High-Throughput Sequencing for Analysis of Small RNAs in Transgenic Cassava Resistant to Cassava Brown Streak Disease

Plants employ RNA-mediated gene silencing to combat viral pathogens. RNAi resistance based on hairpin RNA transcript is effective, especially against RNA viruses. In order to gain better understanding of RNA-mediated gene silencing against cassava brown streak disease (CBSD), the expression profiles of small-interfering RNA (siRNA) before and after viral infection were studied in both wild type and transgenic plants by small RNA high-throughput sequencing. Cassava plants were transgenic for an inverted repeat construct consisting of fused coat protein (CP) regions from Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Mapping analysis shows that siRNA were expressed from almost the entire transgene (>99% breadth of coverage). Distribution of siRNA peaks was not uniform but concentrated in hotspots in patterns differing between the corresponding regions of CBSV and UCBSV. Data confirms that in CBSD resistant transgenic cassava line 5001-5 small RNAs derived from the engineered CP inverted repeat construct constitute 6% of total siRNA. siRNA were seen to be derived from both CBSV-CP and UCBSV-CP regions but UCBSV-CP was the major contributor. Sorting of siRNA based on nucleotide-sequence-length showed that 21 and 22-nt predominated (~70%) and mapped preferentially to the antisense strand of the virus. Contrary to virus-derived siRNA, of which ~1% were 24-nt in size, 25% of total siRNAs seen in non-infected transgenic cassava were of the 24-nt size.. Sorting on the basis of type of 5’ nucleotide shows that majority of siRNA started with the U nucleotide, indicating activity of AGO1.