The aim of this study was to explore the relationship between serum 25-hydroxyvitamin D [25(OH)D] concentrations and lower extremity arterial disease (LEAD) in type 2 diabetes mellitus (T2DM) patients and to investigate the intervention effect of vitamin D. 145 subjects were assigned to a control group (Group NC), T2DM group (Group DM1), and T2DM complicated with LEAD group (Group DM2); then Group DM2 were randomly divided into Group DM3 who received oral hypoglycemic agents and Group DM4 who received oral hypoglycemic drugs and vitamin D3 therapy. Compared to Group NC, 25(OH)D was significantly lower in Group DM2 and marginally lower in Group DM1. In contrast to baseline and Group DM3, 25(OH)D rose while low density lipoprotein (LDL), retinol binding protein 4 (RBP4), and HbA1c significantly lowered in Group DM4. Statistical analysis revealed that 25(OH)D had a negative correlation with RBP4, duration, HbA1c, homeostasis model assessment for insulin resistance (HOMA-IR), and fasting plasma glucose (FPG). LDL, systolic blood pressure (SBP), FPG, and smoking were risk factors of LEAD while high density lipoprotein (HDL) and 25(OH)D were protective ones. Therefore, we deduced that low level of 25(OH)D is significantly associated with the occurrence of T2DM complicated with LEAD. Lower extremity arterial disease (LEAD) is a common peripheral arterial disease, which seriously affects the patient's functional capacity and quality of life . It is o107 patients  who were recruited from outpatient department were in line with the T2DM diagnosis standard delivered by World Health Organization (WHO) in October 1999. They were hospitalized from October 2012 to January 2013 in the Department of Endocrinology in Anhui Provincial Hospital. This study was conducted in accordance with the tenets of the World Medical Association's Declaration of Helsinki and had been approved by the China Ethics Committee. Written informed consent was obtained from all participants.The subjects who met the following criteria were excluded: (1) individuals who had acute complications of T2DM, retinopathy, nephropathy, and other chronic complications, cancer, vitamin A deficiency, liver and kidney dysfunction, osteoporosis, and other history of bone metabolic disorders; (2) those taking drugs including vitamins, calcium, lipid lowering drugs, estrogen, and other drugs affecting bone metabolism. All the patients were assigned into 2 groups: the one that is without complications  and the other that is complicated with LEAD ; then subjects in Group DM2 were further divided into Group DM3  and Group DM4 . Patients in these 2 groups were both treated with hypoglycemic drugs, and additionally patients of Group DM4 were orally administered vitamin D3 1000 IU daily. In this period 2 subjects in Group DM3 were out of touch and 4 subjects in Group DM4 could not insist on the intervention therapy and quitted. The relevant characteristics were tested and ABI was reexamined in Groups DM3 and DM4 after 12 weeks of therapy. 38 age-sex-matched healthy people with no hypertension or impaired glucose tolerance were chosen at the same period as a control group (Group NC) .The Doppler instrument (Bidop ES-100-v3 produced in Japan) was adapted. All the ABI measurements were performed with the subjects in the supine position, a blood pressure cuff was placed on patients' upper arms, and it was inflated until no brachial pulse was detected by the Doppler device. The cuff was then slowly deflated until the Doppler-detected pulse returned to measure brachial systolic blood pressure (BSBP). This maneuver was repeated on the leg, with the cuff being wrapped around the distal calf and the Doppler device being placed over the dorsalis pedis or the posterior tibial artery to measure ankle systolic blood pressure (ASBP) . Ankle b2) and waist circumference divided by hip circumference, respectively. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using a digital automatic blood pressure monitor. Patients' blood samples were kept frozen at −80°C until analysis. Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL), and high density lipoprotein (HDL) were detected by Hitachi 7600 2020 automatic biochemical analyzer. HbA1C was conducted by high-pressure liquid chromatography . Fasting insulin (FINS) was tested by radioimmunoassay . The homeostasis model assessment for insulin resistance (HOMA-IR) was calculated according to the formula HOMA-IR = FINS (mU/L) × FPG (mmol/L)/22.5. Serum 25(OH)D concentrations were detected via radioimmunoassay . Sufficiency was indicated when 25(OH)D ≥ 30 ng/mL, insufficiency when 20 ng/mL ≤ 25(OH)D < 30 ng/mL, and deficiency when 25(OH)D < 20 ng/mL [All the subjects took balanced diets for 3 days and then fasted overnight for 12 hours. Clinical characteristics including sex, age, diabetes duration, height, weight, hip circumference, blood pressure, and history of smoking were collected. Body mass index (BMI) and waist-hip ratio (WHR) were calculated as weight divided by height squared D and other markers. Multivariable logistic regression analysis was used to calculate the ORs and 95% confidence intervals for LEAD; statistical significance was accepted at P < 0.05.All data were calculated by SPSS17.0 statistical software . Continuous variables are presented as mean ± standard deviation (P > 0.05). Participants in Group DM2 had a longer duration than in Group DM1; the differences were statistically significant (P < 0.05). Serum 25(OH)D concentrations were 24.77 ± 5.9 ng/mL, 17.32 ± 7.42 ng/mL, and 12.63 ± 7.83 ng/mL in Groups NC, DM1, and DM2, respectively; compared with Group NC, 25(OH)D decreased in Groups DM1 and DM2 and the decrease in Group DM2 was more significant (P < 0.05). RBP4 level was lower in T2DM patients with LEAD compared to the patients without LEAD and healthy people (P < 0.05). Raised FPG, FINS, HbA1C, TC, TG, LDL, HOMA-IR, SBP, and WHR and decreased HDL in Groups DM1 and DM2 were exhibited as compared with Group NC (P < 0.05); age, FPG, LDL, SBP, HbA1C, and the rate of smoking increased and HDL decreased in Group DM2 compared with the other 2 groups (P < 0.05).The indexes of the subjects were listed in P < 0.05).As depicted in P < 0.05), while, following 12-week basic therapy of hypoglycemic drugs in Group DM3, only RBP4 and HbA1C decreased (P < 0.05). Supplementation with vitamin D for 12 weeks in diabetics with LEAD significantly raised 25(OH)D level and lowered LDL, RBP4, and HbA1c relative to baseline and unsupplemented diabetics (P < 0.05). Reexamination of ABI by Doppler ultrasonography for subjects in Group DM3 and Group DM4 after 12 weeks showed that there were, respectively, 5 cases in Group DM3 and 10 cases in Group DM4 whose values increased to ≥0.9, and the difference was not statistically significant (P > 0.05) D level increased and LDL, RBP4, and HbA1C decreased significantly when compared with baseline ( > 0.05) .r = −0.663, P < 0.05), HbA1c , RBP4 , FPG (r = −0.229), and HOMA-IR , while there was no significant correlation between 25(OH)D and the remaining indicators (P > 0.05). 25(OH)D had a positive correlation trend with HDL, but the difference was not statistically significant (P > 0.05).As depicted in As shown in γ (PPAR-γ) and the differentiation from preadipocytes to mature adipocytes [RBP4 is a molecule found in the circulation, thought to be secreted mainly by adipose tissue and the liver. Increased serum RBP4 levels have been reported in subjects with obesity, IR, and T2DM and in other insulin-resistant states, such as metabolic syndrome and vascular complications of DM . Cabré eipocytes . This reβ cells to secrete insulin by adjusting vitamin D receptor (VDR) and vitamin-D-dependent calcium-binding protein (DBP) in pancreatic tissue. It can also reduce IR in peripheral tissues by regulating inflammatory cytokines and inhibiting the expression of PPAR-γ [δ) and the gene expression of insulin receptor [α, and C-reactive protein (CRP) [The third national health and nutrition examination survey (NHANES-III) found that, compared with residents with normal concentrations of 25(OH)D, residents with low concentrations of 25(OH)D had a higher incidence of peripheral arterial disease . In thisf PPAR-γ . Besidesreceptor . (2) Calreceptor . (6) Vitreceptor , 24 and in (CRP) . (7) CalA 20-year retrospective study has found that  the inciThere were several limitations in our study. The sample size was relatively small and we did not consider the influence of outdoor activities and seasonal variation on the level of vitamin D. However, the study was conducted in winter so that the impact of sun exposure was minimized. In conclusion, the phenomenon of vitamin D deficiency amongst T2DM patients was more and more prevalent, and, with the advantages of low price and validated long-term safety, the supplementation of vitamin D would be one of the intervening measurements for the presentation of T2DM and its vascular complications.


