Meeting the Challenge of Yellow Rust in Cereal Crops - ICARDA

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Virus-induced gene silencing in wheat
Mahinur S. Akkaya
Middle East Technical University, Ankara, Turkey
Using the powerful Virus-Induced Gene Silencing (VIGS) approach, it is now
possible to silence wheat endogenous genes with Barley Stripe Mosaic Virus
(BSMV). The successful use of VIGS in wheat could develop into a
revolutionary method for wheat functional genomics. Using BSMV-mediated
VIGS, it will be possible to better understand the compatible and incompatible
yellow rust fungal interactions, relatively easier than by other silencing
methods. In this powerful reverse genetic approach, upon infection with virus
vectors carrying homologous plant gene fragments, sequence-specific RNA
degradation occurs in the corresponding mRNA in the infected cells and
spreads systemically throughout the leaves. VIGS is rapid, does not require
development of stable transformants, allows characterization of phenotypes
that might be lethal in stable lines and offers the potential to silence either
individual or multiple members of a gene family. Here, we report the silencing
of phytoene desaturase (PDS) gene, causing photo-bleaching in wheat, using
the constructs prepared by Holzberg and colleagues.
The BSMV vectors were linearized with proper restriction enzymes and
transcribed using Ambion T7 mMessage mMachine Kit. Following the
purification of transcribed messages, RNA was suspended in FES solution and
immediately applied onto the young leaves of 20-day-old wheat plants. GFP
expression was observed in infected and newly growing leaves after 6–7 days
of infection. Photo-bleaching was observed in newly growing leaves after 12–
16 days. We are currently in the process of silencing the genes identified with
DD-RT and performing microarray analyses to confirm their roles in relation to
the other genes by both assessing level of silenced genes and their expressional
affects on the other genes of interests by Real Time RT-PCR (qRT-PCR).