Graphical abstract

Figure S1: Optimisation of the Litterbox system. A. Effect of zeolite amount on plant weight (fresh weight aboveground plant parts) of six-week-old plants grown on coarse (35 g or 70 g) zeolite covered by fine ground (20 g) zeolite. B. Effect of zeolite granularity on plant weight of six-week-old plants grown on 90 g of zeolite (70 + 20 g and 90 g). C. Effect of MES buffer (2.5 mM) on plant weight of six-week-old plants grown on coarse zeolite (70 g) covered by fine ground zeolite (20 g). Tukey's boxplots. Figure S2: Effect of agar concentration and tip size of agar filled pipette tips on plant growth. A. Plant weight of six-week-old plants (fresh weight aboveground plant parts) of plants sown either on cut 10 μl or 200 μl pipette tips filled with either 0.6% or 1% agar. Seedlings were transferred seven days after sowing to 70 g coarse zeolite covered by 20 g fine ground zeolite. B. Percentage of plants grown to full size per transplanted seedling. Filled circles represent sample mean, error bars depict standard deviation, dots mark individual plants, thick bar represents median, dotted bars represent quartiles, two-way ANOVA.

Plants are colonised by millions of microorganisms representing thousands of species with varying effects on plant growth and health. The microbial communities found on plants are compositionally consistent and their overall positive effect on the plant is well known. However, the effects of individual microbiota members on plant hosts and vice versa , as well as the underlying mechanisms remain largely unknown. Here, we describe 'Litterbox', a highly controlled system to investigate plant-microbe interactions. Plants were grown gnotobiotically on zeolite-clay, an excellent soil replacement that retains enough moisture to avoid subsequent watering. Plants grown on zeolite phenotypically resemble plants grown under environmental conditions. Further, bacterial densities on leaves in the Litterbox system resembled those in temperate environments. A PDMS sheet was used to cover the zeolite, thereby significantly lowering the bacterial load in the zeolite and rhizosphere. This reduced the likelihood of potential systemic responses in leaves induced by microbial rhizosphere colonisation. We present results of example experiments studying the transcriptional responses of leaves to defined microbiota members and the spatial distribution of bacteria on leaves. We anticipate that this versatile and affordable plant growth system will promote microbiota research and help in elucidating plant-microbe interactions and their underlying mechanisms. Introduction:Plants offer three different habitats to microbes: the endosphere, the rhizosphere, and the phyllosphere. The endosphere encompasses the habitat formed by internal tissues of plants, whereas the rhizosphere and phyllosphere encompass the surfaces of belowground and aboveground plant organs, respectively. Plants host remarkably diverse and complex, yet structured, microbial communities, collectively referred to as the plant microbiota [1][2][3] . Due to the microbiota's diversity and complexity, it is not surprising that the traditional view of host-microbe interactions focussing on plant pathogens, nitrogen-fixing rhizobacteria, and phosphate-mobilizing mycorrhizal fungi, has recently shifted to a holistic view considering the plant and its associated microbiota as a metaorganism or holobiont [4][5][6] . It is widely recognised that members of the microbiota assist in nutrient uptake, promote growth, and protect against biotic and abiotic stresses [7][8][9][10][11][12][13][14] . To harness these positive impacts of plant-associated microbiota, the use of synthetic microbiota has been proposed [15][16][17] . The prospect of using synthetic microbial communities to promote sustainable agriculture is leading to a growing appreciation of plant microbiota research.Generally, plant microbiota research aims to understand 1) plant-microbe and microbe-microbe interactions ranging from the individual microorganism to the microbial community resolution, 2) the underlying molecular mechanisms of these interactions, and 3) their contribution to microbial community structure. Thus far...

Leaves are colonised by a complex mix of microbes, termed the leaf microbiota. Even though the leaf microbiota is increasingly recognised as an integral part of plant life and health, our understanding of its interactions with the plant host is still limited. Here, mature, axenically grown Arabidopsis thaliana plants were spray-inoculated with six diverse leaf-colonising bacteria. The transcriptomic changes in leaves were tracked over time and significant changes in ethylene marker (ARL2) expression were observed only two to four days after spray-inoculation. Whole transcriptome sequencing revealed that four days after inoculation, leaf transcriptional changes to colonisation by non-pathogenic and pathogenic bacteria differed in strength but not in the type of response. Inoculation of plants with different densities of the non-pathogenic bacterium Williamsia sp. Leaf354 showed that high bacterial titers caused disease phenotypes and led to severe transcriptional reprogramming with a strong focus on plant defence. An in silico epigenetic analysis of the data was congruent with the transcriptomic analysis. These findings suggest (1) that plant responses are not rapid after spray-inoculation, (2) that plant responses only differ in strength and (3) that plants respond to high titers of non-pathogenic bacteria with pathogen-like responses.

Leaves host remarkably diverse microbes, collectively referred to as the leaf microbiota. While many beneficial functions have been attributed to the plant microbiota, the individual contributions of leaf-colonising bacteria range from pathogenic to mutualistic interactions. Omics approaches demonstrated that some leaf-colonising bacteria evoke substantial changes in gene expression and metabolic profiles in the plant host, including plant immunity. While omic approaches provide a system level view on cellular functions, they are costly and laborious, thereby severely limiting the throughput of the number of bacterial strains that can be tested in planta. To enable cost-effective high-throughput screens, we have developed a plant protoplast-based assay to measure real-time target gene expression changes following bacterial inoculation. Here, protoplasts were isolated from leaves of stable transgenic plants containing a pPR1:eYFP-nls construct. Changes in yellow fluorescence were captured for up to 96 treatments using a plate reader. This allowed the monitoring of changes in the salicylic acid-dependent plant immune response over time. Protoplast isolation per se evoked mild fluorescence responses, likely linked to endogenous salicylic acid production. This is advantageous in a bacterial assay, as bidirectional changes in PR1 expression can be measured. Plate reader-generated data were validated via fluorescence microscopy and RT-qPCR. Fluorescence microscopy further demonstrated heterogeneity in the response of individual protoplasts, which is potentially linked to differences in cell-type. In summary, the protoplast assay is an affordable and easily up-scalable way of measuring changes in target gene expression to bacterial colonisation.