The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. We are in the final stages of rhizotron construction to build enough for the experiments. Rhizotron construction was slightly delayed because of the late Valencia harvest this year for other projects combined with an unexpected loss of a staff member that will soon be replaced. The Masters student has assisted a member of Tripti Vashisth’s lab with the 2nd repetition of the experiment that created the foundation of this project to become familiar with techniques that will be important for maintaining pH and collecting data. We expect to initiate treatments before the end of May.
The goal is to understand how citrus interacts with Candidatus Liberibacter asiaticus (Las) infection. To achieve the goal of this research, we are conducting the following objectives: Objective 1. Identification of the receptors for Las PAMPs in susceptible and tolerant citrus varieties21 outer membrane proteins have been cloned and the putative targets in citrus are being identified using Yeast 2 hybrid system. Potential PAMPs from Las (either homologous to known PAMPs or pilin genes) LasFlaA (flagellin), LasEF-Tu, LasCSP (cold shock protein), LasSSBP (single strand binding proein) and pilin assembly genes (named LasPil85, LasPil95, LasPil105 and LasPil115) were cloned under 35S promoter and the Arabidopsis phloem specific promoter SUC2 and introduced into Agrobacterium. We will be testing their receptors in Tobacco and citrus. Objective 2. Generate transgenic/cisgenic citrus expressing PAMP receptors recognizing LasWe are cloning and overexpressing the selected PAMP receptors.Objective 3. Investigate the roles of effectors in HLB disease developmentFor the 10 selected SDEs, we have conducting Y2H and identified their targets in Valencia sweet orange. We are in the process of confirming the targets using other approaches such BiFC and co-IP assays. We will conduct Y2H and SPR assays to identify their targets in Poncirus.
1. Continuuing to improve the defined medium (Cruz-Munoz et al. 2018) for the culture of Liberibacter crescens, the cloest cultured relative of the citrus greening pathogen. An analysis of the amino acid requirements of L. crescens shows that only a few are required for growth. Deletion of the other amino acids from the medium results in growth but it is reduced. During the growth of L. crescens, we found that the pH of the medium increases by over 1 pH unit. Buffering this pH change reduces growth. The cause of the pH increase is under investigation. Nabil Killiny has published that the pH of citrus phloem increased from 5.7 to 6.2 after infection. As a result, we believe that studying this phenomenon in L. crescens may give us translatable results to understanding the cause of disease symptoms. 2. Monitoring of citrus groves for non-target antibiotic resistance prior to and after application of streptomycin and oxytetracycline. We have a method for the rapid detection of streptomycin in the field. We now need to test it in the field. 3. Developing second-generation antimicrobial treatments for citrus greening disease. A new antimicrobial Presto-Blue assay was developed for L. crescens on M15 defined medium. It is being tested on compounds we believe may be important for HLB control. If we have success with these, we will inform CRDF. At the moment, one compound looks to be promising. We are moving forward with a test to determine the spontaneous resistance rate in L. crescens for this compound. 4. Phosphate utilization as a strategy for HLB-disease management A greenhouse experiment is still in progress to determine whether foliar phosphate fertilization can recude citrate levels in phloem. Citrate is a preferred nutrient for Liberibacter. Phosphate fertilization is expected to reduce those levels in phloem sharply, thereby starving the pathogen. The phosphate foliar treatments are provided three times per week in the citrus macrophylla seedlings. When the plants flush, we will move them to a psyllid room in Lake Alfred. Cruz-Munoz, M., Petrone, J. R., Cohn, A. R., Munoz-Beristain, A., Killiny, N., Drew, J. C., & Triplett, E. W. (2018). Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 9, 668. Killiny, N. (2017). Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiol. Mol. Plant Pathol. 97, 20-29. doi: 10.1016/j.pmpp.2016.11.004
1. Developing a culture medium for Liberibacter asiaticus through comparative multiomics analysis with its closest cultured relative, L. crescens: Determined the optimal pH for the growth of L. crescens in M15 defined medium is 5.92, whereas in BM-7 is 6.5. Liberibacter crescens grows well in a pH range of 5.8 to 6.2. This is close to the pH of citrus phloem which is between 5.0 and 5.74, (Killiny. 2017). The level of Ca. L. asiaticus in the citrus phloem might be associated with the pH. During the culture of L. crescens, the pH of the medium rises dramatically. We are concerned that this rise may be limiting growth. As a result, we are conducting experiments to learn the source of the pH rise so that it can be mitigated. Chemically defined medium paper accepted for publication in Frontiers in Microbiology (Cruz-Munoz et al. 2018). Using several media based on M15 for culturing for Ca. L. asiaticus. In addition, various insect cell media are being tried. A cell line of the Asian citrus psyllid has been developed to determine whether Ca. L. asiaticus can be co-cultured with the insect cells. 