Long-Term Consequences of PTI Activation and Its Manipulation by Root-Associated Microbiota

Abstract

In nature, plants are constantly colonized by a massive diversity of microbes engaged in mutualistic, pathogenic or commensal relationships with the host. Molecular patterns present in these microbes activate pattern-triggered immunity (PTI), which detects microbes in the apoplast or at the tissue surface. Whether and how PTI distinguishes among soil-borne pathogens, opportunistic pathogens, and commensal microbes within the soil microbiota remains unclear. PTI is a multimodal series of molecular events initiated by pattern perception, such as Ca²⁺ influx, reactive oxygen burst, and extensive transcriptional and metabolic reprogramming. These short-term responses may manifest within minutes to hours, while the long-term consequences of chronic PTI activation persist for days to weeks. Chronic activation of PTI is detrimental to plant growth, so plants need to coordinate growth and defense depending on the surrounding biotic and abiotic environments. Recent studies have demonstrated that root-associated commensal microbes can activate or suppress immune responses to variable extents, clearly pointing to the role of PTI in root–microbiota interactions. However, the molecular mechanisms by which root commensals interfere with root immunity and root immunity modulates microbial behavior remain largely elusive. Here, with a focus on the difference between short-term and long-term PTI responses, we summarize what is known about microbial interference with host PTI, especially in the context of root microbiota. We emphasize some missing pieces that remain to be characterized to promote the ultimate understanding of the role of plant immunity in root–microbiota interactions.

Introduction

Plants are in intimate association with a wide variety of microbes, including serious pathogens that can trigger devastating plant diseases. Plants have therefore acquired a sophisticated molecular system known as plant immunity to cope with these pathogens and secure plant growth. Pathogen-induced plant diseases have a severe impact on the economy, and more than a century of effort has been invested toward the ultimate understanding of plant–pathogen interactions—ever since the discovery of Phytophthora infestans by Heinrich Anton de Bary in 1861 as the virulent agent of potato late blight (de Bary 1861). To date, researchers have revealed a highly robust and tunable molecular network that enables plants to fight against pathogens, securing their survival under highly demanding natural conditions. For instance, a group of receptor proteins located on the plasma membrane (cell surface) operates as the first layer of a surveillance system that recognizes certain molecular patterns shared among a wide range of microbes (microbe-associated molecular patterns; MAMPs), such as the flg22 peptide derived from a bacterial flagellar protein (Boutrot and Zipfel 2017). The second layer is mediated by another group of proteins localized in the cytoplasm, namely, nucleotide-binding oligomerization domain-like receptors (NLRs). Microbial effector proteins that are directly injected into host cells are recognized by NLRs, which then induce strong and sustained immune responses, such as cell death (Saur et al. 2021). Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) not only constitute two essential layers of defense (Jones and Dangl 2006) but also potentiate or antagonize each other in the broader framework of plant innate immunity (Hatsugai et al. 2017, Ngou et al. 2021, Pruitt et al. 2021, Tian et al. 2021, Yuan et al. 2021).

Studies of beneficial microbes have also been pursued extensively. Nitrogen-fixing nodule symbiosis was discovered by Martinus Willem Beijerinck in 1888 (Beijerinck 1888), while arbuscular mycorrhization was described as early as 1842 by Karl Wilhelm von Nägeli (Nägeli 1842). We now know that arbuscular mycorrhizal fungi and rhizobia, the microbial partners in mycorrhization of a wide range of plants and nodule symbiosis with legumes, respectively, exploit overlapping sets of host pathways to establish mutual engagements with plants (Oldroyd 2013). More recently, there has been a focus on microbes that substantially colonize plant tissue but are not obviously or explicitly pathogenic or beneficial, referred to as commensal microbes. The plant-associated microbial community composed of a mixture of these microbes in different lifestyles is known as the plant microbiota. Many of these non-pathogenic microbes also possess MAMPs that potentially induce plant immunity, raising the question of how plant immunity distinguishes pathogenic and non-pathogenic microbes within the natural microbial community.

Over the last decade, a number of independent culture collections composed of microbes isolated from healthy-looking plants (i.e. the constituents of the plant microbiota) have been established, enabling experimental testing of hypotheses about plant–microbiota interactions. Interestingly, multiple independent groups reported that plant microbiota members interfere with host PTI responses (Garrido-Oter et al. 2018; Yu et al. 2019a, Ma et al. 2021, Teixeira et al. 2021, Pfeilmeier et al. 2023, Entila et al. 2024), illustrating the crucial role of plant immunity at the plant–microbiota interface. Revealing and manipulating the molecular systems by which commensal microbes interfere with host immunity will provide key insights into how plant immunity regulates interactions with microbiota. However, we noted that some studies focused on the PTI responses that occur in minutes to hours (the ‘short-term’ responses) as their readout, while others monitored the long-term consequence of PTI activation, typically over days to weeks. This inconsistency among studies makes it challenging to understand the overall molecular framework. In this review, we first summarize what is known about short-term and long-term PTI responses, highlighting their differences and commonalities. We then review past studies of how microbes with different lifestyles manipulate host immune responses, which helps identify the missing pieces needed to draw an integrated framework. Lastly, we propose several future perspectives on how we can fill the gaps in our understanding of the role of plant immunity in root–microbiota interactions. Although we cite as many original articles as possible, due to space constraints, we occasionally cite excellent reviews that provide the details. Of note, we primarily focus on studies using Arabidopsis thaliana as the host species. It is widely appreciated that the molecular framework underlying plant immunity is highly diversified across plant species (Schornack and Kamoun 2023), so putting other plant species on the same table would not help to fill the gaps but would only increase the number of missing pieces in the bigger picture we aim to draw. Nevertheless, it is more than likely than what we highlight, namely, the difference between short-term and long-term PTI responses and how PTI might regulate plant–microbiota interactions, is conceptually applicable to many different plant species.

