the ctEDS1 gene in the binary vector pBINplusARS, we selected the T0 seeds and obtained so far 15 independent T1 transgenic plants. The T2 plants will be planted to select for homozygous lines and also for an initial disease resistance test. For ctNDR1 transformation of the ndr1-1 mutant, we currently obtained 11 homozygous lines. We reported earlier that some of the lines showed enhanced disease resistance. Now we are at a stage to systematically analyze the defense phenotypes of the ctNDR1 overexpressing transgenic plants, using the homozygous lines that we have. For ctPAD4 + pad4-1 and ctEDS5 + eds5-1 plants, we have obtained over 10 and 3 independent T1 transformants, respectively. We have planted the T2 seeds and will test the segregating T2 plants for disease resistance and harvest seeds from multiple individual plants for selection of homozygous lines. For ctEDS5 + eds5-1, we are also selecting more T0 seeds in order obtain additional transformants. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. We have moved the ctNHL1 cDNA fragment from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. For ctSID2, we only obtained the cDNA clone in the pGEM T-easy vector. However, we have had some trouble in moving this fragment into pBINplusARS. We are currently trying a few different approaches to address this problem. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes. The cloning of some of these genes is underway.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic plants containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well transgenic control plants are growing in the laboratory of Dr. Orbovic at the UF Citrus Research Facility (Lake Alfred) until they are ready for the next level experiments. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to a single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. Results: The vast majority of T1 and T2 transgenic Arabidopsis plants expressing snc1 and ssi4 mutant coding sequences under the control of the AtSUC2-940 promoter have wild type phenotypes. Although the AtSUC2 promoter has been reported to be phloem-specific, we have found that it often does not maintain this tissue-specific pattern of expression in transformed Arabidopsis. However, despite the likelihood of expression in tissues other than phloem, only a few transformants showed any negative developmental or growth abnormalities. This lack of a negative phenotype in Arabidopsis provides a basis for optimism for similar results in transformed citrus. Our working hypothesis is that expression of the constitutive R proteins (mutants) in the phloem will active components of the innate immunity response to provide enhanced protection from Liberibacter infection in phloem cells. In order to monitor the activation state on the innate immunity system, we will cross the R protein transformants with transformed Arabidopsis lines containing pathogen-inducible promoters driving GUS reporter genes. We cloned the PR2 (also known as BGL2), and PR5 pathogen-inducible promoters in front of the GUSplus gene in pCAMBIA 2301. They were sequenced, transformed via electroporation into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis (strain GV3101) through the floral dip protocol in order to generate stable transgenic lines. We currently await the T-1 seeds from these transformations. In parallel, we acquired BGL2-GUS (in pBI101 vector; from Dr. Xinnian Dong from the Duke University) stable transgenic line to use as an alternative donor. The introduction of our R protein constructs into reporter lines by crosspollination will be faster and more efficient than transformation by agrobacterium. Being able to monitor constitutive activation of the innate immunity system by GUS will provide a test of the hypothesis that our constructs will activate pathogen-inducible promoters and will allow us to select lines that have strict phloem-specific expression for further study.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
Research on LAS in this quarter has focused on monitoring viable LAS concentrations over time in different culture treatments. Replicate culture treatments were created and inoculated with a LAS suspension obtained from seed of infected pomelo fruits. Monitoring has been conducted with two methods: 1) quantitative polymerase chain reaction (qPCR) of cells treated with ethidium monoazide (EMA) and 2) microfluidic chamber observations. Several LAS experiments have been conducted using three different culture treatments The three different culture treatments are created in duplicate culture flasks. One contains only King’s B (KB) media at a 1/3 diluted concentration. The other two treatments contain the same media with 25% or 50% juice solution. The juice solution is obtained by grinding the pulp from the infected fruit with deionized water and filter sterilizing the resulting solution. Each culture medium is then inoculated with a bacterial suspension created from infected seeds. At the time of inoculation, samples are collected, either treated with EMA or not treated, and frozen. Samples are then collected every two days from that point and treated in the same manner. DNA is extracted from frozen samples and analyzed by qPCR to determine the numbers of viable and non-viable LAS cells. Samples are also collected and injected into the microfluidic chambers at the time of culture inoculation, using the same media described above. The chambers are monitored daily with a microscope for visible cells and any signs of bacterial aggregation or biofilm formation. After cells are monitored for ~1 week, remaining cells are eluted from the chambers and tested with EMA-qPCR as was done for the culture flask samples. Initial results indicated that LAS cells may lose viability more slowly in media containing juice. However, these results came from ripe pomelo fruits that were collected in early summer and represented the last of last year’s fruit. Now, the fruits that can be collected are this year’s immature green fruits. These results have not been replicated with the new fruits. The new fruits have much higher initial cell titer as well as cell viability (~20%) compared to the older ripe fruits (~5%). However, the immature fruits seem to have some endophyte that grows in the cultures as a contaminant. This contamination causes the cultures to be unusable after ~3-5 days post-inoculation. We are working on a way to deal with this problem. In the microfluidic chambers, cells similar in size to that reported for LAS cells have been observed aggregating in some experiments. We eluted them to try to determine if they were LAS cells. Using EMA-qPCR, the samples sometimes have detectable LAS DNA, but no viable LAS DNA. It is unclear if this is because there are not viable cells in the chambers or if it is because they are below the limit of detection. Future work will try to clarify this.