2. Monitoring of citrus groves for non-target antibiotic resistance prior to and after application of streptomycin and oxytetracycline. A high throughput approach for the rapid assessment of streptomycin resistance has been developed and is now being tested with soil samples for citrus groves. Samples have been collected from four sites for this purpose. Streptomycin resistant bacteria have been isolated from these groves to test the efficacy of this method. To date, about 12% of soil bacteria appear to be resistant to streptomycin. More work is needed to test the level of streptomycin resistant levels in the pathogen in groves where streptomycin is being used compared to sites where it is not being use. 3. Developing second-generation antimicrobial treatments for citrus greening disease. A new antimicrobial Presto-Blue assay was developed for L. crescens on M15 defined medium. This approach was shared with representatives from Bayer who are developing their own high throughput assay against L. crescens. The new defined medium will greatly reduced the cost these assays and they should be more reproducible than BM-7 medium. We have learned that the undefined ingredients of BM-7 medium are quite variable among the manufacturers. 4. Phosphate utilization as a strategy for HLB-disease management A greenhouse experiment is in progress to determine whether foliar phosphate fertilization can recude citrate levels in phloem. Citrate is a preferred nutrient for Liberibacter. Phosphate fertilization is expected to reduce those levels in phloem sharply, thereby starving the pathogen. The phosphate foliar treatments are provided three times per week in the citrus macrophylla seedlings. Cruz-Munoz, M., Petrone, J. R., Cohn, A. R., Munoz-Beristain, A., Killiny, N., Drew, J. C., & Triplett, E. W. (2018). Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 9, 668. Killiny, N. (2017). Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiol. Mol. Plant Pathol. 97, 20-29. doi: 10.1016/j.pmpp.2016.11.004
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
The goal of this project is to determine whether pathogen or dsRNA exposure primes the ACP immune system to resist future infection by pathogens, including Las, and whether this effect is multigenerational. We have previously characterized the specificity and efficacy of the immune priming response in ACP (Obj. 1), characterixzed the effect of prior immune challenge on transmission (Obj. 3) and determined the transgenerational effect of pathogen-induced immune priming on Las acquisition. The current report describes our ongoing efforts to quantify the effect of RNAi-induced priming on Las acquisition (Obj 4). Adults of ACP collected from a laboratory colony free of CLas infection were starved for 3 hr, and then were subjected to dsRNA ATPase and sucrose. For each treatment, 5 cages including 10 insects in each were used. Insects were fed on diet solution consisting of 10, 100, and 1000 ng. l -1 dsRNA ATPase, 20% sucrose, and 0.5 green food coloring dye. As a control, adult of ACP was fed on sucrose 20%+ 0.5 green food dye. A cage for artificial feeding was prepared by stretching parafilm membranes on the bottom of plastic petri dish arenas. The parafilm surface was sterilized with ethanol and dried for 5 min under a sterile hood and layered with 400 L of diet solution including dsRNA, 20% sucrose, and green food coloring dye. The liquid was then covered with a second layer of stretched parafilm. During the feeding, the cages were placed in a growth chamber at 28oC. Insect were collected after 24 hrs and 5 days feeding and stored in -80 C for RNA extraction. Insects primed by exposure to artificial diet solutions with dsRNA for 24 hours were transferred to separate branches of a potted citrus plant (var. “Swingle”). Seven days after priming, insects were removed from plants, starved for 3 h, and either injected (experiment 1) with a lethal dose of S. marcescens or control treatment. After feeding or injection, the ACP that survived were allowed to reproduce on healthy of CLas-infected hosts. DNA was extracted from D. citriusing Qiagen DNeasy Blood and tissue kits (Qiagen, Hilden, Germany) per manufacture recommendations. Quality and concentration of DNA was assessed after extraction on a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA), then standardized to 10ng/ l. CLas titers were assessed by the detection of the 16S rDNA gene by qPCR methods described by Coy et al. (2014). Plants were tested to ensure infection with CLas by qPCR following methods described by Li et al. (Li et al., 2006). No significant differences in reproductive success or acquisition by offspring were detected to date, which suggests that immune priming does not occur in response to RNAi, and that this response is unlikely to affect CLas transmission.
