Driving factors of epiphytic bacterial communities: A review
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Driving factors of epiphytic bacterial communities: A review. 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.
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Driving factors of epiphytic bacterial communities: A review. 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..
Journal of Advanced Research 19 (2019) 57–65
Contents lists available at ScienceDirect
Journal of Advanced Research
journal homepage: www.elsevier.com/locate/jare
Driving factors of epiphytic bacterial communities: A review Rudolf O. Schlechtera,b, Moritz Miebacha, Mitja N.P. Remus-Emsermanna,b,⇑
a School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
b Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand
h i g h l i g h t s g r a p h i c a l a b s t r a c t
The physicochemistry of leaves is unique and is a major driver of leaf colonisation.
Competition and cooperation may be major drivers of bacterial colonisation.
Leaves respond to bacterial colonisation locally and systemically.
How leaf responses shape bacterial colonisation patterns is unclear.
Plant-microbe interaction should be studied at the micrometer resolution.
a r t i c l e i n f o a b s t r a c t
Received 12 December 2018 Revised 12 March 2019 Accepted 13 March 2019 Available online 14 March 2019
Keywords: Assembly processes
Microbial communities Single cell Phyllosphere
Bacteria establish complex, compositionally consistent communities on healthy leaves. Ecological pro-cesses such as dispersal, diversiﬁcation, ecological drift, and selection as well as leaf surface physico-chemistry 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 con-ﬁned, 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.
2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer review under responsibility of Cairo University. ⇑ Corresponding author.
E-mail address: email@example.com (M.N.P. Remus-Em-
All the aboveground surfaces of a plant that represent microbial habitats are referred to as the phyllosphere . In particular, leaf
surfaces host a dense population of bacteria (i.e., epiphytes) esti-
2090-1232/ 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
58 R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65
mated to reach 107 bacteria per cm2 of leaf surface . Despite the
high cell density, leaf surfaces are a challenging ecosystem to colo-
charides, salts, lipids, volatile compounds, and proteins, the func-
tions of which are associated with plant-plant, plant-insect and
nise and grow on. Epiphytes must cope with constant ultraviolet plant-microbe interactions [18,19]. The functions of non-
(UV) radiation exposure, low water and nutrient availability and large temperature ﬂuctuations throughout the day, making leaves an extreme environment .
Recent culture-independent sequencing methods have shown that leaves host bacterial communities that are compositionally consistent within a plant species [4–6]. However, little is known about the factors that shape these communities. Although there is increasing evidence that non-pathogenic leaf-colonising bacteria may stimulate plant growth and provide protection against differ-ent stresses [7–14], the functions of most of these bacteria, their dynamics at the community level, and their interactions with the plant host remain largely unknown.
The leaf surface
The leaf is a highly structured and multi-layered plant organ (Fig. 1). Its microtopography is determined by the ﬁrst cell layer, namely, the epidermis, which consists of different cell types that regulate many aspects of leaf physiology, such as gas exchange, temperature regulation, and water and secondary metabolite secretion .
The most common cell type in the epidermis is the pavement cell, which contributes to leaf shape. Within the layer of pavement cells, more specialised epidermal cell types are embedded . Stomata, which are pores formed by two guard cells that act as turgor-driven valves to regulate gas exchange and transpiration, are an important feature of the epidermis . Some plants develop modiﬁed stomata called hydathodes, which are pores found at the end of the vasculature on leaf margins . Because these structures cannot regulate their pore aperture, hydathodes maintain a continuous pathway for water and solute secretion, a process known as guttation . Another type of specialised epi-dermal cell are outgrowths called trichomes, which are either glan-dular or non-glandular . Glandular trichomes are secretion
organs that release a wide spectrum of exudates, such as polysac-
glandular trichomes may include water retention and absorption, light reﬂection to reduce the impact of UV radiation and heat, and increased freezing as well as drought tolerance .
The epidermis is covered by a cuticle, i.e., a waxy layer that pro-vides a physical barrier against abiotic and biotic stresses and determines the physicochemical properties of the leaf surface. The cuticle is formed by an extracellular polymer membrane com-posed of a matrix of cross-linked polyhydroxy fatty acids and glyc-erol called cutin. This matrix is interspersed with polysaccharides and a complex mixture of long-chain aliphatic compounds, which are overlaid on and/or impregnated in the matrix (cuticular waxes) . Aliphatic compounds render the cuticle hydrophobic and determine the physicochemical properties of the leaf surface, such as its permeability and wettability, which limits water and solute diffusion from inner cell layers to the leaf surface and the adher-ence of particles to the surface [21–23].
Impact of leaf topology and physicochemistry on microbial life on leaves
Theorganisationofleafepidermal celltypesdeﬁnesleafphysiol-ogyandshapesanintricatemicrotopologythatinﬂuencesthedistri-bution and abundance of microorganisms on the leaf surface [24– 26]. The establishment of these microhabitats depends on the physicochemical properties of the leaf surface and the ability of microorganisms to adapt and modify this environment . Epi-phytesareoftenfoundinaggregatesorbioﬁlms,likelybecausethese microenvironmentsprotectbacteriafromharshenvironmentalcon-ditions[24,28].Largebacterialaggregateshavebeenpredominantly found at the bases of trichomes, above veins, and in epidermal cell grooves [29,30], where water and nutrients are more prevalent.
The permeability and wettability of the leaf cuticle is likely to be one of the most important properties of the leaf surface that inﬂuences the ability of microbes to colonise this habitat .
Cuticular permeability determines the diffusion rate of compounds
Fig. 1. (A) Representation of an Arabidopsis thaliana leaf and its main features. (B) Cross-section and (C) top-view representations of the leaf surface, including different epidermal cell types that make up the leaf’s surface relief.
R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65 59
from the apoplast onto the leaf surface, while wettability inﬂu-ences the retention of water droplets on the leaf surface [22,32].
Permeation plays an important role in the growth and survival of epiphytes by allowing the leaching of water and compounds to the phyllosphere, making nutrients accessible for microorganisms. An aqueous pathway contributes to compound permeation across the cuticle with facilitation from aqueous pores preferentially found on cuticular ledges of guard cells, at the base of trichomes, and over the cuticle of anticlinal cell walls . Sites on the leaf surface that are characterised by higher permeation rates are also more densely colonised by bacterial communities . Bacteria can modulate cuticular permeability and wettability through the production of biosurfactants such as syringafactin, which is released by Pseudomonas syringae [31,35,36]. Increased cuticular permeability not only affects water diffusion but also alters sugar availability for sustained epiphytic growth . In situ fructose availability to the leaf-colonising bacterium Pantoea eucalypti 299R (formerly known as Erwinia herbicola and Pantoea agglomer-ans [38,39]) in the bean phyllosphere was found in sites containing aqueous pores [32,40,41]. The patchy distribution of carbon sources on bean leaves promotes differentiation of the P. eucalypti population into subpopulations differing in access to fructose . Thus, permeation of photosynthates across the cuticle is exploited by epiphytic microorganisms, allowing them to survive and thrive. Besides modulation of leaf physicochemistry by phyllosphere-associated microbes, changes in leaf chemistry can also be attribu-ted to abiotic and biotic soil conditions [42–44]. However, the extent to which plant-soil feedbacks inﬂuence the assembly of phyllosphere microbial communities are yet to be determined.
The topography and physicochemical properties of leaves ren-der the phyllosphere an oligotrophic and heterogeneous habitat for epiphytes. It is also possible for microorganisms to construct niches on the leaf surface in response to interaction with the plant . Although discrete hotspots on the leaf form microhabitats in which bacterial populations can be sustained , the impact of these microhabitats on the assembly and establishment of bacte-rial communities is not yet understood.
Composition of phyllosphere-associated microbial communities
Advances in cultivation-independent methods and next-generation sequencing techniques have led to a better understand-ing of the composition and diversity of plant microbiota. Although plants host a wide variety of microbes, bacteria are far more abun-dant than eukaryotes and archaea [47,48]. Bacterial communities on plants are dominated by only four phyla: the Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes [1,49,50]. Although bacterial community composition and abundance are host speciﬁc, members of the Alphaproteobacteria are predominant and ubiqui-tous in phyllosphere microbiotas, and within this class, the genera Methylobacterium and Sphingomonas are consistently detected among different hosts . Plant-colonising bacteria are thought to share mechanisms of adaptation to leaf surfaces, considering the high overlap between the proteome of phyllosphere microbio-tas and the identiﬁcation of a core set of genes potentially involved in adaptation to plant colonisation in over 3000 plant-associated bacterial genomes [47,51,52].
