Abstract
Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp . carotovora ( Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp . pekinensis ( Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem–loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3′ end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.
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