Early Events in PTI

PTI is the first layer of the surveillance system. Perception of MAMPs by cell surface pattern recognition receptors (PRRs) often leads to the recruitment of their co-receptor kinases and the formation of heteromeric receptor complexes. These so-called receptor complexes then transfer the information from the extracellular space to the receptor-like cytoplasmic kinases in the cytoplasm. For instance, flg22, a well-characterized MAMP derived from the bacterial flagellar apparatus, is recognized by its cognate receptor FLAGELLIN SENSING 2 (FLS2) that forms a receptor complex with BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). The kinase activity of BAK1 then triggers a cascade of downstream phosphorylation reactions, including but not limited to phosphorylation of BOTRYTIS-INDUCED KINASE 1 (BIK1), a receptor-like cytoplasmic kinase (RLCK subfamily) and the MITOGEN-ACTIVATED PROTEIN KINASE (MAP kinase) cascade. Other MAMPs, such as chitin, elf18 peptide derived from EF-Tu, and peptidoglycans, are perceived by other cognate PRRs (Boutrot and Zipfel 2017) and trigger similar immune reactions, collectively called PTI.

PTI reactions occur in a very short time frame after MAMP perception (Fig. 1). For instance, the production of reactive oxygen species (ROS), known as the ROS burst, and the increase of cytosolic free Ca²⁺ can be detected within a few minutes and reach a peak no later than 30–40 min after plants are treated with flg22 or other MAMPs. These rapid responses, mutually facilitating each other (Tian et al. 2019), are mediated by the NADPH oxidase respiratory burst oxidase homologues D (RbohD) and the gated calcium channels cyclic nucleotide-gated channel (CNGC) 2 and 4, respectively, in A. thaliana leaves. All these proteins are direct targets of the BIK1 kinase (Kadota et al. 2014, Li et al. 2014, Tian et al. 2019). The FLS2/BAK1-triggered phospho-cascade also phosphorylates MITOGEN-ACTIVATED PROTEIN KINASES (MAP kinases) within 5–10 min after MAMP perception and triggers a dynamic transcriptional reprogramming that occurs over minutes to hours. A recent study revealed an immediate-early transcriptional response (within 5–10 min) that is congruent among a wide range of MAMP stimuli (Bjornson et al. 2021), most likely triggered by rapid removal of transcriptional repressors (Jacob et al. 2018). This is followed by further transcriptional responses occurring within hours (30–180 min) in a diverse manner depending on the MAMP species. This transcriptional reprogramming ultimately alters the metabolic status of plants, resulting in increased production of bioactive secondary metabolites, such as phytohormones and phytoalexins, typically recorded a few days after MAMP treatment. POWDERY MILDEW RESISTANCE 4 (PMR4) is among the genes induced during PTI and is responsible for the deposition of beta-1,3-glucan, also known as callose, which occurs 12–24 h after PTI activation (Vogel and Somerville 2000) and is dependent on the MAP kinase cascade (Ostergaard et al. 2002).

Notably, the early PTI responses are not a linear succession but a dynamic multimodal and multifactorial wave of different molecular events: After the first rapid production of ROS, another ROS burst initiates in 2 h and continues for up to 8 h. When ETI is simultaneously induced with PTI, a third ROS burst is detectable in 6–16 h (Ngou et al. 2021). A sharp increase of [Ca²⁺]_cyt is also known to continue and repeat periodically (Kwaaitaal et al. 2011, Ranf et al. 2011, Keinath et al. 2015). In contrast, MAPK phosphorylation is very rapid and transient under PTI conditions partly due to active dephosphorylation by HIGHLY ABA-INDUCED phosphatases in a manner dependent on abscisic acid (Mine et al. 2017). Likewise, PTI triggered by elf18, another peptide MAMP derived from bacterial elongation factor EF-Tu, appears to have two independent phases that are equally important for resistance against pathogens (Lu et al. 2009). Moreover, it has been reported that flg22 peptide variants with diverse amino acid sequences triggered distinctive cellular responses (Vetter et al. 2016), even when perceived by the same FLS2 receptor protein (Colaianni et al. 2021, Parys et al. 2021). These studies, along with many others, illustrate that PTI is not a homogenous response. Rather, it is a complex regulatory circuit that is heterogeneous in terms of its spatiotemporal dynamics as well as its molecular orchestration.

Long-Term Consequences of PTI

Compared to the detailed knowledge of the early PTI responses, it remains less well understood how these molecular responses persist and change over longer periods (from days to weeks). Here, in this review, we define ‘short-term response’ as the responses that occur within the first 3–4 d, typically evaluated by molecular responses such as ROS burst, calcium influx, and MAPK phosphorylation. ‘Long-term consequence’ is defined as the phenotypes observed after chronic exposure to MAMPs for weeks, mainly monitored by macroscopic plant growth phenotypes (Fig. 1). Pattern-triggered growth inhibition is among the well-established long-term consequences of chronic exposure to peptide MAMPs, such as flg22 and elf18 (Gomez-Gomez et al. 1999, Zipfel et al. 2006). Interestingly, the cognate receptors FLS2 and EFR are rapidly (within 30–40 min) internalized and degraded in an endocytosis-dependent manner (Robatzek et al. 2006, Mbengue et al. 2016), and plants become insensitive to flg22 after FLS2 degradation until this protein is de novo synthesized again after 3–4 h (Smith et al. 2014). This leads to the question of how these receptor proteins behave after days or weeks of chronic exposure. It is even unclear to what extent the short-term responses persist after weeks of exposure. Many short-term responses are known to diminish gradually after hours of treatment, but the responses after days or weeks of treatment remain largely uncharacterized.

The pattern-triggered growth inhibition is often explained by the ‘growth–defense trade-off’, which is a conceptual idea according to which plants need to prioritize growth or defense depending on the surrounding environments (Monson et al. 2022). Resource limitation has been believed to be one of the direct causes of this trade-off; however, an increasing body of evidence demonstrates that the growth–defense trade-off cannot be solely explained by resource limitation but is a consequence of active coordination between two independent pathways (Kliebenstein 2016, He et al. 2022). Of note, while being almost the only phenotypic consequence of long-term PTI activation reported thus far, experimental evidence that explains MAMP-triggered growth inhibition at the molecular level remains limited (Fig. 2). One of the few examples is Brassinosteroids (BRs), a group of polyhydroxylated steroid phytohormones that regulate multiple aspects of plant growth and development (Clouse and Sasse 1998, Wang et al. 2022a), which have been suggested to play a key role in the growth–defense trade-off. The flg22 MAMP receptor FLS2 and the BR receptor BRI1 form hetero-dimeric receptor complexes with BAK1 (Nam and Li 2002, Chinchilla et al. 2007) and BR responses negatively affect short-term PTI responses (Albrecht et al. 2012, Belkhadir et al. 2012, Lozano-Duran et al. 2013). These findings suggested a role for BRs in coordinating the growth–defense trade-off (Huot et al. 2014), although a number of unanswered questions remain about the underlying molecular mechanisms (Ortiz-Morea et al. 2020). For instance, while BRs negatively regulate PTI responses, whether and to what extent PTI responses require the BR pathway remains unclear. Besides BRs, many other phytohormones, such as auxin, ethylene, gibberellins and cytokinins, have been proposed as key factors in the growth–defense trade-off (Huot et al. 2014, He et al. 2022, Monson et al. 2022), while no direct evidence has been provided to support the indispensability of the synthesis of or responses to these hormones for pattern-triggered growth inhibition (Fig. 2). This gap can be explained by the fact that the effects of phytohormones on immunity are usually evaluated using the short-term responses as the readout, implying the existence of an additional layer of the trade-off affecting PTI-triggered growth inhibition. Overall, despite a number of studies describing factors involved in both growth and defense, likely operating as parts of the signal integrator, the precise molecular mechanisms by which MAMP chronic exposure results in severe restriction of root and shoot growth remain unknown. Given the heterogeneity and the diversity of short-term PTI responses, we call for more appreciation of the fact that the molecular mechanisms that mediate MAMP-triggered growth inhibition, i.e. the molecular processes that translate chronic PTI inputs into active attenuation of growth, remain almost entirely unknown. Disentangling these processes is crucial for understanding plant growth and immunity under natural conditions, where plants are chronically exposed to the huge repertoire of MAMPs in soils.

Microbial Interference with Immune Responses

One of the key aspects of plant innate immunity is its vulnerability to microbial counteraction (Fig. 3). Many pathogenic bacteria suppress plant immune responses, as do symbiotic microbes such as arbuscular mycorrhizal fungi and nodule-forming rhizobia (Cao et al. 2017). It has also become evident that commensal bacteria, i.e. bacteria that constitute plant microbiota, affect host immune pathways in different manners in both directions (Garrido-Oter et al. 2018, Yu et al. 2019a, Hucklenbroich et al. 2021, Ma et al. 2021, Teixeira et al. 2021). Yet, despite their ability to manipulate it, plant-associated microbes are still significantly influenced by plant innate immunity irrespective of their lifestyles (Nobori et al. 2018, Tzipilevich et al. 2021). This leads to a model according to which plant immunity is not a one-dimensional mechanism to restrict microbial invasion/colonization but rather a dynamic molecular system that enables plants to manage how they interact with each microbial unit, fine-tuning immune activities in a spatiotemporally regulated manner (Hacquard et al. 2017). The context-dependent spatiotemporal heterogeneity of plant immunity was elegantly shown by a pair of recent studies (Nobori et al. 2023, Zhu et al. 2023), while that of root immunity remains elusive. Here, in this section, we first give an overview of the molecular mechanisms by which pathogenic and symbiotic bacteria interfere with host immunity while leaving the details to other excellent review articles (Jones and Dangl 2006, Gourion et al. 2015, Cao et al. 2017, Yu et al. 2019b, Wang et al. 2022b). We then summarize what has been described about how commensal microbes interfere with host immunity, mostly focusing on the root–soil interface.

