Cloning of short interfering RNAs from virus-infected plants

During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as ‘aberrant’ signals and short interfering RNA (siRNA) molecules of 19 - 25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc., provides valuable insights to plant-virus interactions. In this chapter we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5’ and 3’ adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been applied to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses.