Gene expression and protein synthesis related modifiers function in RNA metabolism, ribosome biogenesis, and protein synthesis. This is consistent with reduced translation increasing C. elegans lifespan, perhaps by activating a physiological state with increased stress resistance and folding capacities 49 51. Post-translational control modifier genes are involved in chaperone-assisted folding, such as HSP70 superfamily members, DNAJ co-chaperones and cyclophilins, and post-translation modifying enzymes such as SUMO and E3-ubiquitin ligases.
The role of chaperones on protein solubility, misfolding and aggregation has been well established 5253and an imbalance in certain co-chaperones has been suggested to alter chaperone activity in the cell and folding 54. Signaling RNAi-targeted genes included nuclear hormone receptors, G-protein-coupled receptors, C-type lectins endocytic receptorsor calcium export and channel-transport activity.
These regulators affect reproduction, growth, morphogenesis developmentand locomotion by altering signaling pathways involved in neuronal and muscle function. Genetic Screens for Enhancer and Suppressor Modifiers of PolyQ Aggregation. Information retrieved from comparative analysis of genetic screens of different misfolded proteins can provide important insights to identify both common and protein-specific pathways for conformational disorders Table S3.
Together, these genome-wide screens identified 341 genetic modifiers that cluster into the same functional classes and pathways, but correspond to distinct genes within these pathways. Specific modifier genes may interfere with the misfolded species at different stages of the aggregation process with opposite outcomes, consistent with the functional properties of a network, where different components within a process can shift the equilibrium in opposing directions to alter the stability of the proteome, to intensify or suppress the polyQ phenotype.
While it might seem counter-intuitive that molecular chaperones could suppress polyQ aggregation when knocked-down, this is consistent with observations that proteome stability can be enhanced or suppressed by changing the composition of the cellular chaperome 52 55. For example, reducing the expression of cyn-11 and cyn-12 cyclophilin D isoforms and dnj-5that function primarily as co-chaperones to regulate Hsp70 and Hsp90 activities 56promotes a polyQ soluble state.
These co-chaperones could function as negative regulators of chaperone function, and their down-regulation results in enhanced chaperone activities leading to suppression of misfolding 525758. This would be consistent with evidence that Hsp70 folding activity is negatively regulated by co-chaperones and co-factors such as CHIP 59 and BAG-1 60. Therefore, enhancement of folding can be achieved by both positive and negative regulation of chaperones or by a compensatory response that up-regulates other chaperones.
Highly relevant to this point is our ability to compare the modifier genes identified in this study with a previous complementary screen using the same threshold Q-length properties of the Q35 model to identify genes that when knocked-down by RNAi led to premature onset of polyQ aggregation 35. Suppression of aggregation by knockdown of cell cytoskeleton proteins, such as intermediate filaments MUA-3 and MUA-6 and filamin, is supported by experimental evidence that the dynamics of aggregation rely in part on translocation of proteins into inclusions.
Another functional class common to both Q35 screens is the protein trafficking and cell matrix. For example, the active transport of Htt-exon1 along microtubules has been shown to be required for inclusion body formation 61. In contrast, premature aggregation was observed when the expression of vesicle proteins involved in protein trafficking was knocked-down 35including TFG-1 COP-II complex, APT-3 and APT-1, and cell membrane assembly proteins SNAP-25.
Interference with these processes can disturb essential steps of the folding and secretory pathways, increase the load of misfolded proteins in the cell and lead to premature polyQ aggregation. Suppression of PolyQ Aggregation and Toxicity Can Be Uncoupled. An important observation from these studies is that the Class A PN modifiers Figure 3C, Table 1 were highly effective to suppress polyQ aggregation, and yet only 42 of these modifiers also reduced toxicity, with approximately equal numbers of modifier genes with either no effect on toxicity or even enhancing toxicity Figure 5A.
These results provide independent evidence that suppression of aggregation alone does not predict that the physiological health of the cell will be restored. From a mechanistic perspective, it is increasingly clear that a series of events are associated with the conversion of the nascent polyQ protein into different oligomeric states, immobile aggregate species and inclusion bodies 62 65.
We conclude that the genetic suppression of aggregation can occur via a wide range of mechanisms that are dissociated from the effect on toxicity, consistent with previous observations that interference with the aggregation process could in some cases enhance the formation of toxic oligomeric species 13 1966 68. The demonstration that protein aggregates are uncoupled from cellular toxicity has implications for the understanding of the PN and for development of therapeutics.
