The Lifecycle of the Plant Immune System

Abstract

Throughout their life span, plants confront an endless barrage of pathogens and pests. To successfully defend against biotic threats, plants have evolved a complex immune system responsible for surveillance, perception, and the activation of defense. Plant immunity requires multiple signaling processes, the outcome of which vary according to the lifestyle of the invading pathogen(s). In short, these processes require the activation of host perception, the regulation of numerous signaling cascades, and transcriptome reprograming, all of which are highly dynamic in terms of temporal and spatial scales. At the same time, the development of a single immune event is subjective to the development of plant immune system, which is co-regulated by numerous processes, including plant ontogenesis and the host microbiome. In total, insight into each of these processes provides a fuller understanding of the mechanisms that govern plant-pathogen interactions. In this review, we will discuss the “lifecycle” of plant immunity: the development of individual events of defense, including both local and distal processes, as well as the development and regulation of the overall immune system by ontogenesis regulatory genes and environmental microbiota. In total, we will integrate the output of recent discoveries and theories, together with several hypothetical models, to present a dynamic portrait of plant immunity.

I. An introduction to plant biotic interactions

In natural ecosystems, most plants are resistant to most pathogens, a phenomenon whose mechanism is undoubtedly one of the holy grails in plant pathology-to understand and harness the ability of a plant to respond to, and successfully defend against, pathogen invasion (Staskawicz, 2001). Indeed, the abundance of host, pathogen, and climatic diversity provides a rich source of broad-spectrum resistance, the result of which is a naturally selected balance of genetically diverse plant and pathogen/pest populations. Therefore, epidemics in ecosystems are rare, and when they do occur, they are typically restricted to a specific geographical region, climate, or a combination of both. In the case of natural resistance, typically referred to as non-host resistance, the breadth of genetic diversity represented in the host population is often sufficient to limit infection(s), resulting in the evolution of what is referred to as nonadapted pathogens (Stam et al., 2014). Conversely, the selection for and enrichment of pathogens that are adapted to their host has resulted in the establishment of ecosystems where pathogen virulence and disease are more often the norm than the exception.

Plants begin and end their lifecycles in a single geographical location; however, the environment around plants is in constant flux. In response to these changes, and ultimately, to survive and thrive, plants must sense, respond, and adapt to an endless barrage of external perturbations, such as biotic and abiotic threats. Thus, it is not surprising that an emerging theme in plant pathology is the contribution and influence of the environment on immune system maturation. Herein, while we periodically invoke the concept of environmental impact on the plant immune system, we will not delve too deeply into this body of work, as the complexity and breadth of research in this area is broad, fast moving, and requires a dedicated narrative to appropriately cover this topic. Rather, we point the reader to several recent comprehensive reviews on this topic (Chappelka and Grulke, 2016; Morris et al., 2017; Cheng et al., 2019; Corredor-Moreno and Saunders, 2020).

To successfully respond to and defend against biotic threats, plants have evolved highly complex pathogen defense systems, or surveillance networks, which function in a manner similar to the innate immune systems of humans. The defense signaling acts cooperatively with numerous cellular processes, and together, the sum of these interactions imparts the ability to recognize a vast array of biotic threats (e.g., pathogens, pests, and viruses) and distinguish self from nonself (Sanabria et al., 2008). As described in greater detail below, underpinning the function and activity of the plant immune system is a complex network of preformed and inducible signaling processes, which provides unfettered access to both external and internal (i.e., systemic) cues.

In this review, we describe the events, in a broadly temporal fashion, that encompass the lifecycle of the plant immune system. From perception and recognition, to the multitude of signaling events that require the plant cell to either respond or not to pathogenic microbes, our understanding of the activation and attenuation of immune signaling remains incomplete. Moreover, we highlight recent studies describing the molecular-genetic processes that define how the plant immune system matures and during its development, how it integrates into a multitude of host signaling processes that regulate plant development and response to the environment. To do this, we primarily focus on the dynamics of plant immune signaling as a function of time and scales; from pathogen perception and virulence, to the activation of local and distal defense signaling. In short, we aim to describe and illustrate the development, activation, and ultimate function of the immune system as an extension of whole plant physiology, growth, development, and reproduction.