Pathogens and Symbionts Interfere with Short-Term Immune Responses

Recognition of bacterial flagellin via FLS2 mounts an effective immunity against pathogenic bacteria, and pathogens have evolved multiple mechanisms to evade this recognition (Sanguankiattichai et al. 2022). These include diversifying the amino acid sequence of their flagellin proteins (Vetter et al. 2016, Parys et al. 2021) and secreting extracellular enzymes to degrade free epitopes in the host apoplast (Pel et al. 2014), constituting the first layer of bacterial counteraction against plant immunity. When the MAMP repertoire fails to evade the recognition and initiates PTI, the pathogens try to suppress downstream PTI components by, for instance, producing metabolic cocktails with diverse molecular functions. For example, Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine and auxin to mimic the phytohormone jasmonic acid isoleucine (JA-Ile) (Zhao et al. 2003) and directly activate the auxin pathway (McClerklin et al. 2018), respectively. These activities inhibit the full execution of PTI that is dependent on the salicylic acid pathway. Phevamine A and exopolysaccharide (EPS) are also known as extracellular virulence factors. Phevamine A is a conjugate of phenylalanine, valine and amidino-spermidine that suppresses PTI potentiation by spermidine and l-arginine, possibly in an antagonistic manner (O’Neill et al. 2018). EPS chelates calcium ions to suppress the calcium influx needed for PTI activation (Aslam et al. 2008). Beyond this apoplastic battlefield, the intracellular injection of effectors by type III secretion systems (T3SS) becomes crucial, as has been well demonstrated (Buttner and He 2009). It is now widely recognized that the T3SS is among the most important factors for bacterial virulence, given the highly sophisticated mechanisms plants use to recognize these type III effectors with NLRs that trigger ETI.

In contrast to the clear immunosuppressive capacity of pathogens, how rhizobia overcome host immunity is less well understood. These nodule symbionts initially trigger immune responses in the host, as do pathogens. However, these responses diminish quickly as the symbiotic process proceeds. For example, transient induction of immune-related gene expression has been observed in Lotus japonicus, Medicago truncatula, and Glycine max in interactions with their cognate symbionts (Kouchi et al. 2004, Libault et al. 2010, Lopez-Gomez et al. 2012). It is also known that alfalfa (Medicago sativa) roots transiently produce ROS when inoculated with their symbiont Sinorhizobium meliloti (Santos et al. 2001). Interestingly, this ROS production appears to play a positive role in nodule symbiosis, as its removal by overexpressing a catalase in rhizobia or by inactivating the NADPH oxidase RbohA in M. truncatula resulted in reduced nodule formation (Jamet et al. 2007, Marino et al. 2011). These studies imply that ROS may not necessarily be an agent explicitly restricting microbial colonization but rather an active regulator of microbial behavior. Some rhizobial molecules have been shown to suppress host immunity, including lipopolysaccharide (LPS), a group of membrane-bound polysaccharides important for legume–rhizobia symbiosis. Sinorhizobium meliloti LPS suppresses MAMP-induced defense gene expression in M. truncatula suspension culture cells (Tellstrom et al. 2007), although whether this activity is associated with its importance for symbiosis remains unclear. On the other hand, EPS, a free-form extracellular polysaccharide that initiates symbiotic engagement (Cheng and Walker 1998, Kawaharada et al. 2015), is required for maintaining a high level of root immunity at the early stage of symbiosis (Jones et al. 2008). A previous study reported that MAMP responses in A. thaliana leaves are suppressed by co-treatment with nod factor (Liang et al. 2013), the primary factor that engages legumes and rhizobia to initiate the symbiosis (Oldroyd et al. 2011). Interestingly, similar to pathogens, many symbiotic rhizobia possess type III or type IV secretion systems that deliver effector proteins such as NopP and Bel2-5, although the roles of these proteins appear to be in specifying host range rather than dampening host immunity (Okazaki et al. 2013, Nelson and Sadowsky 2015, Sugawara et al. 2018, Ratu et al. 2021).

Overall, pathogens and symbionts are capable of weakening or dampening PTI both before and after physical attachment to the host cells, deploying arsenals of extracellular and intracellular molecules in a very short time frame (in minutes to hours for pathogens and in a day for symbionts) (Fig. 3). On the other hand, we emphasize that although much is known about how short-term immune events are suppressed by pathogens and symbionts, little is known about how they interact with long-term responses, perhaps because the pathogenic and symbiotic engagements are typically established within a few days.

