Additional file 1 of Conserved methylation signatures associate with the tumor immune microenvironment and immunotherapy response

Additional file 1: Fig S1. Distribution of DMPs’ median-∆β values between tumor and normal tissues at the pan-cancer level. Fig S2. Top 10 significant Hyper-DMPs across 9 cancer types. Fig S3. Top 10 significant Hypo-DMPs across 9 cancer types. Fig S4. Identification of conserved differentially methylated probes at the pan-cancer level. Fig S5. NMF identifies three hypermethylation signatures and seven hypomethylation signatures. Fig S6. Comparison of Hypo-MSs using β or 1-β values as input. Fig S7. Characterization of DNA methylation signatures. Fig S8. Methylation signature activities’ association with age. Fig S9. Analysis of the correlations between overall survival, cancer stages and methylation signature activities. Fig S10. The relationship between methylation signature activities and tumor immune microenvironment in cancers. Fig S11. The relationship between Tumor mutation burden, neoantigen load, tumor progression and Hypo-MS4 activity. Fig S12. Analysis of the correlations between deterministic genes and Hypo-MS4 activity. Fig S13. Analysis of the intersection of deterministic genes and Hypo-MS4 activity. Fig S14. Analysis of overall survival and ICI response of Hypo-MS4 with deterministic genes status

Enhanced High-Temperature Cyclic Stability of Al-Doped Manganese Dioxide and Morphology Evolution Study Through in situ NMR under High Magnetic Field

In this work, Al-doped MnO₂ (Al-MO) nanoparticles have been synthesized by a simple chemical method with the aim to enhance cycling stability. At room temperature and 50 °C, the specific capacitances of Al-MO are well-maintained after 10 000 cycles. Compared with pure MnO₂ nanospheres (180.6 F g^–1 at 1 A g^–1), Al-MO also delivers an enhanced specific capacitance of 264.6 F g^–1 at 1 A g^–1. During the cycling test, Al-MO exhibited relatively stable structure initially and transformed to needlelike structures finally both at room temperature and high temperature. In order to reveal the morphology evolution process, in situ NMR under high magnetic field has been carried out to probe the dynamics of structural properties. The ²³Na spectra and the SEM observation suggest that the morphology evolution may follow pulverization/reassembling process. The Na⁺ intercalation/deintercalation induced pulverization, leading to the formation of tiny MnO₂ nanoparticles. After that, the pulverized tiny nanoparticles reassembled into new structures. In Al-MO electrodes, doping of Al³⁺ could slow down this structure evolution process, resulting in a better electrochemical stability. This work deepens the understanding on the structural changes in faradic reaction of pseudocapacitive materials. It is also important for the practical applications of MnO₂-based supercapacitors

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development

<div><p>The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that <i>Rice dwarf virus</i> (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of <i>OsIAA10</i> enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.</p></div

The RDV P2 protein stabilizes OsIAA10 by inhibiting OsIAA10/OsTIR1 interaction.

<p>(A) Interaction between MBP-OsIAA10 and HA-OsTIR1 is disrupted by MBP-P2 (1–786). MBP-OsIAA10 protein combined with MBP-P2(1–786) or MBP was incubated with immobilized HA-OsTIR1. The immunoprecipitated fractions were detected by anti-MBP antibody. HA-OsTIR1 input is shown in the lower panel. (B) <i>In vitro</i> interaction between GST-OsIAA10 and MBP-OsTIR1 is weakened by GST-P2 (1–786) in a dose dependent manner, revealed by pull-down. GST-OsIAA10 protein combined with GST-P2(1–786) or GST was incubated with immobilized MBP-OsTIR1. The immunoprecipitated fractions were detected by anti-GST antibody. The gradient indicates increasing amount of GST-P2(1–786). MBP-OsTIR1 input is shown in the lower panel. (C) P2 affects dynamic association between OsTIR1 and OsIAA10. Data were collected from microscale thermophoresis (MST) assays as described in Materials and Methods. Experiments repeat for three times and Error bars indicate SD. Fnorm, normalized fluorescence. (D and E) Cell-free degradation assay of MBP-OsIAA10 in mock- or RDV- infected rice extracts (D) or <i>N</i>. <i>benthamiana</i> leaf extracts (E). Mock in (D) indicates healthy rice extracts; Mock in (E), extracts of leaves infiltrated with <i>pWM101</i> vector as a negative control; HA-P2, extracts of leaves infiltrated with <i>pWM101-HAS2</i> that express HA-P2. Rubisco large protein (RuL) was used as a loading control of total plant protein. On the right was a normalized plot for the degradation of MBP-OsIAA10 of the left. The details for quantification and normalization are described in Materials and Methods. Error bars indicate SD. (F) Western blot showing OsIAA10 protein levels in mock and RDV-infected WT rice plants. Actin was used as a loading control. And the Histogram underneath represents the relative protein level. Experiments repeat for three times and Error bars indicate SD. Significant differences were indicated (**<i>P</i><0.01) based on Student’s <i>t</i>-test. (G) qPCR showing <i>OsIAA10</i> transcript levels, respectively, in mock and RDV-infected WT rice plants. <i>OsEF1a</i> was used as the reference. Values are mean ± SD (n = 3 biological replicates). n.s. indicates no significant difference based on Student’s <i>t</i>-test. (H) Effects of P2 and P2Δ(1–90) on the accumulation of OsIAA10 in <i>N</i>. <i>benthamiana</i> after auxin treatment. The three upper panels show protein levels on Western blots and the three lower panels show mRNA levels revealed by RT-PCR. GusA was expressed and loaded as a reference control.</p

