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Elegans muscle cells was performed using the commercial RNAi library, with bacteria expressing dsRNA for 87 of the predicted C. elegans genes GeneService, USA 3540. A semi-automated high throughput setup system was used, consisting of a robotic device Biomek FX Liquid Handler, Beckman Coulter, USA programmed to add bacteria and age-synchronized animals in liquid culture to 96-well plates.
RNAi bacterial cultures were grown for approximately 8 h in LB-ampicillin 50 µg ml 65 µlat 37 C with continuous shaking at 315 rpm Orbital shaker, GeneMachines HiGro, Genomic Solutions, USAand induced with 0. 5 mM isopropyl β-D-thiogalatoside IPTG, Sigma for 3 h at 37 C. To obtain an age synchronized population of L1 larvae first larval state post egg hatchingQ35 gravid adults were bleached with a NaOCl solution 250 mM NaOH and 1 4 v v dilution of commercial bleach and the eggs hatched in M9 buffer overnight at 20 C.
Day 1 is defined as 18 h following NaOCl age-synchronization and animals are said to be 1 day old L1 stage. 10 to 15 animals were added to each well in the 96-well plate in a volume of 50 µl of M9 plus M9, 1 µg ml cholesterol, 50 µg ml ampicillin, 10 µg ml tetracycline, 0. 1 µg ml fungizone and 170 µg ml IPTG and incubated at 20 C with continuous shaking at 200 rpm Innova 4430 Incubator Shaker, New Brunswick, USA.
Animals were scored 5 days later 6 days old for reduction in the number of fluorescent foci using the stereomicroscope Leica MZ16FA equipped for epifluorescence Leica Microsystems, Switzerland. As a negative control, animals were fed bacteria carrying the L4440 empty vector EV. Suppression of aggregation was scored positive when more than 50 of the animals had a 50 or higher reduction in foci number relative to the EV control, without loss of YFP fluorescence, changes in growth rate or development of the animals.
The candidate positive hits were re-screened n 3then tested in the Q24 soluble control strain, and counter screened in Q37 animals 5 days old zoom iq option SOD1 G93A animals 5 days old. In Q37 and SOD1 G93A animals, suppression of aggregation was scored positive when more than 50 of the animals showed a reduction in foci number 25. RNAi was always added on day 1. The identity of the RNAi-targeted genes was verified by sequencing of the dsRNA plasmids, followed by Blast analysis in the NCBI and Wormbase databases revealing high specificity of genomic sequence targeting.
Gene-knockdown by the respective RNAi was also confirmed for a representative group of hits by rtPCR data not shown. For RNAi assays on plates for foci scoring, FRAP and motility analysis, to collect animals for western blot and real-time qPCR, and for TS assaysNGM media was supplemented with 100 µg ml ampicillin, 1 mM IPTG and 12 µg ml tetracycline Sigmaand seeded with overnight 16 h RNAi bacteria cultures, pre-induced with IPTG 1 mM, 3 h.
One day old L1 animals 15 to 20 animals were transferred onto NGM-RNAi bacteria seeded plates and grown at 20 C, and at the time indicated aggregation was scored in at least 50 animals, for each condition n 3. Aggregates were defined as discrete, bright foci that can be distinguished from their surrounding fluorescence by increased brightness intensity.
The detection limit for these foci, measured with the higher resolution Zeiss Axiovert 200 microscope, is in the order of 3 µm in length for elongated foci in Q35 and 7 µm 2 in area for round fociwith the microscopy tools and fluorescence exposure utilized in the genetic screen Leica MZ16FA. Data collected from different experiments was compiled to calculate aggregate number averages relative to the control in EV RNAi. Fluorescent microscopy images were taken using an Axiovert 200 microscope with a Hamamatsu digital camera C4742-98 Carl Zeiss, Germany.
All assays were performed blind as to the identity of the RNAi by attributing to each modifier a number corresponding to a well with the dsRNA bacterial stock, in a 96-well plate. Fluorescence Recovery after Photobleaching Analysis. To examine the biophysical properties of polyQ protein, animals were subjected to FRAP analysis. Animals were mounted on a 3 w v agar pad on a glass slide and immobilized in 2 mM levamisole.