II. Pathogen invasion

A. Host invasion by filamentous pathogens

During fungal colonization of plants, the transition from external to internal growth and proliferation begins with germination of a spore and formation of the penetration-specialized architecture, the appressoria (Yi and Valent, 2013; Ryder and Talbot, 2015). In short, this process enables “forced entry”, or direct penetration, of the outer physical barriers of the plant, a common yet diverse invasion strategy among filamentous pathogens (see Figure 1). For instance, the model foliar powdery mildew pathogen Golovinomyces orontii uses its appressoria to forcibly invade into leaf epidermal cells by breaking the cuticle and cell wall (Braun et al., 2019). Such a strategy is also common in soilborne pathogens, including, for example, Phytophthora sojae, which invades the roots of soybean (Fawke et al., 2015). Alternatively, pathogens do not necessarily need to directly penetrate into a live cell at the onset of the interaction; case-in-point, the oomycete pathogen Hyaloperonospora arabidopsidis (Coates and Beynon, 2010) penetrates the cuticle and grows into the apoplast, the space between the junction of two pavement cells (Underwood, 2012), which potentially benefits the pathogen by delaying the full engagement with plant immune system. As a point of strategy, while the “forced entry” model greatly expands the opportunity for filamentous pathogens to successfully invade the host, the “passive entry” mechanism (e.g., through natural openings or wounds) presents less of a challenge to the pathogen as a function of reduced physical barriers and defense response. One of such examples is the invasion of Colletotrichum species, which causes anthracnose diseases. Outside plant, they generate nonpenetrative appressoria, from which undifferentiated germ-tubes extend and search for stomata to enter, resulting in host colonization via intercellular hyphae development (Latunde-Dada et al., 1999).

Figure 1.

Invasion strategies by phytopathogens. To promote infection, both phyllospheric and rhizospheric pathogens must overcome physical barriers on the plant surface. Filamentous pathogens typically infect their host using the appressorium to invade living cells. During infection, the germinating spore (S) forms an extended tube-like structure (i.e., germination tube, GT), which then develops into an appressoria (A) that promotes the entry into plant. Appressorium can either directly penetrate into epidermis cells by breaking through the cuticle surface and cell wall, or enter through the apoplast, the space between cells. Additionally, wounds or natural openings (i.e., stomata) on the plant surface provide easy entry into the intercellular space. Once inside the host, filamentous pathogens use a root-like structure (i.e., haustoria, H) to obtain host-derived nutrients, resulting in the establishment of the pathogen-host interface. The invasion of bacterial phytopathogens, unlike filamentous pathogen, highly depends on natural openings to enter the plant host.

Of the numerous systems that have advanced our understanding of the processes underpinning appressorium-mediated penetration, the interaction between rice and the fungal pathogen Magnaporthe grisea represents one of the better understood examples of this virulence mechanism. As demonstrated using a combination of genetics-, cell biology-, and classical plant pathology-based methods, M. grisea initiates appressorium development upon the perception of the hydrophobic leaf surface environment, in combination with contact of the wax cuticle (Ryder and Talbot, 2015; Anjago et al., 2018). Upon contact and assessment of the leaf surface environment by the developing fungus, physical penetration is mediated by the establishment and maintenance of cellular turgor pressure, which comes from elevated concentrations of glycerol in the appressoria, as well as a semi-permissive melanin barrier at the host-fungal interface (Chang et al., 2014; Ludwig et al., 2014). During this stage of infection, appressorial growth and development is facilitated by the assembly of a condensed septin-actin network, a mechanism hypothesized to enhance mycelia growth and trafficking during the maturation of pathogen infection (Van Ngo and Mostowy, 2019). In addition to the early stages of fungal development and infection, the pathogen secretes a battery of virulence-associated enzymes to promote infection, including cutinases (Kebdani et al., 2010; Auyong et al., 2015), cellulases (Kebdani et al., 2010; Van Vu et al., 2012), and pectinases (Kebdani et al., 2010), which target host cell wall components to promote further ingress. In total, the integrity of the whole penetration-facilitating system of filamentous pathogen is a pre-requisite of successful and efficient invasion. Indeed, mutants with reduced turgor pressure or an absence of cell wall degrading enzymes display reduced penetration capabilities (Skamnioti and Gurr, 2007; Auyong et al., 2015; Paccanaro et al., 2017; Tang et al., 2018).