Immunization with the C3-binding motifs protected mice against challenge with a lethal dose of non-invasive M49 strain GAS but not invasive M1 strains. To achieve higher efficiency against invasive M1 GAS infection, a combination of synthetic peptides derived from C-terminal epitope of streptolysin S (SLSpp) and from the major C3-binding motif of SPE B  was used to elicit specific immune response to those two important streptococcal exotoxins. Death rates and the severity of skin lesions decreased significantly in PP6/SLSpp-immunized mice that were infected with invasive M1 strains of GAS. These results indicate a combination of the C3-binding motif of SPE B and the protective epitope of SLS could be used as a subunit vaccine against invasive M1 strains group A streptococcal infection.Group A streptococcus (GAS) is an important human pathogen that produces several extracellular exotoxins to facilitate invasion and infection. Streptococcal pyrogenic exotoxin B (SPE B) has been demonstrated to be an important virulence factor of GAS. Our previous studies indicate that SPE B cleaves complement 3 (C3) and inhibits the activation of complement pathways. In this study, we constructed and expressed recombinant fragments of SPE B to examine the C3-binding site of SPE B. Using enzyme-linked immunosorbent assays and pull-down assays, we found that the C-terminal domain, containing amino-acid residues 345–398, of SPE B was the major binding site of human serum C3. We further identified a major, Ala Streptococcus pyogenes) causes pharyngitis, tonsillitis, cellulitis, scarlet fever, myositis, necrotizing fasciitis, puerperal sepsis, and streptococcal toxic shock syndrome. Post-streptococcal glomerulonephritis and rheumatic fever are serious post-infectious immune sequelae following scarlet fever or repeated GAS infection [Group A streptococcus , an extracellular cysteine protease, contributes to increasing GAS invasion and infection , 9. SPE The complement system plays an essential role in the early phase of the host defense against bacterial infection. Three different complement pathways converge in the generation of complement 3 (C3) convertase to cleave C3 to activate the complement cascades. Activation of C3 plays a key role in eliminating bacteria and initiates the common pathway, although the initiation stages in the classical, lectin and alternative complement pathways are diverse. GAS uses several virulence factors to block the activation of complement and prevent complement-mediated killing, including M protein, protein H, C5a peptidase, streptococcus inhibitor of complement (SIC) and SPE B , 17, 18.in vivo infection model [et al. reported that SPE B degrades the assembled membrane attack complex C5b-C9 and complement regulator C1-esterase inhibitor, which contributes to increasing GAS survival in human serum [In our previous study, we found that SPE B could effectively bind and degrade properdin and C3 and led to a decrease of opsonophagocytosis-mediated killing by neutrophils , 20. A son model . Honda-Oan serum . These rIn the present study, we further indicated that SPE B binds to human serum C3 through its C-terminal domain, and the major C3-binding motif is located between residues 376 and 398 in SPE B. Furthermore, immunization with a 23mer synthetic peptide that consisted of residues 376–398 of SPE B protected mice from a lethal GAS invasive infection.The M49 GAS strain NZ131 was a gift from Dr. D. R. Martin, New Zealand Communicable Disease Center, Porirua. Two clinical M1 GAS strains A1 and A20, isolated from culture of blood from patients with necrotizing fasciitis in National Cheng Kung University Hospital, were kindly provided by Dr. J. J. Wu . Cultivation and quantification of bacteria were carried out as previously described .speB gene was amplified by PCR to produce the inactive SPE B point mutant C192S as described previously [BamH1 and Xho1 restriction sites for making the SPE B truncated mutants are listed in BamH1 and Xho1 restriction sites of the pET-42a vector. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) pLyS strains, and the recombinant His-tagged, glutathione S-transferase (GST)-conjugated SPE B mutant proteins were produced by growing the transformants in LB broth containing kanamycin (50 μg/ml) at 37°C. The expression of recombinant proteins was induced by adding of 0.5 mM isopropyl-β-D-thiogalactopyranoside and cells were grown at 37°C for a further 4 h. The cells were harvested by centrifugation and lysed by sonication, the lysate was centrifuged at 12,000 × g for 15 min, and the supernatant was filtered through a 0.45-μm filter. SPE B truncated mutants were purified using Ni2+-chelating column , and the purified recombinant proteins were further examined using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting using rabbit anti-SPE B serum as previously described [146–268 and N-terminally truncated SPE B mutant SPE B269–398 were kindly provided by Dr. W. Huang .The genomic DNA of M1 GAS strain A20 was extracted and the eviously . DNA seqescribed . The C-tThe synthetic peptides PP1 to PP6, which contain the C-terminal region from residues 301 to 398 of SPE B, and SLSpp, which contains amino acid residues 33 to 53 of streptolysin S, were synthesized by Genemed Biotechnologies, Inc. All synthesized peptides were purified using high performance liquid chromatography to a purity greater than 95%. The sequences of the synthetic peptides used for this study are shown in A650 (recombinant SPE B protein)) / (A650 (C192S protein) [The wells of microtiter plate (NUNC) were coated with 50 μl of equal moles of the different SPE B recombinant proteins, including the point mutant C192S and the SPE B truncated mutants at 37°C for 1 h. The plates were washed and then blocked with 200 μl of 1% bovine serum albumin (BSA)/PBS at 4°C overnight. After washing, fifty microliters of PBS-diluted human sera (1:1000) were added to the plates and incubated at 4°C overnight. Unbound serum C3 was washed out by 0.1% Tween-20 in PBS (PBS-T). Anti-human C3 polyclonal antibody  (1:5000) was added and incubated for 1 h at 37°C. After washing, peroxidase-conjugated rabbit anti-goat IgG antibody  (1:10000) was added to the wells for 1h at 37°C. Next, the 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Vector Laboratories) was added, and absorbance values were read at 650 nm. In each assay, the maximal absorbance value of the C192S mutant bound to serum C3 was set as 100%. The relative binding activity of each recombinant protein to serum C3 was calculated as follows: binding activity = 100% × (protein) . In the 345–398 at 37°C for 30 min and these mixtures were then added to the C192S-coated plates and incubated at 4°C overnight. After several washes with 0.1% PBS-T, the anti-human C3 polyclonal antibody (1:5000) and peroxidase-conjugated rabbit anti-goat IgG antibodies (1:10000) were added sequentially, and then developed with TMB substrate at 650 nm as described above. In each assay, the maximal absorbance value of the C192S mutant bound to serum C3 was set as 100%. The percent inhibition of synthetic peptides or SPE B345–398 was calculated as follows: 100% × [1- (A650 (serum incubated with synthetic peptides or SPE B345–398))/ (A650 (serum without incubation with synthetic peptides or SPE B345–398)).Microtiter plates were coated with 1 μg of the SPE B point mutant C192S in 50 μl of coating buffer at 37°C for 1 h. The plates were washed and then blocked with 1% BSA in PBS. The PBS-diluted human sera (1:1000) were incubated with 1 or 10 μM of the control peptides, synthetic peptides PP4, PP5, PP6 or recombinant mutant SPE Bin vitro interaction between the GST-tagged recombinant SPE B mutants and human C3 were assessed using GST pull-down assays. Equal moles of SPE B recombinant proteins, including SPE B146–319, SPE B146–358 and SPE B146–398, were gently mixed with 1:20-diluted human serum in binding buffer  at 4°C for 0.5 h. Then, fifty microliters of glutathione-sepharose beads that were pre-equilibrated in TEE buffer  were added into the serum-SPE B mixtures and gently mixed at 4°C for 2 h. The beads were collected and washed 5 times with TEE buffer containing 1% Triton X-100 and then suspended in 25 μl of 2X SDS loading buffer and boiled for 5 min. The bound complexes were separated by 12% SDS-PAGE and analyzed using Western blotting with goat anti-human C3 antibody.The 8 colony forming units (CFU) of M49 strain S. pyogenes NZ131, M1 clinical strain A1 or M1 clinical strain A20. The infected mice were monitored twice a day, and mice survival rates as well as the tissue lesions were examined for a total of 14 days. During the observation time, the degrees of skin lesion were photographed and measured using the ImageJ software  as previously described [2) inhalation. The air pouch exudates collected from the GAS-infected mice were analyzed using Western-blot analysis for the C3 levels with goat anti-human C3 antibody. During the experimental periods, the mice that manifested clinical signs, including dyspnea, cyanosis, complete anorexia, and 25% weight loss, were humanely sacrificed using CO2 euthanasia to avoid unnecessary suffering. At day 15 post-infection, all the mice were sacrificed by CO2 euthanasia and also received cervical dislocation to ensure death. All procedures, care and handling of the animals was reviewed and approved by the Institutional Animal Care and Use Committee at I-Shou University (IACUC-ISU-99026).Seven- to 8-week old male BALB/cByJNarl mice were used in all experiments, which were purchased from the National Laboratory Animal Center in Taiwan. The mice were maintained on standard laboratory chow and water ad libitum in the animal center at I-Shou University. The synthetic peptides (1 mg/ml) were mixed with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich), and 0.1 ml of the emulsion was inoculated intraperitoneally into the mice under anaesthesia with 5% isoflurane inhalation. The mice then received 3 booster immunizations of the synthetic peptides (25 μg of each was emulsified with Freund’s incomplete adjuvant) at two-week intervals. The sera of the mice were collected under isoflurane anaesthesia, and the titer of the anti-SPE B antibody of each