FRAP was measured using the Zeiss LSM510 confocal microscope Carl Zeiss, Germanyand the 63 objective lens at 5 zoom power, with the 514 nm line for excitation. 623 µm 2 was bleached for 35 iterations at 100 transmission, after which time an image was collected every 123. Relative fluorescence intensity RFI was determined as previously described 4375. SDS-PAGE, Native-PAGE, and Western Blotting Analysis.
For SDS-PAGE analysis, 6 day old animals grown on RNAi-seeded NGM plates were collected and resuspended in PELE buffer 20 mM Tris pH7. 4, 10 glycerol, 2 Triton X-100, 0. 5 mM PMSF, 1 µg ml leupeptin, 1 µg ml pepstatin, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail tablet Roche Diagnostics 11836170001. These nine gene modifiers of BWM protein homeostasis represent core components of the PN that evoke a robust and effective improvement of disease-related and endogenous metastable protein folding.
Lysis of 100 animals was accomplished by a combination of 4 cycles of freeze-thaw, grinding with a motorized pestle Kontes 749541-000 and 749520-0000followed by 8 min sonication Sonicator Bath Branson 1510, Branson. To dissolve the polyQ aggregates, SDS was added to a final concentration of 5. 5 v v and samples were boiled for a total of 10 min. Total protein concentration was determined using the Bradford assay Bio-Rad 500-0006. 15 µg for Q35 or 20 µg for SOD1 of total protein, in the linear range for YFP detection 43were analyzed on a 10 SDS-PAGE followed by Western blotting.
For YFP polyQ and SOD1 detection, blots were probed with the anti-GFP IR800 conjugated antibody 1 5,000 dilution; Rockland Immunochemicals 600-132-215. For α-tubulin detection, blots were probed with the anti-α-tubulin primary antibody 1;4,000 dilution; Sigma T-5168 followed by the secondary antibody Alexa Fluor 680 goat anti-mouse IgG 1 10,000 dilution; Molecular probes A-21057. Antibody binding was detected with the Odyssey Infrared Imaging System LI-COR Biosciences, USA.
The ratio between band intensities YFP α-tubulin was calculated for each sample Adobe Photoshop 7. 0, arbitrary units and compared to the EV control relative. A representative group of modifiers was tested 3 biological replicates. Statistically significant changes in protein amounts were considered if p 3. C SDS-PAGE and western blot analysis of protein samples from animals 6 days old expressing polyQ-YFP protein, immunoblotted with anti-YFP top and anti-α-tubulin bottom antibodies.
YFP tubulin ratios were calculated from protein band intensities total YFP and are shown relative to Q0 SD. D Motility measurements in body length per second BLPS of 6 day old wt and polyQ animals show that Q35 and Q37 aggregation in BWM cells causes a motility defect SEM, n 3, Student t-test p wt adopts a diffuse soluble fluorescent pattern E II and III are zoom in of the boxed areas on Imutant SOD1 G93A displays a pattern of small foci F II and III are zoom in of the boxed areas on I.
PolyQ mRNA levels in RNAi treated animals. q35-yfp mRNA levels from RNAi-treated animals 6 days old analyzed by reverse transcriptase PCR amplification top. Control corresponds to EV and yfp -RNAi is the positive control for reduced q35-yfp mRNA levels. Actin mRNA bottom is the control for total mRNA levels. Ratio q35-yfp actin are calculated from band intensities, and averaged from 3 biological replicates.
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J Biol Chem 281 33182 33191. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. A paratransgenic strategy to block transmission of Xylella fastidiosa from the glassy-winged sharpshooter Homalodisca vitripennis. Arthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture.
Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control. Earlier, we identified Pantoea agglomeransa bacterial symbiont of the GWSS as the paratransgenic control agent. Here, we report a paratransgenic strategy that targets transmission of Xylella fastidiosaa leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter GWSSHomalodisca vitripennis. We genetically engineered P.
Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti- Xylella activity at concentrations that did not kill P. agglomerans to express two antimicrobial peptides AMP -melittin and scorpine-like molecule SLM. Using these AMP-expressing strains of P. agglomeranswe demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels. This is the first report of halting pathogen transmission from paratransgenically modified insects.
