Use of microRNA‐encoded peptide miPEP172c to stimulate nodulation in soybean

Current worldwide agriculture requires huge amounts of chemicals for increasing yields, and this situation will be dramatically degraded by the increasing world population (Velivelli et al., 2014). However, the main problems are that chemicals might be expensive, highly polluting and dangerous for human health, and that some of them, such as phosphates, are available to plants in limited quantities on Earth (Amundson et al., 2015). The search for sustainable alternatives to these chemicals is one of the major challenges of the actual research in plant biology (Oldroyd & Dixon, 2014; Velivelli et al., 2014; Delaux et al., 2015). Another extensively used strategy for improving plant yield is based on conventional breeding methods that require extensive and long-term efforts to identify and introduce favorable traits in crops. While being slightly faster, the use of transgenic methods might nowadays face societal acceptance (Leyser, 2014), but it still needs years of research before its use in agriculture (Oldroyd & Dixon, 2014; Delaux et al., 2015). Another limit of transgenic methods is based on the capacity, and more importantly, the efficiency, of some crops to be regenerated or not. MicroRNAs (miRNAs) are short regulatory RNAs that regulate the expression of their target genes at the post transcriptional level, by cleaving their mRNA or inhibiting their translation. In animals, as in plants, they are involved in most of the developmental processes (Rogers & Chen, 2013). MiRNAs are transcribed by RNA polymerase II as long molecules, called primary transcripts (pri-miRNA). These molecules undergo two successive steps of maturation leading to the formation of the mature, active miRNA. Using miR165a and miR171b in Arabidopsis thaliana and Medicago truncatula, respectively, we have recently shown that these two pri-miRNAs encode for peptides, called miPEPs. These two miPEPs are produced at the same time and location as the primary transcript they come from. MiPEPs increase the quantity of their respective mature miRNA by stimulating the transcription of the associated pri-miRNA (Lauressergues et al., 2015). Finally, it appears that miPEPs are very specific to their associated miRNAs, which might represent a significant advantage in agronomy. MiPEPs have also been shown to modify root development by the external application of synthetic peptides specific to M. truncatula miR171b and A. thaliana miR165a (Lauressergues et al., 2015). These data strongly suggest that miPEPs could be used as tools to increase the agronomical traits of crops (Couzigou et al., 2015). Legume plants can fix atmospheric nitrogen through their symbiotic interaction with soil bacteria commonly known as rhizobia. This interaction takes place in symbiosis-specific organs, the nodules, which are generally formed on the host roots (Ferguson et al., 2010). The main advantage of this interaction is that this symbiosis fixes large amounts of organic nitrogen from atmospheric nitrogen, resulting in reduced necessity for nitrogen fertilization in legume fields. Schematically, rhizobia synthesize lipochitoolichosaccharides, known as Nod Factors (NF), which are, in turn, perceived by plant receptor-like kinases (RLKs), such as NFR1 and NFR5 in soybean (Indrasumunar et al., 2010, 2011). NF perception will lead to the activation of nodule formation through the nodulation signaling pathway activation. However, the nodules, which share common features with lateral roots (Couzigou et al., 2013), must also be harmoniously regulated through a process known as autoregulation of nodulation (AON; Reid et al., 2011). Beyond the well-known regulators of AON such as the homologs of CLAVATA 1 RLK in legumes NARK/HAR1/SUNN (Reid et al., 2011), several miRNAs affecting nodule number have been identified recently (Li et al., 2010; Turner et al., 2013; Wang et al., 2014, 2015; Yan et al., 2015). In these studies, overexpression of several miRNAs has positive or negative effects on nodule formation in soybean. In particular, miR172c overexpression acts positively on nodulation whereas reduced activity of miR172c decreases the nodule number in Glycine max (Wang et al., 2014). This effect has been shown to be mediated through repression of the miR172c target gene, the AP2 transcription factor NODULE NUMBER CONTROL 1 (NNC1), that in turn directly targets cis-elements in the early nodulin ENOD40 promoters (Wang et al., 2014). Additionally, miR172c transcription is activated by NFR1/5 and inhibited by AON (Wang et al., 2014). In this context, we investigated whether miPEP172c would stimulate miR172c expression and consequently, the nodulation process in soybean. We first identified the miPEP172c (see Supporting Information Methods S1) and watered plants with 0.1 μM synthetic miPEP172c or with 0.1 μM scrambled miPEP172c peptide three times a week during nodulation. Our results showed that, in the same conditions of lack of external nitrogen fertilization, treatment with miPEP172c significantly increased the nodule number observed per plant (Fig. 1a), without affecting root development (Fig. 1b). An increased number of ineffective nodules was often observed as a compensation mechanism in consequence of reduced nitrogen fixation. Analysis of the expression of NifH by RT-qPCR (Fig. 1c) and nifD::LacZ fusions (Fig. 1d,e) favors the hypothesis of efficient nitrogen fixation in miPEP172c treated plants. In parallel, we observed that miR172c transcripts were approximately three times more abundant in the roots treated with miPEP172c compared to the scrambled miPEP172c-treated roots (Fig. 1c). Accordingly, NNC1 expression was significantly reduced in the miPEP172c-treated roots (Fig. 1c). Finally, we analyzed the expression of several marker genes of nodulation in both conditions, and we observed that NSP1, NIN, ENOD40-1 and Hb2 (Wang et al., 2014) expression was higher in plants treated with miPEP172c compared to control plants, showing that the symbiosis is more active in these plants (Fig. 1c). Collectively, all these results showed that miPEP172c treatment mimicked the effect of miR172c overexpression at both phenotypic and molecular levels according to the symbiotic phenotypes of Yan et al. (2013), Wang et al. (2015) and Nova-Franco et al. (2015). In this study, we showed that exogenous application of synthetic miPEP172c could be used to stimulate miR172c expression. The miR172c activation results in a more active repression of its target gene, the AP2 transcription factor NNC1. The miPEP172c treatment ultimately leads to the stimulation of nodulation in soybean, which is one of the most cultivated legumes is the world. We show here for the first time that exogenous treatment with well-chosen and synthetic peptides (miPEPs) could have an important and predictable impact on plant development and plant–microbe interactions. Finally, as the roles of all miRNAs are not yet known, we can expect identification of miRNAs, and then miPEPs, able to regulate the development of plants, by stimulating the development of cultured plants and inhibiting the growth of adventives. In this context, the specificity of miPEPs potentially may represent an advantage. This work was funded by the French ANR project miRcorrhiza (ANR-12-JSV7-0002-01). This work is also supported by Toulouse Tech Transfer (http://www.toulouse-tech-transfer.com) for valorization and transfer. The authors thank Prof. Hans-Martin Fischer for providing B. diazoefficiens nifD-lacZ strain. J-P.C. designed the research; J-M.C., O.A., B.G., M.A. and J-P.C. performed the experiments; J-M.C. and J-P.C. wrote the paper. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.