After we received the notes from the Evaluation Committee on May 10, 2011, thirty-seven compounds from the listed 48 were ordered. Eleven compounds are not available, and they should be supplied by the participators. One additional compound sent by Syngenta was received for a test. Thirty-three compounds have been used to treat the HLB-affected scions (Lime) and grafted onto the healthy rootstocks (Duncan). Thirty scions were treated for each compound. All the grafted scions grew well now. More than 300 rootstock seedlings have also been purchased and planted in the greenhouse for further test.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. 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 is much shorter that we expected. 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 also 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 anti-psyllid 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. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. 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. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids.
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 anti-psyllid 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 now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 30 new peptides that are now being screened. ‘ We have greatly improved our efficiency of screening . ‘ We have greatly improved the CTV vector. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
Work with alternative hosts of the HLB associated Candidatus Liberibacter asiaticus continued this quarter with a focus on new hosts and the identification of infections with the traditional 16s primers and probes and a recently developed genus specific primers and probe. Since psyllid transmission tests were successful from Severinia buxifolia to citrus psyllid transmissions of the bacterium from infected citrus to S. buxifolia were attempted to determine the success rate. Transmissions were successfully done at a rate of 75%. New alternative hosts of the bacterium were discovered this quarter. These included Calamondin, Zanthoxylum fragara, Citropsis gilletiana, Esenbeckia runyonii, and Chiosya ternata and C. aztec. Not all of these hosts are good reproductive hosts for the psyllid (see 2010-11 annual report) however transmission does occur using infective psyllids. Ca. Liberibacter asiaticus infected S. buxifolia and citrus plants have now been monitored for one year to determine the live bacterial populations in the plants. This was done using a newly devised qPCR test. As reported earlier it appears that the bacterium does not survive well in S. buxifolia without reinoculation using infective psyllids. A manuscript will be submitted this month showing the new genus specific primers and probe which was utilized to verify the qPCR done using the traditional 16s primers and probe.
The following was presented at the 2011 FSHS annual meeting and submitted for publication in the proceedings: In Florida nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of ‘Candidatus Liberibacter asiaticus’ (Las) by insecticide applications. In 2008, a survey identified two ‘Carrizo’ citrange trees as HLB symptomatic. The potential risk for seed transmission and introduction of Las into nurseries by seed from source trees was recognized early on, so assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 1557 seedlings germinated from ‘Pineapple’ sweet orange seeds from trees in Collier Co. were assayed by qPCR using 16S rRNA gene primers. Of these seedlings, a single plant was Las+, although additional tests were negative. In 2009, no Las+ plants were detected among 332 ‘Murcott’ tangor seedlings from trees in Hendry Co. that were tested at least twice. From nurseries in 2008, one suspect Las+ ‘Carrizo’ seedling was detected in 290 seedlings from fruit located on symptomatic branches of two ‘Carrizo’ citrange trees, but its positive status was not confirmed after repeated testing. In 2009, a single questionable Las+ result was obtained for one of 100 Cleopatra mandarin seedlings, whereas no Las+ seedlings occurred among 125 seedlings from seeds from two trees of ‘Swingle’ citrumelo, 649 seedlings from four trees of ‘Kuharske’ citrange, or 100 seedlings from one tree of ‘Shekwasha’ mandarin. Despite the occasional Las+ qPCR test results, no plants developed HLB symptoms and repeated testing confirmed only transient presence of Las DNA in seedlings grown from seed obtained from HLB-affected trees.