It is also the first demonstration of paratransgenic control in an agriculturally important insect vector. Plant diseases caused by pathogens that are transmitted by insects such as leafhoppers, planthoppers, aphids, whiteflies and thrips have profound implications on food security 2,3,4. Despite advances in public health, arthropod vectors continue to exact a toll, either directly through transmission of human pathogens or indirectly by transmitting pathogens to animals and agricultural crops 1.
The vector borne diseases are managed mainly by controlling insect populations using insecticides. The side effects of chemical pesticides, including secondary pest outbreaks and selection for insect resistance, have confounded efforts to control these diseases and underscore the need to develop new approaches to pathogen control 5. Paratransgenesis, the modification of symbiotic microorganisms associated with insects, has been developed for several vectors of human pathogens such as triatomine bugs, tsetse flies, sandflies and mosquitoes i.
This strategy relies on delivery of anti-pathogen molecules within the insect vector via engineered symbiotic bacteria to make the insect incompetent to carry and transmit the pathogen 6. Several models of paratransgenic insects have been developed but none to date has been validated as a method to block transmission of a pathogen and prevent disease in a target host. Here, we report the paratransgenic manipulation of an agricultural pest, Homalodisca vitripennis the Glassy-Winged Sharpshooterto block transmission of the bacterial pathogen, Xylella fastidiosato grape plants.
fastidiosa is currently a leading agricultural pathogen globally, as the causative agent of Pierce s disease PD of grapevines, citrus variegated chlorosis CVC of citrus crops and olive quick decline of olive trees 10,11,12. Xylem-feeding sharpshooters and spittlebugs are the known vectors of X. fastidiosa 10, 13. vitripennis commonly known as the Glassy-Winged Sharpshooter GWSS due to its long-range mobility and high fecundity, is the most important vector in California 14. We recently identified Pantoea agglomerans as a symbiotic bacterium of H.
vitripennis and, using an EPA-approved non-pathogenic variant of Pantoeareported both paratransgenic manipulation and a field-applicable strategy to target GWSS with engineered bacteria 15. Using this platform, we have engineered lines of P. agglomerans that secrete antimicrobial peptides AMP that kill X. vitripennis that is unable to infect target plants.
Selection of melittin and scorpine-like molecules SLM as effector molecules. Melittin, a 26 amino acid-long peptide having an alpha- helix structure, is found in honeybee venom and kills cells through pore formation or by inducing apoptosis 16. SLM dbEST accession JZ818337 is an AMP found in the venom gland transcriptome of the scorpion Vaejovis mexicanus 17.
SLM is a 77 amino acid-long peptide and its amino-terminal region is similar to peptides of the cecropin family. fastidiosa and report here, for the first time, a pathogen-refractory H. I-TASSER predicted that SLM is composed of three coil-helix structures Additional file 1 Figure S1 18, 19. We tested activity of both peptides against X. fastidiosa as well as P. Melittin killed X. fastidiosa at a concentration of 5 μM, which was 20 of the concentration needed to kill P.
agglomerans 25 μM Fig. Toxicity of melittin and SLM against P. agglomerans and X. 10 5 10 6 CFUs of P. fastidiosa were treated with each AMP. 600 was measured 24 h after treatment of P. agglomerans with each AMP. Given the slow growth rate of X. fastidiosa, this organism was cultured 24 h after treatment with each AMP and CFUs were counted. agglomerans O.
600 after treatment with - a melittin, c SLM; X. fastidiosa CFUs counts after treating with - b melittin, d SLM. Both melittin and SLM exerted greater toxicity toward X. fastidiosa than P. All values in each graph are combined results from two independent experiments. Similarly, SLM killed X. fastidiosa at a concentration of 25 μM; it had no effect on P.
agglomerans even at a concentration of 75 μM Fig. The selective toxicity of these molecules to X. fastidiosa renders them ideal effectors for paratransgenic manipulation of H. Generation of AMP-expressing P. agglomerans strains. It is imperative that melittin and SLM interact with X. fastidiosa directly to kill it. To achieve this, P.
