Background: How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 to dissolve disordered protein aggregates is unknown.Results: Conserved distal loop residues in the Hsp104 and ClpB middle domain contact NBD2 and enable Hsp70-dependent protein disaggregation.Conclusion: Distal loop does not project out into solution and Hsp104 and ClpB are tuned differently for Hsp70 collaboration.Significance: Understanding how protein disaggregases operate may empower strategies to counter protein-misfolding disorders. + proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.The homologous hexameric AAA HspA and B) A 15)..+ ATPaseHsp104 and ClpB are often assumed to share a common mechanism of action due to high sequence homology in their NBDs and because some Hsp104-ClpB chimeras retain partial disaggregase activity –20. Hsp1Thermus thermophilus and Ehrlichia chaffeensis disaggregate disordered aggregates without cofactors (E. coli) and its obligate co-chaperone Hsp40 (DnaJ in E. coli)  , 6, 40, in vitro , 32, 41.Precisely how Hsp70 and Hsp104 coordinate substrate handling in protein disaggregation is unclear . Hsp104 B and C). Artificial Hsp104 chimeras containing the ClpB MD (in place of the Hsp104 MD) weakly collaborate with DnaK to disaggregate disordered aggregates, whereas artificial ClpB chimeras containing the Hsp104 MD can interface with eukaryotic Hsp70 (e.g. Y503A) abolished ClpB collaboration with DnaK  in this region with glutamate or alanine ablates Hsp104 disaggregase activity in the presence of Hsp70 . The function of the MD distal loop has been largely ignored and its location in the hexamer remains controversial due to radically different placements in conflicting hexameric models of Hsp104 and ClpB  but are unable to collaborate with Hsp70. This distal loop function is conserved and enables ClpB collaboration with DnaK. We show that the distal loop does not stably contact Hsp70, but regulates Hsp104 and ClpB activity via an autoinhibitory intramolecular contact with NBD2. This NBD2-MD contact is inconsistent with structural models of ClpB and Hsp104 hexamers in which the distal loop projects out into solution away from the hexamer  between helix 1 and 2 of the MD of Hsp104 , B–D. Thand ClpB , 48–50. and ClpB , 49–52.  hexamer  or invar6-tagged constructs from a pDS56/RBSII plasmid in M15 cells (Qiagen). Hsp104 variants, including isolated MD (amino acids 407–527), were expressed and purified as N terminally His6-tagged constructs , resuspended in lysis buffer , 2 mm β-mercaptoethanol, 5 μm pepstatin, and 1 Mini-EDTA free protease tablets per 50 ml (Roche Applied Science)), and lysed via treatment with 20 mg of hen egg lysozyme per 1 liter of cells followed by probe sonication. Cell debris was removed via centrifugation at 16,000 × g at 4 °C. The supernatant was applied to 3 ml of Affi-Gel Blue (Bio-Rad) per 1 liter of cell culture. Supernatant and resin were rotated at 20 rpm at 4 °C for 3 h. Resin was then washed 4 times with wash buffer , 2 mm β-mercaptoethanol). Hsp104 was eluted with high-salt buffer (wash buffer with 1 m KCl). Hsp104 was then purified further by Resource Q anion-exchange chromatography and size-exclusion chromatography . Firefly luciferase was from Sigma. Hsc70, Hdj2, DnaK, DnaJ, and GrpE were from Enzo Life Sciences. Unless otherwise stated, ClpB and Hsp104 concentrations refer to the hexamer.ClpB and Hsp104 mutants were generated via QuikChange Lightning mutagenesis (Agilent). ClpB variants were expressed as C terminally Hisnstructs  or as unnstructs  was trantography . The isotography  to yieldm) in luciferase refolding buffer  with 6 m urea was incubated at 30 °C for 30 min. The sample was then rapidly diluted 100-fold into luciferase refolding buffer, divided into 100-μl aliquots, snap frozen in liquid N2, and stored at −80 °C. For reactivation assays, aggregated luciferase (50 nm) was incubated with Hsp104 (1 μm), Hsc70 (1 μm), and Hdj2 (1 μm) plus ATP (5.1 mm) and an ATP regeneration system ) for 90 min at 25 °C. In some experiments, Hsc70 and Hdj2 were replaced with 4 mm ATPγS and 1.1 mm ATP. In other experiments, isolated His6-MD (6 or 36 μm) was added. For luciferase reactivation by ClpB, Hsp104, Hsc70, and Hdj2 were replaced with ClpB (0.167 μm), DnaK (0.167 μm), DnaJ (0.033 μm), and GrpE (0.0167 μm) and the reaction was incubated for 60 min at 25 °C. In some experiments, DnaK, DnaJ, and GrpE were replaced by 2.5 mm ATPγS and 2.6 mm ATP. For experiments with disulfide cross-linked Hsp104 or ClpB variants, luciferase aggregates were assembled and reactivated as above except that DTT was omitted from all buffers. Luciferase activity was assessed with a luciferase assay system (Promega). Luminescence was monitored on a Tecan Safire2 or Infinite M1000 plate reader. For doping experiments, the conditions for ClpB and Hsp104 were kept as similar as possible. Thus, GrpE was present (0.167 μm) or absent from the reaction and 1 μm ClpB, 1 μm DnaK, and 1 μm DnaJ were used. Hsp104, Hsc70, and Hdj2 were used as described above. Total Hsp104 or ClpB was comprised of WT or mutant or a 1:5, 2:4, 3:3, 4:2, and 5:1 mixture of the two. To ensure statistical subunit mixing, WT and mutant Hsp104 or ClpB mixtures were equilibrated for 15 min on ice prior to addition to the reaction  yeast were transformed with a centromeric plasmid bearing the HSP104 promoter, pHSE, or pHSE encoding Hsp104 or the indicated Hsp104 variant . Immunoblot showed that each Hsp104 protein was expressed at similar levels.W303 Δm Na3PO4, 50 mm K3PO4, and 10 mm MgAOc, pH 7) and the concentration was adjusted to 1 μm monomer. Data were collected on an Aviv Biomedical Inc. CD spectrophotometer (model 410). Samples were scanned from 260 to 190 nm with a 1-nm step size and a 15-s averaging time at 25 °C.Hsp104 variants were dialyzed into CD buffer . The Hsp104 concentration was adjusted to 35 μm monomer and 100 μl of sample was fractionated by a Superose 6 10/300 column .Hsp104 variants were exchanged into running buffer (40 mm monomer) were incubated for 12 min at 25 °C (for Hsp104) or 30 min at 25 °C (for ClpB) with ATP (1 mm). ATPase activity was determined by the amount of inorganic phosphate released using a malachite green phosphate detection kit (Innova).Hsp104 or ClpB (0.25 μm) in storage buffer , 1 mm DTT). Recovered beads were washed 4 times in storage buffer, with the second wash containing 1 mm ATP, and then eluted with storage buffer plus 20 mm reduced glutathione. Samples were processed for SDS-PAGE and Coomassie staining.GST or GST-MD was coupled to glutathione-Sepharose at 2.5 μg/μl and incubated for 15 min on ice with Hsc70  to ensure that all proteins were hexameric, even in the absence of nucleotide . Samples were excited at 295 nm (4-mm bandwidth) and emission was collected from 305 to 505 nm (3-nm bandwidth) with a 1-nm step size using a Fluorolog-3–21 Jobin-Yvon Spex Instrument SA . Spectra of buffer matched controls were subtracted.Hsp104 variants were dialyzed into low salt buffer . Oxidation of Alexa 488 (Invitrogen) was used to determine optimal exposure time for each buffer and provide normalization constants. Samples were exposed to a mirror-focused synchrotron x-ray beam  at the X28C beamline of the National Synchrotron Light Source at Brookhaven National Laboratory for 0–20 ms. Exposure time was controlled by flow rate through the flow cell of a KinTek  stopped flow apparatus (m) and samples were snap frozen in liquid N2. Irradiated protein samples were thawed on ice, diluted to 1 μm in 5 mm HEPES-NaOH, pH 7.0, 140 mm KCl, 10 mm MgCl2, 1 mm DTT, 1% TFA, and injected into an on-line fragmentation-separation/MS analysis system. Samples were passed through an immobilized pepsin column onto a C18 trap column. Following a 3-min wash, peptides were eluted with a non-linear elution gradient optimized for the highly charged Hsp104 peptide fragments (6 μl/min non-linear 2–50% acetonitrile gradient with 0.1% formic acid at pH 2 and 0 °C) and passed through an analytical C18 HPLC column before injection by electron spray ionization (ESI) to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). To enhance peptide identification, selected peptide ions (the four most abundant in each scan) were fragmented by CID and measured in the LTQ stage. A “peptide pool” of unmodified peptides was obtained by combining high probability peptides identified from 0-ms MS runs. A program used for the identification of H/D exchange data, ExMS  equilibrated in 50 mpparatus . Oxidatita, ExMS , was modta, ExMS , and theta, ExMS  analysis6CS, where all six of the native cysteines of Hsp104  are mutated to serine. Engineered cysteines were introduced into this background. For ClpB, native cysteines were not mutated to serine. After purification, samples were exchanged into running buffer without DTT. Protein concentrations were adjusted to 60 μm and samples were incubated with 1 mm diamide for 30 min at room temperature. Samples were exchanged into running buffer without DTT to remove excess diamide. Disulfide cross-link formation was assessed via SDS-PAGE on 4–20% Tris-HCl non-reducing gel in comparison to non-cross-linked samples (Bio-Rad). Gels were Coomassie stained and intramolecular disulfide formation caused proteins to migrate more slowly (6CS/A430C/F630C according to the manufacturer's instructions (Thermo Scientific Pierce). For all activity assays with disulfide cross-linked proteins, DTT was omitted unless specifically stated.For