AbstractLeaves are colonized by a complex mix of microbes, termed the leaf microbiota. Even though the leaf microbiota is increasingly recognized 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‐colonizing bacteria. The transcriptomic changes in leaves were tracked over time and significant changes in ethylene marker (ARL2) expression were observed only 2–4 days after spray inoculation. Whole‐transcriptome sequencing revealed that 4 days after inoculation, leaf transcriptional changes to colonization by nonpathogenic and pathogenic bacteria differed in strength but not in the type of response. Inoculation of plants with different densities of the nonpathogenic bacterium Williamsia sp. Leaf354 showed that high bacterial titers resulted in disease phenotypes and led to severe transcriptional reprogramming with a strong focus on plant defense. 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 nonpathogenic bacteria with pathogen‐like responses.

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. Plain Language Summary Plants are colonised by diverse bacteria affecting many aspects of plant life. Here we show that plants do not differentiate between different bacteria but measure their quantities to keep bacterial numbers in check.

ABSTRACT 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.

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, otherwise sterile, on zeolite-clay, a soil replacement that retains enough moisture to avoid subsequent watering. Litterbox-grown plants resemble greenhouse-grown plants more closely than agar-grown plants and exhibit lower leaf epiphyte densities (106 cfu/g), reflecting natural conditions. A polydimethylsiloxane (PDMS) sheet was used to cover the zeolite, 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.

Bacteria establish complex, compositionally consistent communities on healthy leaves. Ecological processes such as dispersal, diversification, ecological drift, and selection as well as leaf surface physicochemistry and topology impact community assembly. Since the leaf surface is an oligotrophic environment, species interactions such as competition and cooperation may be major contributors to shape community structure. Furthermore, the plant immune system impacts on microbial community composition, as plant cells respond to bacterial molecules and shape their responses according to the mixture of molecules present. Such tunability of the plant immune network likely enables the plant host to differentiate between pathogenic and non-pathogenic colonisers, avoiding costly immune responses to non-pathogenic colonisers. Plant immune responses are either systemically distributed or locally confined, which in turn affects the colonisation pattern of the associated microbiota. However, how each of these factors impacts the bacterial community is unclear. To better understand this impact, bacterial communities need to be studied at a micrometre resolution, which is the scale that is relevant to the members of the community. Here, current insights into the driving factors influencing the assembly of leaf surface-colonising bacterial communities are discussed, with a special focus on plant host immunity as an emerging factor contributing to bacterial leaf colonisation.