OsIAA10 accumulation enhances RDV pathogenicity.

<p>(A) Phenotypes of RDV-infected WT, L12, L20, M7, and M9 rice plants. Photos were taken 4 weeks after RDV inoculation. The areas of white specks on the leaves represent the degree of disease symptoms. Scale bars: 10 cm (upper panel) and 1 cm (lower panel). (B) Schematic representation of plant height for the plants in (A). The average (±SD) values were obtained from three biological repeats, with 15 plants from each line in every repeat. Different letters indicate significant differences (<i>p</i> < 0.05) based on the Tukey-Kramer HSD test. (C) qRT-PCR assay showing the relative expression level of <i>OsIAA10</i> and RDV RNAs (<i>S2</i> and <i>S11</i>) in plants in (A). The expression levels were normalized using the signal from <i>OsEF1a</i>, and values are mean ± SD (n = 3 biological replicates). Different letters indicate significant differences (<i>p</i> < 0.05) based on the Tukey-Kramer HSD test. (D and E) Northern (D) and Western (E) blots showing the accumulation of RDV RNAs and proteins in RDV-infected WT, L12, L20, M7 and M9 rice lines. rRNAs were used as a loading control for RNA and Actin was used as a loading control for proteins. (F) Time course of symptomatic plants (%) of WT, L12, L20, M7 and M9 from one week-post-inoculation (wpi) to 8 wpi. Inoculation assays were repeated three times, respectively. The error bars indicate SD. L12 and L20 are transgenic rice lines overexpressing OsIAA10; M7 and M9 are transgenic rice lines overexpressing OsIAA10P116L.</p

OsIAA10P116L-overexpressing transgenic rice plants phenocopy RDV-infected rice plants.

<p>(A) Morphologies of mock-inoculated M7 and M9 as well as WT-RDV plants at maturity stage. Bar: 15 cm. (B) Schematic representation of the tiller number, plant height, grain number per panicle, and rate of avoltive grain (%) for the above plants at maturity stage. The average (±SD) values were obtained from three biological repeats, with 15 plants from each line in every repeat. Significant differences were indicated (*<i>P</i><0.05, **<i>P</i><0.01) based on Student’s <i>t</i>-test. (C and D) Phenotypes (C) and schematic representation (D) of the length of the crown roots of 6-week-old mock-inoculated WT and OsIAA10P116L-overexpressing (M7 and M9), as well as RDV-infected WT, rice plants. Bar: 10 cm. The average (±SD) values were obtained from three biological repeats, with 15 plants from each line in every repeat. Significant differences were indicated (**<i>P</i><0.01) based on Student’s <i>t</i>-test. (E) Lengths of crown roots of 6-week-old mock-inoculated WT, M7 and M9 as well as RDV-infected WT rice seedlings cultured in a liquid nutrient containing the indicated concentration of NAA. The average (±SD) values were obtained from three biological repeats, with 15 plants from each line in every repeat. (F) qPCR analysis of auxin-induced gene expression after IAA treatment in mock-inoculated WT and M7 as well as RDV-infected WT rice seedlings. The expression levels were normalized using the signal from <i>OsEF1a</i>, and values are mean ± SD (n = 3 biological replicates).</p

The RDV P2 protein interacts with OsIAA10.

<p>(A) RDV P2 interacts with OsIAA10 in yeast. Yeast transformants were spotted on the control medium (SD-Leu/-Trp (SD-L-W)) and selection medium (SD-Leu/-Trp/-His/-Ade (SD-L-W-H-Ade)). AD, activating domain; BD, binding domain; SD, synthetic dropout. (B) Co-immunoprecipitation confirms the interaction between P2 and OsIAA10 in RDV-infected FLAG-OsIAA10 overexpressing (OsIAA10-OE) rice. WT, wild type rice; Mock, mock-inoculated rice; RDV, RDV-infected rice. (C) LCI assay shows interaction between P2 and OsIAA10 <i>in vivo</i>. The left diagram indicates the leaf panels that were infiltrated with <i>A</i>. <i>tumefaciens</i> containing the different combinations of indicated constructs. Cps indicates signal counts per second. (D) P2 interacts with domain II of OsIAA10. (E) Determination of the functional domains of P2 that interact with OsIAA10. The prey protein AD-OsIAA10 was expressed with the indicated bait proteins in yeast AH109 cells. Interaction was indicated by the ability of cells to grow on medium SD-L-W-H-Ade.</p

RDV infection disturbs auxin pathway in rice.