cross-linking, we employed an Hsp104 variant, Hsp104e slowly . Quantifq (q = 4πsin(θ)/λ, where 2π is the scattering angle and λ is the wavelength). The x-ray wavelength was 0.855 Å and the angular range collected was 0.00550 ≤ q ≥ 1.0060. The sample cell contained a glass capillary sealed across the evacuated chamber. Protein samples and matching buffer solutions were flowed through the capillary during exposure to reduce radiation damage. For data collection, 30 μl of the protein sample at concentrations between 1.5 and 6 mg/ml or matching buffer  was exposed for 180 s, subdivided into three 60-s exposures of 10 μl. After each measurement the capillary was washed thoroughly and purged with compressed nitrogen.SAXS and WAXS data were collected simultaneously at beamline X9 at the National Synchrotron Light Source  at 10 °C by two overlapping detectors, a Mar 165 CCD SAXS detector 3.4 m from the sample, and a custom built Photonic Science CCD WAXS detector. The two-dimensional scattering images collected on the CCD detectors were circularly averaged using software developed at the beamline to yield one-dimensional scattering profiles as a function of momentum transfer gR) were initially calculated using Guinier plots (P(r) distance distribution functions were calculated by the program GNOM (Dmax) was determined by examining the quality of fit to the experimental data for a Dmax range of 180 to 280 Å, varied in 5-Å increments. Fits were performed to maximize the total estimate figure, to minimize the discrepancy between calculated and experimental profiles, and to optimize the visual properties of the shape distribution function. Values for gR were computed from the second moment of the P(r) and compared favorably to those calculated using Guinier plots.The raw scattering data were scaled and buffer was subtracted using the program PRIMUS . Each iner plots . P(r) diram GNOM  using anP is the probability that a hexamer  contains x mutant subunits and p is the probability that a mutant subunit is incorporated. Experiments demonstrated that mutant and WT subunits have a similar probability of being incorporated into a hexamer . Activity versus p plots (B) could then be generated assuming each WT subunit makes an equal contribution to the total activity (one-sixth per subunit). Consequently, if subunits within the hexamer operate independently then activity should decline in a linear manner upon incorporation of defective mutant subunits. Conversely, if subunits are coupled then a specific number of subunits will be sufficient to eliminate activity. Thus, zero activity is assigned to hexamers that are in breach of a specific threshold number of mutant subunits. In this way, we can generate activity versus p plots if we assume that 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more mutant subunits are required to eliminate activity  in induced thermotolerance in yeast . After 20 min at 50 °C, Hsp104Y507A conferred survival levels similar to the vector control (A). However, Hsp104Y507A was not completely inactive and after 10 min at 50 °C provided significant thermotolerance similar to WT Hsp104 (A). WT Hsp104 and Hsp104Y507A were expressed at similar levels (data not shown). Hsp104Y507A reactivated aggregated luciferase in vivo although not as effectively as WT Hsp104 (B). Thus, contrary to expectations from data obtained with ClpBY503A . This finding explains why Hsp104Y507A is partially active in vivo (A and B). However, Hsp104Y507A was also highly active in the presence of ATP in the absence of Hsp70 and Hsp40, whereas WT Hsp104 was inactive (C). In the presence of ATP, Hsp70 and Hsp40 only modestly stimulated (∼10%) Hsp104Y507A disaggregase activity (C). Moreover, substituting a permissive ratio of ATPγS:ATP in place of Hsp70 and Hsp40 inhibited Hsp104Y507A, whereas WT Hsp104 was activated (C). Thus, the Y507A mutation is pleiotropic and causes alterations in the intrinsic disaggregase activity of Hsp104 as well as reduced ability to collaborate with Hsp70.Can Hsp104nditions C. This f in vivo , A and Binactive C. In theactivity C. Moreovctivated C. Thus, Y503A is selectively impaired in the ability to collaborate with DnaK, DnaJ, and GrpE (KJE). ClpBY503A has minimal activity in the presence of ATP and absence of DnaK, DnaJ, and GrpE (D). Note that the relative activation of ClpB by ATP:ATPγS is low compared with ClpB activity in the presence of DnaK, DnaJ, and GrpE (D), whereas in Hsp104 it is considerably higher (C). Thus, for optimal activity ClpB requires Hsp70 more stringently than Hsp104. These findings indicate that mutation of the conserved tyrosine in helix 3 of the MD  to alanine has different functional consequences for Hsp104 versus ClpB.By contrast, ClpBand GrpE , 47, andand GrpE D. Note tand GrpE D, whereay higher C. Thus, C) might be important for Hsp70 collaboration because in another Hsp100, ClpC, this region contacts co-factors (C and D).We sought non-pleiotropic mutations in Hsp104 that specifically ablated collaboration with Hsp70, whereas having no effect on the intrinsic disaggregase activity of Hsp104 against disordered aggregates. Such Hsp104 variants would be unable to disaggregate disordered aggregates in the presence of Hsp70 and Hsp40. However, they would still disaggregate disordered aggregates in the presence of ATPγS:ATP alone and be inactive with ATP alone without Hsp70. We hypothesized that the distal loop (residues 430–446 in Hsp104) between helix 1 and 2 of the MD C might b-factors . Moreove-factors , C and D430, Leu431, and Asp438, because they are highly conserved (B–D). Initially, we made a drastic change and introduced the bulky, hydrophobic residue, tryptophan, in an effort to perturb function. Importantly, CD revealed that Hsp104A430W, Hsp104L431W, and Hsp104D438W were very similar to WT Hsp104 in terms of secondary structure (data not shown). Moreover, the isolated MD bearing the A430W mutation had similar secondary structure to WT MD (data not shown). Hsp104A430W, Hsp104L431W, and Hsp104D438W assembled into hexamers just like WT Hsp104 (data not shown). Thus, these mutations do not grossly perturb the Hsp104 structure.We focused on three distal loop residues: Alaonserved , B–D. InA430W and Hsp104D438W retained WT levels of intrinsic disaggregase activity in the presence of a permissive ATPγS:ATP ratio (A). Intriguingly, Hsp104L431W displayed elevated intrinsic disaggregase activity with ATPγS:ATP (A). Moreover, like WT Hsp104, these Hsp104 variants possessed no disaggregase activity in the presence of ATP and absence of Hsp70 and Hsp40 (B). Thus, in contrast to Hsp104Y507A, in the absence of Hsp70 the distal loop variants Hsp104A430W, Hsp104A430Y, and Hsp104D438W are activated by ATPγS:ATP and inactive with ATP alone similar to WT Hsp104.Hsp104TP ratio A. IntrigTPγS:ATP A. Moreovnd Hsp40 B. Thus, A430W, Hsp104L431W, and Hsp104D438W were unable to collaborate with Hsp70 and Hsp40 to reactivate aggregated luciferase (C). Indeed, in the presence of Hsp70 and Hsp40 plus ATP, Hsp104A430W, Hsp104L431W, and Hsp104D438W were completely inactive (C). We obtained similar results when we substituted these positions with tyrosine instead of tryptophan (A–C). Hsp104A430Y and Hsp104D438Y retained WT levels of intrinsic disaggregase activity in the presence of ATPγS:ATP, whereas Hsp104A431Y disaggregase activity with ATPγS:ATP was elevated (A). Moreover, Hsp104A430Y, Hsp104A431Y, and Hsp104D438Y were completely inactive in the presence of ATP alone (B), or when combined with Hsp70 and Hsp40 (C). These are the first Hsp104 mutants that are: (a) unable to collaborate with Hsp70 and Hsp40; (b) inactive with ATP alone; and (c) retain disaggregase activity elicited by ATPγS:ATP. Thus, conserved MD distal loop residues are critical for Hsp70 collaboration.In striking contrast to WT Hsp104, Hsp104ciferase C. Indeedinactive C. We obtyptophan , A–C. Hselevated A. MoreovTP alone B, or whend Hsp40 C. These 430 (C and D) on intrinsic disaggregase activity and ability to collaborate with Hsp70 and Hsp40. Interestingly, Hsp104A430D and Hsp104A430L displayed reduced disaggregase activity elicited by ATPγS:ATP (A) and reduced Hsp70-dependent disaggregation (C). By contrast, Hsp104A430V was active in the presence of Hsp70 and Hsp40 (C), and was also slightly active in the presence of ATP alone . However, Hsp104A430V had lost intrinsic disaggregase activity in the presence of ATPγS:ATP . Thus, depending upon the precise substitution, mutation of Ala430 can inhibit Hsp70 collaboration (e.g. Trp), inhibit intrinsic disaggregase activity in the presence of ATPγS:ATP , or both (e.g. Asp or Leu). These findings suggest that Ala430 plays a key role in regulating Hsp104 disaggregase activity.Next, we explored the effect of different missense mutations at position Ala430 , C and DTPγS:ATP A and redregation C. By connd Hsp40 C, and wa431 (C), which is highly conserved (D). Against disordered luciferase aggregates, Hsp104L431A displayed lowered activity in Hsp70-dependent disaggregation (C) and Hsp70-independent disaggregation elicited by ATPγS:ATP (A). The reduction was more severe for intrinsic disaggregase activity elicited by ATPγS:ATP (A and C). By contrast, Hsp104L431D displayed mildly reduced Hsp70-dependent disaggregase activity (C), whereas the intrinsic disaggregase activity in the presence of ATPγS:ATP was not significantly affected (A). Interestingly, Hsp104L431D exhibited some ability to reactivate aggregated