agglomerans should be transformed in a way that the molecules are excreted rather than contained within the bacterial cytoplasm. agglomerans to accomplish the goal of AMP secretion 9, 20. An Escherichia coli hemolysin secretion system that has earlier been used to secrete active proteins into the outside environment of Gram-negative bacteria, was used to genetically engineer P.
coli hemolysin secretion system has two components HlyA secretion signal and two pore forming proteins, HlyB and HlyD. Peptides with HlyA secretion signal at the carboxyl end are recognized by the pores formed by HlyB and HlyD and are secreted out of the cytoplasm. We introduced genes encoding melittin or SLM in the plasmid, pEHLYA2-SD at the 5 end of the E-tag, which was in-frame with the HlyA secretion signal Additional file 2 Figure S2b.
Once the AMP genes were cloned into the pEHLYA2-SD plasmid, P. 3, a plasmid with HlyB and HlyD genes, and pEHLYA2-SD or pEHLYA2-SD-Mel or pEHLYA2-SD-SLM See Methods for details. agglomerans were transformed with pVDL9. The spent medium from P. agglomerans culture was tested for AMP production via Western blot using anti-E tag antibodies, which demonstrated accumulation of melittin conjugated with HlyA secretion signal.
29 kDaSLM conjugated with HlyA secretion signal. 34 kDa and HlyA secretion signal peptide alone. We also confirmed melittin expression using an anti-melittin bleed, which bound to melittin conjugated to HlyA secretion signal. 29 kDa as well as to synthetic melittin. 3 kDa Additional file 3 Figure S3a. a Western blot showing secretion and accumulation of melittin and SLM conjugated to HlyA secretion signal by transformed P.
agglomerans lines in spent media. Spent media from transformed P. agglomerans lines were concentrated using Micron 10 kDa filters. Concentrated spent medium was tested using an anti-E-tag antibody. Lane 1 ladder; lane 2 Wild type P. agglomerans ; lane 3 HlyA secretion signal only; lane 4 melittin conjugated to HlyA secretion signal; lane 5 SLM conjugated to HlyA secretion signal. bc Western blots showing secretion and accumulation of melittin and SLM conjugated to HlyA secretion signal by transformed P.
agglomerans lines in the GWSS gut. Extracts from homogenized GWSSs were tested for presence of AMPs using an anti-E-tag antibody. b Lane 1 ladder; lane 2 GWSS fed on P. agglomerans expressing melittin conjugated to HlyA secretion signal; lane 3 GWSS fed on wild type P. agglomerans c Lane 1 ladder; lane 2 GWSS fed on P. agglomerans expressing SLM; lane 3 GWSS fed on wild type P. Five insects were tested individually for accumulation of SLM and melittin, and two insects were found positive for presence of both AMPs.
Blocking transmission of X. fastidiosa from H. Results from two independent experiments were pooled after confirming that the experiments did not affect the outcome using a generalized linear mixed model. GWSS that harbored AMP-producing P. agglomerans were refractory to X. fastidiosa acquisition; insects that carried melittin- or SLM-secreting P. agglomeranson an average, had X.
fastidiosa burden that was 4. 2respectively, of the pathogen burden in control insects p Fig. Graphs showing a decrease in X. fastidiosa acquisition by paratransgenic GWSSs. agglomerans was painted on grape stems after mixing with guar gum. PA WT - wild type P. agglomerans ; PA HlyA - P. agglomerans expressing HlyA secretion signal only; PA Melittin - P. agglomerans expressing melittin conjugated to HlyA; PA SLM - P. agglomerans expressing SLM conjugated to HlyA. The GWSSs were allowed to feed on Pantoea -painted plants for 48 h before putting them in a cage containing X.
fastidiosa- infected plants for 48 h. Subsequently the GWSSs were collected and two GWSSs were caged per single naive grape plant for 24 h. These GWSSs were surface sterilized and X. fastidiosa presence was assayed using rt-PCR. fastidiosa CFUs per insect head; b Percent of GWSSs carrying X. These are pooled results from two independent experiments. The paratransgenic GWSS that acquired melittin- and SLM- producing P.
fastidiosafailed to transmit X. fastidiosa to the naïve grape plants, indicating decreased acquisition of X. fastidiosa by H. agglomerans strains prior to acquisition of X. vitripennis resulted in decreased pathogen transmission to naïve grape plants Fig. Control GWSS and GWSS carrying wild type P. agglomerans transmitted X. fastidiosa 16. 7 and 20 of the time, respectively.