To determine the optimal concentration of dsRNA priming, ACP were exposed to a series of dsRNA concentrations (10, 100, 1000 ng. l-1) in artificial diet for 24 hrs, and 5 days. ACP were subsequently transferred to artificial diet containing S. marcescens for 4 days, then transferred to C. macrophylla for 14 days. Survival of ACP was recorded every 24 hrs for 14 days. ACP survival in response to S. marcescens was lowest after feeding on 100 ng. l-1 of dsRNA T7_pGEMT for 24hrs, as compared with control (no dsRNA priming) treatments. The survival rates of ACP exposed to S. marcescens or sucrose following control (no dsRNA priming) were not significantly different. Eighty percent of ACP survived after feeing on 100 ng. l-1 dsRNA ATPase for 5 d prior to being transferred to sucrose, and was significantly higher than among ACP that fed only on sucrose prior to S. marcescens exposure. This suggests that initial (24 h) exposure to dsRNA may increase susceptibility of ACP to pathogens. Among the insects that did not survive pathogen challenge, S. marcescens was detected in 79% of ACP following priming with 100 ng. l-1 dsRNA T7_pGEMT100 fior 24 h, as compared with 9-15% of ACP not primed with dsRNA. Insects primed for 5 days on dsRNA followed by exposure to S. marcescens, 75% of insects fed with 100 ng. l-1 dsRNA ATPase were infected with the pathogen. No bacterial infection was observed in insects fed on 1000 and 10 ng. l-1 of T7_pGEMTdsRNA , or control (no dsRNA priming) treated insects. The high percentage of bacterial infection in the dsRNA-treated insects indicates that dsRNA may contribute to bacterial loads in ACP, although this effect appears to be dose-dependent. Target gene expression decreased among ACP that were primed with 1000 ng. l-1 T7_pGEMT or 100, 1000 ng. l-1 of dsRNA ATPase prior to S. marcescens exposure but this reduction was not statistically different from untreated (no dsRNA priming) ACP. It is expected that the time between feeding dsRNA and quantifying mRNA at the end of the feeding bioassay is too long long to detected dsRNA-associated changes in expression; therefore, experiments are underway to evaluated changes in expression following 24 h and 5 d exposure to dsRNA. In addition, subsequent analyses will be conducted to determine whether priming facilitates Liberibacter acquisition.
