New approaches for the analysis of Arabidopsis thaliana small RNAs

We are interested in the study of small RNAs (sRNAs) that over-accumulate during the plant hypersensitive response (HR), a form of programmed cell death that occurs in and around the site of infection when plants are challenged by pathogens. For this purpose, we have constructed, by subtractive hybridization, a cDNA library of Arabidopsis sRNAs that are enriched during HR. Sequencing of randomly chosen clones provided evidence for the specific accumulation of several microRNAs as well as previously unidentified sRNAs. In a second approach, we have tested the possibility to hybridize labelled cDNAs derived from sRNAs to a DNA tiling array. We could reproducibly hybridize a custom-made tiling array covering Arabidopsis chromosome 4, with small cDNAs as targets. Furthermore, we have found that the distribution of hybridized fragments with sRNAs extracted from control leaves is in good agreement with the abundance of Arabidopsis sRNAs that correspond to this chromosome as determined by massive parallel sequence signature (MPSS).