Graft‐transmissible movement of inverted‐repeat‐induced siRNA signals into flowers

Abstract

SUMMARYIn plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 (DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells.

Mobile small RNAs regulate genome-wide DNA methylation

“…Nonetheless, those genes that did exhibit mobile sRNA-dependent regulation had diverse functions, indicating they may have significant influence in the correct conditions. Moreover, mobile sRNAs are able to move into both meristematic and meiotically active tissues, where they can alter DNA methylation and gene expression (37,52). In these tissues it is essential to protect genome stability by repression of TEs, so that gametes and developing organs are not harmed (53,54).…”

tRNA-Related Sequences Trigger Systemic mRNA Transport in Plants

“…The DN DMC1 coding sequence was fused to graft-mobile potato BEL5 sequences or phloem tRNA Met at the 39 UTR to evaluate their potential to trigger DN DMC mRNA transport over graft junctions. (B) to (E) Fertile anthers of wild-type tobacco plants show regular pollen production with minimal abnormally shaped pollen (2 to 3%), whereas hpDMC1 siRNA transgenic tobacco plants produce high numbers of abnormally shaped pollen and are sterile as previously described (Zhang et al, 2014). YFP-DN DMC1 transgenic plants have normal pollen production similar to the wild type because the N-terminal YFP fusion abolishes the dominant-negative effect of truncated DMC1.…”

“…Lack of a functional DMC1/RAD51 complex induces achiasmatic meiosis resulting in the formation of anomalously shaped pollen containing an aberrant number of chromosomes and, consequently, is necessary for proper pollen development (Bishop et al, 1992;Zhang et al, 2014). Thus, production of misshaped pollen in anthers and decreased fertility indicate the presence of either DMC1 siRNA (Zhang et al, 2014) (Figures 1B and 1C) or the product of translation of the dominant-negative DN DMC1 mRNA. To implement a reporter system for mRNA mobility, we produced transgenic tobacco (Nicotiana tabacum) lines expressing YFP-DN DMC1 fusion proteins as a fluorescent reporter (Figures 1B and 1C) to test for DMC1 silencing (Zhang et al, 2014) potentially induced by the transgenic DN DMC1 constructs.…”

“…Thus, production of misshaped pollen in anthers and decreased fertility indicate the presence of either DMC1 siRNA (Zhang et al, 2014) (Figures 1B and 1C) or the product of translation of the dominant-negative DN DMC1 mRNA. To implement a reporter system for mRNA mobility, we produced transgenic tobacco (Nicotiana tabacum) lines expressing YFP-DN DMC1 fusion proteins as a fluorescent reporter (Figures 1B and 1C) to test for DMC1 silencing (Zhang et al, 2014) potentially induced by the transgenic DN DMC1 constructs. We also generated lines expressing DN DMC1 mRNA fused to the full-length potato BEL5 transcript, which is known to be mobile (Cho et al, 2015) ( DN DMC1:BEL5) as a positive control ( Figure 1A).…”

“…DMC1 is a specific meiotic cell cycle factor and a member of the highly conserved RecA-type recombinase family of DNA-dependent ATPases active during meiosis in sporogenic cells (Doutriaux et al, 1998). Lack of a functional DMC1/RAD51 complex induces achiasmatic meiosis resulting in the formation of anomalously shaped pollen containing an aberrant number of chromosomes and, consequently, is necessary for proper pollen development (Bishop et al, 1992;Zhang et al, 2014). Thus, production of misshaped pollen in anthers and decreased fertility indicate the presence of either DMC1 siRNA (Zhang et al, 2014) (Figures 1B and 1C) or the product of translation of the dominant-negative DN DMC1 mRNA.…”