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Abstract

Background: Nicotiana attenuata is an ecological model plant whose 2.57 Gb genome has recently been sequenced
and assembled and for which miRNAs and their genomic locations have been identified. To understand how this
plant’s miRNAs are reconfigured during plant-arbuscular mycorrhizal fungal (AMF) interactions and whether hostplant
calcium- and calmodulin dependent protein kinase (CCaMK) expression which regulates the AMF interaction also
modulates miRNAs levels and regulation, we performed a large-scale miRNA analysis of this plant-AMF interaction.
Results: Next generation sequencing of miRNAs in roots of empty vector (EV) N. attenuata plants and an isogenic line
silenced in CCaMK expression (irCCaMK) impaired in AMF-interactions grown under competitive conditions with and
without AMF inoculum revealed a total of 149 unique miRNAs: 67 conserved and 82 novel ones. The majority of the
miRNAs had a length of 21 nucleotides. MiRNA abundances were highly variable ranging from 400 to more than
25,000 reads per million. The miRNA profile of irCCaMK plants impaired in AMF colonization was distinct from fully
AMF-functional EV plants grown in the same pot. Six conserved miRNAs were present in all conditions and
accumulated differentially depending on treatment and genotype; five (miR6153, miR403a-3p, miR7122a,
miR167-5p and miR482d, but not miR399a-3p) showed the highest accumulation in AMF inoculated EV plants
compared to inoculated irCCaMK plants. Furthermore, the accumulation patterns of sequence variants of
selected conserved miRNAs showed a very distinct pattern related to AMF colonization - one variant of
miR473-5p specifically accumulated in AMF-inoculated plants. Also abundances of miR403a-3p, miR171a-3p
and one of the sequence variants of miR172a-3p increased in AMF-inoculated EV compared to inoculated
irCCaMK plants and to non-inoculated EV plants, while miR399a-3p was most strongly enriched in AMF
inoculated irCCaMK plants grown in competition with EV. The analysis of putative targets of selected miRNAs
revealed an involvement in P starvation (miR399), phytohormone signaling (Nat-R-PN59, miR172, miR393) and
defense (e.g. miR482, miR8667, Nat-R-PN-47).
Conclusions: Our study demonstrates (1) a large-scale reprograming of miRNAs induced by AMF colonization
and (2) that the impaired AMF signaling due to CCaMK silencing and the resulting reduced competitive
ability of irCCaMK plants play a role in modulating signal-dependent miRNA accumulation.