Virus-induced gene silencing of wheat ATG8

The barley stripe mosaic virus (BSMV)-based VIGS method was used to create gene knockdown plants (Ma et al., 2012 (link); Dong et al., 2019 (link)). Briefly, a 283-bp fragment of wheat ATG8 from the conserved coding sequence was amplified and purified, with a same-sized fragment of GFP used as a control. The γ strand of BSMV was then digested in XmacI and fused with the ATG8 or GFP fragment to form the vectors BSMVγ-ATG8 and BSMVγ-GFP, respectively. BSMV-α was then linearized with MIuI, BSMV-β was linearized with SpeI, and BSMVγ-ATG8 and BSMVγ-GFP were linearized with BssHII then the linearized vectors were transcribed in vitro to produce 5ʹ-capped infectious BSMV RNA molecules using the RiboMAX Large-Scale RNA Production-T7 Kit (Promega, Madison, WI, United States), with a cap analog added to the transcription mixture. They were then mechanically infected with a 1:1:1 mixture of RNAα, RNAβ and RNAγ-ATG6, or RNAγ- GFP in 1 × GKP buffer (50 mM Gly, 30 mM K2HPO3, 1% bentonite and 1% kieselguhr). In the field, inoculation of BSMV was performed at the heading stage by inoculating 50 spikes with 20 µL of BSMV-ATG8 or BSMV-GFP transcript mixture, respectively.