B. Bacterial pathogen invasion

In the case of phytopathogenic bacteria, the transition from epiphytic/saprophytic growth to infection is hypothesized to be induced by external signals, including those emanating from the host, abiotic environment, and the microbial community (i.e., microbiome composition, quorum sensing, etc.) (Baker et al., 2010; Leonard et al., 2017; Xin et al., 2018). In the case of leaf-attached bacterial colonies, communities may persist as noninfective entities as a consequence of low surface humidity. Such “dormancy” on the host surface is mediated by a humidity-regulated quorum sensing system that inhibits the transition to an infection phase, as indicated by bacterial mobility, exopolysaccharide production, and pathogen secretion system maturation (Quiñones et al., 2005; Dulla and Lindow, 2008; Cheng et al., 2016). Once the stimulus is perceived by potential pathogenic microorganisms, as described in the case of the model bacterial phytopathogen P. syringae (Ortiz-Morea et al., 2016), the bacteria enters infection phase. In short, this process coincides with the rapid expression of core pathogenesis regulons, including hrp/hrc, hrpA, hrpL, and hrpR. In turn, this leads to the activation of signaling associated with the production of key virulence factors, including toxins (Brooks et al., 2005; Baker et al., 2010; Geng et al., 2012), and the induction of signaling responsible for the production of the type III secretion system (TTSS) (Tang et al., 2006). In another example for soilborne bacteria, Ralstonia solanacearum perceives oleanolic acid (Wu et al., 2015) and ferulic acid (Zhang et al., 2017) as critical host-released virulence inducive signals, potentially via PrhA-PrhR receptor complex. This is significant, because these compounds are directly released into the soil matrix, and R. solanacearum, like other soilborne pathogens, may induce transitions to pathogenesis and gain higher virulence before host invasion.

In the case of bacterial pathogens, bypassing the surface barrier via natural opening or wound is necessary. Among the best characterized modes of phytobacterial invasion is through stomata, the opening between two guard cells that functions as the site of transpiration and gas exchange. For nearly 30 years, research in the area of plant-pathogen interactions has led to the accumulation of strong evidence demonstrating that a range of pathogens, including bacteria and fungi, utilize stomata as points of host entry. However, it is only within the past decade or so that our understanding of the molecular mechanisms underpinning this interaction has been realized (Melotto et al., 2006; Melotto et al., 2017). Although stomatal aperture is dominantly regulated by light and the internal circadian clock (Hubbard and Webb, 2015), plants can also activate stomatal closure outside of the standard circadian rhythms of daylight hours in response to pathogen infection (Melotto et al., 2006). To counter the defense-induced stomata closure, pathogens, such as P. syringae, have evolved mechanisms to re-open stomata through the process of defense hormone mimicry to ensure successful and efficient invasion (Geng et al., 2014). This paradigm, the back-and-forth of virulence and defense through stomatal gating, is referred to as stomatal immunity (Melotto et al., 2017) and represents a new battle-ground in the field of plant-pathogen interactions and immune signaling.

C. Virulence in advance of entry

As a foundation describing molecular plant-pathogen interactions during host immune signaling and defense, it is important to clarify the status of pathogen virulence in advance of host infection. Current models portray pathogen virulence, in the most generalizable terms, as a process activated upon host contact. In this context, and herein, we too will define contact between a pathogen and the apoplast or living cell as “time zero” in the chronology of the activation of plant immunity. This leads to an essential question related to the entire process of plant immunity - are pathogens already capable (i.e., competent) of interfering with immune signaling at time zero? For bacteria, as discussed above, they are capable of entering the infection phase and activating the effector/toxin secretion systems before time zero. For filamentous pathogens, spore germination, per se, is a hallmark of the initiation of the infection phase, which activates a virulent secretome before penetration (Kleemann et al., 2012). Hence, it is reasonable to hypothesize that pathogens have already obtained the ability to inhibit the impending defense response by host before confronting with plant immune system. This temporal advance is critical for pathogenesis because it ensures that the secretion of effectors or other defense-inhibitory compounds to host is, at a minimum, simultaneous with pathogen perception, if not in advance.

III. Temporal development of local immunity

A. The timing of local immune activation

In plants, local immunity describes a fundamental concept founded on the basic principle that an independent live cell is immuno-totipotent, or possessing the full capability of the immune response, independent of additional signal input(s) from other host-associated components (Verdeil et al., 2007). Previous work has generalized a canonic model to describe local immunity with two primary nodes, namely PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Thomma et al., 2011). PTI, as mechanism of basal defense, is activated following host perception of microbial PAMPs (pathogen associated molecular patterns), the conserved organismal motifs required for the survival and lifestyle of the microbe (e.g., flagellin, chitin). As an immune response that best illustrates the initiation and integration of complex host signaling and innate immunity, PTI follows a classic cellular signaling model comprised of receptors, cascades, and defense executors. Here, we describe the temporal function(s) of each of these three components (i.e., receptors, cascades, and defense executors) as they correlate with the initiation of basal defense signaling (Figure 2).

Figure 2.