GWSS that carried P. agglomerans, which secreted only the HlyA signal protein and not the AMP molecules also failed to transmit X. fastidiosa to the naïve plants. Decrease in X. fastidiosa transmission to grape plants by paratransgenic GWSSs. agglomerans were painted on grape stems after mixing with guar gum.
The GWSSs were allowed to acquire P. agglomerans from P. agglomerans -painted plants for 48 h before an acquisition access period of 48 h on X. fastidiosa -infected grape plants. Subsequently the GWSSs were collected and two GWSSs were then confined per naive grape plant. After 24 h of inoculation access, the insects were removed and the plants were kept in a greenhouse for 30 weeks before testing them for presence of X. fastidiosa using rt-PCR. GWSSs that acquired P. agglomerans expressing HlyA secretion signal, melittin conjugated to HlyA secretion signal and SLM conjugated to HlyA secretion signal did not transmit X.
Expression of AMP within H. GWSS that fed on AMP-expressing P. agglomerans were tested for presence of recombinant AMP molecules to confirm that decrease in Xylella transmission to grapevines was a result of AMP activity in the insect gut. Western blot analysis confirmed presence of both melittin and SLM with attached HlyA secretion signals within the insects Fig.
Further, we confirmed presence of melittin using anti-melittin serum Additional file 3 Figure S3b. Prior studies with paratransgenic insect vectors demonstrated reduction or elimination of pathogens in the insects 6, 9. Here, we report a paratransgenic strategy that completely eliminates the detectable transmission of a pathogen from an arthropod to a target plant.
Three molecules- the HlyA protein, melittin and SLM- when expressed in the GWSS via engineered P. agglomeransblocked transmission of X. fastidiosa to grape plants. Melittin and SLM decreased Xylella CFUs in paratransgenic GWSS to levels that should eliminate pathogen transmission even during periods of feeding that exceed the 24 h window used in our experimental model. Additionally, under field conditions, several GWSS may feed on a single plant, unlike our experimental model in which only 2 insects were placed on target plants.
Again, the level of elimination of X. fastidiosa in the insect achieved with melittin and SLM should block transmission under such real world conditions. HlyA alone did reduce X. fastidiosa acquisition by the GWSS and eliminated transmission in our study. Similar results were also observed by Wang et al. 9 in paratransgenic mosquitoes, wherein they observed a 32 decrease in Plasmodium prevalence in mosquitoes carrying HlyA secretion signal-expressing P.
agglomeransthough this reduction was not significant statistically. We believe that the impact of HlyA on X. fastidiosa may be more pronounced than the effect on Plasmodia due to greater susceptibility of the bacterial cell membrane. However, the degree of X. fastidiosa reduction in the insect that is due to HlyA alone may not prevent transmission of the pathogen under conditions of prolonged feeding under field settings.
Almeida and Purcell 21 reported transmission efficiency of 35. 3 after zoom iq option h of acquisition and inoculation access using a model with a single GWSS per plant. However, we used two insects per plant with an acquisition and inoculation access of 48 h and 24 h, respectively, and a transmission of 20 was observed in the control group. Future studies will require varying acquisition and inoculation access times and increased number of insects per plant to simulate vector pressure under field conditions to understand the overall impact of transformed bacteria on acquisition and inoculation efficiency of the insect.
Our lab has also developed single chain antibodies scFvs specific to the X. fastidiosa surface protein, mopB 22. These antibodies can be expressed in tandem with active AMPs or as antibody AMP chimeras to increase killing efficacy and further reduce target resistance. We are also working on developing antibodies targeting different membrane proteins and pili present on the surface of X.