Media continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from citrus phloem to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including carbohydrates (a-ketoglutarate, galactose, glucose, fructose, maltose, sucrose, xylose), amino acids (alanine, arginine, asparagine, aspartate, cysteine, 2-aminobutyrate, glutamate, glycine, isoleucine, phenylalanine, proline, serine, threonine, and valine), sugar alcohols (iso-inositol, sorbitol, xylitol), organic acids (citrate, fumarate, malate, and succinate), micronutrients (B, Cu, I, Mn, Mo, Zn), vitamins (ascorbate, cobolamine, diaminopemilate, pyridoxal phosphate, riboflavin, thiamine). In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. For our defined media for L. crescens, we have always used a commercial source of Grace’s insect medium as part of the formulation. In the past, we mistakenly assumed that the levels of compounds reported by the manufacturers are the actual levels present in the Grace’s medium. The components of three commercial sources of Grace’s medium were examined by metabolomics and none of them were very close to the standard composition of Grace’s medium as listed on their labels. One of them, made by a manufacturer in Mubai, India, was very different and provided remarkable growth of L. crescens with no other added components. The growth obtained with this medium was very similar to that obtained by culture in BM-7, the standard, undefined medium used for this organism. The two other sources of Grace’s medium from Gibco and Sigma did not support L. crescens growth and a version of this maedium made by our lab with ingredients from Sigma also did not support growth. We were unable to culture CLas on the Indian-made Grace’s medium, referred to as Hi-GI. Metabolomics analysis showed that Hi-GI medium contains 10-fold higher levels of various vitamins compared to what would be expected in Grace’s medium. Sugars and organic acids (including glucose, fructose, sucrose, turanose, maltose, fumarate, alpha-ketoglutarate, malate, maleate and succinate) were also higher. To date, we haven’t been able to make a defined medium based on Hi-GI that provides the same high level of growth as does Hi-GI. This implies the chemically defined medium still requires improvements. Liberibacter crescens requires compound or combinations of compounds for optimal growth and these have not yet been discovered. Meawhile, we have a large stock of Hi-GI on hand and are modifying it in order to culture CLas.
Meida continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from the haemolymph of the Asian citrus psyllid to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including heptadecanoic acid, n-methyl-L-proline, methyl-maleic acid, homoserine, pyroglutamic acid, arabinopyranose, D-glucopyranoside, fucose, azaleic acid, arabinofuranose, iso-citric acid, n-acetylglucosamine, alpha-D galactoside, inositol-2-phosphate , trehalose, citrulline, ornithine, AMP, CMP, NADP, GMO, IMP, and FAD. In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. During this period, we continued to follow-up on our discovery that citrate is a preferred carbon source of L. crescens. We discovered that the optimal level of citrate required for growth was 2.5 g/l or 13mM. We also found that the pH of the medium increases dramatically during growth from 5.9 to 7.6. This increase in pH is expected from the metabolism of citric acid. We are now testing medium with more buffering capacity to maintain pH near 6.0 during culture growth. This is being tested with higher levels of phosphate buffer and ACES buffer. During this period we have considered ideas on whether CLas can be starved of citrate by modifying the nutrition of citrus saplings. The first experiment tested whether changes in nitrogen nutrition (potassium nitrate, sodium nitrate, or ammonium nitrate) can alter the levels of citrate in phloem. These nutritional changes are expected to alter the ionic balance in the plant. Sodium nitrate was expected to reduce the level of citrate in phloem. However, after 60 days of treatment in the greenhouse, we saw no difference in citrate phloem levels between these nutritional treatments. We are now testing the notion that adeqaute phosphate nutrition is required to reduce citrate levels in phloem. Under conditions of phosphorous deficiency, plants load citric acid into the phloem in order to exude citric acid from roots (Gaume et al. 2001. Plant and Soil 228:253-264). It is also known that the exuded citric acid can then solubilize rock phosphate (P2O5) or other forms of bound phosphate for use by plants (Chen and Liao 2016. J. Genetics & Genomics 43:631-638). Zhao et al. (2013, Molec. Plant 6:301-310) reported that certain small RNAs made in response to HLB infection were involved in P nutrition. They also found a 35% reduction of P in CLas-positive citrus trees compared to healthy trees. Applying phosphate to HLB-positive sweet orange trees in southwest Florida reduced HLB symptom severity and significantly improved fruit production in a 3-year field trial. So our hypothesis is that the severity of citrus greening disease can be greatly reduced with application of phosphate (not rock phosphate as is sometimes recommended). Added phosphate will improve the nutrition of the tree and lower citrate loading into the phloem. The reduced citrate levels in the phloem is expected to starve Liberibacter. These ideas will be proposed in a grant proposal to the USDA SCRI program with Killiny, Wang, and Vincent from the CREC as co-PIs with Triplett.