luciferase with ATP alone in the absence of Hsp70 and Hsp104 (∼12.2% of WT-Hsp104 with Hsp70 and Hsp40), whereas WT Hsp104 is completely inactive under these conditions (B). Thus, Leu431 also regulates Hsp104 disaggregase activity.We next explored different missense mutations at Leu431 C, which onserved D. Againsregation C and HspTPγS:ATP A. The reTPγS:ATP , A and Cactivity C, whereaaffected A. Interenditions B. Thus, 438 variants (C). Mutation of Asp438 to Trp, Tyr, Leu, or Ala had the same effect: Hsp70-dependent reactivation of aggregated luciferase was ablated, whereas intrinsic disaggregase activity in the presence of ATPγS:ATP or ATP alone was unaltered (A–C). Thus, Asp438 plays a critical role in Hsp70 collaboration, but can be substituted and not affect the ability of Hsp104 to process aggregated substrates per se. These studies suggest that Ala430, Leu431, and Asp438 all make important contributions to Hsp70 collaboration. Moreover, Ala430 and Leu431 tightly regulate Hsp104 disaggregase activity.We also explored Aspvariants C. Mutatinaltered , A–C. ThA430W, Hsp104L431W, and Hsp104D438W, because they are specifically defective in Hsp70 collaboration (A–C). We tested whether these Hsp104 variants could confer induced thermotolerance in vivo. In preconditioned Δhsp104 cells, Hsp104A430W, Hsp104L431W, and Hsp104D438W were severely defective in thermotolerance (D and E). Indeed, Hsp104A430W and Hsp104L431W were completely inactive and conferred thermotolerance levels comparable with the vector control (D and E). By contrast, Hsp104D438W afforded some thermotolerance, but was greatly impaired compared with WT Hsp104 (E). Importantly, tryptophan substitutions in the distal loop or elsewhere in the MD did not impair thermotolerance: Hsp104A437W, Hsp104A453W, Hsp104Y466W, Hsp104Y497W, and Hsp104Y507W (C) all conferred thermotolerance comparable with WT Hsp104 (D) . Thus, conserved residues in the MD distal loop of Hsp104 enable collaboration with Hsp70 and induced thermotolerance in vivo.We focused on the tryptophan variants, Hsp104boration , A–C. Weolerance , D and E control , D and ET Hsp104 E. Import104Y507W C all conT Hsp104 D 63). T. TA430W,A428W, which is analogous to Hsp104A430W (D). The intrinsic disaggregase activity of ClpBA428W was similar to WT ClpB. Indeed, ClpBA428W was slightly more active than WT ClpB in reactivating aggregated luciferase in the presence of ATPγS and ATP (F) and inactive in the presence of ATP (G). However, ClpBA428W was unable to collaborate with DnaK, DnaJ, and GrpE (H). Thus, the A428W mutation specifically ablates ClpB-DnaK collaboration. These data suggest that the function of the MD distal loop in Hsp70 collaboration is conserved across two billion years of evolution.To test conservation in ClpB, we generated ClpB104A430W D. The in and ATP F and inae of ATP G. Howeveand GrpE H. Thus, e.g. Hsp104A430W, Hsp104L431W, and Hsp104D438W, to collaborate with Hsp70 could have at least three causes: (a) these distal loop variants may have an impaired ATPase cycle that precludes collaboration with Hsp70. Indeed, some ATPase-defective ClpB variants are less able to synergize with DnaK (b) by analogy with the ClpC distal loop, which directly engages MecA (c) mutations in the distal loop might alter an intramolecular Hsp104 interface that regulates Hsp70 collaboration. In fact, one hexameric model of Hsp104 and ClpB posits that the distal loop lies in close proximity to NBD2  by ges MecA , the dis to NBD2 –52. ThusA428W (A). The majority of Hsp104 distal loop mutants had mildly impaired ATPase activity, ranging from ∼30% (Hsp104L431D: ∼3.04 ± 0.5 min−1) to ∼85% (Hsp104D438A: ∼11.2 ± 0.96 min−1) of WT Hsp104 activity (∼13.1 ± 0.93 min−1). In fact, impairment was statistically significant with the exception of Hsp104D438A (A). Thus, at first glance, impaired ATPase activity of these mutants might explain defective collaboration with Hsp70 (A). However, upon closer inspection there is no simple linear correlation between ATPase activity and the ability to collaborate with Hsp70 . For example, Hsp104D438A has ∼85% WT Hsp104 ATPase activity, but is unable to collaborate with Hsp70 , whereas Hsp104L431D has the lowest ATPase activity but can collaborate with Hsp70 in luciferase reactivation (C and 4B). Moreover, ClpBA428W, which is unable to collaborate with DnaK (H), did not have lower ATPase activity than WT ClpB . In fact, if simply decreasing ATPase activity led to defective Hsp70 collaboration, we would expect all distal loop variants to be deficient in Hsp70 collaboration. However, this is not the case. For example, Hsp104A430V can collaborate effectively with Hsp70 (C and 4B). Thus, the moderately impaired ATPase activity of these distal loop variants does not explain their inability to collaborate with Hsp70.We determined the ATPase activity of each Hsp104 distal loop mutant, as well as WT ClpB and ClpBA428W A. The ma104D438A A. Thus, th Hsp70 A. Howevetivation C and 4B.ith DnaK H, did noth Hsp70 C and 4B.C, linear regression, R2 = 0.012). Thus, the ATPase activity of these Hsp104 variants provides little information about their disaggregase activity in the presence of Hsp70 or ATPγS and ATP.There was also no simple linear relationship between the Hsp104 variant ATPase activity and ability to reactivate luciferase in the presence of ATPγS and ATP . These experiments do not exclude the possibility that the MD of Hsp104 interacts directly with Hsp70. However, the isolated MD appears insufficient to recapitulate the Hsp104 and Hsp70 interaction.Another possibility for how specific distal loop mutations impair Hsp70 collaboration is that mutation in this region disrupts a direct interaction between this region of Hsp104 and Hsp70. Indeed, in peptide arrays Hsp70 interacts with a distal loop peptide (residues 435–448) in this region . HoweverT. thermophilus has been solved  indicate a more hydrophobic environment, e.g. regions that are shielded from solvent (466 position (C), which is buried in both hexameric models and the Tyr497 position (C), which is solvated in both models to ensure that we could accurately determine the solvation state (Y466W and Hsp104Y497W confer WT levels of thermotolerance in vivo (D). As expected, in each nucleotide state tested  Hsp104Y466W had blue-shifted emission maxima of ∼330 nm indicating a buried region, whereas Hsp104Y497W had red-shifted emission maxima of ∼350 nm, indicating a solvated region (A–D).To assess the positioning of the MD within the Hsp104 hexamer, we exploited the fact that Hsp104 has no natural tryptophans, which makes it well suited for site-specific tryptophan fluorescence spectroscopy. This sensitive technique measures the polarity of the microenvironment within 10 Å of a Trp residue , 65. Red solvent , 65. We position C, which position C, which on state , 52. Imp in vivo D. As expd region , A–D.A430W, Hsp104L431W, and Hsp104D438W exhibited emission maxima between ∼335 and 340 nm in each nucleotide state (A–D) indicating that the distal loop is partially shielded from solvent. These findings were not due to studying defective Hsp104 variants because Hsp104A437W, which displays WT levels of thermotolerance (D), also exhibited emission maxima between ∼336 and 339 nm (A–D). Although fluorescence intensity changed in response to nucleotide for all the distal loop variants, we observed only minor shifts in emission maxima in response to different nucleotides, with the exception of Hsp104L431W where the emission maximum remained at ∼335 nm for all states (A–D). For example, the emission maximum of Hsp104A430W in the absence of nucleotide and in the presence of ADP was more red-shifted , whereas the spectra of ATP and ATPγS were more blue-shifted . Likewise, the emission maximum of Hsp104D438W in the presence of ADP was more red-shifted , whereas in the presence of ATP or ATPγS it was more blue-shifted . Although minor, these shifts indicate that Ala430 and Asp438 are more solvated in the apo and ADP states and more buried in the ATP and ATPγS states.Hsp104de state , A–D indolerance D, also ed 339 nm , A–D. All states , A–D. Fo∼337 nm; , C and D∼341 nm; D, whereaA430W, Hsp104L431W, Hsp104A437W, and Hsp104D438W indicate that the distal loop is unlikely to always be solvent exposed, which is inconsistent with a model where the MD projects out from the hexamer into solvent in all nucleotide states examined  the distal loop is partially sequestered from solvent all of the time; and 2) the distal loop moves between two different conformations, one of which is solvated and one of which is buried. Hsp104 hexamers are strongly asymmetric with each protomer adopting a distinct conformation . Thus, texamined , 49, 53.i.e. water). These radicals oxidatively modify solvent-exposed protein side chains in well defined ways, although Ala, Asp, and Asn have lower reactivity and Gly behaves like the main chain . In XF, millisecond bursts of x-rays generate hydroxyl radicals from solvent molecules . In agreement with the Hsp104Y466W fluorescence data (C), peptide-(456–476) was not modified in the presence of ATPγS suggesting that this region is solvent inaccessible. In the presence of ADP, peptide-(456–466) was modified at time points longer than 2.5 ms, whereas peptide-(467–474) was unmodified. Although Tyr466 is predicted to be buried by both models . The rate of modification is ∼7-fold less rapid in ATPγS  than in ADP . Thus, the MD distal loop of WT Hsp104 is less solvent accessible