Abstract
BackgroundNicotiana 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.ResultsNext 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).ConclusionsOur 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.
Highlights
Nicotiana attenuata is an ecological model plant whose 2.57 Gb genome has recently been sequenced and assembled and for which Micro RNA (miRNA) and their genomic locations have been identified
N. attenuata silenced in the expression of calcium and calmodulin dependent protein kinase due to an inverted repeat construct and empty vector (EV) plants were germinated on Gamborg B5 medium after surface sterilization with a 2% (w/v) aqueous solution of sodium dichloroisocyanuric acid (DCCS) and treatment with a smoke solution and gibberellic acid for 1 h [57]. 12 days after germination seedlings were transferred to 2 L pots filled either with living or autoclaved Rhizophagus irregularis inoculum (Biomyc Vital)
Profiles of conserved and novel miRNAs identified in N. attenuata roots After further cleaning of the data, we identified 211 reads mapped to 67 conserved miRNAs (Additional file 1: Table S1), 136 of these reads were identified as miRNA stars (Fig. 2a)
Summary
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 modulates miRNAs levels and regulation, we performed a large-scale miRNA analysis of this plant-AMF interaction. Root colonization by arbuscular mycorrhizal fungi (AMF), which belong to the phylum Glomeromycota [1], is wide-spread across almost all plant families, and it is estimated to occur in more than 70% of all terrestrial plants [2] This interaction improves the plant’s uptake of nutrients from the soil, in particular of inorganic phosphorus and nitrogen, while in exchange, the plants provide carbohydrates to the fungus [3]. Many miRNAs are conserved across angiosperms and seem to play similar roles in different plant species
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