Spatially explicit ecology of bacteria on leaves
Due to the leaf’s heterogeneous nature, the composition and abundance of bacterial communities at the whole-leaf scale are not sufﬁcient to understand the drivers of community assembly
. Therefore, the importance of spatial information becomes
increasingly apparent for understanding community structure (Fig. 2). In the example given in Fig. 2, the same community com-position on a whole-leaf scale can be explained by different inter-species correlations such as different levels of co-aggregation, segregation, or random distributions (Fig. 2C, D, or E, respectively). Fluorescence in situ hybridisation (FISH), a method commonly used to visualise and identify microorganisms in their environment, has been used to describe the distribution patterns of taxonomically different bacterial groups within the phyllosphere . This study, the ﬁrst investigation of the spatial distribution of phylogenetically different taxa colonising leaf surfaces under different environmen-tal conditions, estimated the likelihood of bacterial taxa co-aggregation in the Arabidopsis thaliana phyllosphere. Bacterial aggregates can be either monoclonal or polyclonal. Monoclonal aggregates represent the offspring of single cells, while polyclonal aggregates are formed by the aggregation of multiple cells in one location . Distantly related taxa can form mixed aggregates, even though members of the same phylogenetic group have the highest probability of co-aggregation. However, due to the techni-cal limitations of the FISH probes used in that study, it is unclear which individual species contribute to the observed aggregation patterns and whether co-aggregation of the same phylogenetic groups is a result of local monoclonal aggregate formation of an individual species or mixed populations. In general, aggregation between different taxa is observed at a distance of less than 5 mm. A similar approach taken to study the spatial distribution and colonisation patterns of two ﬂuorescently tagged bacterial strains (P. eucalypti 299R and P. syringae B728a) on bean leaves provided similar results . In an investigation of the spatial aggregation between the bacterial strains and topological features of the leaf, the strongest correlations were found between bacterial colonies and epidermal cell grooves within distances of up to 12 mm, and adjacent to glandular and non-glandular trichomes within 60 and 120 mm, respectively. Closer examinations of spatial relationships between bacterial species and their surroundings could shed light on the functional and metabolic diversity within aggregates and communities in the phyllosphere.
Individual bacterial cells can signiﬁcantly change the concen-tration of solutes, such as nutrients in their environments, within a distance of approximately ten times their cell’s diameter. This distance can be effectively interpreted as an ‘‘interaction distance”, where ﬂuxes of compounds and metabolites can diffuse from cell to cell . This interaction distance of cells without direct physi-cal contact is in good agreement with the observed scales of bacte-rial aggregation in the phyllosphere. Recently, cell-to-cell interactions on porous surfaces were found to be higher when the aqueous phase was fragmented, which increased the probabil-ity of direct physical interactions between cells . These ﬁndings can be extrapolated to the leaf surface, as its segregated nature should result in a high prevalence of cell-to-cell interactions . Evidently, community assembly on leaves is strongly determined by factors acting at a micrometre resolution .
Ecological drivers of bacterial community assembly
Recently, a framework of community assembly was proposed for microbial communities in the phyllosphere . The frame-work proposes that community structure is driven by four main processes: (1) dispersal, (2) diversiﬁcation, (3) ecological drift, and (4) selection (Fig. 2A). First, dispersal is the immigration of microorganisms onto the leaf surface, which can occur via seed inoculation, rainfall, animal transmission vectors, bud burst coloni-sation, and bioaerosols . Second, the emergence of new genetic variation through evolutionary diversiﬁcation may affect commu-
nity diversity. As UV radiation and/or reactive oxygen species can
60 R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65
Fig. 2. Relevance of spatial patterns in bacterial community structure on the leaf surface. (A) Considering a plant host with a deﬁned microbial composition and relative abundance of species 1 and 2, factors driving community structure, i.e., dispersal, diversiﬁcation, ecological drift, and selection, can lead to different aggregation patterns of these species while maintaining the same bacterial diversity and abundance. (B) Bacterial community of species 1 and 2 with a strong spatial co-aggregation pattern inﬂuenced by, for example, cooperation, resource partitioning, stochastic processes, and/or priority effects (niche modiﬁcation). (C) Bacterial community of species 1 and 2 with a strong spatial segregation pattern inﬂuenced by resource overlap-driven exploitation competition, antibiosis (interference competition), stochastic processes, and/or priority effects (niche preemption). (D) Bacterial community of species 1 and 2 showing a random distribution, which might indicate benign interactions between the species.