in the presence of ATPγS and more solvent accessible in the presence of ADP, which is consistent with Hsp104A430W and Hsp104D438W fluorescence (C and D). The MD distal loop of Hsp104 likely shifts from a more buried position to a more solvent exposed position upon ATP hydrolysis. These XF and tryptophan fluorescence data are consistent with a hexameric model in which the MD distal loop is positioned in a way that could make an intramolecular contact with NBD2  becomes oxidized indicating that it must be partially exposed to solvent E. The raith NBD2 –52.Using tryptophan fluorescence and XF, we have established that the distal loop of Hsp104 can become shielded from solvent. Size exclusion chromatography coupled to dynamic light scattering reveal that Hsp104 hexamers are monodisperse in solution (data not shown). Thus, the distal loop of the MD is likely to contact another domain within the Hsp104 hexamer. Where does the distal loop become buried?N728A hexamer in the presence of ATP, where the distal loop is in close proximity to NBD2 (F and G) (G), we site specifically mutated proposed contact residues between MD and NBD2 to cysteine and assessed the proximity by attempting to form disulfide cross-links. This strategy has confirmed that helix 3 of the MD can contact NBD1 in ClpB .To answer this question, we examined an optimized structural model of the Hsp104 to NBD2 , F and GF and G) –52. BaseF and G) G, we sit in ClpB , 47. Fir6CS background to test the proposed proximity of A430C(MD)/F630C(NBD2)  and K451C(MD)/E790C(NBD2)  (A and B) as well as between MD residue K451C and the NBD2 residue E790C (A and C). Both disulfides were intramolecular as no higher order multimeric species were observed in single or double cysteine variants. In both cases, the disulfide formed more readily in the presence of AMP-PNP  or ATP than in the presence of ADP . Thus, upon ATP binding the distal loop (Ala430) and helix 2 (Lys451) of the MD contact NBD2. Upon ATP hydrolysis, the MD is in less close contact with NBD2, which is in accord with tryptophan fluorescence (C and D) and XF data (E). These findings argue against hexameric models where the MD invariably projects away from the hexamer out into solution (We introduced cysteine pairs into the Hsp104esidues) –52. Disuesidues) . Single rescence , C and D XF data E. These solution , 49, 53 solution , which wE, yellow residue) readily formed a disulfide with L817C , which is located in the NBD2 small domain (E and F). Unlike Hsp104, we observed maximal disulfide formation in all nucleotide states . Thus, the MD is oriented differently in Hsp104 and ClpB. In comparison to Hsp104 the MD of ClpB seems to be rotated ∼90° anticlockwise around helix 2. Nonetheless, the MD distal loop also contacts NBD2 in ClpB, which further argues against a hexameric model where the MD invariably projects away from the hexamer out into solution  and F621C (F630C in Hsp104) in ClpB. By contrast, in ClpB, A428C  crystal structure (A) (B) (A) /K480C(MD) and R366C(NBD1)/V540C(NBD1), which correspond to V350C(NBD1)/Q467C(MD)  and R355C(NBD1)/E520C(NBD1)  in tClpB, respectively , but not the other model , whereas R366C(NBD1)/V540C(NBD1) are predicted to form a disulfide in both . In agreement with data from tClpB (6CS readily formed a disulfide within NBD1 (C and D). Thus, two β-strands of the NBD1 small domain, which are separated in primary sequence by the entire MD, can be linked by a disulfide in Hsp104 and ClpB (C and D). By contrast and as predicted by the Hsp104 cryo-EM model  (C and E). Thus, NBD1-MD domain contacts are not absolutely conserved between ClpB and Hsp104.The NBD2-MD contacts in Hsp104 and ClpB are not anticipated by a hexameric model of Hsp104 based on the tructure A  , but areture (A) B 50–52)–52T. the (A) (B) A (14). TG) , D247C(NBD1)/H471C(MD) , D247C(NBD1)/E475C(MD) , and E320C(NBD1)/N467C(MD)  in Hsp1046CS. We also assessed H362C(NBD1)/D484C(MD) , which are not predicted to be in close enough proximity to form disulfides (C and F). In the presence of ADP or ATP, K182C(NBD1)/E485C(MD), D247C(NBD1)/H471C(MD), and D247C(NBD1)/E475C(MD) all formed intramolecular disulfides, although the efficiency was ∼15–20%, indicating that on average only one subunit per hexamer could make this cross-link . By contrast, the E320C(NBD1)/N467C(MD) disulfide formed with greater efficiency in ADP (∼40%) than ATP (∼15%) (C and J). None of these disulfides are expected by a hexameric model where the MD projects out into solution  –52G)  , 49, 53.6CS/A430C/F630C and ClpBA428C/L817C. We also cross-linked A430C to F630C in the Hsp1046CS background with the longer and more flexible 11-Å linker, BMB. Before cross-linking, Hsp1046CS/A430C/F630C and ClpBA428C/L817C displayed luciferase reactivation levels comparable with WT . After disulfide cross-linking, Hsp1046CS/A430C/F630C and ClpBA428C/L817C had very low luciferase reactivation activity, suggesting that the cross-links nixed disaggregase activity . Importantly, reduction of the Hsp1046CS/A430C/F630C and ClpBA428C/L817C disulfides restored disaggregase activity . Thus, the cross-linking procedure itself does not result in impaired activity. We confirmed by SDS-PAGE that BMB induced an upshift indicative of an intramolecular cross-link in ∼85% of total Hsp1046CS/A430C/F630C. In contrast to disulfide cross-linked Hsp1046CS/A430C/F630C, BMB cross-linked Hsp1046CS/A430C/F630C retained some disaggregase activity, whereas Hsp1046CS was unaffected . Thus, a longer, more flexible cross-link between NBD2 and the distal loop permits some disaggregase activity.The cross-linking data suggested that the MD populates two different conformations that result in the distal loop being in two different environments. The first conformation (populated in the ATP-bound state) results in a more solvent-inaccessible distal loop that is juxta-NBD2 and thus able to form a covalent cross-link between the two domains. The second conformation (populated in the ADP-bound state) leaves the distal loop more solvent accessible and not as closely associated with NBD2. To probe the functional importance of the NBD2-MD contact we assessed the luciferase disaggregase and ATPase activity of disulfide cross-linked and uncross-linked Hsp1046CS/A430C/F630C and ClpBA428C/L817C. Hsp1046CS/A430C/F630C displayed an elevated ATPase rate (∼25 ± 0.67 min−1) compared with WT Hsp104 (∼13.1 ± 0.93 min−1) . Disulfide cross-linked Hsp1046CS/A430C/F630C had reduced ATPase activity (∼9.54 ± 1.63 min−1) . Inhibition was relieved upon reduction of the Hsp1046CS/A430C/F630C disulfide (∼27 ± 0.67 min−1), indicating that the NBD2-MD disulfide contact inhibits ATP hydrolysis . BMB cross-linked Hsp1046CS/A430C/F630C was less inhibited (∼15.2 ± 0.3 min−1) . By contrast, disulfide cross-linked ClpBA428C/L817C had similar ATPase activity to uncross-linked ClpBA428C/L817C . Thus, in ClpB, constraining the distal loop contact with NBD2 inhibits disaggregase activity  without significantly affecting ATPase activity. Collectively, the foregoing data suggest that the distal loop of the MD and NBD2 of the same subunit are in close proximity and can form an autoinhibitory contact that tightly constrains the disaggregase activity of Hsp104 and ClpB. Based on these observations, we suggest that a critical step in the activation of Hsp104 and ClpB for protein disaggregation lies in the separation of these autoinhibitory NBD2-MD contacts.To determine how the disulfide cross-link between MD and NBD2 inhibits disaggregase activity, we examined the ATPase activity of Hsp104Dmax) and radius of gyration (gR) of WT Hsp104 and Hsp104A430W hexamers in the presence of ATP . SAXS exploits the fact that x-ray scattering by a particle at very low angles contains information about particle size and shape. The pairwise distance distribution (P(r) curve), which represents the distances between pairs of atoms within a given volume, reveals that Hsp104A430W has a smaller Dmax than WT Hsp104 (205 Å compared with 230 Å for WT Hsp104). Moreover, the gR, which is the root mean square average of all of the interatomic distances with respect to the center-of-mass, is shorter for Hsp104A430W than for WT Hsp104 (compare 64 to 68.2 Å) . Thus, Hsp104A430W occupies a smaller spatial extent in solution than WT Hsp104 (A430W is evident in the P(r) curves where WT Hsp104 exhibits a decrease in smaller interatomic vectors near the gR (the peak) and an increase in larger interatomic vectors . The Porod volume of Hsp104A430W is also smaller than that of WT Hsp104  , Fig. 8ET Hsp104 . The smaT Hsp104 . Porod-DT Hsp104  indicatee.g. Hsp70 collaboration). These mixtures rapidly form heterohexamers containing both WT and mutant subunits according to the binomial distribution (A) (e.g. Hsp70 collaboration) (B). For example, by mixing WT Hsp104 with Hsp104A430W or WT ClpB with ClpBA428W in known and established ratios we were able to determine how many WT subunits per hexamer are required for Hsp104 or ClpB to collaborate with Hsp70 during disaggregation. We conducted doping experiments  with Hsp104A430W and ClpBA428W. ClpB was more sensitive to the Hsp70 collaboration-deficient mutant than Hsp104. In fact, ClpB required between 3 and 4 WT subunits per hexamer to disaggregate luciferase , whereas Hsp104 only required 2 WT subunits per hexamer .Hsp104 and ClpB form dynamic hexamers, which exchange subunits rapidly and randomly. Thus, we can use a mutant-doping strategy to ask how many