Objective 1 (confirm biofilm formation by X. citri subsp. citri (Xcc) in comparison with other bacteria that are well known to form biofilms). In previous work using stable and labile Gfp expressing strains and confocal laser microscopy and a colorimetric assay involving crystal violet staining, aggregation and biofilm formation were confirmed for wide (XccA) and limit host range strains (XccA* and XccAw) of Xcc. Higher aggregation and biofilm formation was demonstrated for Xcc-A than XccA-Aw or XccA-A*. This higher biofilm formation of XccA was shown in vivo and in vitro after propagation on several culture media. However, further evaluation using the media XVM2, a medium commonly used to simulate plant conditions for gene expression analysis, behavior of 306 strain (XccA) and 407 (XccA*) was reversed. Aggregation by 407 strain in XVM2 medium was promoted compared to Xcc-A 306. To determine which component of XVM2 medium is involved in this biofilm response of XccA*, aggregation of strain 407 was screened in LB by omitting each of the components (casein, fructose, MgSO4, (NH4)2SO4, NaCl, CaCl2, FeSO4, KH2P4, K2HP4). MgSO4 was identified as the component that most affected biofilm formation and bacterial survival. Mg has been associated with flagellar integrity as well as aggregation processes in other bacterial models, whereas, our results reveal that flagella-like structures are related to the enhancement of biofilm formation. Greater flagellation and presence of swarming cells (with high ability for movement) in the XccA strains compared toXccAw demonstrates that flagella-like structures may account for the difference in biofilm formation between these strains. When other compnents were removed from XVM2 medium, less survival or biofilm in strain 407 indicates that biofilm formation process is a complex system related to more than one environmental variable. Influence of Mg+2 is being currently studied in order to elucidate a possible role in expression of genes related to biofilm formation as well as flagellation and bacterial motility in Xcc. Differences in biofilm formation and motility among wide and limit host range strains may be at least one of the bases for their difference in virulence. Regarding gene expression analysis new assays confirmed higher presence of the GumD mRNA in biofilm than in bacterial suspension. In contrast FleN, a flagellation regulator, is more highly expressed in bacterial suspension than biofilm in Xcc. FleN has been described as a negative regulator for multiflagelation in other bacterial models and our results point out its possible role in formation of biofilm cells in Xcc. Objective 2 (biofilm formation under different conditions). In previous assays biofilm disruption was evaluated in vitro after treatments with peroxide, CuSO4, SOPP, NaCl and NaClO at different concentrations by colorimetric assay. CuSO4 and peroxide were not able to disrupt and remove the bacteria from the surface. Experiments with the limited host range strains giving similar results. Viability of these remaining aggregates is being currently evaluated to determine if those compounds, that do not disrupt the biofilm, are still lethal to the bacteria. Biofilm disruption is also being evaluated in vivo. To do so, biofilm formation is induced in leaf dishes on petri plates under controlled conditions in a growth chamber. After 72 hours leaf incubation with XccA, dish leaf surfaces were sprayed with the different bactericide compounds previously tested. In the first assay already performed with high concentration of those compounds, living bacteria were found after NaCl, SOPP and peroxide treatments. New experiments are in progress with a range of concentrations of the bactericides.
Over the past quarter, we have made progress in the following areas: 1. We have continued testing TAL effector and promoter constructs in a Nicotiana benthamiana system to examine effector specificity for induction. 2. We have isolated TAL effectors from new citrus canker strains. Newly isolated variants will be sequenced, compared to known citrus TAL effectors, and tested for their ability to trigger our engineered resistance approach. 3. We have been continuing the molecular characterization of transgenic lines and testing of response to bacterial infiltration. 4. We have undertaken transformation of additional citrus varieties important to the Florida citrus industry, specifically sweet orange and red grapefruit. We continue to test additional promoter constructs in Duncan grapefruit. 5. We have begun planning for field trials of transgenic material.
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