Commensals Interfere with Immune Responses

Our understanding of the role of plant immunity in plant–commensal interactions is still in its infancy (Hacquard et al. 2017). It is probable that commensal bacteria also possess a plethora of MAMPs and induce immune responses, similar to pathogens and symbionts. In fact, more than 30% of flg22 sequences found in commensal bacterial genomes induced plant immune responses to various extents (Colaianni et al. 2021), and the commensals often induce immune responses in A. thaliana roots when they are applied as heat-killed MAMP cocktails (Garrido-Oter et al. 2018) or as a synthetic community (Wippel et al. 2021). Of note, it is frequently the case that commensal bacteria, whose heat-killed cell debris induces immune responses, do not activate immune signaling pathways when inoculated as viable bacterial cells (Millet et al. 2010, Garrido-Oter et al. 2018). Also, many root-associated bacteria suppress short-term induction of immune marker genes by flg22 (Millet et al. 2010, Yu et al. 2019a, Teixeira et al. 2021, Liu et al. 2023). These observations point to the presence of mechanisms by which commensals actively alleviate immune elicitation. Moreover, a series of recent studies have provided experimental evidence that commensal bacteria can suppress MAMP-triggered growth inhibition after chronic exposure for weeks (Garrido-Oter et al. 2018, Ma et al. 2021, Teixeira et al. 2021). On the other hand, a large-scale meta-analysis of public RNA-seq experiments showed that commensal microbes also induce the expression of a group of immune-related genes (Hucklenbroich et al. 2021), which can be further facilitated when exposure coincides with cell damage (Zhou et al. 2020). A study using L. japonicus and A. thaliana as well as commensal bacteria isolated from their roots, revealed that the commensal-induced activation of an immune sector is a characteristic of ‘native’ interactions (i.e. A. thaliana inoculated with A. thaliana-derived commensals and L. japonicus inoculated with L. japonicus–derived commensals) but not non-native cross-inoculations (Wippel et al. 2021). These recent findings all point to the idea that root immunity plays a key role in root–microbiota interactions and that root commensals are equipped with the ability to manipulate it. In fact, Trp-derived phytoalexins, such as camalexin, indole glucosinolates, and other yet-to-be-identified metabolites that constitute an essential layer of root immunity, are needed for maintaining ‘healthy’ interactions with the root-associated microbial community (Wolinska et al. 2021; Koprivova et al. 2023, Basak et al. 2024), which is somewhat similar to the role of immunity in shaping leaf-associated microbiota (Chen et al. 2020, Pfeilmeier et al. 2021, 2023, Entila et al. 2024). Interestingly, ROS production triggered by commensal colonization, which is recognized as an immune response and negatively regulates colonization by Pseudomonas commensal bacteria (Song et al. 2021), has a positive impact on colonization by Bacillus velezensis FZB42 in an auxin-dependent manner (Tzipilevich et al. 2021). In a community context, i.e. in the presence of other microbes in the same niche, loss of the ability to suppress host short-term immune responses results in reduced competence in roots (Yu et al. 2019a), while co-inoculation with its respective wild-type (WT) strain did not trans-complement the root competence of the mutant strain (Ordon et al. 2024). These obeservations clearly demonstrate the complexity of the effects of root immunity on microbial behavior. To understand root immunity and its impact on root microbiota, it is crucial to understand the molecular mechanisms by which commensals manipulate root immunity and whether and how this affects overall root microbiota homeostasis and plant health.

Molecular Mechanisms by which Commensals Affect Root Immune Responses

A handful of studies have identified genes needed in commensal bacteria to suppress host immune responses, which we summarize here, although there are a few points that should be kept in mind to correctly capture the overview of this molecular dialog. First, the plant immune response is not a single process that occurs continuously and ubiquitously but rather an ensemble of different sectors at both the phenotypic and genetic levels, whose orchestration is precisely and adaptively arranged in a spatiotemporal manner [see earlier; excellently reviewed in Tsai et al. (2023)]. Second, leaves and roots are exposed to fundamentally different biotic conditions: while foliar encounters with microbes usually occur upon rain or physical contact with the soil surface, roots are continuously in contact with an immense diversity of soil-borne microbes (Bulgarelli et al. 2013). This might explain why expression of the flg22 receptor FLS2 is generally low and only induced upon tissue/cell damage (Zhou et al. 2020). It is also important to note that foliar pathogens, and perhaps leaf endophytes to some extent, usually exploit stomata to invade the leaf interior (Melotto et al. 2006), whereas root pathogens, symbionts, and endophytes need to find alternative gateways, such as lateral root emergence sites, infection threads, wounded sites, or dead cells. This might have caused plants to develop multiple layers of root apoplastic physical barriers, including Casparian strips and suberins (Schreiber et al. 1999). Hence, it is of crucial importance to appreciate the differences in experimental set-ups, particularly the time points, tissues, and phenotypic readouts, in order to avoid inappropriate integration of information that actually concerns different molecular processes.