A schematic map of plant local immunity. Invasive pathogens are recognized by plant PRR (pattern recognition receptor) proteins, which results in the activation of broad spectrum of downstream signaling, such as Ca²⁺ influx, the accumulation of H₂O₂ generated by RbohD (respiratory burst oxidase homolog protein D), and kinase cascading, which includes signaling pathways mediated by MAPKs, CPKs, and other additional kinases. As depicted, various kinases may also engage in a highly coordinated cross-talk during signal amplification and attenuation. These immune signals, amplified by kinase cascades, trigger a variety of defense responses, including cytoskeletal remodeling, activation of defense function in organelles, and transcriptional reprograming through the activity of pro-immune transcription factors (TF). In total, the sum of this highly coordinated signaling functions to promote plant defense signaling and pathogen resistance. Concomitant with the activation of defense signaling, the attenuation of key immune pathways occurs, a process hypothesized to function in rebalancing of immunity and growth pathways occurs. To cope with plant immunity, pathogens have evolved mechanisms to deliver effector proteins into plant cell, which target and inhibits immune signaling, as well as to subvert immunity through targeting of critical host cellular processes. In response, plants utilize NLR (nucleotide-binding leucine-rich-repeat proteins) proteins to recognize certain effectors through sensing pathogen modification of surveilled host processes (i.e., guardee), resulting the activation of robust immune signaling and cell death (i.e., ETI; effector-triggered immunity). As a potential mechanism to activate ETI, cell membrane (PM)-associated NLRs (in most instances, possessing a coiled-coil domain, i.e, C-NLR), can form a channel-like structure following activation, which presumably functions to mobilize additional defense signaling molecules. NLRs containing a Toll/interleukin-1 receptor-like domain (T-NLRs) at the C-terminus are typically associated with a nuclear subcellular localization, and in large part, function as sensors (i.e., sNLR) that activate helper NLRs (hNLR) to form channels within the PM. As an additional hypothesized mechanism, activated nuclear NLRs may regulate specific defense genes functioning in ETI, by interacting with TFs. Dashed in indicate putative/hypothesized processes.

Plant pattern recognition receptors (PRRs) perceive a wide range of elicitors, including pathogen-derived cell wall/membrane components (e.g., peptidoglycan, chitin), pathogen-associated proteins (e.g., flagellin, effectors), and host-derived danger associated molecular patterns (DAMPs; e.g., cuticle) (Ziv et al., 2018). Though diverse, these receptors share certain features: they are single-transmembrane receptor-like kinases or receptor-like proteins, containing a leucine-rich repeat (LRR), LysM, EGF-like, or lectin domain for ligand binding within the apoplast (Boutrot and Zipfel, 2017). For most cases, evidence supports a general mechanism wherein a core receptor and their associated kinases form the primary receptor complex, in association with additional regulators, mediate pathogen recognition and the initiation of downstream signaling. Herein lies one of the key remaining questions: How fast does PRR activation occur following pathogen perception?

While no technical approaches currently exist which can directly measure the speed of PRRs activation, this question can be answered by correlating the timing of measurable downstream outputs, such as the generation of apoplastic reactive oxygen species (ROS) - one of the earliest measurable defense responses (Torres et al., 2006; Lehmann et al., 2015). In Arabidopsis, the PTI-triggered apoplastic ROS burst is generated by respiratory burst oxidase homolog protein D (RBOHD) (Kadota et al., 2015), a plasma membrane (PM)-associated NADPH oxidase that generates H₂O₂ as secondary signaling messenger. As one example of the link to PRR signaling complex activation, RBOHD is phosphorylated and activated by BIK1 (Botrytis-induced kinase 1), a core signaling kinase within the FLS2 (flagellin sensitive 2)-associated PRR complex (see Figure 2; Kadota et al. 2014). As an indicator of the timing of this response, the rate of ROS accumulation (i.e., d[ROS]/dt) reaches saturation at approximately 3 min after flg22 (elicitor that activates FLS2) stimulation (Nühse et al., 2007), with complementary data demonstrating a maximum accumulation approximate 25 min following P. syringae infection (Smith and Heese, 2014). Taken together, the initial activation of PRRs occurs within the first few minutes following pathogen perception (Figure 3); the rapidity of this process further illustrates the role of ROS as second messenger in downstream immune signaling, including regulating Ca²⁺ influx.

Figure 3.

Dynamics of signaling processes associated with local immunity. The signaling processes associated with local immune signaling can largely be described in a temporal fashion; for the sake of comparison, we suppose “Time 0” = PRR activation. To estimate the signaling dynamics (i.e., timing of initiation, sustained saturation, peak of increasing speed, and termination), pub lished data recording the development of immune processes following elicitor treatment or pathogen infection are collected, analyzed, and translated into this figure. Dashed lines in indicate estimation without direct evidence.