subunits per hexamer are required for a given aspect of Hsp104 or ClpB activity , 38. Herribution A  B. For exA428W doping experiments, but omitted GrpE. Here, we observed an even more deleterious effect upon titration of ClpBA428W. In the absence of GrpE, ClpB required between 5 and 6 WT subunits per hexamer to accomplish disaggregation . Thus, more ClpB subunits per hexamer must be competent to collaborate with Hsp70 than Hsp104 subunits for successful disaggregation. Since these mutant doping studies of in vitro luciferase reactivation by Hsp104 and ClpB accurately model physiological conditions  unable to collaborate with Hsp70? These mutants form hexamers, retain WT secondary structure, possess robust ATPase activity, and disaggregate disordered aggregates in the presence of permissive ATPγS:ATP ratios. The MD distal loop does not engage Hsp70 directly . Thus, ClpB has a more rigid requirement for Hsp70 collaboration than Hsp104, and this requirement is intensified in the absence of GrpE. This result was surprising because it indicated that Hsp104 and ClpB differ fundamentally in their mechanism of collaboration with Hsp70. This fundamental difference between Hsp104 and ClpB, defined here with pure components, is likely to explain differences between Hsp104 and ClpB observed under physiological conditions. More ClpB subunits per hexamer must be competent to interact functionally with Hsp70 than Hsp104 subunits. This distinction likely reflects important differences in how substrate handover from Hsp70 to ClpB and Hsp104 is coordinated. For example, more ClpB subunits per hexamer are likely required to wrench substrate from Hsp70, particularly in the absence of GrpE. Moreover, ClpB is more dependent upon Hsp70 for optimal activity than Hsp104 (C and D). These differences between Hsp104 and ClpB likely reflect differential tuning for the respective challenges of yeast and bacterial proteostasis.We used Hsp104eriments  to detereriments C. Thus, n Hsp104 , C and D


Plasmodium falciparum H2B.Z localizes to euchromatic intergenic regions throughout intraerythrocytic development and together with H2A.Z forms a double-variant nucleosome subtype. These nucleosomes are enriched in promoters over 3′ intergenic regions and their occupancy generally correlates with the strength of the promoter, but not with its temporal activity. Remarkably, H2B.Z occupancy levels exhibit a clear correlation with the base-composition of the underlying DNA, raising the intriguing possibility that the extreme AT content of the intergenic regions within the Plasmodium genome might be instructive for histone variant deposition. In summary, our data show that the H2A.Z/H2B.Z double-variant nucleosome demarcates putative regulatory regions of the P. falciparum epigenome and likely provides a scaffold for dynamic regulation of gene expression in this deadly human pathogen.Histone variants are key components of the epigenetic code and evolved to perform specific functions in transcriptional regulation, DNA repair, chromosome segregation and other fundamental processes. Although variants for histone H2A and H3 are found throughout the eukaryotic kingdom, variants of histone H2B and H4 are rarely encountered. H2B.Z is one of those rare H2B variants and is apicomplexan-specific. Here we show that in  Plasmodium falciparum is the protozoan parasite responsible for almost 1 million malaria deaths annually   encodes for a 123-amino-acid protein that differs from canonical H2B (PF3D7_1105100) mostly in its N-terminal domain , but differs substantially from T. brucei H2B variant H2BV (only 37% identity and 81% similarity).P. falciparum, we generated a parasite line (3D7) carrying an episomal Ty1-tagged copy of PfH2B.Z  of mono-nucleosomal DNA fragments, followed by linear amplification for deep sequencing (LADS) , tandem (neighboured by one 3′ and one 5′ end) or as divergent (neighboured by two 5′ ends) high occupancy is most prevalent in promoter containing regions (tandem or divergent). On the contrary, low H2B.Z occupancy is almost exclusively observed at convergent regions (two 3′ ends) B. This f regions B – table regions D.Visual inspection of the coverage plots furthermore suggested that H2B.Z is primarily present at regions with AT content higher than 80% A. To expP. falciparum genome where they display highest occupancies in promoter regions. Furthermore, both H2B.Z and H2A.Z histone variant occupancy displays a clear positive correlation towards genomic AT content.In conclusion, we have shown that histone variants H2A.Z and H2B.Z form a double-variant nucleosome subtype and locate to euchromatic intergenic regions of the P. falciparum, similar to T. gondii H2A.Z/H2B.Z  indicate that variant nucleosomes in both species are less stable compared with their canonical counterparts. Consequently, double-variant nucleosomes residing on regulatory DNA sequences might be more readily displaced by chromatin remodelers or transcription machinery. Alternatively, these nucleosomes might display a reduced residence time (e.g. a higher ‘off’ rate) on the DNA, thereby temporally exposing regulatory sequences for transcription factor binding. In either of these models, the H2A.Z/H2B.Z double-variant nucleosome subtype likely plays a supportive function in transcriptional initiation in line with their increased levels on promoter regions  genes  further supporting the hypothesis on the AT content driven deposition of these nucleosomes. On the contrary, however, dynamic deposition of H2A.Z  has been observed at the promoter region of the single active var gene. This stage-specific deposition of double-variant nucleosomes associated with the temporal activity of this active var. Therefore, it seems that although AT content might act as a targeting mechanism for H2A.Z/H2B.Z nucleosomes, other mechanisms are in place to fine-tune and modulate the localization pattern of this special nucleosome subtype.As described above, double-variant nucleosomes are generally absent from the heterochromatic domain and within the euchromatic regions their level is largely invariable between the stages of intraerythrocytic development. Intriguingly, H2A.Z/H2B.Z nucleosomes also ubiquitously occupy the highly AT-rich introns of antigenic variation  under the control of the chloroquine resistance transporter promoter  using BWA . Coverage plots shown in et al. . For all et al.,  and HP1 R2 of square-rooted values were determined in R using the ‘lm’ function.For scatterplot analysis, the number of uniquely mapped tags were counted in each intergenic region of the H2B.Z-Ty1 genome. H2B.Z square-rooted signals were plotted against H2A.Z square-rooted occupancy values in R for ring, trophozoite and schizont stages. et al.  with coding body of 1–10 kb  and a minimum of 800 bp intergenic regions on both sides (to minimize overlap with flanking region of nearby genes) as in Bartfai et al. . Genes w . For To determine a correlation between H2B.Z or H2A.Z occupancy and genomic AT content B, ChIP-oet al., For Supplementary et al., To define the correlation between AT content of the promoter region and gene expression, the AT content of the 600 bp sequence upstream of 1855 euchromatic genes (the same as used for the generation of average gene profiles) was defined by a self-written script. This was correlated to the maximum expression level of the gene across 8 stages intraerythrocytic development [defined by the highest number of RNAseq reads per 1000 bp coding sequence . The membrane was probed with mouse αTy1  and rabbit αH2A  and secondary αMouse IRDye 680  and αRabbit IRDye 800CW  and measured on the Odyssey system (LI-COR Biosciences). Co-immunoprecipitations were performed on mono-nucleosomes harvested from H2B.Z-Ty1 schizont stages as for native ChIP using 2 μg of BB2, 1 μl of αhttp://www.plasmodb.org) and will be available at Gene Expression Omnibus (reference number: GSE39702).All data files have been submitted and are available at PlasmoDB (


Mayetiola destructor) is one of the most destructive pests of wheat. The genes encoding 12-oxo-phytodienoic acid reductase (OPR) and lipoxygenase (LOX) play critical roles in insect resistance pathways in higher plants, but little is known about genes controlling resistance to Hessian fly in wheat.Hessian fly (6:8 recombinant inbred lines (RILs) generated from a cross between two cultivars, ‘Jagger’ and ‘2174’ of hexaploid wheat , were used to map genes associated with resistance to Hessian fly. Two QTLs were identified. The first one was a major QTL on chromosome 1A (QHf.osu-1A), which explained 70% of the total phenotypic variation. The resistant allele at this locus in cultivar 2174 could be orthologous to one or more of the previously mapped resistance genes  in tetraploid wheat. The second QTL was a minor QTL on chromosome 2A (QHf.osu-2A), which accounted for 18% of the total phenotypic variation. The resistant allele at this locus in 2174 is collinear to an Yr17-containing-fragment translocated from chromosome 2N of Triticum ventricosum  in Jagger. Genetic mapping results showed that two OPR genes, TaOPR1-A and TaOPR2-A, were tightly associated with QHf.osu-1A and QHf.osu-2A, respectively. Another OPR gene and three LOX genes were mapped but not associated with Hessian fly resistance in the segregating population.In this study, 154 FOPR and LOX genes in wheat.This study has located two major QTLs/genes in bread wheat that can be directly used in wheat breeding programs and has also provided insights for the genetic association and disassociation of Hessian fly resistance with  Mayetiola destructor (Say)] is one of the most destructive pests of hexaploid wheat  in the United States and worldwide [Hessian fly [Hf, orldwide . HessianH1 to H32 and Hdic[. Among these Hessian fly resistance genes, however, only 8 genes  were identified in hexaploid wheat [H6, H9–H11, H14–H20, H28, H29, H31, and Hdic) identified in tetraploid wheat species Triticum turgidum subsp. durum  [H6, H9-H11, H31, and Hdic) have been introgressed from tetraploid to hexaploid wheat [H13, H22, H23, H24, H26, and H32) from Aegilops tauschii  [H21 and H25) from Secale cerale L.  [H27) from Aegilops ventricosa  from Aegilops triuncialis  [At least 33 Hessian fly resistance genes have been identified, designated as 4 × =28) , and 6 oid wheat -20. Othe2 × =14) -25, two 2 × =14) , one genH5, H9, H10, H11, H16, H17, and Hdic) were mapped on chromosome 1A, and these genes may comprise a cluster (or family) of Hessian fly resistance genes in the distal gene-rich region of wheat chromosome 1AS [H3, H6, H12, H14, H15, H19, H28, and H29) on chromosome 5A [H24, H26, and H32) on chromosome 3D [H18, H20 and H21) on chromosome 2B [H13 and H23) on chromosome 6D [H7 on chromosome 5D [H22 on chromosome 1D [H25 on chromosome 6B [H31 on chromosome 5B [H27 on chromosome 4D [Simple sequence repeat (SSR) or microsatellite markers were used to map Hessian fly resistance genes in previous studies. Seven genes , are useful for designing primers specific to a homoeologous gene for genomic mapping. With the integration of these techniques and tools, mutants of several genes including VRN-A1[VRN-D3[PPD-D1[Lr34-D[Yr17[Pm3[Recent progress in the application of high-throughput sequencing technologies and development of sophisticated genomic mapping tools has accelerated identification of agriculturally important genes in wheat. Numerous wheat ESTs assigned to wheat deletion bins have facilitated the physical mapping of a gene ,46. The istachyon, and synistachyon,49 have ng VRN-A1, VRN-D3[A1[VRN-D3, PPD-D1[D3[PPD-D1, Lr34-D[D1[Lr34-D, Yr17[52[Yr17[Pm3 have beePlants possess multiple defense mechanisms in response to mechanical damage due to insect attack. Jasmonic acid (JA) and its conjugates, jasmonates, play a central role in regulating defense responses of plants to insect herbivores . In highOPR genes and 3 LOX genes in a population of recombinant inbred lines (RILs) that was generated from a cross between two locally adapted winter cultivars, ‘Jagger’ and ‘2174’, and showed demonstrable segregation for resistance to Hessian fly. The implication in association and disassociation of the QTLs for resistance to Hessian fly with these OPR and LOX genes is discussed.In this study, we mapped 3 6:8 RILs generated by crossing the parental cultivars.Jagger was highly susceptible and 2174 was highly resistant to Hessian fly biotype GP when the two parental lines were tested with susceptible (Karl 92) and resistant (WGRC42) cultivars as controls   genes.Using 154 RILs of the Jagger × 2174 population and genome-wide SSR markers , we mappentified . The LODQHf.osu-2A)  in Jagger [QHf.osu-2A was 3.5 and this QTL accounted for 18% of the total phenotypic variation. Phenotypic data showed that those RILs which carried the Jagger allele had resistance at the level of 23.1%, whereas those RILs that carried the 2174 allele had resistance at the level of 51.9%. The resistant allele at this locus in 2174 is collinear to an Yr17-containing-fragment translocated from chromosome 2N in Jagger. Jagger attained the translocated chromosome 2N fragment conferring resistance to stripe rust [OPR2-A gene on chromosome 2A in 2174.The second QTL was a minor QTL on chromosome 2A , and the wheat sequences retrieved from the databases were grouped based on sequence alignments of the homoeologous and paralogous OPR genes. Primers were designed for each group of these genes and tested for specificity to N1AT1D, N1BT1D, and N1DT1B of ‘Chinese Spring’ (CS) nullisomic–tetrasomic (NT) lines [OPR genes were identified specific to chromosome 1A  lines . PrimersOPR gene TaOPR1-A, the first OPR gene to be mapped on chromosome 1A of T. aestivum. The primers amplified a 337-bp fragment that was digested with KpnI into 213 bp and 124 bp for the Jagger allele (GenBank: KF035075) and into 36 bp, 177 bp, and 124 bp for the 2174 allele (GenBank: KF035076) , showed complete linkage with a group of markers for Yr17 translocated from chromosome 2N and under the peak of QHf.osu-2A. The wheat EST BE403717 that was mapped in 1AS-3 FL 0.86 deletion bin was used to blast against GenBank Nucleotide Collection (nr/nt) databases, and it showed 82% identity within a range of 318 bp (E = 1e-83) to an orthologous OPR gene in rice BAC from chromosome 6 (GenBank: AP004741). Specific primers were designed based on the grouped sequences retrieved from the wheat genomic DNA databases. The primers OPR22-C1-F3 and OPR-R2 amplified a 634 bp fragment that was digested with HincII into 20 bp, 321 bp and 292 bp for the Jagger allele (GenBank: KF035084) and into 342 bp, 215 bp, and 77 bp for the 2174 allele (GenBank: KF035085)  was analyzed and used to search for the wheat orthologous OPR genes, because the rice BAC could be in a collinear region to wheat chromosome 1A. Specific forward primer OPR1-C1F1 was used to combine with conserved reverse primer OPR-R1 to amplify a single copy of the OPR gene mapped on chromosome 7B (TaOPR7-B). The chromosomal location of TaOPR7-B was confirmed by using CS nullisomic-tetrasomic lines  and 452 bp but was not digested for the Jagger allele (GenBank: KF035086)  that was mapped in the 1AS-3 FL 0.86 deletion bin was used to blast against wheat gDNA databases  and GenBank EST databases for the homoeologous and paralogous LOX genes. This wheat EST was expected to reside in a region collinear with rice chromosome 5[LOX genes in rice, with 87% identity in a range of only 294 bp (E = 5e-96) to one in chromosome 2 (GenBank: AP004184), with 72% identity (E = 2e-38) to two genes on chromosome 8 (GenBank: AP005816). Homoeologous chromosomes of hexaploid wheat could have three genes for each orthologous gene in rice. Primers were designed specifically to each of the groups retrieved from the sequences by BF482663.Three omosome 5. It showLOX gene was mapped using primers LOX-C5-F5 and LOX-C5-R6 specific to chromosome 1A  compared with the Jagger allele (GenBank: KF035088)  but outside of the QHf.osu-1A region  and 272 bp in 2174 (GenBank: KF035092). The sequencing results confirmed that these PCR products were derived from part of a LOX gene, with an 8 bp deletion for the Jagger allele or an 8 bp insertion for the 2174 allele  was sequenced with low coverage to find genes that could be used to determine collinear regions of rice and Brachypodium. Four OPR genes , and three genes encoding stress-induced receptor-like kinase (Srk) (GenBank: KF035093), catalytic domain of protein kinases (Pkc) (GenBank: KF035099), and calcineurin-like phosphoesterase-like (Clp) (GenBank: KF035096) respectively, were present in the wheat BAC clone  and the 2174 allele (GenBank: KF035095), the Clp gene for the Jagger allele (GenBank: KF035097) and the 2174 allele (GenBank: KF035098), and the Pkc gene for the Jagger allele (GenBank: KF035100) and the 2174 allele (GenBank: KF035101). Howerver, no crossover was found between the OPR genes and these linked genes in the the QHf.osu-1A region.TaOPR1-A displayed the highest identity to orthologous OPR genes in a rice BAC from chromosome 6 (GenBank: AP004741), which contains 7 OPR genes arranged in tandem  were not present in the flanking regions of the OPR genes in the two BAC clones shown in Figure OPRs in rice. TaOPR1-A displayed the highest identity to three orthologous OPR genes in Brachypodium  were not observed in the collinear region of Brachypodium. The orthologous genes in Brachypodium were not present in the flanking regions of the OPR genes in rice either.H5, H9, H10, H11, H16, H17, and Hdic[T. durum. Our study is the first report that a resistance gene against Hessian fly exists in the short arm of chromosome 1A in bread wheat. The resistance gene at the QHf.osu-1A locus observed in hexaploid wheat cv. 2174 could be orthologous to one or more of the previously mapped resistance genes in tetraploid wheat. This study provides an effective resistance source to manage biotype GP that frequently damage bread wheat cultivars in the southern Great Plains. Further studies need to test if the QHf.osu-1A gene is allelic to genes H16 and H17 that confer resistance against Hessian fly biotype L, the most virulent and prevalent biotype in the eastern USA [QHf.osu-1A in 2174 and its derived cultivars can be immediately utilized to control Hessian fly biotype GP in winter wheat improvement programs in the southern Great Plains in USA. The gene may also be useful for pyramiding to prolong the effective periods of other resistance genes.A resistance gene against Hessian fly has been repeatedly mapped to the end of the short arm of chromosome 1A in previous studies, and it was suggested that this genomic region contained a cluster of major dominant resistance genes against multiple Hessian fly biotypes, including  and Hdic,20,30,31QHf.osu-1A showed that it is closely linked with Pm3a. Previous studies indicated that Pm3 was linked to H9 at a genetic distance of 4.5 cM [Pm3 gene [Lr10, which has three paralogous genes and is effective against Puccinia triticina Eriks, was also mapped in the same chromosomal region on 1AS [QHf.osu-1A. An effort was made to construct a fine physical map for QHf.osu-1A. However, low collinearity of the gene order in this region among wheat, rice, or Brachypodium indicates that the fine physical map for QHf.osu-1A cannot be established by using genome information from rice or Brachypodium only. The recent sequencing of wheat genomes may provide a powerful tool in cloning QHf.osu-1A.Mapping of f 4.5 cM . SeveralPm3 gene . Leaf run on 1AS ,65. The OPR genes were identified in association with two QTLs for resistance against Hessian fly, but we cannot yet decisively conclude if either of the candidate OPR genes is responsible for QHf.osu-1A. The two QTLs were discovered in the same population, and resistance to Hessian fly is inseparable between the two QTLs. Two specific RIL lines (#23 and #26) have been selected to backcross with the 2174 parental line to generate progeny that will segregate for a single Hessian fly gene. In these backcross populations, QHf.osu-1A will occur in the heterozygous state, whereas QHf.osu-2A will be fixed in a homozygous genetic background. We expect that the two alleles of QHf.osu-1A will produce clear segregation for resistance to Hessian fly. BC1F2-3 populations will be used also to determine degree of dominance for the Hessian fly gene.Two TaLOX6-B was mapped between Xwmc398 and Xbarc198 toward the centromere of wheat chromosome 6B. It was reported that H25 was translocated from rye to the long arm of chromosome 6B of wheat [TaLOX6-B is not allelic to H25. TaLOX2-B is located between Xwms388 and Xwms120 on the long arm of chromosome 2B. H20 was transferred from T. durum to chromosome 2B, and H21 was translocated from rye to chromosome 2B in wheat. However, more information is needed to further clarify any allelic relationship of TaLOX6-B with either H20 or H21. A recent study indicates that a novel wheat gene encoding a lectin-like protein (Hfr-3) was associated with response to Hessian fly [Hfr-3 genes are located on group 7 chromosomes of the hexaploid wheat. Yet, no Hessian fly resistance gene was reported on chromosome 7B where TaOPR7-B was mapped.of wheat , and thusian fly . The homLr37, Yr17 and Sr38 conferring resistance to leaf rust, stripe rust, and stem rust, but Jagger lost the allelic region containing the resistance gene on QHf.osu-2A against Hessian fly. The molecular marker can greatly facilitate the identification of such a candidate gene for the QHf.osu-1A locus, enabling opportunities for functional analysis which can then be used to better understand the defense mechanisms of wheat plants in response to this major insect.In summary, a frequent objective of wheat breeding is to pyramid multiple genes for resistance to diseases and insects into a single cultivar. Compared with 2174, Jagger attained a translocated fragment that contains A Kansas population of Hessian fly consisting of predominantly biotype GP  was used6:8 recombinant inbred lines (RILs) were used to map genes associated with resistance to Hessian fly. Wheat genomic DNA was extracted from leaf tissue of each RIL plant as described previously [Based on observations on seedling plants, Jagger is susceptible to Hessian fly biotype GP infestation whereas 2174 is resistant; therefore, the Jagger × 2174 population of 154 Feviously .6:8 RILs were evaluated for phenotypic reaction to Hessian fly infestation in growth chambers at 18 ± 1°C with a 14 h:10 h (light: dark) photoperiod as described previously [Parental lines and 154 Feviously ,34,69. Beviously . HessianHdic), were also planted in each flat. Wheat plants were infested at one leaf-stage (the second leaf just emerged) with Hessian fly adults under a cheese cloth tent. Females deposit eggs on the adaxial surface of the first leaf. Infestation was stopped when the egg density reaches 8 per leaf, which usually results in ~5 larvae per plant. Resistant and susceptible plants were phenotyped three weeks after infestation. Phenotypes of resistant and susceptible plants were distinct and easy to score. Resistant plants grew normally with light green color and susceptible plants were stunt with dark-green color. Plants with a resistant phenotype were further dissected to check for dead larvae. Plants without dead larvae were taken as escapes and were excluded from the data set.Resistance screening was carried out in greenhouse as described previously . SpecifiA total of 404 SSR markers were assembled in linkage groups using MapMaker 3.0 program, and a QTL was identified using the WinQTLCart 2.5 program as previously described ,70. OtheOPR and LOX genes in rice are available in GenBank databases, including OPR genes in rice BAC from chromosome 1 (GenBank: AK100034), chromosome 6 (GenBank: AP004741), and chromosome 8 (GenBank: AP004707); and LOX genes from chromosome 2 (GenBank: AP004184), chromosome 5 (GenBank: AC136525), and chromosome 8 (GenBank: AP005816). Rice BACs and wheat EST sequences were used to blast against wheat gDNA databases  to retrieve homoeologous and paralogous sequences for each TaOPR and TaLOX genes. Multiple sequence alignments showed that the homoeologous and paralogous TaOPR and TaLOX genes are variable, which made it possible to design genome-specific primers for each gene. Gene markers developed were used to analyze the RIL population and evaluated for linkage (Table Wheat ESTs encoding LOX (GenBank: BE403717) and OPR (GenBank: BE482663) were found present in the deletion bin (1AS-3 FL 0.86) on chromosome 1A, and these ESTs were used to develop markers for mapping. The genomic sequences of the orthologous All PCR reactions were performed in a 25 μl reaction under the following conditions: 1 denaturation cycle at 94°C for 5 min; 40 cycles of 94°C for 30 s, 52-62°C  for 30 s, and 72°C for 30 s – 2.5 min (based on fragment length), then a final extension step at 72°C for 10 min before cooling to 4°C. PCR amplified fragments were separated on 2% agarose gels through electrophoresis in 1× TAE buffer or 6% acrylamide gel in 0.5× TBE buffer. DNA banding patterns were visualized under UV light with ethidium bromide staining.OPR: 12-oxo-phytodienoic acid reductase; LOX: Lipoxygenase; RILs: Recombinant inbred lines; QTL: Quantitative trait loci; Hf: Hessian fly; SSR: Simple sequence repeat; JA: Jasmonic acid; OPDA: 12-oxo-phytodienoic acid; AOS: Allene oxide synthase; OPR3: 12-oxo-phytodienoic acid reductase 3; GP: Great plains; EST: Expressed sequence tag; BAC: Bacterial artificial chromosome; gDNA: Genomic DNA; Srk: Induced receptor-like kinase; Pkc: Catalytic domain of protein kinases; Clp: Calcineurin-like phosphoesterase-like.The authors declare that they have no competing interests.CTT performed the experiments and analyzed data. BFC provided plant materials for the project and helped edit the manuscript. MSC performed phenotype experiments for Hessian fly resistance. YQG performed analysis of comparative genomics and provided BAC clones. CTT and LY designed the project and wrote the manuscript. All authors read and approved the manuscript.Sequence comparison of TaOPR1-A and TaOPR2-A.Click here for fileSequence comparison of a LOX gene between wheat and rice.Click here for file


Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16.Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses.The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16.EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials.NCT01268787ClinicalTrials.gov __ Therefore, EV71 vaccine is urgently needed, and promising candidates are being evaluated in human phase I clinical trials Enterovirus 71 (EV71) infections is the leading cause of hand, foot and mouth disease (HFMD) in Asian children that can result in severe neurological complications and death Picornaviridae, and contains a plus sense ssRNA (7.5–8.5 kb) and four structural proteins: VP1, VP2, VP3 and VP4 EV71 belongs to a non-enveloped RNA virus of the family The inactivated EV71 virion produced in Vero cells has been shown to elicit more efficacious immune responses than those obtained from recombinant VP1 protein or DNA vector vaccines in the mouse challenge model The protocol for this trial and supporting CONSORT checklist are available as supporting information; see In this study, we report the results of human immunogenicity of EV71vac at 5- and 10-µg doses in 60 healthy volunteers according to NHRI clinical protocol #QCR10013. This study is registered with ClinicalTrials.gov #NCT01268787. The protocol was approved by Taiwan FDA #9917719 and conducted in compliance with National Taiwan University Hospital (NTUH) IRB# 201007056M and Veteran General Hospital Taipei IRB#201008015MA. The trial was started in December 2010 and completed in March 2012 for patient recruitment and follow-up. At the screening visit (Visit 1), the investigator verbally reviewed the written, informed consent form  with each volunteer to ensure he/she understanding the study and the study procedures. Signed informed consent was obtained before performing any procedures related to the study and after the volunteers had received sufficient information about the study and had had the opportunity to ask any questions and consider the options. A copy of the signed ICF was provided to the subject and the original document retained in the study file.in vitro neutralization assay were grown in