mouse was determined using ELISA, as previously mentioned [Four days after the final booster injection, mouse serum was collected, and the levels of serum creatinine (CRTN) and blood urea nitrogen (BUN) were measured using the Beckman Image System . The results of CRTN and BUN are presented as mg/dl. Skins, kidneys, and hearts from the control and immunized mice were collected for histopathologic analysis. The tissue samples were fixed in 10% neutral buffered formalin solution, embedded in paraffin, sliced into 4-μm-thick sections, and stained with hematoxylin-eosin (H&E).P < 0.05.The survival curves of mice were compared for significance using the log-rank test. Statistical analysis was performed using ANOVA. Differences were considered significant at 146–398 represented a mature form of SPE B (mSPE B) that lacks protease activity; SPE B146–358 and SPE B146–319 lack the C-terminal domain of mSPE B; and SPE B281–358 lacks the C-terminal and N-terminal domains of mSPE B  was inoculated into the air-pouches of synthetic peptide-immunized mice or control mice. The tissues surrounding the air pouch were collected, formalin-fixed, paraffin-embedded at 48 h after GAS infection, and then examined by H&E staining. The H&E stained skin tissues from GAS-infected control mice showed sever necrosis of epidermis, dermis and subcutaneous fat, which did not present in the tissues from the GAS infected synthetic peptide-immunized mice. The numbers of infiltrating cells in inflamed skin tissues of the PP4- or PP6-immunized mice were much higher than tissues from control mice . These results indicate that immunization with PP4 or PP6 was able to protect mice from GAS infection, but could not induce autoimmune-like symptoms.Previous reports suggest that production of SPE B and elevation of anti-SPE B antibody are both involved in the pathogenesis of post-streptococcal glomerulonephritis , 28, 29.8 CFU of A1 or A20, and the survival rates of the PP6-immunized mice elevated to 44% in the A1- and A20-infected groups  isolates from patients with necrotizing fasciitis to evaluate the protective efficiency of immunization with the synthetic peptides. The control mice all died within 4 days after being inoculated with 2 × 10d groups . In contd groups  while prd groups . The skid groups . These rOur previous study shows that SPE B and SLS have a synergistic effect on the pathogenesis of GAS invasive infection . To achiSPE B, an important virulence factor of GAS, cleaves a number of host proteins that are important for the immune response against group A streptococcal infection . Previou146–358 and SPE B146–398 proteins, but not the C-terminal truncated SPE B146–319 mutant, were able to bind with human serum C3 , contains amino acid residues of the glyicne-rich C-terminal loop of SPE B and effectively interferes with the interaction of C3 and SPE B at a concentration of 1μM. This major C3-binding motif (Ala376-Pro398) of SPE B contains these two key residues, Ala376 and Gly385, and play an important role to interfere with the complements activation (346-Gly360) and PP5 (Asn356-Ser375) both contained residues Trp357 and Trp359 of SPE B, which previously reported for substrate binding [Staphylococcus aureus binder of immunoglobulin (Sbi) and Efb homologous protein (Ehp) are the C3-binding proteins produced from S. aureus [346-Gly360) contains Arg350, Val355, and Asn356, which are highly conserved in the C3-binding motifs (RxxxxVN) of Efb, Sbi and Ehp [231 and Asn238, that are important for the Sbi and C3 interaction [350 and Asn356 of SPE B for C3-binding need further investigation.González-Páez and Wolan reported that the residues Alaof SPE B . In thistivation . At a hitivation . Althougtivation 6-Gly360 d PP5 Asn6-Ser375  and Ehp . Analysieraction . However345–398 protein could efficiently block cleavage of immunoglobulins by SPE B and interfere with SPE B-induced inhibition of complement activation. Immunization with SPE B345–398 protected mice from a lethal-dose infection of M49 GAS [346-Gly360 or SPE B Ala376-Pro398, did not induce autoimmune-like symptoms.In our previous study, we found that the recombinant SPE B M49 GAS . In the  M49 GAS , 27, 28.The synthetic peptide PP6 that covers the last 23 C-terminal amino-acids of SPE B, which was distinct from previous identified autoepitope (amino-acid residues 296–310) of SPE B . HoweverS1 Fig(TIF)Click here for additional data file.S2 FigBALB/c mice were immunized with control peptide (A-B), synthetic peptide PP4 (C-D) or PP6 (E-F), and their hearts  and kidney  sections were stained with hematoxylin-eosin.(TIF)Click here for additional data file.(PDF)Click here for additional data file.


Arabidopsis thaliana root meristem where they positively regulate root hair cell development. Here we show that there are three RSL class I protein coding genes in the Brachypodium distachyon genome, BdRSL1, BdRSL2 and BdRSL3, and each is expressed in developing root hair cells after the asymmetric cell division that forms root hair cells and hairless epidermal cells. Expression of BdRSL class I genes is sufficient for root hair cell development: ectopic overexpression of any of the three RSL class I genes induces the development of root hairs in every cell of the root epidermis. Expression of BdRSL class I genes in root hairless Arabidopsis thaliana root hair defective 6 (Atrhd6) Atrsl1 double mutants, devoid of RSL class I function, restores root hair development indicating that the function of these proteins has been conserved. However, neither AtRSL nor BdRSL class I genes is sufficient for root hair development in A. thaliana. These data demonstrate that the spatial pattern of class I RSL activity can account for the pattern of root hair cell differentiation in B. distachyon. However, the spatial pattern of class I RSL activity cannot account for the spatial pattern of root hair cells in A. thaliana. Taken together these data indicate that that the functions of RSL class I proteins have been conserved among most angiosperms—monocots and eudicots—despite the dramatically different patterns of root hair cell development.Genes encoding ROOT HAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix loop helix proteins are expressed in future root hair cells of the  Arabidopsis thaliana. Root hair cells form in discrete longitudinal files in cresses: there are stripes of root hair cells that alternate with stripes of hairless epidermal cells. Root hair cells alternate with hairless epidermal cells in a chessboard pattern in the root epidermis of grasses. We show that the pattern of RSL class I gene expression defines the pattern of root hair cell differentiation in the root epidermis of the grass Brachypodium distachyon but not in the cress Arabidopsis thaliana; ectopic expression of RSL genes can transform every cell into a root hair cell in the grass but not in the cress. Despite these differences in development we also show that the function of RSL class I genes has been conserved since these genes last shared a common ancestor approximately 200 million years ago.Root hairs are tubular extensions that extend from specialized cells in the root surface. They take up nutrients and water from the soil and tether the root to its substrate. The differentiation of root hair cells in the cress family is controlled by a group of regulators called RSL class I transcription factors. The spatial arrangement of root hair cells in grasses is very different from cresses like  Root hairs are filamentous extensions of epidermal cells that extend the absorbing surface of roots into the surrounding soil. They play essential functions in nutrient acquisition and are particularly important for the uptake of nutrients with limited soil mobility such as phosphate ,2. The sA. thaliana is positively regulated by the activity of ROOT HAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix transcription factors in future root hair cells [GL2 is not expressed [GL2 expression is determined by a signaling system, which produces a transcriptionally active complex—containing the WEREWOLF (WER) Myb transcriptional activator—in the future hairless epidermal cell files that promotes GL2 expression and an inactive complex  in the future hair cell files [The differentiation of root hair cells in ir cells . The strxpressed . The spall files –15.A. thaliana RSL class I genes are expressed in future root hair cells located in the meristem [AtRSL4, which is sufficient for root hair elongation; loss of AtRSL4 function results in the development of fewer and shorter root hairs while constitutive expression results in the constitutive elongation of root hair cells [meristem . The expir cells .Brachypodium distachyon this alternating pattern is the result of asymmetric mitoses which form smaller daughters cell that differentiate as a root hair cells, and larger cells that differentiate as hairless epidermal cells [O. sativa. ROOT HAIRLESS1 (OsRHL1) encodes a group XI basic helix loop helix transcription factor that is required for root hair cell development. Plants homozygous for loss of function Osrhl1-1 mutations initiate root hairs but they do not elongate [Lotus japonicus and A. thaliana [OsRHL1 promotes the expression of genes required for the growth or root hairs. B. distachyon TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS RELATED2 (BdTAR2) is required for auxin biosynthesis in the root and for root hair elongation; Bdtar2 loss of function mutants develop shorter root hairs than wild type [The grass (Poaceae) root epidermis comprises files of cells in which hair cells alternate with hairless epidermal cells. In al cells ,17,18. Gelongate . Closelythaliana ,21. It iild type .in A. thaliana we tested the hypothesis that RSL genes positively regulate root hair development in the grass B. distachyon. We show here that class I RSL genes promote root hair development and expression is sufficient for root hair development. This suggests that the function of RSL genes in promoting root hair cell differentiation is conserved among monocots and eudicots,Given the central regulatory role played by RSL class I genes during root hair development B. distachyon we searched for similar genes in the genomes of members of the grass family (Poaceae)   were most closely related to the previously characterized RSL class I genes AtRHD6 and AtRSL1 of A. thaliana. BdRSL1, BdRSL2 and BdRSL3 are respectively 84%, 89% and 73% identical to AtRHD6 in the bHLH-RSL domain  . We discPoaceae) . The top BdRSL3)  and threL domain  but therne pairs . RSL claA. thaliana root meristem and mRNA disappears from cells before root hair initiation. We set out to determine if this expression pattern is conserved in B. distachyon. First, using RT-PCR we detected BdRSL1, BdRSL2 mRNA only in roots while BdRSL3 mRNA transcript was present in the roots and in the shoot apical meristem . Plants lines in which each of the RSL class I genes was overexpressed were identified  B. distachyon RSL class I genes being functionally different from A. thaliana RSL class I genes, or (ii) the presence of an activity that represses root hair cell development in A. thaliana despite the high levels of RSL class I gene activity in the experimental transgenic plants. It was possible to rule out the former because while expression of B. distachyon RSL class I genes in the A. thaliana Atrhd6 Atrsl1 double mutants was sufficient to restore root hair development in root hair cells, their expression was not sufficient for root hair development in the cells in the hairless epidermal cell position [A. thaliana in developing hairless epidermal cells that are missing from B. distachyon. Taken together these data suggest that the mechanism controlling root hair cell differentiation is conserved between B. distachyon and A. thaliana, but the mechanism that controls the spatial patterning of the two cells types in the root epidermis is different between the two species.Despite the conservation of RSL class I function between position . This suB. distachyon and A. thaliana, their expression patterns differ dramatically. BdRSL1, BdRSL2 and BdRSL3 are expressed at or soon after the formative asymmetric cell division that forms the future root hair cells. That is, these genes are expressed in the elongations zone and in root hair cells in the differentiation zone, but there is no expression in the meristem. By contrast, AtRHD6 and AtRSL1 are expressed in the meristem and no expression of these RSL class I genes is detected in the developing root hair cells in the elongation or differentiation zones [A. thaliana, where files of relatively short cells are destined to develop root hairs and relatively long cells are destined to become hairless epidermal cells. Therefore, morphological differentiation is first visible in the meristem in A. thaliana and in the elongation zone of B. distachyon and this mirrors the patterns of RSL class I gene expression in the two species. Consequently we can reconcile the different expression patterns if RSL class I genes are expressed in future hair cells as soon as they become morphologically different from neighbouring future hairless epidermal cells. Since this event occurs in the meristem of A. thaliana, RSL class I genes are expressed in the meristem and since this event occurs after the last cell division in B. distachyon, the genes are in the elongation zone. However this does not explain why the RSL class I genes continue to be expressed during root hair elongation in the grass. It is possible that the continued expression of BdRSL1, BdRSL2 and BdRSL3 in these cells maintains the growth program active.While RSL class I genes positively regulate root hair cell development in both on zones . This diB. distachyon which demonstrates that the mechanism of RSL-regulated root hair differentiation has been conserved between Brassicaceae and Poaceae which last shared a common ancestor some time around 200 million years ago [We conclude that RSL class I genes positively regulate the development of root hair cells in ears ago . We alsoBrachypodium distachyon (line Bd21) caryopses (grain) were sterilized in 10% NaClO and 0.1% Triton X-100 with gentle shaking for 15 minutes, then washed with sterilized water 5 times. Grain were then placed on wet filter paper in a Petri dish sealed with cling film and incubated for 2 days in the dark at 5°C (B. distachyon) to ensure that all grain germinated at the same time. Grain were placed in Petri dishes containing media as in described previously [Arabidopsis thaliana wild type plants of the ecotype Columbia-0 (Col0) were used. Arabidopsis thaliana seeds were sterilized in 5% NaClO for 5 min and washed with sterilized water for 5 times. Seeds were sown and germinated in Petri dishes containing sterilized media as in Ma et al. [eviously  but modia et al.  modifiedhttp://mafft.cbrc.jp/alignment/server/index.html). The bHLH region was then extensively manually aligned in BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Unambiguous aligned positions were used for the subsequent phylogenetic analyses (http://www.atgc-montpellier.fr/phyml/) and thMaximum Likelihood method with the following parameters: SPR &NNI, aLRT SH-like. The constructed tree file was visualized using MEGA [http://www.megasoftware.net/index.html). Logos of amino acid sequence alignments were created with WebLogo (http://weblogo.berkeley.edu/). The multiple alignment to score similarity of amino acid was carried out Mview program (http://www.ebi.ac.uk/Tools/msa/mview/).Protein sequences were pre-aligned using Multiple Alignment using Fast Fourier Transform (MAFFT) , and the ORFs in both O. sativa and B. distachyon genome were identified using a BLAST filtering approach . All predicted gene sequences were searched against all available databases of fully sequenced genomes .Synteny B. distachyon overexpression constructs were based on pVec8- GFP [ZmUBI promoter and Nos terminator to make the pVec8-overexpression vector. For the constitutive expression of RSL class I genes in B. distachyon, the coding sequences of RSL class I were amplified from root cDNA with primer pairs that added a KpnI recognition site at both ends: BdRSL1 F (KpnI): 5’-CGGTACCCTCATGGCAAGCAGGCACG-3’, BdRSL1 R (KpnI): 5’-CGGTACCGCATCATGCATGGCCTAGG-3’ for BdRSL1 (836bp); BdRSL2 F (KpnI): 5’-CGGTACCCTGATCATGGCATTAGTGCG-3’, BdRSL2 R (KpnI): 5’-CGGTACCGGTACGTGTTTCCTTCTAGC-3’ for BdRSL2 (951bp); BdRSL3 F (KpnI): 5’-CGGTACCGCCATGGCTCTAGTGGGTC-3’, BdRSL3 R (KpnI): 5’-CGGTACCGCTATATACTGCTAGCTCC-3’ for BdRSL3 (1157bp) (the KpnI site is underlined). The PCR products were subcloned into the pGEM-T Easy Vector (Promega), and sequenced using SP6 and T7 primers to verify the PCR product. Cloned PCR fragments were digested with KpnI and ligated into KpnI-digested pVec8-overexpression plasmid. Sense orientations were confirmed by sequencing with the forward primer 5′-TGATGGCATATGCAGCAGC-3’ from the ZmUBI promoter.ec8- GFP  (a gift ZmUBI promoter and Nos terminator. RNA silencing constructs carrying the 3’ region of each class I RSL gene were made using the modified pVec8 dsRNAi vector to silence the endogenous class I RSL gene. 50 ng cDNA was used for PCR amplification with each of the following gene specific primer sets (underlines are attB site to create entry clones using the BP recombination reaction) BdRSL1 dsRNA F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAATCAATCCTAGGCCATG, BdRSL1 dsRNA R: GGGGACCACTTTGTACAAGAAAGCTGGGTCAGTGACCAGAACTCCGAAC for BdRSL1 dsRNAi (239bp); BdRSL2 dsRNA F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGGCAATTCAACTGCTCCAG, BdRSL2 dsRNA R: GGGGACCACTTTGTACAAGAAAGCTGGGTCCAGGAGTACTACCTACTAC for BdRSL2 dsRNAi (273bp); BdRSL3 dsRNA F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCAGAAGCCTCGTCAGTG, BdRSL3 dsRNA R: GGGGACCACTTTGTACAAGAAAGCTGGGTCAGTTCGGATGGCAAGGTG for BdRSL3 dsRNAi (207bp) and then PCR product subcloned into the pDONR207 donor vector by a BP clonase reaction (Invitrogen). The final RNA silencing vectors were produced by an LR clonase reaction (Invitrogen) between an entry vector and the modified pVec8 dsRNAi vector. The gateway system (Invitrogen) was used to construct the GFP fusion vector. For the BdRSL class I promoter, the genomic DNA was amplified with each of the following gene specific primer sets (attB site underlined to create entry clones using the BP recombination reaction) and cloned into pDONR207. BdRSL1pro F:GGGGACAAGTTTGTACAAAAAAGCAGGCTTC GTTACTATGGGCTACTGGG, BdRSL1pro R:GGGGACCACTTTGTACAAGAAAGCTGGGTC GAGCTAGGCAGCTATAACAC, for BdRSL1 promoter, BdR2pro F:GGGGACAAGTTTGTACAAAAAAGCAGGCTTC GCAACATGATCGAGGATCC, BdR2pro R:GGGGACCACTTTGTACAAGAAAGCTGGGTC GATCAGTTGATCACGTATCAATC, for BdRSL2 promoter, BdR3pro F:GGGGACAAGTTTGTACAAAAAAGCAGGCTTC CGTATCCGGTGAAAACGAC, BdR3pro R:GGGGACCACTTTGTACAAGAAAGCTGGGTC GGCGGTGGGACTAAGCGG, for BdRSL3 promoter. After sequencing, to confirm the insertion, a GATEWAY LR reaction was then performed with pVEC8-R1-CmR-ccdB-R2:sGFP vector.The dsRNAi site of pANDA vector , containB.distachyon plants were generated using Agrobacterium tumefaciens mediated transformation method as described previously [Transgenic eviously . Immatureviously . The traA. thaliana, BdRSL1, BdRSL2, and BdRLS3 cDNAs were used and subcloned into the pGEM-T Easy Vector (Promega) (see above). cDNA was inserted into the pCAMBIA1300 binary vector. All binary vectors were transferred into Agrobacterium strain GV3101. Arabidopsis plants were transformed using a modified version of the floral dip method [Arabidopsis plants were dipped into the bacterial suspension for 60–90 seconds and covered with a plastic bag for 24 hour to maintain humidity. After removing the bag, plants were returned to normal greenhouse conditions for seed harvesting. Transformants were selected on kanamycin (50 μg/ml).For overexpression in http://rsb.info.nih.gov/ij/) and Adobe Photoshop CS4.Images of root hairs of 5 day old seedling were visualized using a Nikon Eclipse E600 epifluorescence light microscope  with Nikon Plan Apo objectives and bright field optics, and a Leica M165 FC stereo microscope and images captured with a Leica DFC310 FX camera. Images were postprocessed with ImageJ  and reverse transcribed using the Super Script III First-Strand Synthesis System for RT-PCR using oligo dT primer (Invitrogen). Semi quantitative RT-PCR was routinely carried out for 30 to 35 cycles, depending on the linear range of PCR amplification for each gene. Each PCR cycle included incubations at 98°C for 10 s, 58°C for 20 s, and 72°C for 30 s. Specific primers were designed to generate RT-PCR products: BdRSL1 RT: 5’-GAAGAAGCAGTGTGGAGGAAG-3’ and 5’-GACCATGTCAACCTTTGTGCC-3’; BdRSL2 RT: 5’-GACGTTGCAGGAGATGGTG-3’ and 5’-CGAGCACCTTGACTTGCAG-3’; BdRSL3 RT: 5’-CAAGCTGCCAAGAGCCGTC-3’ and 5’-CTGTCTCGAGCACCGTGAG-3’; BdUBI RT: 5’-CAATGTCAAGGCGAAGATCC-3’ and 5’-GGTCTTCACGAAGATCTGC-3’; AtRHD6 RT: 5’-CCTAAATCCGCTGGAAACAA-3’ and 5’-CTCTTCGATTCTTGGCTGCT-3’; AtRSL1 RT: 5’-CCCTAAACTGGCTGGCAATA-3’ and 5’-TCTTGGCTGCTAGGCTTTGT-3’; AtRSL2 RT: 5’-TCCCCAATGGAACAAAGGTC-3’ and 5’-TCTCGGTGAGCTGAGACCAA-3’; AtRSL3: 5’-GGAGCCAGAAATGCGTAGAG-3’ and 5’-GTCTCCACCGTTTGATTCGT-3’; AtRSL4 RT: 5’-GTGCCAAACGGGACAAAAGT-3’ and 5’-TTGTGATGGAACCCCATGTC-3’; AtRSL5 RT: 5’-GCAGGAACTTCACGTAATGGA-3’ and 5’-TATACGCTAGGAAACGAAGAGAAA-3’; EF1α: RT: 5’-GGTGGTGGCATCCATCTTGTTACA-3’ and 5’-TGAGCACGCTCTTCTTGCTTTCA-3’Total RNA was isolated from http://rsb.info.nih.gov/ij/) and Adobe Photoshop CS4.Roots were mounted in 10 ng/ml propidium iodide (Sigma) solution. Confocal images of root epidermal cells were obtained by Leica TCS SP5 confocal laser scanning microscopes using 543-nM and 490-nM laser line for excitation of PI and GFP, respectively. Images were post-processed with ImageJ (http://rsb.info.nih.gov/ij/) and Adobe Photoshop CS4 were used to process the images.For cryo-scanning electron microscopy, seedlings were placed on moist nitrocellulose paper, mounted on a stub and immersed in liquid nitrogen slush. Roots were transferred to a cold stage. After removal of water by sublimation, roots were sputter coated with gold and observed with a Philips XL30 FEG scanning electron microscope equipped with an Oxford Instruments CT1500 HF cryo-stage at -147°C. Digital images were captured with the maximum pixel density. Image J (RSL gene were made by amplifying the full coding sequence including the 5’ UTR and 3’ UTR by PCR using full length cDNA primers (see above). PCR fragments were cloned into pGEM-T Easy Vector (Promega). To generate specific antisense probes, full length cDNA was transcribed from the terminal SP6 or T7 RNA polymerase priming sites of these vectors, then treated DNase and precipitated RNA by ethanol. Full length RNA was hydrolyzed by carbonate buffer  to create 150 bp fragments for hybridization. In vitro transcription, digoxigenin labeling of RNA probes, tissue preparation, and in situ hybridization were carried out as described in Drea et al. [et al. [In situ hybridization was used to determine the cells in which RSL class I mRNA accumulated. Antisense probes for the class I a et al.  and Derb [et al. .S1 Fig(A) RSL sequences from B. distachyon, O. sativa, T. aestivum and H. vulgare (AtIND and OsLAX. (B) Alignment of RSL class I and class II gene sequences used for phylogenetic analysis. vulgare . The lis(TIF)Click here for additional data file.S2 FigBdRSL1pro:sGFP transgene in root hair cells. Scale bar 50 μm.Fluorescence produced from the activity of the (TIF)Click here for additional data file.S3 Fig(A)BdRSL class I cDNAs  were inserted between the ZmUBI promoter and the Nos terminator. Arrows indicate restriction sites and the names of the respective restriction enzymes. (B) Root hairs are longer in lines transformed with ZmUBIpro:BdRSL1, ZmUBIpro:BdRSL2 and ZmUBIpro:BdRSL3 than wild type. Asterisks indicate statistically significant differences between overexpression lines and wild type at **P<0.01 (t-test). Error bar indicates SD.(TIF)Click here for additional data file.S4 FigBdRSL1 and BdRSL2 mRNA are lower in Bdrsl1 Bdrsl2 double knockdown lines than in wild type. Steady state levels of BdRSL2 and BdRSL3 mRNA are lower in Bdrsl1 Bdrsl2 double knockdown lines than in wild type. Error bar is SD.Steady state levels of (TIF)Click here for additional data file.S5 Fig(A) Wild-type (Col-0) A. thaliana transformed with 35S:BdRSL1, 35S:BdRSL2 and 35S:BdRSL3. Scale bar 500 μm. (B) Mean root hair number mm-1 (± SD) in lines transformed with 35S:BdRSL1, 35S:BdRSL2 and 35S:BdRSL3 is indistinguishable from wild-type.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Here we map the silky-feather locus to chromosome 3 by linkage analysis and subsequently fine-map it to an 18.9 kb interval using the identical by descent (IBD) method. Further analysis reveals that a C to G transversion located upstream of the prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) gene is causing silky-feather. All silky-feather birds are homozygous for the G allele. The silky-feather mutation significantly decreases the expression of PDSS2 during feather development in vivo. Consistent with the regulatory effect, the C to G transversion is shown to remarkably reduce PDSS2 promoter activity in vitro. We report a new example of feather structure variation associated with a spontaneous mutation and provide new insight into the PDSS2 function.Silky-feather has been selected and fixed in some breeds due to its unique appearance. This phenotype is caused by a single recessive gene . Silkie chicken from China is one of the breeds showing the fascinating silky-feather phenotype and the breed has been known for hundreds of years. In this study, we mapped the silky-feather locus to an 18.9 kb interval and identified a single nucleotide polymorphism (SNP) completely associated with silky-feather. The causative mutation is located 103 base pairs upstream of the coding sequence of prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2). The expression of the PDSS2 gene is decreased in silky-feather skin during feather development in vivo. The silky-feather allele also reduces the PDSS2 promoter activity in vitro. This is the first report of feather structure variation associated with PDSS2 and provides new insight into molecular signaling in the late development stage of feather morphogenesis.The feather is an excellent model for evolution and development due to its complex structure and vast diversity. Some chickens have silky-feather because of a loss of hooklets in pennaceous feathers, while most chickens have the wild-type normal feather. Hooklets are formed in the last differentiation stage of the life cycle of a pennaceous feather. Chickens with silky-feather are homozygous for a recessive allele (hookless,  The development of feathers, beginning in the embryo and continuing through several cycles of regeneration after hatching, has been one of the most challenging subjects in the field of avian morphology since the early 19Variations in contour feather structure usually occur in the distal barbules part of the vane and disturb the interlocking barbules. The structure variations in domesticated birds have been summarized frizzle feather locus is inherited in an autosomal incomplete dominant manner and caused by the KRT75 gene deletion mutation lace-feathering locus) in domestic pigeons (e.g. Silky Fantail) and Ring-neck Doves is completely different with the silky-feather in chicken lace-feathering locus is controlled by an autosomal gene with incomplete dominance A few variations of feather vane structure are related with hooklets in birds. One variation is the chicken frizzle feather which curves backward and mainly alters rachis structure. The mature frizzle feather structure also exhibits other modifications such as thickening of the barbs and barbules, alteration of the hooklets and other structural abnormalities, however without losing any morphological component et al.et al.Some signaling molecules involved in feather morphogenesis have been studied, and some feather-branching morphogenesis models are proposed involving the expression of sonic hedgehog (SHH), bone morphogenetic protein (BMP), Noggin, etc. PDSS2 gene.In the present study, we show that the silky-feather phenotype is associated with a single regulatory SNP modulating the expression of the silky-feather locus was based on a genome-wide set of 125 microsatellite markers previously used for mapping growth trait QTLs in the CAU resource population (CAURP) comprised of a Silkie x White Plymouth Rock intercross silky-feather locus was mapped to the SF01–SF06 (GGA3:69.28–71.63 Mb) interval on chromosome 3 and showed tight linkage with the SF03 marker (70.41 Mb) with a LOD score of 30.1, consistent with the results in another resource population silky-feather