Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.

<p>A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a>) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.</p

Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.

<p>A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a> for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.</p

Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.

<p>A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.</p

Yeast Cof1 mutants Cof1-5 and Cof1-22 can depolymerize F-actin with or without tropomyosin bound.

<p>A, B, C) F-actin (10 µM) polymerized without Tpm1p was incubated with 0, 10, 20 µM Cof1, Cof1-5 and Cof1-22 respectively and with 10 µM Tpm1p was incubated with 0, 2.5, 5, 10 and 20 µM Cof1, Cof1-5 and Cof1-22 respectively. The supernatants and pellets after ultracentrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a>) were analyzed on an SDS-PAGE gel. D) The cofilin/actin molar ratios in the pellet from two independent co-sedimentation experiments using 10 µM actin and 10 µM cofilin. Ratios were determined by densitometry of Coomassie-stained SDS-gels shown in (A), (B) and (C).</p

High-scoring binders of Tpm1 affinity purifications identified by MudPIT.

a<p>Proteins were first ranked based on their frequency of detection in the Tmp1 affinity purifications and their absence (or presence at much lower levels based on NSAF values) in the BSA negative controls. Proteins were then ranked based on their NSAF values averaged across the three Tmp1 experiments.</p>b<p>NSAF (normalized spectral abundance factor) values measured in the Urea or KCl eluted samples and negative controls, as well as NSAF values calculated when spectral counts and proteins from all four runs were merged (ALL_NSAF).</p>c<p>All_P, All_sS, and All_uS are respectively the peptide, shared spectral, and unique spectral counts when the four runs were merged, while All_SC is the final sequence coverage.</p

Yeast Cofilin binds Tpm1.

<p>A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#pone.0003641-Moon1" target="_blank">[39]</a>. B) Determination of dissociation constant K_d between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated K_d was 3.34±0.12 µM (mean±SD).</p

Summary of cofilin depolymerization activity toward tropomyosin decorated actin filaments.

<p>Various sources of actin, cofilin and tropomyosin were tested using the co-sedimentation assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#pone-0003641-g002" target="_blank">Figure 2</a> legend. ‘+’ refers to cofilin co-sedimenting with F-actin without causing depolymerization; ‘+ +’, ‘+ + +’ and ‘+ + + +’ refer to weak, moderate and strong, respectively, depolymerization of F-actin by cofilin.</p

Table_5_Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.XLSX

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem–loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA–gene axis for further investigation.</p

Data_Sheet_1_Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.PDF

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem–loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA–gene axis for further investigation.</p

Table1_CircRNA-regulated immune responses of asian honey bee workers to microsporidian infection.XLSX

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana workers’ midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 days (AcT1) and 10 days (AcT2) post inoculation days post-inoculation with N. ceranae. PCR amplification result verified the back-splicing sites within three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691, and 3723 respectively. Further, 83 52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group. Source genes of DEcircRNAs in workers’ midgut at seven dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA (ceRNA) network analysis showed that 23 13) DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group could target 18 14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 164) internal ribosome entry site and 54 26) ORFs were identified from DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at seven dpi with N. ceranae were associated with several cellular immune pathways including endocytosis and ubiquitin-mediated proteolysis. Ultimately, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers’ midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.</p