Relationship between Symptoms and Gene Expression Induced by the Infection of Three Strains of Rice dwarf virus

BACKGROUND: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which often results in severe yield losses of rice in East Asian countries. The disease symptoms are stunted growth, chlorotic specks on leaves, and delayed and incomplete panicle exsertion. Three RDV strains, O, D84, and S, were reported. RDV-S causes the most severe symptoms, whereas RDV-O causes the mildest. Twenty amino acid substitutions were found in 10 of 12 virus proteins among three RDV strains. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the gene expression of rice in response to infection with the three RDV strains using a 60-mer oligonucleotide microarray to examine the relationship between symptom severity and gene responses. The number of differentially expressed genes (DEGs) upon the infection of RDV-O, -D84, and -S was 1985, 3782, and 6726, respectively, showing a correlation between the number of DEGs and symptom severity. Many DEGs were related to defense, stress response, and development and morphogenesis processes. For defense and stress response processes, gene silencing-related genes were activated by RDV infection and the degree of activation was similar among plants infected with the three RDV strains. Genes for hormone-regulated defense systems were also activated by RDV infection, and the degree of activation seemed to be correlated with the concentration of RDV in plants. Some development and morphogenesis processes were suppressed by RDV infection, but the degree of suppression was not correlated well with the RDV concentration. CONCLUSIONS/SIGNIFICANCE: Gene responses to RDV infection were regulated differently depending on the gene groups regulated and the strains infecting. It seems that symptom severity is associated with the degree of gene response in defense-related and development- and morphogenesis-related processes. The titer levels of RDV in plants and the amino acid substitutions in RDV proteins could be involved in regulating such gene responses

Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4

Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4's Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro

Arabidopsis C-Terminal Domain Phosphatase-Like 1 Functions in miRNA Accumulation and DNA Methylation

<div><p>Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that <i>cpl1</i> was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of <i>ros1</i> suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, <i>cpl1</i> reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.</p></div

The <i>cpl1</i> mutation influences DNA methylation.

<p>(A) Bisulfite sequencing results of endogenous <i>RD29A</i> and <i>RD29A-LUC</i> transgene promoters. The ratio of cytosine methylation in percentage was determined at CG, CHG, and CHH sites on endogenous (left) and transgenic (right) <i>RD29A</i> promoters. H represents A, T, or C. (B) PCR-based cytosine methylation assay on RdDM target loci using methylation-sensitive enzymes. The amplifications using undigested DNA templates (-) were used as controls.</p

CPL1 interacts with HYL1-SE complex in nucleus.

<p>(A) Immunoprecipitation with (+) or without (−) anti-HYL1 was performed using a crude extract of calli containing <i>gCPL1-FLAG</i> transgene. CPL1-FLAG was detected by immunoblot using anti-FLAG1 antibody. (B) Immunoprecipitation with (+) or without (−) anti-FLAG was performed using a crude extract of calli containing <i>gCPL1-FLAG</i> transgene. HYL1 was detected by immunoblot using anti-HYL1 antibody. (C) BiFC visualization of CPL1-HYL1 interaction. Epifluorescence (YFP) and bright field images of protoplasts that were transfected with plasmids encoding nYFP-CPL1 and cYFP-HYL1 fusion proteins and NLS-RFP. NLS-RFP was used as a positive control for nuclear localization. Yellow signals on merged images indicate co-localization of YFP and nuclear-localized RFP proteins. Scale bars indicate 10 µm. (D) Luminescence images of <i>N. benthamiana</i> leaves infiltrated with NLUC-HYL1 (top panel) or NLUC-SE (bottom panel) with CLUC-CPL1 fragments. LUC images were obtained 3 days after infiltration. MYB75, CLUC-MYB75 used as a negative control. (E) Yeast two-hybrid assay. Growth of PJ69-4A co-expressing GAL4 DNA binding domain (BD) fused with CPL1 (BD_CPL1) and GAL4 activation domain (AD) fused with HYL1 and SE (AD_HYL1 and AD_SE). Cells were grown on synthetic dropout (SD) media lacking uracil and leucine (-UL) or SD medium lacking uracil, leucine, histidine and adenine (-ULHA). 2×10⁵ cells were used for (1) and diluted 10-fold for (1/10). Photographs were taken after incubation at 28°C for 48 hours. ADv and BDv indicate vector controls.</p