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.
Ho
Thien X
TX
NERC/Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford OX1 3SR, UK.
Rusholme
Rachel
R
Dalmay
Tamas
T
Wang
Hui
H
D003001:Cloning, Molecular / Q000379:methods; D010935:Plant Diseases / Q000821:virology*; D010944:Plants / Q000821:virology; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D012330:RNA, Double-Stranded; D018749:RNA, Plant; D034741:RNA, Small Interfering; D012367:RNA, Viral