Manipulation of short-term immune responses by commensals

One of the major phenotypic readouts used to study immuno-manipulation by commensals is the transcriptional response at the root tip (root elongation zone), namely, the altered promoter activities of PTI marker genes, such as CYP71A12, MYB51, and WKRY11. It was first utilized in A. thaliana by Millet et al. using the β-Glucuronidase staining method to describe root immune activities and, ever since then, has been a major asset for studying root immune responses (Millet et al. 2010). In addition, a set of fluorescence transcriptional marker lines reporting PTI have been established and used to observe spatiotemporal dynamics of root immunity in living cells (Poncini et al. 2017, Zhou et al. 2020, Emonet et al. 2021). These experimental systems allowed high-throughput screening of bacterial mutants impaired in host immune manipulation. For example, Yu et al. and Liu et al. performed random transposon mutant screening in the beneficial bacterial species Pseudomonas capeferrum WCS358 and P. simiae WCS417, respectively, whose WT strains suppress PTI responses triggered by flg22 within 3 h (Yu et al. 2019a, Liu et al. 2023). These studies revealed that apoplastic/rhizospheric acidification (a drop in pH), triggered by gluconic acids (GAs) and/or arginine (Arg) produced by these bacteria, is crucial for the suppression of flg22-triggered CYP71A12 expression. Interestingly, WCS358 primarily depends on GA, whereas WCS417 exploits both GA and Arg to lower the environmental pH. This illustrates that, although these two strains target the same molecular process (the rhizospheric pH), the underlying mechanisms differ depending on the microbial lineages. A similar approach was employed by Teixeira et al. to identify the type II secretion system (T2SS) as the causal component for immune suppression by the bacterial commensal Dyella japonica MF79 (Teixeira et al. 2021). One of the possible mechanisms by which apoplastic pH may manipulate immune activity is its influence on cell surface receptor complex dynamics: Liu et al. recently showed that MAMP treatments induce rapid alkalinization in the root meristematic zone and facilitate the interaction between atpep1 peptide, its cognate receptor PEPR1 and its co-receptor BAK1, to strengthen immune responses (Liu et al. 2022).

Besides the role of extracellular pH in interfering with PTI responses, commensal bacteria are also capable of evading recognition by PRRs, as are symbionts, in different manners. The flg22 sequence variants present in A. thaliana commensal bacterial genomes have been tested for their effects on PTI (Colaianni et al. 2021). It was shown that some variants are able to inhibit flg22 responses in a competitive manner, while others are simply non-recognizable by A. thaliana FLS2 protein. Interestingly, while the majority of the antagonistic peptide variants inhibited or destabilized the FLS2-BAK1 receptor complex, a few variants did not affect this complex formation despite their clear antagonistic activity (Colaianni et al. 2021, Parys et al. 2021), indicating that flg22 variants manipulate FLS2 signal transduction at multiple levels. Pseudomonas sp. WCS365 mutant strains lacking morA and spuC genes, encoding putative diguanylate cyclase/phosphodiesterase and putrescine aminotransferase, respectively, strongly activated host immune responses, while the WT strain did not, pointing to an evasive capacity in the WCS365 strain (Liu et al. 2018).

It is now clear that root commensal bacteria are capable of manipulating host immune responses in a short time frame (Fig. 3). Interestingly, while pathogens tend to exploit T3/4SS to manipulate PTI intracellularly, the suppressive factors thus far reported in commensals are restricted in extracellular space, delineating unique characteristics of anti-PTI machinery in pathogens and commensals. It is worth noting that the only common process that contributes to PTI suppression is the extracellular degradation of MAMP epitopes by bacterial proteases (Pel et al. 2014, Ma et al. 2021). Assuming that the pathogenic and symbiotic lifestyles evolved from the commensal lifestyle in bacteria (Garrido-Oter et al. 2018, Wang et al. 2020), this might suggest that the acquisition of a more drastic way of suppressing host immunity has been one of the key drivers for these lifestyle switches. Addressing whether pathogens still retain the commensal-type suppressive mechanisms would further promote our understanding of the role of root immunity in plant–microbe interactions in general and help decipher the co-evolutionary processes mediated by root immunity.

Manipulation of long-term immune responses by commensals

In contrast to the accumulating amount of research about commensal interference with short-term PTI responses (the transcriptional induction of PTI marker genes at the root elongation zone), it remains elusive how the long-term consequences of PTI are impacted by these microbes. PTI-induced growth inhibition is typically recorded by submerging plants in MAMP-containing liquid media, although in some studies, MAMPs were mixed into agar-based solid media. The solid system largely limits the plant exposure to MAMPs to root tissues, which resembles soil conditions better than the liquid system. On the other hand, the liquid system induces more severe growth inhibition, likely due to the direct exposure of shoots to MAMPs. It remains to be addressed whether PTI-triggered inhibition of shoot growth and root growth is mediated by the same (or at least similar) pathways or is a consequence of largely distinct molecular processes. It needs to be noted that flg22 and elf18 exhibit different tissue specificity in their growth inhibition activity when treated in liquid culture (Ranf et al. 2011, Vetter et al. 2016), demonstrating distinctive molecular pathways that mediate growth inhibition triggered by different MAMPs in different tissues. To date, too little is known about the precise and possibly diverse molecular mechanisms by which chronic PTI activation by different MAMPs inhibits plant growth to address this question. A wide-scale survey of the flg22 population with the natural diversity observed in commensal bacteria noted that a handful of flg22 variants (so-termed ‘deviant’ variants) induced a short-term response (ROS burst) but not the long-term consequence (growth inhibition of submerged seedlings), demonstrating that the short-term and long-term responses are at least partly separable (Colaianni et al. 2021).

