The development of SACMV-resistant cassava using pathogen-derived resistance strategies, targeting the DNA - a component
Date
2009-09-09T12:38:32Z
Authors
Taylor, Sarah Helen
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Abstract
South African cassava mosaic virus (SACVM) is a cassava mosaic
disease- causing virus, belonging to the genus Begomovirus of the Geminiviridae
family. It is a bipartite geminivirus, having two circular single-stranded DNA
components, designated DNA-A and DNA-B. The AC1 open reading frame on
the DNA-A component encodes for the Rep protein, which is absolutely required
for virus replication. Plant virus silencing strategies exploit the use of the viralderived
sequences to confer resistance towards that particular virus, a concept
termed pathogen derived resistance (PDR). PDR resistance strategies used to
engineer resistance towards geminiviruses include the expression of viral
proteins, the use of naturally occurring defective interfering molecules which
delay and attenuate infection symptoms, the sequence specific degradation of
viral transcripts in the host cytoplasm by inducing RNA silencing with sense,
antisense or invert-repeat transgenes and the use of double-stranded RNA
molecules to direct methylation of virus promoter sequences. In this study
SACMV AC1-derived sequences in sense and invert-repeat transgene constructs were used to induce RNA silencing against SACMV infection in Nicotiana
benthamiana. The replication of SACMV DNA-A was reduced by almost 100% in
two N. benthamiana lines expressing untranslatable SACMV AC1 sense
transcripts compared to antisense and control lines. Two size classes of
transgene derived siRNAs were isolated from unchallenged leaf tissue; one size
class being unique, amongst the sense lines, to the replication knockdown lines.
Similarly, a sense arm sequence-modified invert-repeat construct resulted in
almost 100% SACMV replication reduction in six transgenic lines. This construct
was produced using a technique for the production of stable RNA silencing
constructs. Unmodified, spliceable intron invert-repeat transgene constructs transcribed to produce hairpin RNA molecules of 572-nt and 193-nt arm length,
resulted in SACMV replication knockdown of 60 and 80% respectively. Towards
the production of SACMV resistant cassava cultivars, TMS60444 FEC and MTAI
16 cotyledons were transformed with an invert-repeat RNA silencing construct,
however regeneration of transformed plants failed. Cassava transformation and
regeneration is inherently difficult and it has been proposed here that a minimal
cassette technology-based method can be applied to achieve transformation of
cassava with the RNA silencing constructs that showed significant SAMCV
silencing in this study.