Nitrogen-fixing root nodules on legumes result from two developmental processes, bacterial infection and nodule organogenesis. To balance symbiosis and plant growth, legume hosts restrict nodule numbers through an inducible autoregulatory process. Here, we present a mechanism where repression of a negative regulator ensures symbiotic susceptibility of uninfected roots of the host We show that microRNA miR2111 undergoes shoot-to-root translocation to control rhizobial infection through posttranscriptional regulation of the symbiosis suppressor TOO MUCH LOVE in roots. miR2111 maintains a susceptible default status in uninfected hosts and functions as an activator of symbiosis downstream of LOTUS HISTIDINE KINASE1-mediated cytokinin perception in roots and HYPERNODULATION ABERRANT ROOT FORMATION1, a shoot factor in autoregulation. The miR2111- node ensures activation of feedback regulation to balance infection and nodulation events.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest, enabling the covalent biotin labelling of proteins and subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

Optogenetics, the genetic approach of controlling cellular processes with light, is revolutionizing biological signalling and metabolic studies. It provides unmatched spatiotemporal, quantitative and reversible control, overcoming limitations of chemically-inducible systems. However, optogenetics severely lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue engineering PULSE (Plant Usable Light-Switch Elements), the first optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue light-regulated repressor with a red light-inducible switch. Gene expression is only activated under red light and remains inactive under white light/darkness. Supported by a quantitative mathematical model we characterized PULSE in protoplasts achieving high induction rates, and combined it with CRISPR/Cas9-based technologies to target synthetic signalling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues for plant research and biotechnology. RESULTS Design, implementation, and test of the PULSE system in plant cells PULSE is an integrated optogenetic molecular device, consisting of two components, a module providing activation of gene expression under red light (ROn) and a second one ensuring effective transcriptional repression under blue light (BOff) (Fig. 1). The rationale behind this new conceptual and experimental approach is that the combination of both switches will yield a system that is inactive in ambient growth conditions (light and darkness) and only active upon irradiation with red light. This enables full applicability in plants growing under standard light conditions. We first constructed a blue light-regulated gene repression switch BOff based on the photoreceptor EL222 from the bacterium Erythrobacter litoralis 11 which has a Light-Oxygen-Voltage (LOV) dependent motif and an Helix-Turn-Helix (HTH) domain. Upon blue light it binds as a dimer to the target DNA sequence C120 12 . BOff thus comprises (Fig. 2a): i) the constitutively expressed EL222 fused to a transcriptional repressor domain (REP), and ii) a reporter module driving the expression of a reporter gene (e.g. Firefly luciferase, FLuc) under the control of a synthetic tripartite promoter. The promoter comprises a quintuple-repeat target sequence for EL222, termed (C120)5, flanked by the enhancer sequence of the CaMV35S promoter and the minimal domain of the constitutive promoter hCMV. We evaluated three versions of the blue light-repressor module by fusing either of three different known transrepressor domains to the N-terminus of EL222, one from the human Krüppel Associated Box (KRAB) 13,14 protein, and two from Arabidopsis, namely the B3 repression domain (BRD) 15 and the EAR repression domain (SRDX) 15 (Fig. 2a). The functionality of the BOff optoswitches was assayed by transient cotransformation with the repor...

SummaryLegumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation.We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infectionresponsive sRNAs, followed by candidate-based functional characterization.Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves.The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis.

35Proximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a 36 peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and 37 allows subsequent capture and identification of interacting and neighbouring proteins without 38 the need for the protein complex to remain intact. To date, only few papers report on the use of 39 PDL in plants. Here we present the results of a systematic study applying a variety of PDL 40 approaches in several plant systems using various conditions and bait proteins. We show that 41TurboID is the most promiscuous variant in several plant model systems and establish protocols 42 which combine Mass Spectrometry-based analysis with harsh extraction and washing 43 conditions. We demonstrate the applicability of TurboID in capturing membrane-associated 44 protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. 45We further benchmark the efficiency of various PBLs in comparison with one-step affinity 46 purification approaches. We identified both known as well as novel interactors of the endocytic 47Protein-protein interaction (PPI) studies often fail to capture low-affinity interactions as these 53 are usually not maintained following cell lysis, protein extraction and protein complex 54 purification. Particularly, this is the case for PPI's of integral membrane proteins because of 55 the harsh conditions during protein extraction and purification. Proximity-dependent biotin 56 labelling (PDL) on the contrary, uses covalent biotinylation of proteins that are interactors or 57 near-neighbours of a bait protein of interest in vivo (Varnaite and MacNeill, 2016). Hence, to 58 identify interactions, they do not need to remain intact during purification. Although biotin is 59 an essential cofactor for a small number of omnipresent biotin-dependent enzymes involved 60 mainly in the transfer of CO2 during HCO3 --dependent carboxylation reactions, biotinylation 61 is a relatively rare in vivo protein modification. Moreover, biotinylated proteins can be 62 selectively isolated with high affinity using streptavidin-biotin pairing. PDL, therefore, permits 63