Testing the efficacy of artificial microRNAs to control cassava brown streak disease
Abstract
204 words
Cassava brown streak disease (CBSD) is a major constraint to
cassava production in East Africa. It is caused by at least two
single stranded (+) RNA viruses (CBSV and CBSUV). To date,
only a few genetic sources of resistance to the disease in cassava
are known. Artificial microRNAs (amiRNAs) have in the recent
been employed to control plant viruses. In this study, 21 nt of
the A. thaliana pre-mi159a were replaced with 21 nt conserved
sequences selected across the CBSV and CBSUV genomes,
generating 11 amiRNA constructs targeting different CBSV
genes (P1 {CBSV-Ug and CBSV-Tz}, P3, CI, NIb, CP and
3’UTR).The modified precursors were then sub-cloned in a
shuttle vector CGT11003-I and subsequently cloned into a binary
vector AKK1420 with an RNAi cassette targeting the green
fluorescent protein (GFP), as an internal silencing control.
Transient studies of these amiRNA constructs in transgenic N.
benthamiana (16C) using Agrobacterium (GV3001 strain),
showed that constructs targeting CBSV-P1, CBSUV-P1, NIb,
CP and UTR expressed miRNAs specific to their target
nucleotides of CBSUV. Transient protection studies showed varied levels of resistance to the homologous virus roughly
corresponding to the level of siRNA accumulation using Northern
analysis. Artificial microRNAs (amiRNA) therefore have the
potential to control the spread of CBSUV.
Subjects
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