Post-transcriptional Gene Silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation

A multiplex microsatellite marker kit for diversity assessment of large cassava (Manihot esculenta Crantz) germplasm collections

Current methods for molecular fingerprinting of cassava (Manihot esculenta Crantz) have limited throughput or are costly, thus preventing the characterization of large germplasm collections such as those held by the International Agricultural Research Centers or National Research Institutions, which comprise hundreds to thousands of accessions. Here, we report the development of a fluorescence-based multiplex simple sequence repeat (SSR) marker kit that enables accurate and cost-effective cassava fingerprinting. The kit comprises 16 SSR markers assembled into five multiplex panels and was tested on 21 cassava cultivars alongside one accession of Manihot epruinosa, a wild relative. A total of 68 alleles were detected with, on average, 4.25 alleles per locus and a polymorphism information content of 0.53. The marker kit reported here is comparable to previously published amplified fragment length polymorphism and SSR marker systems in terms of discriminating power and informativeness while offering significant advantages in speed and cost of marker analysis. Previous molecular genetic diversity studies have suggested that cassava germplasm collections contain duplicate entries based on the occurrence of identical genetic profiles. Using the newly developed microsatellite kit, three out of six putative duplicate accessions could be readily differentiated, showing that these are distinct genotypes. The relevance of these findings with respect to the characterization and management of large cassava germplasm collections is discussed

RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires MetI for maintenance

BACKGROUND: The association between DNA methylation and gene silencing has long been recognized; however, signals that initiate de novo methylation are largely unknown. In plants, recognition of RNAs that are inducers of posttranscriptional gene silencing (PTGS) can result in sequence-specific DNA methylation, and the aim of this work was to investigate whether heritable epigenetic changes can occur by this mechanism and if the Met1 methyltransferase is required. RESULTS: RNA-directed DNA methylation (RdDM) was initiated in 35S-GFP transgenic plants following infection with plant RNA viruses modified to carry portions of either the 35S promoter or the GFP coding region. Targeting of the promoter sequence resulted in both methylation and transcriptional gene silencing (TGS) that was inherited independently of the RNA trigger. Targeting the coding region also resulted in methylation; however, this was not inherited. Expression of Met1 was suppressed in order to investigate its role in initiation and maintenance of RdDM. Initiation of RdDM was found to be Met1-independent, whereas maintenance of methylation and TGS in the subsequent generations in the absence of the RNA trigger was Met1-dependent. Maintenance of methylation associated with systemic PTGS was also found to be Met1-independent. CONCLUSIONS: RNA-triggered events can lead to heritable changes in gene expression, and it is possible that initiation of other epigenetic phenomena such as trans-silencing and paramutation may have an RNA component

De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus.

The relationship between co-suppression and DNA methylation was explored in transgenic plants showing inducible co-suppression following infection with a cytoplasmically replicating RNA virus. Induction resulted in a loss of transgene mRNA and resistance to further infection, factors typical of post-transcriptional gene silencing. In infected plants, de novo methylation of the transgene appeared to precede the onset of resistance and only occurred in plants where the outcome was co-suppression. The methylation was limited to sequences homologous to the viral RNA and occurred at both symmetric and non-symmetric sites on the DNA. Although methylation is predicted to occur in mitotic cells, the virus was found not to access the meristem. A diffusible sequence-specific signal may account for the epigenetic changes in those tissues