Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

Abstract

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

Double-Stranded RNAs (dsRNAs) as a Sustainable Tool against Gray Mold (Botrytis cinerea) in Grapevine: Effectiveness of Different Application Methods in an Open-Air Environment

“…A possible explanation for this phenomenon is linked to the specificity of fungal RNAi machinery [53]. It has been demonstrated that fungal sRNA patterns differ from that of plants: the typical plant siRNA sizes are conserved and range between 21-22 and 24 nucleotides in length [58][59][60]. In fungi, the siRNA lengths range from 21 up to 30 nucleotides in length, also within the same fungal family [38,61,62].…”

Small RNA-Omics for Plant Virus Identification, Virome Reconstruction, and Antiviral Defense Characterization

“…Such RNAi-transgenic plants are resistant and, in some cases, immune to infection with the corresponding virus, and the immunity correlates with transgenic production of phloem-mobile 24-nt siRNAs (reviewed in Pooggin, 2017 ). Interestingly, abundance of transgene-derived 24-nt siRNAs appears to correlate with immunity or increased resistance not only to DNA viruses such as tomato yellow leaf curl virus ( Begomovirus, Geminiviridae ) in tomato ( Leibman et al, 2015 ; Fuentes et al, 2016 ; see Pooggin, 2017 ), but also RNA viruses such as zucchini yellow mosaic virus ( Potyvirus, Potyviridae ) in cucurbits ( Leibman et al, 2011 ), prunus necrotic ring spot virus ( Ilarvirus, Bromoviridae ) in sweet cherry ( Zhao and Song, 2014a , b ) and lettuce infectious yellows virus ( Crinivirus, Closteroviridae ) in N. benthamiana ( Qiao et al, 2018 ).…”

RNA Interference: A Natural Immune System of Plants to Counteract Biotic Stressors

“…The 24 nt siRNA produced by DCL3 is not enough against invading RNA, and, thus, the production of 21- and 22 nt species is needed for an effective silencing process. However, accumulation of the 24 nt transgene-derived siRNAs (t-siRNAs) are associated with plant immunity against crinivirus [94], which shows that DCL3 play minor role in antiviral defense against RNA viruses.…”