Media continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from citrus phloem to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including carbohydrates (a-ketoglutarate, galactose, glucose, fructose, maltose, sucrose, xylose), amino acids (alanine, arginine, asparagine, aspartate, cysteine, 2-aminobutyrate, glutamate, glycine, isoleucine, phenylalanine, proline, serine, threonine, and valine), sugar alcohols (iso-inositol, sorbitol, xylitol), organic acids (citrate, fumarate, malate, and succinate), micronutrients (B, Cu, I, Mn, Mo, Zn), vitamins (ascorbate, cobolamine, diaminopemilate, pyridoxal phosphate, riboflavin, thiamine). In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. For our defined media for L. crescens, we have always used a commercial source of Grace’s insect medium as part of the formulation. In the past, we mistakenly assumed that the levels of compounds reported by the manufacturers are the actual levels present in the Grace’s medium. The components of three commercial sources of Grace’s medium were examined by metabolomics and none of them were very close to the standard composition of Grace’s medium as listed on their labels. One of them, made by a manufacturer in Mubai, India, was very different and provided remarkable growth of L. crescens with no other added components. The growth obtained with this medium was very similar to that obtained by culture in BM-7, the standard, undefined medium used for this organism. The two other sources of Grace’s medium from Gibco and Sigma did not support L. crescens growth and a version of this maedium made by our lab with ingredients from Sigma also did not support growth. We were unable to culture CLas on the Indian-made Grace’s medium, referred to as Hi-GI. Metabolomics analysis showed that Hi-GI medium contains 10-fold higher levels of various vitamins compared to what would be expected in Grace’s medium. Sugars and organic acids (including glucose, fructose, sucrose, turanose, maltose, fumarate, alpha-ketoglutarate, malate, maleate and succinate) were also higher. To date, we haven’t been able to make a defined medium based on Hi-GI that provides the same high level of growth as does Hi-GI. This implies the chemically defined medium still requires improvements. Liberibacter crescens requires compound or combinations of compounds for optimal growth and these have not yet been discovered. Meawhile, we have a large stock of Hi-GI on hand and are modifying it in order to culture CLas.
Meida continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from the haemolymph of the Asian citrus psyllid to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including heptadecanoic acid, n-methyl-L-proline, methyl-maleic acid, homoserine, pyroglutamic acid, arabinopyranose, D-glucopyranoside, fucose, azaleic acid, arabinofuranose, iso-citric acid, n-acetylglucosamine, alpha-D galactoside, inositol-2-phosphate , trehalose, citrulline, ornithine, AMP, CMP, NADP, GMO, IMP, and FAD. In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. During this period, we continued to follow-up on our discovery that citrate is a preferred carbon source of L. crescens. We discovered that the optimal level of citrate required for growth was 2.5 g/l or 13mM. We also found that the pH of the medium increases dramatically during growth from 5.9 to 7.6. This increase in pH is expected from the metabolism of citric acid. We are now testing medium with more buffering capacity to maintain pH near 6.0 during culture growth. This is being tested with higher levels of phosphate buffer and ACES buffer. During this period we have considered ideas on whether CLas can be starved of citrate by modifying the nutrition of citrus saplings. The first experiment tested whether changes in nitrogen nutrition (potassium nitrate, sodium nitrate, or ammonium nitrate) can alter the levels of citrate in phloem. These nutritional changes are expected to alter the ionic balance in the plant. Sodium nitrate was expected to reduce the level of citrate in phloem. However, after 60 days of treatment in the greenhouse, we saw no difference in citrate phloem levels between these nutritional treatments. We are now testing the notion that adeqaute phosphate nutrition is required to reduce citrate levels in phloem. Under conditions of phosphorous deficiency, plants load citric acid into the phloem in order to exude citric acid from roots (Gaume et al. 2001. Plant and Soil 228:253-264). It is also known that the exuded citric acid can then solubilize rock phosphate (P2O5) or other forms of bound phosphate for use by plants (Chen and Liao 2016. J. Genetics & Genomics 43:631-638). Zhao et al. (2013, Molec. Plant 6:301-310) reported that certain small RNAs made in response to HLB infection were involved in P nutrition. They also found a 35% reduction of P in CLas-positive citrus trees compared to healthy trees. Applying phosphate to HLB-positive sweet orange trees in southwest Florida reduced HLB symptom severity and significantly improved fruit production in a 3-year field trial. So our hypothesis is that the severity of citrus greening disease can be greatly reduced with application of phosphate (not rock phosphate as is sometimes recommended). Added phosphate will improve the nutrition of the tree and lower citrate loading into the phloem. The reduced citrate levels in the phloem is expected to starve Liberibacter. These ideas will be proposed in a grant proposal to the USDA SCRI program with Killiny, Wang, and Vincent from the CREC as co-PIs with Triplett.
