RNA‐directed DNA methylation efficiency depends on trigger and target sequence identity

Abstract

SUMMARYRNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2). The two viroid sequences exhibit 81% sequence identity. Nt-TASVd and Nt-SB2 plants were genetically crossed. In the progeny plants (Nt-SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt-SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis-RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans-RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger-target sequence identity and RdDM efficiency. Importantly, trans-RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger-target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24-nt RNAs) trigger RdDM.

Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants

“…In this regard, recent data have demonstrated that DNA methylation can also be modulated by DCL-independent siRNAs ( Yang et al. , 2016 ; Dalakouras et al. , 2016 ; Ye et al.…”

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

“…Indeed, siRNAs derived from both nuclear and chloroplastic viroids can direct silencing of the host plant mRNAs through sequence-specific cleavage and degradation ( Navarro et al, 2012 ; Adkar-Purushothama et al, 2015 , 2018 ; Adkar-Purushothama and Perreault, 2018 ), although viroids can also indirectly impact on the host gene expression at both post-transcriptional and transcriptional levels ( Castellano et al, 2015 ; Tsushima et al, 2015 ; Torchetti et al, 2016 ; Zheng et al, 2017c ). Notably, nuclear viroids can also trigger de novo RNA-directed DNA methylation of the transgenes containing viroid sequences ( Wassenegger et al, 1994 ; Dalakouras et al, 2016 ). Whether or not viroid-derived 24-nt siRNAs are involved in this RdDM process remains unclear ( Dalakouras et al, 2016 ).…”

“…Notably, nuclear viroids can also trigger de novo RNA-directed DNA methylation of the transgenes containing viroid sequences ( Wassenegger et al, 1994 ; Dalakouras et al, 2016 ). Whether or not viroid-derived 24-nt siRNAs are involved in this RdDM process remains unclear ( Dalakouras et al, 2016 ).…”

New discoveries generate new questions about RNA-directed DNA methylation in Arabidopsis

“…RdDM efficiency is affected by the quality of the trigger RNA, i.e. sequence identity between the trigger and the target [18]. Interestingly, in contrast to the dramatic reduction in DNA methylation in dcl2/3/4 compared to the WT, P4 RNA levels in dcl2/3/4 are comparable to 24-nt siRNA levels in the WT [4,6–8].…”