lead to increased mutation rates, low-abundance taxa might be a genetic reservoir for horizontal gene transfer; furthermore, bacte-ria can exhibit dormancy . Third, ecological drift relates to changes in the abundance of taxa due to stochastic events. This process is assumed to have greater effects on low-abundance taxa, which may become extinct at local scales . Lastly, selection is the deterministic ﬁtness differences between species within a community, which can be due to internal and external determi-nants, such as species interactions and environmental factors, respectively.
In general, the effect of microbial interactions in shaping com-munity structure can be divided into cooperation, parasitism and competition. Cooperation describes interactions that are beneﬁcial to at least one species and do not cause harm to the other, while the latter type refers to interactions that are detrimental to at least one species. Mutualism is a type of cooperative interaction in which both species beneﬁt from each other, while commensalism is an interaction between two organisms in which one partner ben-eﬁts while the other is not impacted. Microorganisms would be commensals when, for example, one microbe produces non-metabolisable substrates and/or growth factors that positively affect the commensal [60,61]. However, to be considered a true commensal, the organism should not inﬂuence their interaction partner at all, which although theoretically possible, is highly unli-kely in practice. The alternative term ‘‘tritagonist” has been pro-posed for these organisms instead . The effect of cooperative interactions in shaping the structure of phyllosphere bacterial communities has not yet been investigated. However, an example of cooperation has been observed using synthetic communities from the maize rhizosphere, in which the removal of a keystone species led to the collapse of other bacterial populations . Another kind of mutualism may occur between fast growing bacte-rial species and fungal pathogens infecting host plants, as the latter seemed to increase bacterial richness and diversity . However,
this depends on speciﬁc microbe-microbe interactions, as the fun-
gal and oomycete species Dioszegia sp. and Albugo sp., respectively, have been shown to decrease the bacterial species diversity of the A. thaliana leaf microbiota .
Parasitism in the phyllosphere is mostly driven by virus-bacterium interactions. Bacterium-infecting viruses (i.e., bacterio-phages or phages) are found in most (if not all) ecosystems and can alter community dynamics by inﬂuencing bacterial diversity, nutrient cycling, and species interactions . Phages impose strong selection on bacterial members of the leaf microbiota at a local scale and in short time periods, affecting the microbiota com-position [66,67].
Competitive relationships involve detrimental effects for at least one species, which may be a result of interference or exploita-tion competition. When competition is the result of active mecha-nisms of species exclusion, this interaction is known as interference. The most common example of interference is antibio-sis, in which a species secretes compounds that are toxic to the other. This effect is also the case for epiphytes; for example, P. agglomerans E325 has been shown to suppress the growth of the phytopathogen Erwinia amylovora on apple ﬂowers through antibi-otic activity . Exploitation competition is the dispute for shared resources, such as nutrients or space. In this relationship, one of the species has compromised population growth, resulting in either complete spatial exclusion or coexistence . Fructose and sucrose, for example, are limiting resources during phyllo-sphere colonisation . In bean leaves, the amount of available sugars on the leaf surface decreases rapidly to a tenth of the initial concentration upon colonisation of Pseudomonas ﬂuorescens A506 . The availability of sugars is also limited by its permeation from the apoplast to the leaf surface, and sugars may not be replen-ished at rates supporting the survival of large bacterial populations .
Effect of resource overlap on species interactions
Cooperative and competitive interactions greatly depend on the metabolic capabilities of the members of a microbial community,
that is, the potential of the microbes to uptake, metabolise, and
R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65 61
secrete an array of nutrients and other compounds (e.g., sidero-phores or antimicrobials). The degree of shared nutritional require-ments between species, i.e., nutrient or resource overlap, may shape microbial communities, as shown by the fact that nutrient overlaps in naturally occurring bacterial communities are higher than expected based on null models . When estimating nutri-ent overlaps between over 6000 pair of bacterial species, Freilich et al. (2011) found a positive correlation with the competition potential between species in a community and negatively with the cooperation potential, suggesting that the higher the nutrient overlap between the species in a community is, the more likely these species are to compete for shared nutrients and, conse-quently, the less likely they are to cooperate with each other . However, nutrient overlaps cannot solely predict the nature
of species interactions e.g. it has recently been shown that close
community [80,85], thereby indicating an active role of the host plant in modulating the composition of its associated microbiota.