<p>(A and B) Aboveground (A) and root (B) phenotypes of mock- and RDV- infected rice plants at 6-week-old seedling stage. Bars: 10 cm. (C and D) Schematic representation of the tiller number and crown roots length of mock- and RDV- infected rice plants in (A) and (B). The average (± standard deviation (SD)) values were obtained from three biological repeats, with 15 plants from each line in every repeat. Significant differences were indicated (*<i>P</i><0.05, **<i>P</i><0.01) based on Student’s <i>t</i>-test. (E) Relative average expression (log2) of auxin-induced genes in RDV-infected rice plants. Data were obtained from qPCR assays and analyzed using 2^-ΔΔC(t) method and the <i>OsEF1a</i> mRNA levels were used as internal controls. Values are mean ± SD (n = 3 biological replicates). Columns with asterisks are statistically different according to Student’s <i>t</i>-test (*<i>P</i><0.05, **<i>P</i><0.01) as compared to their expression in mock-inoculated rice plants. (F and G) RDV-infected rice plants exhibit reduced sensitivity to auxin treatment. Phenotypes (F) and lengths (G) of crown roots of mock- and RDV- infected 4-week-old seedlings cultured in liquid nutrition containing 0 or 0.1 μM NAA for 10 days. Bar: 10 cm. The average (± SD) values were from three biological repeats with 15 plants for each line every repeat. Significant differences were indicated (n.s., no significant, **<i>P</i><0.01) based on Student’s <i>t</i>-test.</p

NS3 interacts with OsDRB1, a pri-miRNA processing factor.

<p>The BiFC assay was conducted in <i>N</i>. <i>benthamiana</i> epidermal cells, mCherry is a nuclear localization marker fused with red florescent protein (RFP). (A) Results of a BiFC assay showing the interaction between NS3 and OsDRB1a. Scale bar = 0.1 μm. (B) Results of a co-immunoprecipitation analysis showing the interaction between NS3 and OsDRB1a. (C) The accuracy of mutation sites of mOsDRB1a. (D) Results of a BiFC assay showing the accuracy of interaction sites between OsDRB1a. Scale bar = 0.1 μm (E) Formation of OsDRB1a dimers <i>in vivo</i>. Total rice protein extracts from the WT and <i>OsDRB1</i>-knockdown lines were treated with ‘‘native” buffer and detected using anti-HYL1antibodies. (F) Results of a BiFC assay showing the accuracy of interaction sites between NS3 and DRB1a. Scale bar = 0.1 μm.</p

Expression of NS3 promotes the accumulation of several miRNAs and reduces the expression of their targets.

<p>(A) Mutation site of the mutant NS3 (mNS3). (B) Measurement of miRNA (miR168, 395, 398, 399, and 528) accumulation in the WT, <i>NS3</i> OX#1, and <i>mNS3</i> OX#1 rice lines by small RNA sequencing. (C) Detection of miRNA (miR168, 395, 398, 399, and 528) accumulations in mock-infected, RSV-infected (RSV), <i>NS3</i> OX#1, <i>NS3</i> OX#7, <i>mNS3</i> OX#1, and <i>mNS3</i> OX#4 rice plants by northern blotting. U6 served as a loading control, the expression levels in the WT-Mock plants are set to a value of 1.0 and the expression levels of in the other plants are relative to this reference value. (D) Relative expression levels of the target genes of the miRNAs (miR168, 395, 398, 399, and 528), including <i>OsAGO1a</i>, <i>OsSULTR2;1</i>, <i>OsCSD1</i>, <i>Os08g45000</i>, and <i>OsRFPH2-10</i> in the mock-infected, RSV-infected (RSV), <i>NS3</i> OX#1, <i>NS3</i> OX#7, <i>mNS3</i> OX#1, and <i>mNS3</i> OX#4 rice plants. (E) Relative expression levels of the miRNA (miR168, 395, 398, 399, and 528) precursors, including <i>pri-miR168a</i>, <i>pri-miR395d</i>, <i>pri-miR398a</i>, <i>pri-miR399b</i>, and <i>pri-miR528</i> in the mock-infected, RSV-infected (RSV), <i>NS3</i> OX#1, <i>NS3</i> OX#7, <i>mNS3</i>OX#1, and <i>mNS3</i> OX#4 rice plants. Average (± SD) values based on RT-qPCR analysis of three biological replicates are shown. ***, <i>P</i> ≤ 0.001; **, <i>P</i> ≤ 0.01; *, <i>P</i> ≤ 0.05.</p