locus to a 380 kb interval between positions 70,201,106–70,581,126 bp (SCML4), Sine oculis-binding protein  and Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2). Refined linkage mapping in another population Our initial linkage analysis to map the 1,126 bp . This insilky-feather locus in the CAURP using the identical-by-descent (IBD) mapping method 0 generation the White Plymouth Rock is fixed for the wild-type allele and the Silkie chicken is fixed for the silky-feather allele. The White Plymouth Rock is not suspected of carrying any silky-feather haplotype as no introgression between these breeds has occurred to the best of our knowledge. The 38 SNP markers genotyped on 12 White Plymouth Rock and 19 Silkie birds were used for the initial IBD mapping. Of the 38 SNP markers, 20 covered the 380-kb  interval without recombination in the linkage analysis   .silky-feather allele and identified 886 polymorphisms in the 70,349,337–70,487,932 bp interval. An estimated 891 bp gap at 70,470,161–70,471,051 bp in the chicken galGal3 genome assembly was re-sequenced successfully in the Red Jungle Fowl BAC  and was determined to be 626 bp (GenBank KC166241).We sequenced one Silkie chicken BAC and one Red Jungle Fowl BAC to identify all sequence polymorphisms associated with the silky-feather haplotype defined in the 70,461,033–70,484,959 bp interval was fixed in all 76 silky-feather chickens  and was not found in the 95 wild-type chickens, indicating that the causative mutation must be located in the 70,460,739–70,504,365 bp region.We genotyped 34 SNP markers within the 70,441,580–70,484,959 bp interval in 12 breeds for further IBD mapping. This breed panel consisted of 76 samples from Silkie, Kuaida Silky and Lanping Silky chickens all with silky-feather, and 95 samples from nine breeds that have the wild-type phenotype. The upstream IBD boundary  was established based on two heterozygous Silkie and one homozygous Lanping Silky chickens . A downs1 heterozygous birds and one F2 silky-feather bird were included. The F2 bird carried an intact Silkie chromosome and a recombined chromosome within the 70,467,968–70,581,126 bp interval that was of Silkie proximal descent and of White Plymouth Rock distal descent. Interestingly, among the nine wild-type birds, one White Leghorn and one White Plymouth Rock had the identical haplotype with silky-feather at 70,472,921–70,484,959 bp  bird showed the same homozygous genotype as silky-feather (h/h) birds. We genotyped ss666793747 in 718 birds from 33 populations and found that all 337 silky-feather chickens were homozygous G/G, 341 wild-type chickens were homozygous C/C and 40 known heterozygous birds were G/C .The candidate mutation ss666793747 is located between the SOBP contains six predicted exons and five introns according to the current annotation of the chicken genome assembly. The coding sequence is well conserved among chicken, human, mouse and other vertebrate species. The human and mouse have a non-coding exon 7. The chicken exon 7 was confirmed by 3′ RACE analysis and the mRNA has been submitted to GenBank with number KC166240. Chicken PDSS2 contains eight exons and the transcript has been submitted to GenBank with number JX982522. To determine whether PDSS2(-103C-G) disrupted the transcription start site (TSS) and the coding sequence of PDSS2, we performed 5′ RACE analysis using dorsal skin from three wild-type and three silky-feather homozygous birds. The most common start site at position −90  downstream of PDSS2(-103C-G) was identical in all the wild-type and silky-feather birds (PDSS2(-103C-G) was found to be present in the 5′ UTR in a few clones  days 60 enotypes . Transcrenotypes  and S6.PDSS2 and SOBP mRNA in dorsal skin tissue at P10, P60, P130 and P200 from two homozygotes. Quantitative RT-PCR analysis showed that the expression of PDSS2 was significantly reduced in silky-feather birds at all stages, but SOBP expression was only decreased at P60 (PDSS2(-103C-G) is a tissue-specific regulatory mutation, the expression analysis was further performed in liver tissue at these four stages. A similar pattern with reduced PDSS2 expression in silky-feather birds at all stages and lower SOBP expression only at P60 was observed  days 9  days 9 .PDSS2 and SOBP expression at P130 in all three genotypes. The expression of PDSS2 in H/h skin was higher than in h/h skin, but showed no difference with H/H skin  is a cis-acting regulatory mutation, consistent with the expression differences in We further compared H/H skin . The exp/h birds . An allefor SOBP . The resSCML4 transcript was detected. Five genes  were differentially expressed between silky-feather and wild-type birds at P10 and two genes (OSTM1 and QRSL1) at P60  rather than long range regulatory effects from PDSS2(-103C-G).We also analyzed the expression in dorsal skin tissue of the other nine genes located in the broad 70,177,177–70,816,125 bp interval. No ) at P60 . The expwith P60 . These iPDSS2 in the skin. An anti-chicken PDSS2 antibody was custom made and its specificity was confirmed by western blotting and immunofluorescence analysis (www.proteinatlas.org/ENSG00000164494/tissue) and mitochondrion according to the UniProt database (www.uniprot.org/uniprot/Q86YH6). We performed immunohistochemical staining with the chicken PDSS2 antibody in both wild-type and silky-feather embryos at day E9, E11, E14 and E17  (30 bp upstream and 30 bp downstream), however, we did not find any significant sequence conservation among birds, reptiles and mammals. Considering the position of PDSS2(-103C-G), we decided to test the promoter effect in “forward” (toward PDSS2) orientation and enhancer effect in “reverse” (toward SOBP) orientation in the pGL3 luciferase vector  and ∼53-fold  whereas the silky-feather vectors increased the activity only ∼8-fold  and ∼9-fold   but not the silky-feather allele We did a BLAST search for the surrounding sequence of e vector . We tranxpressed , and meat, h-SF) . Thus, t in vivo . No enha-feather . The AbdPDSS2 is responsible for silky-feather in chicken based on high-resolution mapping and expression analysis. We mapped the silky-feather locus to a 182 kb region by linkage analysis using two separate mapping populations and then fine mapped it to an 18.9 kb region using IBD mapping. The identification of an overlapping 21.7 kb IBD region in a second population of Silkie chickens is further indication that this region harbours the causal mutation. Within the minimal haplotype region, SNP PDSS2(-103C-G) was completely associated with silky-feather. Of the two flanking genes, PDSS2 showed differential expression in both skin and liver at all stages while SOBP only showed differential expression at P60 and E9. Furthermore, consistent with this finding, the silky-feather allele was associated with reduced promoter activity in a luciferase assay. We conclude that PDSS2(-103C-G) is a cis-acting regulatory mutation that causes altered expression of PDSS2 and is the primary cause for the development of the silky-feather phenotype. We should note that we cannot exclude the possibility that (i) this mutation in the 5′ UTR of PDSS2 can influence gene expression at the level of translation via other mechanisms such as translation efficiency and mRNA stability SOBPSilky-feather is a recessive Mendelian trait found in only a few chicken breeds that dramatically alters the structure and appearance of the juvenile and adult chicken feather. We herein show that a single-base change upstream of silky-feather haplotype from the Silkie. To our knowledge, there is no gene introgression between Jinyang Silky or Lanping Silky and the Silkie breed, and both of these breeds share no other phenotypic characters with Silkie. However, both of them share the 18.9 kb IBD silky-feather haplotype with Silkie. Thus, the same ancestral silky-feather mutation most likely originated before the formation of these breeds.Among silky-feather chicken types, Kuaida Silky chicken was developed by crossing Silkie with Broiler, inheriting the wild-type and silky-feather chickens , is expressed specifically in feather follicles that generate pennaceous barbules but not in follicles that generated plumulaceous barbule BLSK1 might be involved in the formation of pennaceous barbules and hooklets. In silky-feathers, no hooklets form in apical cells of barbules in plumulaceous or pennaceous feathers. The feather follicle forms at the embryonic stage while contour feathers form after the first molting. During development, silky-feathers lack hooklets in pennaceous feathers compared with the wild-type. The expression of PDSS2 was decreased in silky-feather dorsal skin and feathers during all stages  biosynthesis, which is required for mitochondrial respiratory electron transport. In human, missense mutations in PDSS2 were reported to cause Coenzyme Q10 (CoQ10) deficiency with Leigh syndrome with nephropathy 10 decreased to 12% of control cells and ATP synthesis decreased by 51% compared with the control PDSS2 missense mutations and in glomerular podocytes conditional PDSS2 knockout mice, but not in renal tubular epithelium, monocytes, or hepatocytes conditional knockout mice PDSS2 mice, CoQ content in the kidney was significantly lower compared with that in wild-type mice PDSS2 missense mutant mice showed mitochondrial respiratory chain deficiency in all organs studied , another study showed that only the affected kidney organs showed increased reactive oxygen species (ROS) production and oxidative stress PDSS2 dysfunction associated with CoQ deficiency might be responsible for the renal