The plant immune system consists of two layers. The ﬁrst layer of immunity, pattern-triggered immunity (PTI), is elicited by con-served molecular structures such as microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) or damage-associated molec-ular patterns, which are perceived by plasma membrane-localised pattern recognition receptors [86,87]. A prominent example of pat-tern recognition receptors is the receptor ﬂagellin sensing 2 (FLS2), which recognises a 22-amino-acid peptide of bacterial ﬂagellin (ﬂg22) . The signiﬁcance of MAMP/PAMP recognition in limit-ing pathogen growth was shown in a ﬂs2 mutant exhibiting enhanced susceptibility to the bacterial pathogen P. syringae pathovar (pv.) tomato DC3000 (Pst DC3000) . Recently, com-
parison of plant responses to the growth-promoting rhizobac-
relatedness and similarity in gene expression between pairs of terium Pseudomonas simiae WCS417 and its cognate ﬂg22
algal species, and thus similarity in their metabolisms, was corre-lated to facilitation, stabilising coexistence . In addition, other factors such as priority effects can inﬂuence species interactions through, for example, niche preemptions or niche modiﬁcations . Resource utilisation accounts for only a fraction of a species’ niche; thus, the spatial context, environmental conditions, and bio-tic relationships should be considered when positioning species in their ecosystems.
An operational approach for comparing species’ nutrient utilisa-tion proﬁles is to determine a resource overlap index between pairs of species. In phyllosphere studies, Wilson and Lindow intro-duced the niche overlap index in 1994 , which relates to the number of carbon sources utilised by two species as a proportion of the total carbon sources used by one of the species. Two issues arise from this formulation: (1) the index is asymmetrical, such that one species can have total overlap while the other only partial overlap, as is the case for specialist and generalist species, respec-tively; and (2) by measuring only the ability of a strain to consume a carbon source independently, information is lost about the spe-cies’ afﬁnity for that resource, which may lead to false interpreta-tion of the potential for two species to coexist based on their nutrient preferences. Instead, symmetrical indices that account for the proportions of utilised nutrients are more informative and less biased .
The major drivers of community assembly in the phyllosphere are currently unclear. As the phyllosphere is generally oligotrophic, cooperation and competition may have a major impact on commu-nity assemblies in the phyllosphere.
The host as a driver of bacterial community structure
Plants provide habitats that support different bacterial commu-nities [4,76]. Although bacteria share a core set of adaptation mechanisms to colonise and thrive on plants [4,47,51], community composition is, to some degree, host speciﬁc [6,76–79]; thus, changes in bacterial diversity, abundance, and community struc-ture can be attributed to host factors and speciﬁc bacterial adapta-tion mechanisms. For example, bacterial community composition is inﬂuenced by host species, genotype, plant traits (e.g., cuticle composition), leaf age and host developmental stage; the latter two factors are often indistinguishable from seasonal effects [25,79–84].
Plant responses to microbial colonisation
In addition to providing a habitat for microorganisms, plants interact with their associated microbiota. Recent ﬁndings suggest
that the plant immune system plays a role in shaping the bacterial
peptide showed that the whole organism elicited only about half of the plant transcriptional responses compared to the puriﬁed ﬂg22 peptide alone. Genes that were only upregulated in the ﬂg22-treated plants were enriched for defence-related transcrip-tional responses, suggesting that P. simiae WCS417 suppressed a signiﬁcant number of defence-associated genes . Whether plants recognise non-pathogenic bacteria and modulate their response to limit unnecessary costly defence are still unknown.
To subvert PTI, microbial pathogens release so-called effector proteins . Effector proteins from different microbes target overlapping sets of plant proteins. Most of the targeted plant pro-teins are considered hubs of highly interconnected protein-protein interaction networks. Hence, targeting these proteins will likely result in strong perturbations of the host’s immune response [92,93]. As a countermeasure, plants have evolved additional intra-cellular receptors, which are often nucleotide-binding leucine-rich repeat proteins (NLRs), that directly or indirectly recognise effector proteins, thus forming the second layer of plant immunity, desig-nated effector-triggered immunity (ETI) [94,95]. Recently, a study on microbial genes important for adaptation to the plant environ-ment identiﬁed 64 plant-resembling plant-associated and root-associated domains (PREPARADOs). Some PREPARADOs resemble NLR domains. This ﬁnding suggests that bacterial proteins contain-ing PREPARADOs might compete with NLRs for effector binding, thereby restricting bacterial detection by the plant .
Plant responses are ﬁnely tuned
Although the same signaling networks seem to be employed during PTI and ETI, they are used in a different manner, generating speciﬁc outputs of immunity level. While the relationships between different signaling pathways in PTI are partly synergistic and partly compensatory, they are exclusively compensatory dur-ing ETI [96,97]. As MAMPs can be found on pathogenic as well as non-pathogenic bacteria, it seems reasonable that PTI is less robust or more tunable than ETI, allowing the host to avoid recurrent ﬁt-ness costs . Synergism between signaling pathways enables the plant to elevate the output of its immune response when multiple MAMPs, providing more information than a single MAMP, signal a pathogen.
Different MAMPs have been shown to activate different immune signaling pathways with varying strengths, leading to diverse immune outputs (Fig. 3) . Such differential use of the immune signaling network likely allows plants to induce appropri-ate defence mechanisms against pathogens with different lifestyles during PTI. For example, salicylic acid signaling is known to be effective against biotrophs, pathogens that feed on living host tis-sue, and hemibiotrophs, pathogens that ﬁrst feed on living host tis-sue and later feed on dead host tissue. In contrast, jasmonic acid
and ethylene signaling is effective against necrotrophs, pathogens
62 R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65
Fig. 3. Plant responses to bacterial colonisation. Leaf-colonising bacteria elicit local and systemic responses. As shown on the left-hand side, a cell type-speciﬁc response to prevent bacteria from entering the apoplast is stomatal closure. As shown on the right-hand side, plants perceive bacteria via receptors localised in the plasma membrane (A,
B, C, D), which recognise microbe-associated molecular patterns (depicted by hexagons, ovals and stars around the bacterium). Downstream signaling of these receptors is integrated in a highly interconnected immune signaling network. Integration of varying receptor inputs leads to speciﬁc immune outputs (a, b, c).
that feed on dead host tissue . Furthermore, tunability of the immune network presumably enables plants to limit costly immune responses to non-pathogenic colonisers.
Recently, two non-pathogenic bacteria were shown to elicit unique transcriptional and metabolic responses that differed from those of a pathogenic bacterium, indicating that plants differenti-ate bacteria [7,101]. Since the availability of carbohydrates clearly affects bacterial community composition [102,103] and plants actively deprive the apoplast of monosaccharides upon pathogen encounter to limit pathogen growth , plants likely also supply
certain carbohydrates by sugar exporters  to support beneﬁ-
ferently to microorganisms. In a recent study, local responses to the biotrophic pathogen Hyaloperonospora arabidopsis were shown to differ markedly from systemic responses . This ﬁnding highlights the importance of performing -omic studies at a high spatial (potentially single-host cell) resolution to identify the nat-ure of plant responses. To perform cell type-speciﬁc and single-cell studies, the cell type of interest must be isolated from the sur-rounding tissue. Root hairs are likely the simplest cell type to iso-late as they can be separated from frozen root tips by stirring with a glass rod . Other cell types demand more sophisticated tech-
niques for isolation; these types can be divided into two groups.
cial bacterial populations. Such responses are likely to be spatially Microscopy-assisted techniques such as capillary extraction
explicit, as the bacterial composition on the leaf is heterogeneous and the same carbohydrate might promote the growth of spatially separated populations of beneﬁcial and non-beneﬁcial bacteria. However, plant responses at a high spatial resolution are currently underinvestigated in the context of plant-microbe interactions.
