By including mutants defective in strigolactone (SL) or KAI2 ligand (KL) synthesis and/or signaling, these methods can be used as bioassays for different SLs or SL-like molecules. In addition, the techniques described here can be used for studying seedling root system architecture, root skewing, and root hair development in any context.Shoot branching is a highly variable trait that evolves during plant development and is influenced by environmental and endogenous cues such as hormones. In particular, strigolactones (SLs) are hormones that play a key role in the control of shoot branching. Branch primordia, axillary buds formed in the leaf axils, display differential growth depending on their position in the plant and also respond to hormone signaling. In this chapter, we will describe how to quantify the degree of shoot branching in two plant model species, Arabidopsis and pea, commonly used to decipher the control of this complex trait. We will also propose several methods to perform treatments of SL or SL analogs, to investigate their bioactivity and effect on the shoot branching patterns of plants of different genotypes.Current knowledge on the mechanism of strigolactones (SLs) as signaling molecules during specific interactions in the rhizosphere is mainly related to the control of germination of parasitic weed seeds and hyphal branching of arbuscular mycorrhizal fungi. Thus, the role of plant secreted SLs in regulating the growth and development of root-colonizing fungi still remains controversial. Fusarium oxysporum can sense and respond to extracellular signals through oriented germ tube emergence and redirectioning of hyphal growth toward gradients of nutrients, sex pheromones, or plant root exudates. However, chemoattractant activity of SLs against microorganisms living in the soil has not been tested so far. Here we propose a quantitative chemotropic assay to understand if and how soil fungi could sense gradients of SLs and SLs-like sources. In the example case of F. oxysporum, hyphae of fungal representative mutants preferentially grow toward the synthetic SL analog GR24; and this chemotropic response requires conserved elements of the fungal invasive growth mitogen-activated protein kinase (MAPK) cascade.In the Rhizobium-legume symbiosis, strigolactones (SLs) promote root nodule formation; however, the exact mechanism underlying this positive effect remains unknown. The recent finding that an SL receptor legume mutant shows a wild-type nodulation phenotype suggests that SLs influence the symbiosis by acting on the bacterial partner. In agreement with this, the application of the synthetic SL analog GR24 on the alfalfa symbiont Sinorhizobium (Ensifer) meliloti has been shown to stimulate swarming, a specialized bacterial surface motility, which could influence infection of legumes by Rhizobia. Surface motility assays for many bacteria, and particularly for Rhizobia, are challenging. The establishment of protocols to study bacterial surface motility is key to decipher the role of SLs as rhizosphere cues for rhizobacteria. In this chapter, we describe a set of protocols implemented to study the different types of motility exhibited by S. meliloti.Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.Strigolactones are a class of plant hormones involved in shoot branching, growth of symbiotic arbuscular mycorrhizal fungi, and germination of parasitic plant seeds. Assaying new molecules or compound exhibiting strigolactone-like activities is therefore important but unfortunately time-consuming and hard to implement because of the extremely low concentrations at which they are active. Seeds of parasite plants are natural integrator of these hormones since they can perceive molecule concentrations in the picomolar to nanomolar range stimulating their germination. Here we describe a simple and inexpensive method to evaluate the activity of these molecules by scoring the germination of parasitic plant seeds upon treatment with these molecules. Up to four molecules can be assayed from a single 96-well plate by this method. A comparison of SL-like bioactivities between molecules is done by determining the EC50 and the maximum percentage of germination.Strigolactones (SLs) are new plant hormones that play an important role in the control development of plants. They are germination stimulants for seed of parasitic weeds, are the branching factor of arbuscular mycorrhizal fungi and inhibitors for bud outgrowth and shoot branching. Natural SLs contain an annulated system of three rings (ABC scaffold) connected to a furanone (the D-ring) by an enol ether unit. The natural distribution of strigolactones is low, and their synthesis is long and difficult. Therefore, SL analogs are designed to have the same bioactiphore as natural SLs and an appreciable bioactivity. For the design a model is used based on the natural bioactiphore. Typical SL analogs are GR24, Nijmegen-1, and EM1 (derived from ethyl 2-phenylacetate). FL118 The synthesis of these SL analogs is reported together with their stability in aqueous solution.