Exploring the possible cause of different levels of RNA silencing against BNYVV in transgenic sugar beets: a comparative virus transcriptome study.

Sugar beet is one of the most economically important crops because of its high sucrose content. The worldwide production of this crop is severely threatened by Beet necrotic yellow vein virus (BNYVV). Developing resistant transgenic cultivars is a promising approach to control this disease. Our group successfully generated RNA silencing-induced resistance to BNYVV in two transgenic sets of lines named S3 and S6 by introducing constructs carrying sense and antisense 5'-UTR with or without coat protein (CP21)-coding sequences of BNYVV, respectively. However, RNA-silencing-based resistance may be weakened or broken in some transgenic plant progenies. This study employed high-throughput virus transcriptome analyses to investigate the possible cause of differences in BNYVV propagation levels among transgenic sugar beets with the same genetic backgrounds. The RNA-seq and small RNA-seq results demonstrated different levels of viral propagation varied between samples. Quantitative RT-PCR as well as stem-loop PCR showed that RNA silencing machinery can effectively process the transgene transcripts into siRNAs. Subsequently, it was proven that all the infective BNYVV had the same ecotype, A-type, which shared 96.3% and 98.3% sequence identities with S3 and S6 transgenes, respectively. Interestingly, Beet soil-borne virus (BSBV), Beet cryptic virus 2 (BCV2), and Beet satellite virus (BSV) were detected in the transcriptomes of highly infected samples. Detailed analysis revealed that simultaneous BSBV and BSV accumulations in transgenic plants could be linked to the weakening of RNA silencing of BNYVV.