A theoretical concern exists for evolution of resistance amongst target X. fastidiosa populations. These antibodies in combination with other AMPs may slow resistance development. In our experiments, 10 10 CFU of P. agglomerans were painted on each plant. This is, indeed, a high concentration of bacteria but one that was readily administered using a hand-painted approach.
In future applications, different bacterial concentrations need to be tested to determine the threshold CFU required to break transmission cycles. Antibody-AMP chimeras are known to increase the potency of effector molecules 23 and, perhaps, can be used to make the insect incompetent of acquiring the pathogen at lower CFU s. Field collected GWSS have been reported to carry different bacteria within their foregut other than X. fastidiosa 24,25,26 and to our knowledge no adverse effect of these colonizing bacteria on insect health has been reported.
For instance, P. agglomerans expressing EGFP was able to colonize the GWSS gut without impacting the insect s health 15. We also did not observe adverse physiological effects in the GWSS carrying P. agglomerans strains, such as decreased feeding or early mortality. This suggests that paratransgenic GWSS could be able to complete their life cycle without a negative selection pressure from recombinant symbiotic bacteria.
We anticipate that this will allow persistence of recombinant bacteria and, possibly, spread amongst field populations of GWSS. The balance between persistence of recombinant bacteria, spread within a GWSS population and need for repeated applications of engineered symbionts can be addressed in additional field studies. The full potential of the paratransgenic control method under field conditions has not yet been realized, largely due to lack of delivery strategies that target arthropods.
We have recently developed a strategy based on calcium-alginate microparticles to disseminate genetically modified bacteria in the field. These microparticles not only provide a physical barrier between the bacteria and the outer environment to decrease environmental contamination, but also provide protection against desiccation and UV radiation 15. Spread of pathogens that cause Pierce s disease and other vector-borne diseases depends largely on the control of arthropod populations with insecticides.
There are reports of development of resistance in many insect vectors including mosquitoes and triatomine bugs against insecticides 27,28,29,30. Paratransgenic control of these zoom iq option is an alternative, which can be employed in the field to decrease transmission. It can also be included in integrated vector management. The high bacterial concentrations in this study were intended as proof-of-concept.
Paratransgenic control may help to reduce spread of human and plant diseases and may decrease over-reliance on chemical pesticides. The paratransgenic model for PD control may write a new chapter in the control of diseases caused by pathogens carried by agricultural vectors. Whiteflies, aphids, leafhoppers and thrips transmit deadly pathogens to crop plants ranging from cotton to sugarcane to papaya to rice 31,32,33,34,35,36. These insects carry bacterial symbionts that enhance their fitness.
Future directions of paratransgenic control for agricultural diseases may employ these symbionts as Trojan Horses to block transmission of pathogens. Furthermore, we report the first potential agricultural application of paratransgenic control and are confident that transgenic symbiotic bacteria can be used individually or as a component of integrated vector management to protect crops from threats such as Pierce s disease.
The glassy-winged sharpshooters GWSS maintenance. The Glassy-Winged Sharpshooters H. We report, for the first time, protection of a target from a vector-borne disease, using paratransgenic control. vitripennis were collected from crepe myrtle, Lagerstroemia sp. trees planted in parking lot 9 of UC, Riverside. These GWSS were kept on basil plants until they were used. A laboratory-based method for propagation of GWSS is not yet available, necessitating use of field-caught arthropods.
Bacterial strains, culture conditions. Escherichia coli strain XL1-Blue was used to maintain plasmids and for gene cloning. Pantoea agglomerans E325, an EPA approved biological control agent, was used to express and deliver different AMP molecules inside the GWSS gut. agglomerans were grown in Luria Bertani agar or broth. agglomerans and E. coli were cultured on agar plates at 30 C and at 37 C, respectively. Broth cultures were grown at the same temperatures in a shaker incubator 200 rpm.
Carbenicillin or chloramphenicol was added at a concentration of 100 μg mL and 35 μg mL, respectively, when needed. Two plasmids that were used in study pEHLYA2-SD and pVDL9. 3 have carbenicillin or chloramphenicol resistance markers, respectively. fastidiosa Temecula strain was used in this study and was cultured in PD3 agar at 28 C or in PD3 broth at 28 C. The culture was agitated at 175 200 rpm to grow X. fastidiosa in broth culture. Plant inoculations. fastidiosa strain Temecula was grown in PD3 medium using the conditions as described previously.