Once PAMP recognition and PRR associated signaling events are activated, the immune signal is handed off to downstream signaling processes, which serves to not only amplify the initial signal, but importantly, functions as a mechanism to regulate signaling specificity and the activation of defenses that are appropriate to the nature of the stimulus. To accomplish this, plants utilize a complex series of phosphorylation-dependent signaling cascade, the best characterized of which include mitogen-activated protein kinases (MAPK) (Meng and Zhang, 2013) and calcium-dependent protein kinase (CPDK; aka CPK) relays (Singh et al., 2017). To date, one of the best characterized signaling pathways is flg22-triggered PTI, wherein MAPKs are rapidly activated following phosphorylation by FLS2-associated signal regulators - a cascade from MAPKKK3/5 to MAPK3/4/6 (see Figure 2; Mithoe and Menke, 2018). As an illustration of the rapidity of this process, it has been demonstrated that flg22-induced signaling occurs within ~5 min following ligand perception as determined by MAPK3/4/6 phosphorylation; maximal phosphorylation is believed to peak at ~30 min post elicitation (see Figure 3; Frei dit Frey et al., 2014).

Simultaneous with MAPK cascading, CPK-dependent signaling is induced by Ca²⁺ influx, a process that is initiated by gated Ca²⁺ channel(s) downstream of PRRs (also discussed in Section IV). While direct evidence is largely absent which describes the dynamic status of CPK phosphorylation during PTI, the influx of cytosolic Ca²⁺ can be used as an indirect index of CPK activity. Indeed, the accumulation of cytosolic Ca²⁺ obtains maximum speed (i.e., d[Ca²⁺]_cyt/dt) in 1 min after elicitor treatment; the Ca²⁺ concentration reaches the peak in 3 min post-treatment (Qi et al., 2010). Because CPKs are directly activated by elevated concentration of cytosolic Ca²⁺ without intermediate kinases, and CPKs, Ca²⁺ channels, and RBOHD form a positive feedback loop (see Figure 2 and 4), the activation of CPKs is supposed to be slightly faster than MAPKs. This hypothesis is supported by evidence demonstrating that the rice OsCPK18 functions as direct upstream regulator of OsMAPK5 (Xie et al., 2014), which further indicates that CPK-MAPK cross-talk is involved in PTI signaling. In total, these data support a model whereby MAPKs and CPKs work synergistically (Tena et al., 2011), yet nonredundantly (Mehlmer et al., 2010; Li et al., 2018), as pro-immune activators.

Figure 4.

The mechanism of distal immune signal transmission. (A) Biotic stresses trigger systemic immune signaling. Local immunity is induced following local pathogen perception, which results not only in the activation of local signaling, but also the induction of distal signaling within the root parenchyma and/or mesophyll. When the signal(s) reach the vasculature, signal transmission is substantially accelerated until it arrives at the site of distal parenchyma tissues in the leaf and/or root, after which time signal transmission decelerates. (B) Generation of distal signal molecules in local cells. Following immune activation, Ca²⁺ influx is initiated through an unknown Ca²⁺ channel(s) that are directly activated by PRR and/or RBOHD-synthesized H₂O₂. This initial influx activates Ca²⁺-dependent signaling nodules as CPKs and CaM, which further activates additional Ca²⁺ channels such as CGNC2/4, rendering robust secondary Ca²⁺ influxes. Local defense response also leads to biosynthesis of immune hormones such as SA and JA, a partial of which will spread to distal tissues. (C) Transmission of distal signals in parenchyma cells (including mesophyll). Ca²⁺ influx at a given location can activate RbohD via CPKs and presumably CIPKs, a process that results in the generation of H₂O₂ and the further activation of unknown H₂O₂-activated Ca²⁺ channels. Simultaneously, Ca²⁺ influx triggers an unknown glutamate (Glu) efflux pathway that activate glutamate-gated Ca²⁺ channel GRL3.3/3.6. Tonoplast membrane localized TPC1, a Ca²⁺ channel, gated by both Ca²⁺ and the resultant electrical potential may serve to amplify the Ca²⁺ signal. The transmission of signaling molecules is slowed at intercellular junctions as a result of the cell wall. (D) Transmission of distal signals within the vasculature. The mechanisms are the same as those in parenchyma (C), yet the gap of the intercellular junction is relieved via the action of the sieve plate, resulting in a faster speed of signal transmission.