Local plant responses may have important implications for leaf
, atomic force microscopy-based extraction  and laser capture microdissection  allow the isolation of cells or cell material with direct spatial context. With regard to plant-microorganism interactions, capillary extraction was used to study transcriptional changes of barley cells infected by powdery mildew
. Cell/nuclei sorting or afﬁnity puriﬁcation methods such as
surface-colonising bacteria and bacterial community composition. ﬂuorescence-activated cell sorting of protoplasts ,
Local and systemic plant host responses to microorganisms
Plant immune responses to pathogens can be divided into local and systemic responses (Fig. 3). Local immune responses at the infection site comprise early calcium ion (Ca2+) inﬂux and MAP kinase and calcium-dependent protein kinase activation within minutes, followed by reactive oxygen species production, defence gene activation and after several hours, callose deposition and hypersensitivity response resulting in programmed cell death, which is regarded as a hallmark of ETI . A cell type-speciﬁc local response is stomatal closure, which occurs within an hour after bacterial recognition to restrict the bacteria from entering the apoplast (Fig. 3). However, some bacterial pathogens, such as Pst DC3000, are able to modulate stomatal aperture . In addi-tion to local responses, pathogens also elicit systemic responses, such as systemic acquired resistance, conferring broad spectrum resistance against biotrophic pathogens to plant parts that have not been in contact with the pathogen . Recently, the impres-sively fast propagation of systemic Ca2+ signaling throughout the plant in A. thaliana (approximately 1 mm/s) was shown to be medi-ated by glutamate, which is released upon wounding .
Studying plant epigenetic, transcriptional, proteomic, and meta-bolomic changes in microorganisms at the whole-tissue level does not allow us to study local and systemic responses separately.
Moreover, tissues are mixtures of different cell types that react dif-
ﬂuorescence-activated nuclei sorting , isolation of nuclei tagged in speciﬁc cell types , and ‘‘translating ribosome afﬁn-ity puriﬁcation” (TRAP)  allow the isolation of larger numbers of cells or more cell material. However, the spatial information is only indirect, and the techniques rely on cell type-speciﬁc markers. Plants shape their associated microbiotas. Consequently, plants need to be considered as a driving factor of community composi-tion in bacterial ecology studies. To study the role of the plant host in shaping microbiota, new controls must be found that distinguish between fast active plant signaling and comparably slow changes in physicochemical properties of leaves. To that end, studies can be performed on artiﬁcial plants, ranging from plastic tomato plants in a ﬁeld context  to artiﬁcial leaves in the laboratory [120–123]. Such studies should progress to a more reﬁned spatial scale, as bacterial colonisation is heterogeneous, plant responses
are likely also heterogenous.
Conclusions and future perspectives
The relatively static physicochemistry of the leaf surface as well as dynamic microbe-microbe, microbe-plant and plant-mediated microbe-microbe interactions are drivers of plant microbiota com-munity composition. These factors do not homogeneously inﬂu-ence the bacterial communities on leaves; instead, there might
be markedly different communities on the same leaf. Such spatial
R.O. Schlechter et al./Journal of Advanced Research 19 (2019) 57–65 63
heterogeneity of leaf-colonising microbial communities is not only due to the heterogeneity of the leaf surface and heterogeneous microbial colonisation but also likely due to plant responses that may differ between individual cells of the same leaf. Since the plant immune network is highly tunable, it presumably enables plants to limit costly immune responses to non-pathogenic colonisers, and on a more reﬁned spatial scale to respond differ-ently to distinct microbial colonisers. Heterogeneous colonisation patterns are thus likely to translate into heterogeneous plant response patterns.
In the future, more emphasis should be placed on spatial heterogeneous differences in colonisation and plant responses to further our understanding of the underlying mechanisms that drive bacterial community composition and assembly. By resolving (1) the effect of microbe-microbe interactions, (2) the function of non-pathogenic bacteria on the plant and (3) the role of the host in shaping community structure, will give bottom-up insights into the intimate interplay of plant hosts and their bacterial communities.
Conﬂict of interest
The authors have declared no conﬂict of interest.
Compliance with Ethics requirements
This article does not contain any studies with human or animal subjects.
This work was funded by the Royal Society of New Zealand Marsden Fast Start grant (UOC1704) to MR-E. RS was supported by a New Zealand International Doctoral Research Scholarship, and MM was supported by a University of Canterbury Doctoral Scholarship.
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