During the past quarter, we have optimized dsRNA targets and have conducted immune priming assays to investigate the specificity of immune priming in ACP following exposure to dsRNA. The yield of redesigned dsRNA using MEGAscript RNAi Kit transcription was initially low, possibly because the QG buffer in the Zymoclean Gel DNA Recovery Kit may have decreased transcription. In order to avoid using QG buffer in gel purification and increased PCR concentration,gel purification method was modified. PCR products were analyzed on 1% agarose gel and the band was excised, and transferred to a filter tip and centrifuged at 5000 rpm for 10 min. Three l of purified gel product were run on 1% agarose gel which showed a high concentration (based on the intensity of the band) of the product. The remaining purified gel product was precipitated and resuspended in 30 l of RNase-DNase free water, and 2 l of the product were analyzed on agarose gel and directly amplified. PCR products obtained from the modified purification protocol for dsRNA synthesis yielded significantly higher dsRNA concentrations to compared to the kit. Bioassays were conducted with adult uninfected ACP. After 3 hrs of starvation, insects were fed on dsRNA for 24hrs and 5 days. After 24 hrs, insects were starved for 3 hrs and then transferred to a lethal dose of S. marcescens for 4 days. For each dsRNA concentration, two cages were exposed to dsRNA; then either fed on S. marcescens in diet (20% sucrose, and 0.5 green food coloring dye) or diet only (control). Diet containing the pGEM-T easy vector without a T7 tail, a blank sucrose + buffer diet, and container without food were also included as controls in the feeding experiment. The stability of different concentrations of dsRNA on diet solution were confirmed with agarose gel electrophoresis at the end of the 24hr and 5 d feeding periods. DsRNA of 1000 ng/ l and 100 ng/ l were present in agarose gel. Also the survival of S. marcescens after 4 days feeding was checked on nutrient agar plate. S. marcescens from feeding cage was streaked on nutrient agar and kept O/N at 30 C. Result confirmed that bacteria were viable during the feeding assay. Following exposure to bacteria, insect were placed on Citrus macrophylla in the greenhouse, and mortality was recorded daily. After 14 d, live insects from the 24hr and 5 d feeding treatments were collected and laterally bisected. Half of the insects were examined for the presence of S. marcescens, and the remaining insects were used for RNA extraction. To quantify S. marcescens, single insects were smashed in tube with 20 l of nuclease-free water and plated on nutrient agar, and kept at 30 C O/N. Colonies were obtained from most of the insects on nutrient agar. No colonies were obtained from control insects. Conventional PCR confirmed the presence S. marcescens in insects. To study the effect of dsRNA on mRNA levels, total RNA was extracted from single ACP using TRIzol (Invitrogen) and cDNA was prepared. The survival rate of insects after feeding on S. marcescens and from 14 days on C. macrophylla will be analyzed and presented when all replicates are completed; however, initial results indicate thigh mortality after feeding on S. marcescens as compared with those feeding on sucrose, suggesting that dsRNA exposure does not modulate ACP immune responses to S. marcenscens e.g. does not prime ACP to tolerate or mitigate lethal doses of pathogenic bacteria, although it must still be determined whether this response is similar following exposure to sublethal doses of bacteria. Whether this is responses is specific or is generalizable to other Gram negative bacteria, such as Liberibacter, will be determined in the next quarter.