Following MAPK and CPK signaling, the next step is the activation of defense executor proteins, a process leads to nuclear-based transcriptional reprograming (Li et al., 2016), induction of defense hormone accumulation and signaling (Verma et al., 2016), cytoskeleton/organelle remodeling (Li and Day, 2019), regulation of the secretome and cell wall/apoplast composition (Bellincampi et al., 2014; Gupta et al., 2015), and cellular motion (e.g. stomatal closure (Arnaud and Hwang, 2015). As key outputs of defense, the development of each of these cellular processes can be briefly categorized into two distinct phases. The first phase is the fast, pretranscriptional defense responses, which are often directly activated as a by-product of the basal immune signaling cascade. For example, RBOHD, described above, activates a robust ROS burst in the apoplast via the direct activation of PRR complex assembly and activation (Kadota et al., 2014; Kadota et al., 2015); as noted above, the timing of this response is detectable within 3 min of elicitation. Similar rapid signaling responses are also observed in the case of PM-associated ion channels (Jeworutzki et al., 2010). As another example of rapid signaling through PTI executor, PAMP-triggered actin remodeling illustrates the integration of PRR function with broader signaling platforms, as illustrated by the detection of changes in microfilament remodeling within 5–15 min following PTI elicitation (Henty-Ridilla et al., 2014; Li et al., 2015). While the full mechanism(s) underpinning this response is unknown, we posit that it involves the regulation of actin depolymerizing factors (ADFs) by cytosolic kinases at the downstream of PRRs, as well as H₂O₂ and phosphatidic acid (Porter et al., 2012; Li et al., 2015; Li et al., 2017; Li and Staiger, 2018; Li and Day, 2019). Although still largely hypothetical, this model is in agreement with an abundance of data describing fast responses mediated by changes in actin filament organization, including the activation of downstream immune signaling processes.

In order to initiate a large-scale and long-term output of defense, signaling next proceeds to the phase of transcriptional activation of sustained and robust defense processes (Lewis et al., 2015). As indicated by its classification, the foundation of this stage of immunity lies in the activation of stress-responsive transcription factors and the gene networks under their control. In brief, key regulators of this includes AP2/ERF, bHLH, bZIP, MYB, NAC, and WRKY (Tsuda and Somssich, 2015). Here, as a result of MAPK activation (described above), phosphorylation of defense transcription factors by MAPK significantly contributes to plant immunity. For example, in response to necrotrophic fungal pathogen Botrytis cinereal, MAPK3/6 phosphorylates WRKY33 within 0–12 h post-infection (hpi), which has been shown to regulate the overall resistance signaling within 6–24 hpi (Mao et al., 2011). Interestingly, as an example of the dynamic control and specificity of signaling, the WRKY33 mRNA is upregulated in response to flg22 or HrpZ (a bacterial elicitor) elicitation at ca. 1 hpi and subsequently downregulated at 4 hpi (eFP Browser, Winter et al., 2007), suggestive of a negative feedback loop to control signaling (Liu et al., 2015). As such, a single PAMP treatment does not necessarily reflect the true dynamics of TF activity, because pathogens possess multiple elicitors (e.g., PAMPs, effectors) that result in the stimulation of various synergistic signaling cascades. In this regard, the overall dynamics and pattern of defense-induced transcription cannot be measured exclusively by the early (ca. minutes to hours) events, but rather, must be evaluated over the duration of the interaction, which can last days or longer. Thus, as illustrated in Figures 2 and 3, the activation of immunity is not a sequential series of events, but rather, represents a complex network of processes, each of which can be activated or attenuated multiple times during the host-pathogen interaction.

B. The activation of ETI

Effector-triggered immunity (ETI), is a robust and sustained immune response activated following perception of pathogen-secreted effector proteins. As an additional layer of the immune surveillance platform, ETI resembles PTI in many regards, such as the involvement of MAPK signaling cascades (Lu et al., 2018) and defense gene activation. However, distinct from PTI, ETI results in the activation of an apoptosis-like cell death (aka hypersensitive response (HR); Balint-Kurti, 2019), a fast process hypothesized to result in an abrogation of pathogen proliferation. As estimated by the dynamics of electrolyte leakage (indicating cell death) during HR, full intensity of ETI occurs within 2–6 h after inoculation of avirulent (containing effectors that triggers ETI) bacterial pathogens (Mackey et al., 2002; Mackey et al., 2003). Similar to such dynamic pattern, the transcriptome reprograming during ETI reaches the maximum speed during the same period of time (Mine et al., 2018), indicating that ETI is a fast-acting immune response that may overcover the development of basal defense.

