RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM–mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

Double-stranded (ds) RNAs and imperfect hairpin RNAs of endogenous genes trigger post-transcriptional gene silencing (PTGS) and are cleaved by a Dicer-like nuclease into small interfering RNAs (siRNAs) and microRNs (miRNAs), respectively. Such small RNAs (siRNAs and miRNAs) then guide an RNA-induced silencing complex (RISC) for sequence-specific RNA degradation. While PTGS serves as an antiviral defense in plants, many plant viruses encode suppressors as a counter defense. Here we demonstrate that the PTGS suppressor (2b) of a severe strain (CM95R) of cucumber mosaic virus (CMV) can bind to in vitro synthesized siRNAs and even to long dsRNAs to a lesser extent. However, the 2b suppressor weakly bound to a miRNA (miR171) duplex in contrast to another small RNA-binding suppressor, p19 of tombusvirus that can effectively bind miRNAs. Because the 2b suppressor of an attenuated strain of CMV (CM95), which differs in a single amino acid from the 2b of CM95R, could barely bind siRNAs, we hypothesized that the weak suppressor activity of the attenuated strain resulted from a loss of the siRNA-binding property of 2b via a single amino acid change. Here we consider that 2b interferes with the PTGS pathway by directly binding siRNAs (or long dsRNA).

Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction.

Petunia hybrida 'Red Star' is a variety whose flowers exhibit a star-type red and white bicolor pattern. We analyzed the mRNA levels of six genes involved in anthocyanin biosynthesis. Only the level of chalcone synthase (CHS) mRNA was depressed in the unpigmented flower sectors. Both transcriptional activity and the accumulation of short interfering RNA of CHS in the unpigmented sectors were detected. Viral infection blocked the generation of CHS-silenced sectors. These results indicate that sequence-specific degradation of CHS RNA is the primary cause of the formation of white sectors in 'Red Star' flowers.

SUMMARYGene silencing through transcriptional repression can be induced by targeting double-stranded RNA (dsRNA) to a gene promoter. It has been reported that a transgene was silenced by targeting dsRNA to the promoter, and the silenced state was inherited to the progeny plant even after removal of the silencing inducer from cells. In contrast, no plant has been produced that harbors silenced endogenous gene after removal of promotertargeting dsRNA. Here, we show that heritable gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using the Cucumber mosaic virus (CMV)-based vector. We found that efficient silencing of endogenous genes depends on the function of the 2b protein encoded in the vector virus, which has the ability to facilitate epigenetic modifications through the transport of short interfering RNA to nucleus. Bisulfite sequencing analyses on the targeted promoter in the virus-infected and its progeny plants revealed that cytosine methylation was found not only at CG or CNG but also at CNN sites. The observed inheritance of asymmetric DNA methylation is quite unique, suggesting that plants have a mechanism to maintain even asymmetric methylation. This CMV-based gene silencing system provides a useful tool to artificially modify DNA methylation in plant genomes and elucidate the mechanism for epigenetic controls.

To explore the electrophysiological properties of the interstitial cells of Cajal (ICCs) and fibroblast-like cells (FLCs), we developed a new preparation by treating the murine small intestine with collagenase. This thin muscle layer preparation contained at least two types of interstitial cells around the enteric nerve bundles, and the cluster of smooth muscle cells displayed a rhythmic contraction. We morphologically identified ICCs and FLCs and conducted patch clamp experiments on each type of cell. The c-kit-positive CD34-negative ICCs showed spontaneous and rhythmic potential fluctuations, and a large transient inward current was evoked by depolarization under voltage clamp conditions. Once the inward current was triggered, it took a regenerative time course and lasted approximately 500 ms. The current was inactivated by continuous depolarization, and by removal of external Ca 2+ . The application of acetylcholine (ACh) prolonged the duration of spontaneous depolarization as well as the depolarization-induced inward current. This inward current showed a reversal potential of around +3 mV and was considered to be due to non-selective cation channels. The c-kit-negative CD34-positive FLCs showed irregular or regular potential fluctuations, and spontaneous outward current was observed under voltage clamp conditions. This outward current showed a reversal potential of around −80 mV and might be classified as a potassium current. We failed to observe major time-and voltage-dependent currents except the above two currents in the interstitial cells.

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Reactive oxygen species (ROS) are proposed to function as diffusible signaling molecules in plant immune response. Rice respiratory burst oxidase homologs (Osrboh genes) are proposed to play a role in ROS generation. We examined a role in rice immune responses of four Osrboh homologs, OsrbohA, OsrbohB, OsrbohD, and OsrbohE. OsrbohA and OsrbohD transcripts were induced after inoculation with an incompatible N1141 strain of Acidovorax avenae, whereas OsrbohaB and OsrbohE mRNA levels did not obviously change even after inoculation with the incompatible strain. We examined the function of the Osrboh genes in ROS generation and in the plant immune response using RNAi-based knockdown in rice cells. OsrbohA and OsrbohE knockdown lines showed that rapid H 2 O 2 generation is caused by OsrbohA, whereas OsrbohE is involved in late H 2 O 2 production during the immune response. Hypersensitive cell death was decreased only in the OsrbohA knockdown line. We further demonstrated that among immune related genes, the induction of EL2 and LOX genes is controlled by ROS generated by OsrbohE, whereas expression of Cht-1 gene is regulated by both OsrbohA and OsrbohE. These results indicate that the ROS molecules generated by OsrbohA and OsrbohE regulate different signaling pathways in the plant immune response. the Arabidopsis genome (Foreman et al. 2003). Of these rboh in Arabidopsis, AtrbohD and AtrbohF were shown to be necessary for ROS generation in the plant immune responses (Torres et al. 2002). Furthermore, NtbohD from Nicotiana tabacum and NbrbohA and NbrbohB from N. benthamiana were required for ROS accumulation and resistance (Simon-Plas et al. 2002;Yoshioka et al. 2003), suggesting that individual isoforms have different functions and participate in multiple distinct signaling pathways.Acidovorax avenae is a Gram-negative bacterium that causes a seedling disease characterized by the formation of brown stripes on the sheaths of infected plants (Kadota et al. 1991). A. avenae can infect a wide range of monocotyledonous plants, including rice, oats, Italian millet, and maize. However, individual strains of the pathogen can infect only one or a few host species (Nishiyama et al. 1979;Kadota et al. 1991;Kadota et al. 1996). We recently reported that several immune responses, such as H 2 O 2 generation, hypersensitive cell death accompanied by clear 180-bp nucleosomal DNA laddering, and immune-related genes were induced when cultured rice cells were inoculated with a riceincompatible strain of A. avenae. These responses were completely abolished when a rice-compatible strain of A. avenae was used as the inoculating pathogen (Che et al. 1999;Tanaka et al. 2001;Iwano et al. 2002;Tanaka et al. 2003;Fujiwara et al. 2004). To identify the specific elicitor related to the induction of these immune responses in cultured rice cells, a strain-specific antibody was raised against the incompatible strain (N1141) and then absorbed by the compatible strain (H8301). The specific antibody detected flagellin protein, a component of t...