A few recent studies describe the ability of commensal bacteria to interfere with PTI-triggered root growth inhibition (RGI). Understanding the molecular mechanisms by which commensals manipulate the long-term consequence of PTI activation allows us not only to reveal the root immunity-mediated molecular dialog between roots and root-associated microbiota but also to disentangle how chronic PTI activation results in growth inhibition and how this response is relevant to plant adaptation to soils. Commensal bacteria interfering with the RGI triggered by PTI chronic activation were first discovered in a study focusing on the order Rhizobiales. Rhizobiales commensals commonly promoted A. thaliana root growth in a gnotobiotic inoculation assay, and a subset of them were found to interfere with root immune responses. An in-depth analysis of the type strain R129_E revealed that R129_E suppressed RGI triggered by mixing the flg22 peptide into solid media, which is consistent with an RNA-seq experiment that identified the downregulation of flg22-responsive genes, even in the absence of exogenous peptide application (Garrido-Oter et al. 2018). Ma et al. further elaborated on this finding and showed that more than 40% (62 out of 151 strains) of taxonomically diverse root bacterial commensals suppressed flg22-triggered RGI in the same experimental set-up, in both mono-association and synthetic communities (SynCom) (Ma et al. 2021). Interestingly, they recorded pH reductions in culture media when plants were inoculated with either suppressive or non-suppressive commensals, to an even greater extent for the latter, showing that the rhizospheric pH does not fully explain this suppression phenotype. Culture filtrates from two Janibacter strains (in the order Micrococcales) were found to degrade or modify flg22 peptides, similar to what has been shown for pathogens (Pel et al. 2014), while those from the other three tested strains (from the orders Micrococcales, Rhizobiales, and Xanthomonadales) did not. These results again supported the idea that different commensal bacteria utilize different molecular mechanisms to interrupt PTI. A similar study exploiting another independent culture collection identified the same capacity in ∼23% (8 out of 35) of tested commensal strains (Teixeira et al. 2021). They also identified the T2SS as a potential factor manipulating host immunity, although a short-term response (CYP71A12 expression) was used as the readout. Whether the T2SS in the D. japonica MF79 strain is also required for suppressing flg22-triggered RGI remains to be tested.

Garrido-Oter et al. and Ma et al. demonstrated that suppressive capacity could be a dominant trait within a small-scale synthetic community (Garrido-Oter et al. 2018, Ma et al. 2021), although it is easy to imagine that it depends on the microbial combination, as in the case of many other microbial traits. For example, the opportunistic pathogenicity of microbiota members illustrates that pathogenicity is suppressed by other microbiota members (Bartoli et al. 2018, Karasov et al. 2018, Ma et al. 2021, Teixeira et al. 2021). The inability of the WCS358 WT strain to restore the root competence of the WCS358 mutant strain impaired in suppressing short-term PTI responses when they were co-inoculated together with a 15-member SynCom also illustrates the complexity of how PTI influences microbial colonization and how it is suppressed in a community context (Ordon et al. 2024). A more systematic survey of microbe–microbe interactions in the root niche using the suppression of PTI-triggered RGI as a phenotypic readout will provide deeper insight into how individual microbial traits are influenced by the presence of other microbes in the community.

Nevertheless, given the high frequency of suppressive isolates in multiple independent culture collections (Ma et al. 2021, Teixeira et al. 2021), it is reasonable to assume that any plant-associated microbiota would contain microbes with suppressive capability. This, in turn, raises the question of how plants ensure their defense against true pathogens while accommodating a plethora of commensals potentially interfering with their immune responses. Inoculation with suppressive SynCom made host plants susceptible to a subsequent inoculation with opportunistic pathogens, namely Ochrobactrum sp. MF370 and Pseudomonas viridiflava OTU5 (Teixeira et al. 2021) or Pseudomonas sp. Root401 (Ma et al. 2021), showing that these commensals indeed influence root immunity, in addition to their impact on the PTI-influenced root growth regulation. A recent work focusing on leaf–microbe interactions reported that age-dependent immune mounting, the phenomenon in which developmentally older leaves mount stronger immunity also known as age-related resistance (Develey-Rivière and Galiana 2007), is impaired in axenic plants but observed in plants re-colonized by a SynCom (so-termed holoxenic plants) (Paasch et al. 2023). This might be explained by a model in which the holoxenic plants have been continuously exposed to the MAMP repertoire of the colonizing microbes, which in turn helps plants to mount age-dependent immunity. Further studies are needed to test this hypothesis, as well as to determine to what extent this observation is relevant to root–microbiota interactions.

Future Perspectives

Despite the clear experimental evidence for commensals interfering with host growth–defense coordination, the underlying molecular mechanisms remain entirely unknown, except for Janibacter degrading/modifying extracellular peptides. This is in clear contrast to our accumulating knowledge of how commensals interfere with short-term immune responses, and more studies are needed to ultimately understand how plants coordinate their defense and growth in the presence of the soil-borne MAMP repertoire. Unveiling the molecular arsenal that commensal microbes deploy to suppress the host PTI-triggered RGI may pave the way for exploring the immunity-mediated molecular dialog between roots and associated microbiota, given its clear and robust phenotypic readouts. In this last section, we summarize what is needed to achieve this ultimate goal.