In terms of its mechanism, ETI relies on the function of host resistance (R) proteins to survey the cell for perturbations, through what is referred to as the Guard Hypothesis (Van der Biezen and Jones, 1998). As highlighted in reviews by Dangl and Jones (Jones and Dangl, 2006) and Chisholm et al. (Chisholm et al., 2006), the Guard Hypothesis posits that R protein “guards” another host derived protein (guardee), and when a guardee is modified (e.g., cleaved, phosphorylated, etc.) by a pathogen-secreted effector, its associated R protein recognize such modification and triggers downstream signaling. Most of R protein belongs to nucleotide-binding site leucine-rich repeat (NB-LRR or NLR) protein family, which is also the best studied R protein architecture (Monteiro and Nishimura, 2018). According to their distinguishing feature of the structure and activity, NLR proteins have been historically divided into two subgroups, based on the amino-terminal presence of either a coiled-coil (CC) domain or a domain with similarity to the Toll/interleukin-1 receptor (TIR) family of proteins. In total, different domains (e.g., TIR/CC, NB, and LRR) of NLR provide abundant interaction interfaces, which not only supports intramolecular interactions that inhibit NLR activation at the absence of corresponding effector, but also serves as intermolecular adapters to form NLR heterodimers that regulates ETI with higher order of flexibility (Sukarta et al., 2016). Directly related to this feature is the genetic evidence that certain extra downstream NLR(s) may be commonly required for ETI activation mediated by various NLRs (Adachi et al., 2019). This leads to a helper-sensor model where a “sensor” NLR (sNLR; Bernoux et al., 2014) perceives the existence of avirulent effector and activates a “helper” NLR (hNLR; Bonardi et al., 2011), which next processes ETI signaling pathway. Such model potentially explains why NLRs form heterodimer and why ETI is mediated by various R protein shares a unique pattern. However, one of the key questions that remains in ETI is: How does activated NLR protein function in ETI initiation?

Recently, a series of publications offers a mechanistic insight into the biochemical function of activated NLR proteins. To explore the topic, Wang and colleagues (Wang et al., 2019a; Wang et al., 2019b) inspected the protein structure of activated NLR ZAR1 (HOPZ-activation resistance-1) through a combined approach of cryo-EM based modeling and analysis. In brief, the study demonstrated that ZAR1 presents a pentameric-like structure, forming a general funnel shape within the PM (Dangl & Jones 2019; Wang et al., 2019a). Related to its function to activate programed cell death, this conformation is easily associated to mammalian inflammasome complexes, which serve as a cytosolic catalytic center to activate downstream apoptosis (Sharma and Kanneganti, 2016). However, the PM-localized ZAR1 differs from cytosolic inflammasomes with respect to its subcellular localization, which suggests a distinguishing function of ZAR1 as a massive channel that mediates influx of apoplast components (including Ca²⁺) and leakage of cytosol and trigger the downstream signaling of ETI. Interestingly and related to this, co-expression of NAIP (inflammasome structure protein that resembles sNLR and recognize animal PAMP), RPS4^TIR-NLRC4 (inflammasome structure protein that resembles hNLR, and fused with TIR domain of RPS4), and corresponding PAMP (conceptually equal to “effector” in plant immunity) in N. benthamiana can trigger HR-like symptoms (Duxbury, 2016), which suggests that formation of the inflammasome in plants is sufficient to trigger ETI, yet the downstream signaling events in animals and plants may vary.

A general picture of ETI can be further clarified if the sensor-helper model is combined with the hypothesis that NLR functions as a PM localized channel. As described by Jubic and colleagues (Jubic et al., 2019), while some NLR, such as ZAR1, can both perceive effector activity and form up an active pentamer channel on the PM, other NLRs (i.e., absolute sNLR) does not have the second capability due to the lack of corresponding interaction interfaces or PM localization. In this case, an hNLR to be activated by sNLR is required for the assembly of PM-localized channel to activate ETI. Howbeit, it is still not clear whether the vast material transport mediated by the NLR channel is the major process responsible for ETI. Since some NLRs have additional biochemical activity, such as regulating TFs in the nucleus (Sun et al., 2020), it is possible that activated NLR can initiate several relatively independent signaling pathways the synergistically contributes to the development of ETI.