The bacteria were harvested in log phase and washed thrice with PBS before resuspending in PBS and brought to an O. 25, which is an equivalent of 10 8 cells ml. Twenty μL of bacterial suspension was inoculated twice into the vine using a needle. The stem was pricked above the second leaf using the needle and one drop of X. fastidiosa suspension 2X10 6 was placed on the point of inoculation; the negative pressure of xylem internalized the bacterial suspension.
The plants were kept for 15 weeks before they were used. MIC and MBC of AMPs against P. agglomerans was grown in LB broth at 200 rpm in a shaker incubator at 30 C for 16 h. Afterwards P. agglomerans was diluted 1 100 in 3 mL LB broth and grown at 30 C to mid-log phase. At mid-log phase the bacteria were diluted in LB medium to 10 5 10 6 colony forming units mL CFUs mL. Ninety μL of diluted P. agglomerans were pipetted into sterilized 0. 2 mL PCR tubes and to this 10 μL of 10X test concentration of either melittin or SLM both synthesized by China Peptides, Shanghai, China was added.
These tubes were incubated at 30 C and after 16 h of incubation OD 600 was determined to ascertain MIC minimum inhibitory concentration of AMPs against P. fastidiosa strain Temecula was grown in PD3 medium in a shaker incubator at 28 C and 200 rpm until it reached its log phase. Afterwards the X. fastidiosa culture was diluted to a concentration of 10 5 10 6 CFUs mL in PD3 medium. Ninety μL of diluted X. fastidiosa were mixed with 10 μL of 10X test concentration either AMP in a sterilized 0.
2 mL PCR tube and was incubated at 28 C in a shaker incubator for 16 h. fastidiosa is a slow growing bacterium, which makes measuring change in OD 600 of overnight cultures unfeasible. Hence, after treatment with AMPs X. fastidiosa was plated on to PD3 agar to determine MBC minimum bactericidal concentration of AMPs against X. These plates were incubated at 28 C for 10 days and CFUs were counted. The toxicity assays were repeated twice with three replicates for each dose in each experiment.
Plasmid construction. Sense and antisense sequences of the melittin gene with NheI and XmaI overhang were ordered from IDT Coralville, Iowa, USA and were annealed to themselves by lowering the temperature by 1 C min from 95 C to 50 C. Possani using forward primer ScoHlyAF1. Scorpine like molecule SLMan AMP from Vaejovis mexicanus venomgene was amplified from a plasmid kindly provided by Dr.
1 CAGCTAGCGGTTGGATAAGCGAG; and reverse Primer ScoHlyAR1. The product was cut using restriction enzymes NheI and SmaI. 1 TTTTTTATAGGCACGGGGTATACC. The plasmid pEHLYA2-SD kindly provided by Dr. Fernandez, National Center for Biotechnology, Madrid, Spain - having the hlyA secretion signal of the E. coli hemolysin secretion system and bla β-lactamase gene as marker- was also cut using restriction enzymes NheI and SmaI.
Melittin or SLM genes were ligated into linearized pEHLYA2-SD plasmid Additional file 2 Figure S2. The in-frame presence of both melittin and SLM genes was confirmed by sequencing. The in frame insertion of melittin or SLM gene resulted in plasmid pEHLYA2-SD-Mel or pEHLYA2-SD-SLM. agglomerans transformation. agglomerans was cultured in LB broth and grown to an OD600 of 0.
7 mid-log phase. These cells were centrifuged at 4 C and 8000 rpm for 10 mins and supernatant was removed. The cells were washed with ice cold autoclaved water. The final cell pellet of competent cells was re-suspended in 1 mL 10 glycerol. Eighty μL of competent cell suspension were aliquoted into microcentrifuge tubes.
One μL of pVDL9. 3 chloramphenicol resistance as marker plasmid was added to 80 μL of competent cells and transferred to an ice cold 1 mm cuvette. These cells were electroporated at 2. 0 kV, 25 microF. The cells were then plated onto chloramphenicol-containing LB agar.
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