Discussion

73 mycorrhizal phenotypes of the wild-type plants, the nsp1 mutant that still potentially possesses the mimicry sequence and the nsp1 mutant expressing the NSP1 siRNA cassette. In our three repeats, nsp1 mutant silenced for NSP1 displayed a reduced mycorrhization rate compared to either the nsp1 mutant or the WT transformed with an empty vector. This accentuated defect in mycorrhization when both the NSP1 protein is non-functional and the mRNA of NSP1 is not expressed, points out the potential role NSP1 transcripts as target mimicry of the miR171h.

2.4. Prediction of potential new coding target mimics (cTMs).

Because we could show that a coding sequence is able to act as a Target Mimic (TM) we performed a bioinformatic analysis to investigate, in the plant model Arabidospis thaliana and Medicago truncatula, the possibility that other coding sequences could potentially be target mimics. Our screen was set, using the mRNA library from miRBase and the coding genome of both species. We first search for coding mRNA sequences having a 3 nucleotides gap in the critical position 10-11 of a microRNA. We allowed the presence of up to five mismatches in the target mRNA sequences (Axtell & Bowman, 2008; Mallory et al., 2008; Brousse et al., 2014), but we removed those presenting the five mismatches on only one side of the cleaving site (5’or 3’). By performing this analysis on both genomes we could identify thousands of potential cTMs for the whole set of miRNAs, and sometimes several hundred of potential cTMs per miRNA (Table 1). We also performed the same analysis on the non-coding RNA of A.

thaliana and found to a less extent a few TMs for most of the conserved miRNA family.

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Figure 6: Mycorrhizal phenotype of WT and nsp1 mutant expressing or not the RNAi MtNSP1 cassette in M. truncatula chimeric plant 12 weeks after inoculation with R.

irregularis. Mycorrhizal rate measured by the grid-intersect method (Giovannetti and Mosse, 1980). Error bars represent SEM.

Table 1: Summary table representing the number of target mimics found in the in-silico prediction for both the A. thaliana and M. truncatula coding genomes and the A. thaliana non-coding genome.

The 19 conserved miRNA families are represented, and for each genome an arbitrary color scaling has been add showing the abundance of TM found per family (from low to high number of TM, green to red).

0 5 10 15 20 25 30 35 40

A17 ctr nsp1/ctr nsp1/RNAi nsp1

Colonization rate (%)

WtT+ empty vector nsp1+ empty vector nsp1+ NSP1RNAi

# of ncTMs miRNA family A. thaliana M. truncatula A. thaliana

miR156 92 972 2

miR159 488 293 5

miR160 141 165 1

miR162 99 140 0

miR164 516 182 5

miR166 138 401 0

miR167 361 282 3

miR168 247 263 4

miR169 363 559 0

miR171 267 942 0

miR172 1279 1522 9

miR319 134 893 5

miR390 164 38 2

miR393 660 286 6

miR395 93 567 1

miR396 1051 1456 7

miR397 249 234 3

miR398 265 86 6

miR399 283 789 7

# of cTMs

75 the mature miR171h that can modify its concentration over time and space, we figure how dynamic and subtle the spatio-temporal regulation of NSP2 must be. This complex regulation must also play a role in root development, since miR171h has been found to be expressed in the root meristematic and elongation zones like both NSP1 and NSP2 (Untergasser et al., 2012) (Chap1. Fig. 1). As miR171h is expressed at low levels along the root and pNSP1::GUS expression seems to be localized in the central cylinder, where NSP2 is expressed as well (Fig.

S2). We could speculate that an overlapping expression in some regions along the roots is necessary for the subtle regulation of miR171h activity on NSP2.

Moreover, miR171h has been predicted to target at least three other genes, such as a NSP2-like (Medtr5g058860) and tow genes encoding pentatricopeptide-repeat proteins, Mtr.25350.1.S1_at and Mtr.11537.1.S1_at. Their regulation could also depend on the presence of cTMs. From our in-silico analysis, we predicted in Arabidospis 267 potential cTMs of the miR171 family (from which 171h is absent) and 90 miR171h potential cTMs in Medicago.

These results highlight the potential for numerous cross regulations between miRNAs and a consortium of targets and pseudotargets. This high number of potential cTMs for each miRNA raises the intriguing question of the biological and functional relevance of such a system. We hypothesize that during plant evolution miRNAs and natural coding mimics have been concomitantly developed to restrict miRNA activities, where it was biologically relevant.

It has been shown that certain miRNAs can migrate between different cell layers (like miR166 and miR390) but also through the vascular system like miR395 and miR399 (Reviewed in Marín-González & Suárez-López, 2012). Given this natural spreading plants may have developed strategies avoid inappropriate miRNA activities in neighboring cells. We speculate that the high occurrence of cTMs might sustain this strong requirement for a plant to restrict miRNA activities just where they are necessary. The cTMs would mainly be efficient to trap escaping, less concentrated, miRNAs in the neighboring cells. The activity of miRNAs to be restricted in the proper cells would occur where their concentration is the highest, i.e. close to where the pri-miRNAs have been transcribed (Fig. 7).

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miR X Target1

Target 1 degradationRelevant biological effect

mimic 1

mimic 4 mimic Y

Target

Target Target

mimic 4 mimic 1

mimic 3 mimic 2 miR X Target1

Target1Target1 Target 1 degradation

Target 1 protected by mimic targetsNo effect miR X expression

miR X migration

Relevant biological effect

Targets mimic abundance miR X abundance

mimic 2mimic 1 mimic 2mimic 1

mimic 1mimic 2

Figure 7

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