Pie charts of sRNA distribution for four datasets.

<p>All sRNAs are annotated to a specific category, including miRNA, siRNA, exon_sense, intron_sense, rRNA, repeat sequence, tRNA, unannotated sRNA and so on.</p

Alterations in SiRNA and MiRNA Expression Profiles Detected by Deep Sequencing of Transgenic Rice with SiRNA-Mediated Viral Resistance

<div><p>RNA-mediated gene silencing has been demonstrated to serve as a defensive mechanism against viral pathogens by plants. It is known that specifically expressed endogenous siRNAs and miRNAs are involved in the self-defense process during viral infection. However, research has been rarely devoted to the endogenous siRNA and miRNA expression changes under viral infection if the resistance has already been genetically engineered in plants. Aiming to gain a deeper understanding of the RNA-mediated gene silencing defense process in plants, the expression profiles of siRNAs and miRNAs before and after viral infection in both wild type and transgenic anti-<i>Rice stripe virus</i> (RSV) rice plants were examined by small RNA high-throughput sequencing. Our research confirms that the newly generated siRNAs, which are derived from the engineered inverted repeat construct, is the major contributor of the viral resistance in rice. Further analysis suggests the accuracy of siRNA biogenesis might be affected when siRNAs machinery is excessively used in the transgenic plants. In addition, the expression levels of many known miRNAs are dramatically changed due to RSV infection on both wild type and transgenic rice plants, indicating potential function of those miRNAs involved in plant-virus interacting process.</p></div

Identification of siRNA origin by mapping different miRNA species to rice genome and RSVCP sequence.

<p>A. Classification of siRNA among all four libraries when m = 0. Based on its origin, siRNA are classified into three groups, unmapped siRNA, rice derived siRNA and RSVCP derived siRNA. B. Un-mapped sequences analysis using GSNAP with different parameter settings among four datasets.</p

Sequences and restriction sites of PCR primers.

<p>Lowercase indicates a restriction enzyme site.</p

RT-PCR analysis and yeast two-hybrid screen of five virus-binding proteins.

<p>(A) Electrophoresis of gene amplified products was in 1% agarose gel. DNA markers were indicated on the left and genes on the top. (B) Yeast two-hybrid assay of protein-protein interactions between RSV-NCP and five virus-binding proteins (GAPDH3, RACK, RPL8, RPL5 and RPL7a). Yeast cotransformants were incubated on the selective medium (SD/–Ade/–His/–Leu/–Trp plus X-α-Gal) at 28°C for 4 d. 1: positive control; 2: negative control; 3: GAPDH3 & RSV-NCP; 4: RACK & RSV-NCP; 5: RPL8 & RSV-NCP; 6: RPL5 & RSV-NCP; 7: RPL7a & RSV-NCP.</p

The venn diagram for the shared novel miRNAs among four libraries.

<p>The venn diagram for the shared novel miRNAs among four libraries.</p

SDS-PAGE analysis and binding experiments with RSV particles of expression products of virus-binding proteins.

<p>(A) Expression products were separated by 12% SDS-PAGE. (B) Binding experiments with purified RSV particles by far-Western blot. (C) Binding experiments with RSV particles by DIBA. Lane 1, 8: relative molecular weight markers; lane 2, 9, 16: pET32a vector; lane 3, 10, 17: pET-GAPDH3; lane 4, 11, 18: pET-RACK; lane 5, 12, 19: pET-RPL8; lane 6, 13, 20: pET-RPL5; lane 7, 14, 21: pET-RPL7a; lane 15: <i>E. coli</i> BL21 (wt).</p

Identification of <i>L. striatellus</i> proteins using Nano LC-ESI-CID-MS/MS.

<p>Selected protein spots were subjected to in-gel trypsin digestion. Protein fragments were then analyzed by LC-MS/MS on an LTQ Orbitrap linear ion trap mass spectrometer connected with an Agilent 1100 HPLC system. Data analyses were done with Bioworks Browser software and Mascot against NCBInr database. The score is significant if it is higher than 39.</p

Characterization of purified RSV particles and total proteins from non-viruliferous <i>L. striatellus</i>.

<p>(A) Purified RSV particles. (B) Separation of <i>L. striatellus</i> proteins by 12% SDS-PAGE. (C) Separation of <i>L. striatellus</i> proteins by 2-DE. Relative molecular weight markers (M_w) were indicated on the left and pI ladder on the top.</p

Binding experiment of <i>L. striatellus</i> proteins with purified RSV particles.

<p>(A) Binding experiment. M_w was indicated on the left and pI ladder on the top. (B) Selected area of 2-DE gels showing <i>L. striatellus</i> proteins that bound in vitro to RSV particles. 2D spots indicated by arrows were selected for the LC-MS-MS analysis.</p