Abstract:
Under N-limiting soil condition legumes display a fitness advantage, because the plants can acquire nitrogen with help of nitrogen fixing rhizobia inducing symbiosis. During symbiosis a specialized organ called nodule is developed, which is energy intense for the plant. Therefore, a tightly regulated feedback loop termed autoregulation of nodulation (AON) is essential to balance nodule number. One important factor in AON is the shoot-derived inhibitor, which accumulates in AON activated leaf tissue and systemically moves to the roots inhibiting further nodulation. Here we show that Lotus japonicus miR171c fulfills SDI criteria like dependence on the receptor-like kinase gene HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1), expression in shoot phloem and systemic travel. miR171c is further shown to be involved in nitrate mediated inhibition of nodulation.
Using small RNA sequencing, we show that Lotus japonicus miR171c is highly expressed in AON activated (CLE-RS3 OX) and dramatically reduced in AON-inhibited (har1-3) plants. miR171c is specifically expressed in shoot phloem cells and systemically travels to the roots. miR171c loss-of-function (ko) mutants generated using CRISPR/Cas9-technology revealed that miR171c is not only involved in AON but also in nitrate dependent inhibition of nodulation. Specifically, miR171c ko plants are insensitive to nitrate-dependent inhibition of nodulation, developing nodules despite high nitrate concentration. In contrast, miR171c overexpression (ox) plants partially inhibit nodulation and are hypersensitive to nitrate application. Moreover, miR171c is systemically and locally induced in shoots and roots upon nitrate as well as rhizobia treatment. Nitrate and rhizobia induced miR171c in the shoot is HAR1 dependent. Taken together, our data show that miR171c fulfills key SDI criteria and acts in both rhizobia- and nitrate dependent inhibition of nodulation.