Triage Screening Method for Identification of Core Modulators of BWM Proteostasis. Moreover, each modifier gene is certain to function within its own network of interacting partners Table 2revealing an expanding network through which each modifier can suppress aggregation, but with differential effects on toxicity depending on the affected cellular function. We took advantage of C.
elegans models of protein aggregation-toxicity in addition to folding sensor strains harboring TS mutations in endogenous proteins to identify a core group of modulators that improve folding in muscle cells Figure 7A. Our results highlight important aspects of the PN, relevant for both gain-of-toxic function by aggregation-prone proteins, and loss-of-function -derived toxicity due to protein misfolding. The nine PN modifiers that remained at the end of the screening tree function in the mitochondrial respiratory chain and TCA cycle, that regulate metabolism, energy balance and prevention of oxidative stress, in addition to rRNA processing and transcription, that determine gene expression and proteome load Table 2.
While these modifier genes, upon initial inspection, seem not to be directly involved in folding, perturbation of their specific functions and networks of interactions re-adjusts the PN to enhance its capacity, as suggested by activation of the heat shock response and chaperone expression Figure 7B, 7C. Moreover, comparison to other genetic screens performed in Drosophila 323339C. elegans 34 and yeast 3871 Table S3 provides additional insights into the regulation of the PN by these modifiers.
3 encodes an ubiquinol-cytochrome c reductase subunit of the mitochondrial respiratory chain, and F43G9. 1 and T22B11. 5 encode TCA-cycle enzymes isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively Table 2. Disruption of the respiratory chain and energy production has been suggested to have consequences on cellular homeostasis 4748. Intriguingly, knockdown of ucr-2. 3 was shown to enhance the toxicity of human Tau expressed in C.
elegans neuronal cells Table S3 34. This discrepancy may be related to Tau-specific proteotoxicity, not derived from aggregation or misfolding, but associated with microtubule binding and disruption. 1 was also identified as a enhancer of lifespan 70which is consistent with a role in proteostasis. 7 encodes an rRNA processing factor, Y110A7A. 8 is a putative mRNA splicing factor, and R05D11. 4 encodes an RNA helicase required for translation Table 2. Thus, perturbing components of the gene expression machinery can enhance proteostasis, likely by altering the expression load of unstable proteins and activating stress responses to restore proteostasis.
In particular, knockdown of Y110A7A. 8 activates the osmotic stress response 69 and causes premature onset of polyQ aggregation on 3 day old animals 35consistent with an increase in misfolding Table S3. However, on 6 day old animals we show that the number of aggregates is suppressed, which suggests a time-dependent response by the PN to enhance the folding machinery and restore folding.
gei-11 encodes a Myb-family transcription factor proposed to regulate cholinergic receptor function at the BWM cells 72which affects muscle function and homeostasis 4373 ; and let-607 encodes a CREBH ortholog transcription factor predicted to be a component of the C. elegans ER stress response. The role of let-607 is particularly intriguing as it reveals a genetic crosstalk between the cytoplasmic and ER lumen stress pathways. Knockdown of let-607 induces chaperone expression that is not dependent upon HSF-1 Figure 7B, 7Csuggesting that other stress responses such as the ER unfolded protein response may be involved in the suppression of cytosolic protein aggregation.
Taken together, these results emphasize that diverse genetic and cellular mechanisms can restore cellular proteostasis beyond the traditional heat shock response. Identification of these processes is a fundamental step towards identifying new components that constitute the network, and the cellular and organismal mechanisms by which they contribute to protein homeostasis and protect against chronic expression of misfolded toxic proteins.
elegans Strains and Maintenance. Animals were maintained according to standard methods, at 20 C on nematode growth media NGM with OP50 E. The strains utilized in this work, and previously described, are the following wild-type wt Bristol strain N2; polyQ strains Q0 AM134 rmIs126 P unc-54 yfpQ24 AM138 rmIs130 P unc-54 q24 yfp IIQ35 AM140 rmIs132 P unc-54 q35 yfp IQ37 AM470 rmIs225 P unc-54 q37 yfp II Text S1 2635 ; human SOD1 strains SOD1 G93A AM265 rmIs177 P unc-54 sod1 G93A yfp and SOD1 WT AM263 rmIs175 P unc-54 sod1 wt yfp 12 ; temperature sensitive TS mutant strains CB1402 unc-15 e1402CB1157 unc-54 e1157HE250 unc-52 e669su250 and CB286 unc-45 e286 10.
The transgenic polyQ and SOD1 strains had been integrated by gamma-irradiation, 5 times backcrossed, and were previously described 1226. The strain Q37; hsf-1 sy441 was generated by genetic cross of the original strains AM470 and PS3551 hsf-1 sy441 I. RNA Interference Screen. The genome-wide RNAi screen for suppression of aggregation in C. 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 iqoption signals 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 and 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 Iqoption signals 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 iqoption signals 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.
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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 iqoption signals 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 iqoption signals 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.
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