disease phenotype PDSS2 having an important role in CoQ biosynthesis and abnormal PDSS2 expression initiating kidney and nervous system problems in human and mouse, little is known about the effect of PDSS2 mutations in the skin. In this study, we showed that lower expression of PDSS2 in silky-feather chicken skin affected feather development. Mice with keratinocyte-specific deficiency in mitochondrial transcription factor A had an abnormally thick epidermis, lacked hair and showed defects in differentiation PDSS2 locus in the same pedigree, indicating that the silky-feather mutation has no pleiotropic effect on these quantitative traits Despite silky-feather mutation in the feather follicle is independent of the endocrine status. However, some similar but non-genetic silky-feather phenotypes are known. Similar long silky-feathers were first observed in thyroidectomized Brown Leghorn chickens in 1927 Emberiza bruniceps) PDSS2 may decrease the CoQ10 synthesis, which influences the mitochondrial respiratory chain in the feather follicle.Silkie skin transplantation experiments have indicated that the determining factors for hooklet development reside in the feather follicle Protarchaeopteryx showed that remiges and rectrices evolved earlier than flight in theropod dinosaurs AnchiornisPDSS2 contributes to normal hooklet morphogenesis may provide us with a better understanding of the origin and evolution of pennaceous feathers.The evolutionary origin of feathers has been under debate for more than 150 years PDSS2(-103C-G) mutation is presumed to alter the interaction with one or more transcription factors. Regulatory mutations are an important contributor to phenotypic diversity, but it is challenging to establish genotype-phenotype relationships for regulatory mutations and convincingly prove causality cis-regulatory element (CRE) mutations have been found to underlie phenotypic variation in domestic animals and CREs can regulate cell-type-specific expression of genes BCDO2 associated with yellow skin SOX5 with Pea-comb SOX10 underlies the Dark brown plumage phenotype BMP12 causes Naked neck EDN3 causes fibromelanosis MNR2 causes Rose-comb EAV-HP insertion in SLCO1B3 causes Blue-eggshell The All the chickens were fed and handled according to relevant national and international guidelines.0, 19 F1 and 229 F2 individuals that were used for linkage analysis. The F0 generation consisted of 12 White Plymouth Rock chickens and 19 Silkie chickens homozygous for the wild-type and silky-feather alleles, respectively. All 19 F1 birds showed the wild-type phenotype. The observed ratio of 162 wild-type birds and 67 silky-feather birds among the 229 F2 birds, did not deviate significantly from the expected 3∶1 ratio . The USA population used for linkage analysis was derived from a single New Hampshire breed male, homozygous wild-type, and a single Silkie female chicken, homozygous for the silky-feather allele. A single male F1 individual was mated to 10 Silkie females and 180 backcross progeny were used for linkage mapping as previously described The China Agricultural University Resource Population (CAURP) consisted of 31 FThe majority of the DNA samples used for IBD analysis and putative causal mutation genotyping were from Jiangsu Institute of Poultry Science. Additional Silkie samples were from Guangdong and Beijing, China; North Carolina and Wisconsin, USA.Genotyping of the CAURP was performed with 125 microsatellite markers and the Illumina Chicken 60K SNP Beadchip Two BAC clones were resequenced. The RJF BAC CH261-103J18 spanning 70,324,632–70,520,054 bp on chromosome 3 was from BACPAC Resource Center (BPRC), and the Silkie BAC 292C7 spanning 70,349,337–70,487,932 bp was identified by PCR from a Silkie chicken BAC library h/h, and 95 wild-type birds (H/H) representing nine breeds. The samples of Silkie chickens originated from Beijing, Yangzhou and Guangzhou, China. The Kuaida Silky population was generated by crossing Silkie and a commercial Broiler line, followed by selection for Silkie-like appearance and rapid growth for five generations. Lanping Silky originally came from Yunnan, China, and there was no genetic introgression with Silkie as far as we knew. SNPs were genotyped using the Sequenom MassARRAY platform  (ss666793747) and ss666793770 was performed using pyrosequencing. The pyrosequencing PCR assays contained 40 ng gDNA, 1× PCR buffer, 1× Q-solution (Qiagen), 200 µM dNTP, 400 nM of each primer, 2 IU LongAmp Taq (NEB), and H2O was added to achieve a final volume of 25 µl. A touchdown PCR protocol was used, including 94°C for 5 min, 10 cycles of 94°C, 63°C (−1.0°C/cycle), and 65°C for 30 s each, followed by 30 cycles of 94°C, 53°C, and 65°C for 30 s each, and a final extension at 65°C for 5 min. The PCR product was analyzed by 2% agarose gel electrophoresis and used for pyrosequencing according to standard protocol of PyroMark ID (Qiagen).Genotyping of h/h) birds at postnatal (P) days P10 (n = 7), P60 (n = 7), P130 (n = 10), P200 (n = 10) and White Leghorn (H/H) birds at P10 (n = 7), P60 (n = 7), Beijing You (H/H) at P130 (n = 10), Huiyang Bearded (H/H) birds at P200 (n = 10). Heterozygous (H/h) birds at P130 (n = 11) used for allelic expression imbalance originated from a cross between Silkie (h/h) and Youxi Partridge (H/H) chicken. Dorsal skin and liver were collected and stored in liquid nitrogen. Embryonic dorsal skin tissue was collected from Silkie (h/h) and White Leghorn (H/H) birds. Samples from five birds of each genotype were collected at embryonic (E) days 9, 11, 14 and 17. Tissue was homogenized using TissueLyser LT (Qiagen). Total RNA was extracted using TriZol (Ambion). 1 µg total RNA was used in the first strand cDNA synthesis (Promega) with oligo (dT) 18 primers. The details of primer sequence used for RT-PCR were presented in Tissues used for expression analysis were from Silkie (H/H) and three Silkie (h/h) chicken dorsal skin tissues at P100 using Trizol reagent (Ambion). The RNA was treated with DNase I (Qiagen) and purified using RNeasy Mini Kit (Qiagen). 3 µg RNA for each sample was used for reverse transcription in a 25 µl reaction with SuperScript III (Invitrogen), which was incubated at 55°C for 50 min for the high GC content. The original and the nested PCR assays contained 3 µl dC-tailed cDNA (or 1∶100 dilution of primary PCR product), 1× PCR buffer, , 1× Q-solution (Qiagen), 5% DMSO, 400 µM dNTP, 400 nM of each primer, 2.5 IU LongAmp Taq (NEB), H2O was added to give a final volume of 25 µl. A touchdown PCR protocol was used, including 95°C for 3 min, 15 cycles of 95°C for 20 s, 65°C (−1.0°C/cycle) for 30 s, and 65°C for 2 min, followed by 20 cycles of 95°C for 20 s, 50°C for 30 s, and 65°C for 2 min, and a final extension at 65°C for 5 min. The PCR fragments were purified and sub-cloned into the pMD19-T vector. At least 16 clones per sample were selected for sequencing. Primers used for 5′ RACE were presented in The 5′ rapid amplification of cDNA ends (RACE) was performed with the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen). RNA was extracted from three Beijing You , 750 nM of each primer and 250 nM probe in a total volume of 20 µl with a PCR condition of 15 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min. For analysis of SOBP, OSTM1, SEC63, BEND3, C3H6orf203, QRSL1, RTN4IP1, AIM1, and ENSGALT00000037260, 40 ng cDNA, 1× SYBR green mix (Applied Biosystems) and 300 nM of each primer were used in a total volume of 20 µl with a PCR condition of 15 min at 95°C and 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by a melting curve analysis. Expression level data was normalized using GAPDH as endogenous reference gene and calculated using the 2−ΔΔCt method. Student's T-test was used to compare different groups.Quantitative real-time PCR were performed with triplicate on LightCycler 480 (Roche). PDSS2 and SOBP. Total RNA was purified with DNase I (NEB) and minus-RT (not reverse transcribed) PCR products were used as negative controls. Two SNPs in PDSS2 (ss666793773 in exon 8 and ss189596174 in exon 6) and two SNPs in SOBP (ss666793686 and ss666793687 in exon 6) were designed for PCR amplification. The pyrosequencing PCR assays contained 40 ng cDNA (or gDNA), 1× PCR buffer, 200 µM dNTP, 400 nM of each primer, 2 IU LongAmp Taq (NEB), H2O was added to give a final volume of 25 µl. A touchdown PCR protocol was used for the pyrosequencing SNP genotyping test including 94°C for 5 min, 10 cycles of 94°C, 63°C (−1.0°C/cycle), and 65°C for 30 s each, followed by 30 cycles of 94°C, 53°C, and 65°C for 30 s each, and a final extension at 65°C for 5 min. The PCR product was analyzed by 2% agarose gel electrophoresis and used for pyrosequencing according to standard protocol of PyroMark ID (Qiagen). The relative proportion of each allele was obtained using the AQ analysis mode . The details of all primer sequences were given in Pyrosequencing was used to test allelic expression imbalance of 2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g/l of glucose (Gibco) and supplemented with 10% fetal bovine serum (FBS).DF1 (chicken fibroblast cell line) and 293T (human embryonic kidney cell line) cells were cultured at 37°C in a 5% COPDSS2 (cPDSS2) gene by fusion PCR. The eGFP-cPDSS2 was cloned into pcDNA3.1(+) using BamHI and XhoI sites and used for transfection. 