Rhabdomyosarcoma (RD) cells. EV71/E59 and EV71/E36 virus strains were provided by Taiwan CDC All EV71 strains used for Vero cell banks and EV71 virus seed banks (E59 strain) were established according to US FDA cGMP guidelines as described in previous publications 4) and 0.5 mL per dose , respectively. Both groups followed the same visit schedule and were vaccinated intramuscularly with one dose of EV71vac on Day 0 and a second dose on Day 21. Serum samples were collected at Day 0, 21 and 42, and inactivated at 56°C for 30 minutes and stored at −80°C before use.This clinical study was a phase I, uncontrolled, prospective, randomized, open-label, two-center clinical study in Taipei, Taiwan. The safety, reactogenicity and preliminary immunogenicity of EV71vac at 5- and 10-µg doses in sixty eligible healthy volunteers are currently reported elsewhere The cytopathic effect (CPE) assay was used to evaluate virus neutralization titers (Nt) which was defined as the reciprocal of the highest dilution capable of inhibiting 50% of CPE Specific IgG titer against EV71/E59 was determined by enzyme-linked immunosorbent assay (ELISA). Inactivated EV71/E59 (1 g/mL) was first coated onto 96-well microplates and allowed to stand overnight. The microplates were then washed with 0.5% Tween 20 in phosphate buffer saline (PBST) and blocked with 1% bovine serum albumin (BSA) in PBS for 2 hours at room temperature. After washing the microplates with PBST for a second time, 100 µL of two-fold serial diluted clinical serum specimens (beginning with 1∶1000) were added into each well. The microplates were incubated at room temperature for 2 hours, and then washed with PBST. One hundred microliters of the detection antibody  was added into each well and incubated on the microplates at room temperature for 1 hour. After washing the microplates with PBST, 100 µL the chromogenic substrate 2,2-azino-di-(ethylbenzthiazoline-sulfonic acid) (ABTS) was added into each well and incubated at room temperature for 20 minutes. A spectrophotometer measured the absorbance at 405 nm. The IgG titer was defined as the endpoint of serial dilution at which the optical density (OD) value was two-fold higher than the background value that was obtained from the negative control peptide CB82 with sequence (DVSDFTDSVRDPKTS) derived from SARS S protein.All statistical analyses were performed using SAS software and all tests were two-sided when applied. Statistical significance for all comparisons was determined at P<0.05.One of the enrollment criteria was adult with ages between 20 and 60 years. In this trial, the mean age of the volunteers was found to be 29.3±4.9 and 28.6±5.0 for 5 and 10 microgram dosage groups, respectively. One subject withdrew from the study; and one blood sample was misplaced and lost . The ratTwenty-seven out of sixty participants were found to have <8 Nt against vaccine strain in their pre-immune sera (Table S1 in Greater than 90% (29/32) of participants in both groups with pre-existing Nt were found to exhibit a >4-fold increase in Nt after the first vaccination . Their GCareful immunological analyses of the sera showed that 21 participants had no detectable Nt (<8) against EV71 subgenotypes B1 and B5 before vaccination, and that a >4-fold increase in Nt was observed in 17/21 and 18/21 (>80%) of participants after the first vaccination, respectively (Table S2 in Most of participants (57/60) had no detectable Nt against subgenotype C2 before vaccination, and even after two doses of EV71vac only nine of 56 subjects  in both groups mounted a positive neutralizing response  had no detectable Nt against the EV71/E36 strain (subgenotype C4A that circulated in Taiwan in 2004) . After oThe analysis of virus cross-neutralization data against CVA16 revealed that 49/60 (>80%) of the pre-immune sera had no detectable Nt (<8)  against the B4 vaccine strain. Except Subject #18, all produced strong Nt against B4 after one EV71vac immunization . In addiThe lack of an efficacious vaccine against EV71 is a major hurdle in preventing the spread of EV71 in Asia-Pacific regions. A report by Cheng et al. The results from current immunogenicity study indicate that a 5-µg dose of EV71vac is highly immunogenic in the tested volunteers. Several important conclusions can therefore be drawn from the study. (1) Based on the rate of seroconversion , >95% of volunteers sero-converted; 2) EV71vac is capable of eliciting strong VNA responses in naive volunteers ; 3) EV71vac induced very strong VNA responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A; (4) EV71vac elicited weak VNA responses (∼20% of participants) against a C4B and CVA16; (5) >90% (51/56) of vaccinated young adults without pre-existing Nt against C2 did not develop VNA responses (Nt<8) against a C2 strain; (6) EV71vac can boost and significantly enhance VNA responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16; and (7) most volunteers vaccinated twice with EV71vac did not show further increase in Nt. To our surprise, a decrease in the GMT of Nt was observed after the booster injection , 2, 3. T EV71vac  EV71vac Clinical trial results from Li et al. et al.et al.Is the current vaccine derived from an EV71 subgenotype B4 strain the right choice? We did not observe poor VNA responses against C2 in EV71vac immunized mice, rabbits and macaques in pre-clinical studies The weak and poor cross-neutralizing antibody responses against C4B, CVA16 and C2, prompted us to test other EV71 isolates in the laboratories. From the preliminary results, Subjects who were previously found to have Nt<8 against C2 strain were shown to have a >4-fold increase in Nt against another C2 strain (EV71/4643) and another C4B (EV71/1757) strain . To settle these discrepancies, we are currently purifying the virus strains and standardizing their viral titer used in the cross-neutralization assay since we have found that different virus strains could generate different amounts of defective particles that could interfere with the neutralization titer. In our previous studies The current study indicate that preexisting antibody due to stealth infections could interfere the efficacy of the vaccine, so we urgently need a good biomarker to pre-screen naive subjects since most young children in epidemic Asian countries have likely been exposed to and/or infected with EV71 and/or CVA16. The EV71 particle-based specific ELISA may be used as a quick screening test of pre-immune sera from volunteers since at the 1/2000 dilution EV71 negative sera correlates with Nt levels <8.Although animal immunogenicity study in general is used to evaluate the consistency of immunogenicity among different batches of vaccines, it is of interest to know which animal  immunogenicity study can be correlated to the potency of EV71vac. Mice immunized with EV71vac mainly produced antibodies against the immunodominant neutralization epitope VP1–43 corresponding to residues 205–225 (FGEHKQEKDLEYGAC) of VP1 Overall, the current results indicate that EV71vac based on an EV71 subgenotype B4 strain is a good vaccine candidate. It is safe, well tolerated and effectively elicits cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5 and C4A. We should keep in mind that the target population of the EV71 vaccine is young infant and children younger than 5 years old. The cross-neutralization immune responses to EV71vac in children should be investigated. In addition, the vaccination schedule and the long-term immunogenicity should be assessed as well. Therefore, the current results provide a strong rationale for further evaluations of EV71vac in phase II clinical trials.Protocol S1This study is registered with ClinicalTrials.gov with the registration #NCT01268787. The protocol was approved by Taiwan FDA #9917719 and conducted in compliance with National Taiwan University Hospital (NTUH) IRB# 201007056M and Veteran General Hospital Taipei IRB#201008015MA. The inclusion/exclusion criteria for the subject recruitment and randomization were reported in our recent publication (PDF)Click here for additional data file.File S1This file describes the Supplementary Materials of Formalin-inactivated EV71 vaccine candidate induced cross-neutralizing antibody against subgenotypes B1, B4, B5 and C4A in adult volunteers. The contents include: Table S1. Summary of pre- and post-vaccination immune responses from individual subjects. Table S2. Summary of EV71 B genotypes-specific virus neutralizing antibody titers obtained from subjects immunized with the EV71 vaccine. Table S3. Summary of EV71 C4 subgenotypes-specific virus neutralizing antibody titer obtained from human sera immunized with EV71 vaccine. Figure S1. Virus neutralizing antibody responses of young adults immunized with EV71vac against subgenotype B4 (vaccine strain). Figure S1. Virus neutralizing antibody responses of young adults immunized with EV71vac against subgenotype B4 (vaccine strain). Panels A and B show the neutralization titers of sera obtained from volunteers immunized twice with either a 5- or 10-µg dose, respectively. Thirty healthy adults were enrolled in each group. Each spot represents the neutralizing antibody titer for individual sera. The bar corresponds to the geometric mean titer (GMT) of each set of sera.(DOC)Click here for additional data file.Checklist S1CONSORT checklist of a Phase 1, Randomized, Open-Label Study to Evaluate the Safety and Immunogenicity of Enterovirus 71 Vaccine.(DOC)Click here for additional data file.