A notable advantage to this end is the diverse set of plant-derived bacterial culture collections whose whole-genome sequencing data are publicly available (Bai et al. 2015, Levy et al. 2017, Garrido-Oter et al. 2018, Karasov et al. 2018), and a number of comparative genomic studies have identified bacterial genes involved in host–microbiota interactions. A genome-wide association study (GWAS)–like approach is often employed, where it utilizes the association between the functional properties of a set of bacterial strains and the presence and absence of orthologs in their genome sequences (Bai et al. 2015, Levy et al. 2017). It is a powerful tool when the functional diversity is associated with a common molecular pathway; however, as clearly demonstrated by Ma et al. (2021), root-associated commensals possess diverse molecular mechanisms that modulate host immune responses, even though the ultimate macro-phenotypic readout is very similar. Therefore, it is needed to prepare a group of microbes/genomes with (i) a sufficient number of both suppressive and non-suppressive isolates and (ii) a reasonable assumption that the suppressive isolates within this group exploit a common mechanism for identifying genes responsible for their suppressive machinery. For example, deep sampling combined with functional characterization of a specific taxon of bacterial isolates enables us to reconstruct their phylogenetic structures and predict ancestral functional status (Garrido-Oter et al. 2018, Karasov et al. 2018). This, in turn, allows for the identification of a monophyletic group of suppressive isolates along with a non-suppressive outgroup, which is an ideal situation to look for genes whose acquisition or loss correlates with the acquisition of the suppressive activity. We can also take advantage of canonical yet powerful forward genetic approaches, like many of the studies about suppression of short-term PTI responses by commensals (Liu et al. 2018, 2023, Yu et al. 2019a, Teixeira et al. 2021). The low throughput of recording long-term responses might have been an obstacle to this end, although recent efforts have already provided a potentially useful platform for screening the genes responsible for the manipulation of long-term PTI consequences (Ma et al. 2022). Of note, the two strategies we suggest here (lineage-focused GWAS and forward genetics) are for analyzing one specific group of bacteria rather than a comprehensive survey of the entire range of microbiota. It thus calls for a research community-wide effort to analyze each suppressive lineage to reveal individual mechanisms, followed by integration of all the information obtained to build a molecular model in the community context.

The presence of multiple mechanisms raises a number of attractive scientific questions. First, how individual pathways interact with each other, i.e. whether they operate in a redundant, additive, or synergistic manner, is one of the key aspects of host–microbiota interactions. It is possible that individual pathways use different mechanisms but target the same host process, in which case these traits might be simply redundant with each other. An alternative, and perhaps a more likely scenario given the contrasting activities that degrade/modify extracellular peptides (Ma et al. 2021), is that different mechanisms target different host pathways and thereby operate robustly against perturbations by host counteraction or interference from other organisms. This hypothesis can be addressed by answering our second question about spatiotemporal regulation. A microbial community is a highly heterogeneous population of microbial cells and so are plant roots and their immunity. Thus, what is needed for bacteria to suppress local immune responses might depend on the cell type they interact with, which would have introduced distinctive selective pressures on commensals with different colonization patterns and facilitated the acquisition of diverse molecular mechanisms. Moreover, pattern recognition activates PTI not only in local tissues but also in systemic tissues (Vlot et al. 2021), delineating the presence of signals traveling from infected to uninfected tissues and from roots to shoots (Pieterse et al. 2014), and it is intriguing to ask whether suppressive commensals also suppress systemic signaling and to what extent it has an impact on the root growth–defense coordination. To understand the spatiotemporal regulation of suppressive mechanisms, it is necessary to identify plant and bacterial genes involved in this suppressive process and microscopically observe their expression dynamics using transcriptional/translational marker constructs. This leads us to the third question of how commensals have acquired and maintained suppressive activity toward host immune responses. Intriguingly, roughly one-third of bacterial strains from two independent culture collections exhibited the capacity to suppress PTI-triggered RGI. This indicates that this suppressive trait can provide a public good for the community, allowing more than half of the community members to rely on the portion that takes on an important community task. This also implies that root immunity and the PTI-triggered RGI have significant impacts on bacterial fitness in the root niche. Deciphering the evolutionary process through which root immunity and its suppression by commensals have been acquired will directly reveal their adaptive role under ecological conditions.

In order to address these important questions, there is an urgent need to reveal the molecular mechanisms by which commensals interfere with host immunity. To this end, the difference between the short-term PTI responses and the long-term consequences of chronic PTI activation, both of which are strongly influenced by root-associated commensals, needs to be better appreciated and correctly reflected in the collective interpretation. It is conceivable that short-term and long-term responses constitute different sectors of plant immunity and are controlled by overlapping yet distinct regulatory machinery. Long-term responses might become more and more important for agriculture, given the increased demand for microbial agents enabling crops to have better growth and defense simultaneously without the use of chemical fertilizers or pesticides. It is also important to test to what extent our knowledge of A. thaliana and its associated microbiota can be applied to other plant species, including crops. We therefore propose a community-wide effort to tackle this complex problem and disentangle it, piece by piece, into a molecular framework from which we can ultimately build an integrative model that explains root–microbiota interactions from the perspective of root immunity.

Data Availability

No new datasets were generated or analyzed in this study.

Funding

KAKENHI, funded by the Japanese Society for the Promotino of Science (JSPS, Japan) to R.T.N. (22K21367) and to T.S. (22KJ3147).

Disclosures

The authors declare no conflicts of interest.

References