293T and DF1 cells were transiently transfected using FuGENE HD Transfection Reagent (Promega) according to the technical manual.The enhanced green fluorescent protein (eGFP) was fused to the N-terminus of the chicken An anti-cPDSS2 monoclonal antibody was custom made in mouse using the immunizing peptide IGISTWKEQV-amide corresponding to amino acid residues 221–230 .Cells were collected at 48 h after transfection. Protein was extracted from cells with Cell Lysis Buffer (Beyotime). The proteins  were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes according to standard protocols. The membranes were blocked and incubated with anti-cPDSS2 (1∶1000), anti-GFP  and anti-alpha tubulin  antibodies overnight at 4°C. The Membranes were subsequently incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1∶10000) and visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).293T and DF1 cells (transient transfection and wild-type control) were cultured on poly-lysine-coated coverslips for 36 h before staining. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100 for 10 min, and blocked for 1 h in blocking solution . Cells were incubated with cPDSS2 antibody (1∶50) overnight at 4°C. Cells were washed three times with PBS and incubated with Alexa Fluor 594 Goat Anti-Mouse IgG Antibody  for 1 h at room temperature. Cells were washed three times with PBS and treated with DAPI (1 µg/mL) for 5 min. The slides were washed with PBS, mounted with Antifade mounting medium (Beyotime) and imaged with the Olympus Fluoview FV1000 confocal microscope. Images were formatted, resized, enhanced and arranged using FV10-ASW and Adobe Photoshop.The embryonic dorsal skin tissues were fixed in 4% paraformaldehyde in PBS for 3 h, followed by 15 min wash with PBS and incubated in 30% sucrose overnight at 4°C. Tissues were frozen in OCT (Sakura) and sectioned to obtain 10 µm thickness. The sections were rehydrated and blocked for 2 h in blocking solution . cPDSS2 antibody was diluted 1∶100 in blocking solution and incubated overnight at 4°C. The sections were washed three times for 5 min in PBS and incubated with Alexa Fluor 488 Goat Anti-Mouse IgG Antibody  for 2 h at room temperature. The nuclei were stained with DAPI. Samples were analyzed using Olympus Fluoview FV1000 confocal microscope with the same parameters. Images were processed using FV10-ASW and Adobe Photoshop.PDSS2(-103C-G) were generated with PCR and cloned into pGL3 Basic vector (Promega), a longer 643 bp fragment  and a shorter 405 bp fragment  in the “forward” (toward PDSS2) orientation. A longer 752 bp fragment  and a shorter 399 bp fragment  in the “reverse” (toward SOBP) orientation were also amplified and cloned into pGL3 Promoter vector (Promega). NheI and XhoI sites were selected to construct the vector. DF1 cells plated on 24 wells were transfected at 70–80% confluency with 720 ng of the pGL3 reporter plasmid and 80 ng of pRL-TK Renilla luciferase construct by 2 µl Lipofectamine 2000 (Invitrogen) for each well. The luciferase activity was measured 23–24 h after transfection using the Dual-Glo Luciferase Assay System (Promega) and an Infinite F200 Luminometer . Ratios of firefly luminescence/Renilla luminescence were calculated, and normalized to control samples (Basic vector). For each test construct, one expression value was the average of three technical replicates in each plate and three separate operations were carried out to represent the final value. The pGL3 Basic vector, pGL3 Promoter vector and pGL3 Control vector were used as control.Two fragments containing http://www.genome.ucsc.edu .Information on the chicken genome sequence is available at The sequence data presented in this paper have been submitted to GenBank with accession numbers KC166240, KC166241, JX982522 and JX982523.Figure S1silky-feather locus to 56.7 kb interval. The genotypes of 38 SNP markers covering about 770 kb interval  in F0 individuals are shown. The silky-feather is mapped to the 380-kb interval  by linkage analysis. H/H represents wild-type homozygotes and h/h represents silky-feather allele homozygotes. Red and Green: two alternative homozygous genotypes. Yellow: heterozygous genotype. White: missing genotype. Homozygosity for the Silkie birds is limited to two short haplotype blocks marked by black thick lines respectively: the proximal 57.4-kb  and the distal 56.7-kb . The distal block is exclusively shared by Silkie birds.IBD mapping in CAURP narrows the (TIF)Click here for additional data file.Figure S2IBD haplotype detected by whole genome sequencing in Silkies from USA. A single extended genomic region fixed for the variant allele at all detected SNPs and representing an IBD haplotype is found in a pool of 15 Silkie chickens from the USA by whole genome sequencing. Variant allele frequency (left y-axis) is indicated in blue and coverage (right y-axis) is indicated in red. The IBD haplotype in the USA Silkies is 21.7 kb  and completely overlaps the 18.9 kb IBD haplotype identified in Silkies from China.(TIF)Click here for additional data file.Figure S3PDSS2 in skin tissue. Figure shows the DNA, mRNA and protein information of part of PDSS2 in skin. Three wild-type (H/H) and three silky-feather (h/h) dorsal skin tissues are used for 5′ RACE analysis as described in PDSS2(-103C-G)  is highlighted in uppercase and indicated by the red arrow. The most common start site  is highlighted in bold and indicated by the black arrow. Most of the 5′ ends are located between position −81 and −96 around the most common position −90. Specifically, the proportions from all the six samples are 86.4% , 72.2% , 84.2% , 100% , 87.5%  and 100% , respectively. Furthermore, PDSS2(-103C-G) is contained in a few clones. Determining the exact transcription start site of PDSS2 with 5′ RACE is technically challenging due to the high GC content (75.8%) in 339-bp sequence of the presumed 5′ UTR and exon 1 at 70,486,623–70,486,961 bp.The 5′ RACE analysis of (TIF)Click here for additional data file.Figure S4PDSS2 and SOBP. RT-PCR analysis of PDSS2 and SOBP expression levels in multiple chicken tissues in (A) H/H and (B) h/h genotypes.Expression pattern of (TIF)Click here for additional data file.Figure S5PDSS2 gene. (A) Schematic structure of the PDSS2 gene and primers used for RT-PCR. (B) RT-PCR is performed with cDNA from dorsal skin and liver tissues of homozygous H/H and h/h individuals.Spliced transcript analysis of (TIF)Click here for additional data file.Figure S6SOBP gene. (A) Schematic structure of the SOBP gene and primers used for RT-PCR. (B) RT-PCR is performed with cDNA from dorsal skin and liver tissues that are from homozygous H/H and h/h individuals.Spliced transcript analysis of (TIF)Click here for additional data file.Figure S7silky-feather mutation in skin. (A) The schematic structure of the flanking genes around silky-feather mutation PDSS2(-103C-G). Relative mRNA expression of (B) OSTM1, (C) SEC63, (D) SCML4, (E) BEND3, (F) C6orf203, (G) QRSL1, (H) RTN4IP1, (I) AIM1 and (J) ENSGALT00000037260 gene in skin. Relative mRNA expression is compared with GAPDH gene. H/H and h/h represent wild-type homozygous and silky-feather homozygous birds separately. Dorsal skin is from postnatal (P) 10, 60, 130 and 200 birds. * indicates p<0.05 and *** indicates p<0.001. The bar represents standard deviation.Relative mRNA expression levels of the other flanking genes around (TIF)Click here for additional data file.Figure S8The chicken PDSS2 antibody is specific to the PDSS2 protein. (A) The chicken amino acid residues used for generating the anti-chicken PDSS2 antibody and its homologous sequence in human. (B) Western blotting analysis demonstrates that both cPDSS2 and GFP antibodies bind to the eGFP-cPDSS2 fusion protein (predicted molecular weight: 68.1 kDa) from the transfected 293T cells. The cPDSS2 antibody also binds to the endogenous PDSS2 protein from the DF1 (predicted molecular weight of cPDSS2: 41.2 kDa) and 293T cells (predicted molecular weight of human PDSS2: 44.1 kDa). 293T-WT, wild-type 293T cells; DF1-WT, wild-type DF1 cells; 293T-Trans, 293T cells transfected with pcDNA-eGFP-cPDSS2. (C–F) Immunofluorescence experiments in 293T and DF1 cells. The eGFP-cPDSS2 fusion protein (green) is fully consistent with cPDSS2 antibody pattern (red), as indicated by arrows in transfected GFP-positive 293T (C) and DF1 (D) cells. The cPDSS2 antibody also recognizes the endogenous cPDSS2 protein with weak signals (red), as indicated by arrowheads in GFP-negative DF1  cells. Red staining indicates that PDSS2 protein is localized in the cytoplasm. Scale bar, 20 µm.(TIF)Click here for additional data file.Table S1SNP marker information used for mapping.(PDF)Click here for additional data file.Table S2silky-feather in different breeds. The adjacent SNPs, whose alleles are fixed in silky-feather birds, are highlighted in yellow.Frequency analysis of (PDF)Click here for additional data file.Table S3SOBP and PDSS2 and genotype distribution. Four SNPs in the exons of SOBP and nine SNPs in the exons of PDSS2 gene were identified, and genotyped in three different populations with known silky-feather genotypes. None of these SNP genotypes was completely associated with silky-feather phenotype, vice versa.Exon SNP information in (PDF)Click here for additional data file.Table S4PDSS2 in different populations. The PDSS2 exon 5 G682A; Glu228Lys mutation (ss666793770) is predicted to cause a gain of MoRF binding (P = 0.026) and a gain of ubiquitination (P = 0.045) silky-feather. The A mutant allele is present in some heterozygous G/A birds with low frequency (freq A = 0.032) and the heterozygous birds show wild-type normal feather. Thus ss666793770 is excluded for further research.Genotype results for ss666793770 of (PDF)Click here for additional data file.Table S5Primer sequences information.(PDF)Click here for additional data file.