Obj. 4. The purpose of this objective is to determine whether prior pathogen or dsRNA exposure inhibits Las acquisition by psyllids. We investigated if D. citri exhibits immune priming and produces a different response to secondary infections and the specificity of that protection. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Surviving D. citri were moved to a second sachet containing 1e6 cells/mL live S. marcescens where they remained until all D. citri were dead. Mortality was recorded once daily. Transgenerational immune priming bioassays were conducted with psyllids placed on artificial diets containing heat inactivated E. coli, M. luteus, or no bacteria. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Data presented here are preliminary, as additional replicates were collected during February and March 2017. These insects are currently being processed and analyzed via QPCR. Offspring of female D. citri that were fed a diet containing M. luteus (n = 17, 2.60E7 1.35E7) or E. coli (n = 18, 2.09E6 8.40E5) had higher CLas titers than offspring of non-primed females (n = 8, 1.22E5 6.20E4). The Ct of plant material was correlated with the titer of CLas in D. citri (b = -0.140, F1,41 = 4.115, p = 0.050, R2 = 0.09). Previously, we demonstrated that the D. citri immune response is induced due to recognition of Gram-positive bacteria, such as M. luteus. Given that M. luteus protected D. citri from S. marcescens, and CLas is also a Gram-negative bacterium, it was expected that CLas titers would be lower in offspring of M. luteus primed adults. In fact, the opposite was observed. Females fed a diet containing M. luteus prior to mating had higher CLas titers and produced offspring that acquired CLas at a much higher rate than those from control or E. coli fed females. This suggests that immune priming with M. luteus infection may facilitate CLas infection.
A paper was submitted during this period (Cruz-Munoz M, Cohn A, Lai K.-K, Dia, R, Rusoff K, Conrad R, Triplett EW 2016. A chemically defined medium suggests a-ketoglutarate and malate as primary carbon sources for Liberibacter crescens BT-1 growth. Submitted to Applied Microbiology). We continued to improve the defined medium for L. crescens and now use a medium called M12. In addition, we obtained more phloem metabolomics data from Nabil Killiny which was used to create more media formations for CLas. We have also learned that the typical primers used to assess CLas growth in culture give misleading results because they were created to amplify the bacteriophage sequence and not those of the bacterial genome. A new set of primers was designed that sigificatly improve our ability to assess growth. . The metabolomics data for the culture of L. crescens in defined and undefined media are encouraging. The strain grows much faster in BM-7 than in M11n so we are identifying those components that are limiting for growth in the defined medium (M11n). When identified, their concentration will be increased in the CLas medium. We have had difficulty maintaining an infected psyllid colony in Gainesville which has prevented us from studying the heterogeneity of CLas strains
An important breakthrough came for us during this quarter that was quite accidental. We are designing defined media for L. crescens. Our first such medium was made about 9 months earlier and we continue to improve upon it. We want to develop the best defiend medium we can that can then be easily adapted to a CLas medium based on the metabolomics of the psyllid hemolymph and citrus phloem (data from the Killiny lab). The design of these medium is largely based on trying to reproduce the constituents of BM-7, the undefined medium typcailly used to culture L. crescens. One of those constituents is an insect cell culture medium called Grace’s medium. The recipe for Grace’s is well known and the medium is available commercially from at least three sources. We have made Grace’s medium from scratch ourselves and it doesn’t do as well as the commerically obtained Grace’s medium for culturing L. crescens. So there is something awry in the recipes that are published. Furthermore, one of the sources of Grace’s medium we obtained commercially provides excellent growth of L. crescens. This was the case with one batch from this company and wasn’t reproducible in other batches. So we are in the process of performing metabolomics of this purchased medium to learn how it might be doing so well in the culturing of L. crrescens. Once we know that, we should be able to design better media for L. crescens. We are also using this great batch of Grace’s medium in various media to culture CLas.
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