C. Local immune attenuation: rebalancing growth versus defense

The energy distribution of growth versus defense requires a constant balancing of signaling processes, including the simultaneous activation and attenuation of processes that share considerable overlap. Quite obviously, plant defense signaling following pathogen perception requires the rapid engagement and activation of a broad range of immune signaling processes, as described above. At the same time, in the absence of pathogens, plants redirect a considerable amount of energy to processes which downregulate immune signaling (Huot et al., 2014; Karasov et al., 2017). Thus, the attenuation of immune signaling is a critical process of self-defense which likely evolved as a mechanism to protect the host from the ill effects of hyperactivated defenses that down-regulates growth.

In contrast to the events associated with the activation of pro-immune signaling, our current knowledge of con-immune signaling is relatively limited. What we do know, however, is that much like immune activation signaling, MAPK cascade also plays an essential role in this process and represent one of the best characterized con-immune signaling mechanisms known. In Arabidopsis, a well-illustrated example of immune attenuation lies in our understanding of signaling mediated by MAPK3/6, which is activates its own inhibitory, MAPK phosphatase 1/2 (MKP1/2; Jiang et al., 2018). In a detailed and elegant series of temporal gradient analyses focusing on the dynamics of MKP1 activity, it was revealed that MKP1 phosphorylation by MAPK6 is saturated at ~10 min following PAMP treatment, resulting in the stabilization of MKP1 and an increase in MPK1 protein levels (Jiang et al., 2017a). Corelated to this observation, MPK1 and MKP2 mRNAs are nominally upregulated (< 2-fold) in response to biotic stress perception; we surmise that this illustrates a relatively low impact of transcriptional regulation on MPK abundance. Besides MAPKs per se, MKPs may actually dephosphorylates a wide spectrum of immune signaling substrates, as evidenced by the fact that MKP1 regulates thousands of MPK6-independent pro-immune transcriptions within 90 min after flg22 elicitation (Jiang et al., 2017b). Moreover, and consistent with the role for MPK1/2 as broad regulators of immune attenuation, it is note-worthy that previous work has demonstrated that MPK1/2 are negative regulators of defense against biotrophic (e.g., R. solanacearum) and hemi-biotrophic (e.g., P. syringae) pathogen, whereas MKP2 is a positive regulator against necrotrophic (e.g., B. cinerea) pathogens (Lumbreras et al., 2010; Anderson et al., 2011). Taken together, these data illustrate that defense attenuation facilitates host immunototipotency against full spectrum of pathogens.

In addition to MKPs, other protein phosphatases (i.e., PP2A/Cs) also function as known contributors of counteracting kinase activity in immune signaling, and as such, play a substantial role in immune attenuation (Withers and Dong, 2017). For example, recent work has demonstrated that a group of PP2Cs (i.e., HAI1/2/3) quench MAPK3/6 downstream of flg22 triggered ABA signaling - a key virulence mechanism utilized by pathogens to manipulate immune signaling (Mine et al., 2017). Likewise, Arabidopsis AP2C1 (aka PP2C25) dephosphorylates MAPK4/6, which modulates JA- and SA-associated immune signaling. In similar mechanisms, additional kinases also regulate the activation of PP2A/Cs, including the key PTI signaling regulators CPK6, BIK1, and BAK1 (Brandt et al., 2012; Segonzac et al., 2014; Couto et al., 2016). However, evidence indicating phosphatase targets of other immune signaling components, such as receptors, enzymes, channels, and TFs, is still lacking, illustrating a general knowledge gap in the breadth of engagement by the mechanism of immune attenuation.

Another important mechanism for immune signaling attenuation is the degradation of immune signaling components, a process that is typically mediated via the ubiquitin-proteasome system (UPS) (Nandi et al., 2006). In brief, UPS functions through the enzymes E1, E2, and E3, among which E1 and E2 energize and load ubiquitin onto the ubiquitin ligase E3, while E3 determines ubiquitination target specificity (Sharma et al., 2016). As a common mechanism in plant immune signaling, several well-characterized examples of ubiquitin-mediated attenuation exist. For example, FLS2 is targeted by the U-box E3 ligases PUB12/13, resulting in the degradation of FLS2 following flg22 stimulation (Lu et al., 2011). As an illustration of the specificity and rapidity of this response, it was further demonstrated that physical association of PUB13 with FLS2 is initiated at ca. 30 seconds post FLS2 activation, indicating that PUB12/13 promotes rapid quenching of immunity. In contrast, LYK5, a membrane-associated receptor kinase responsible for chitin perception, is also targeted by PUB13, but the activation of LYK5 results in its dissociation from PUB13 and enhances LYK5 accumulation (Liao et al., 2017). In another example, the immune kinase BIK1 is ubiquitinated by U-box E3 ligases PUB25/26, but such process is inhibited by the hetero-trimeric G-protein complex XLG2/3-AGB1-AGG1/2 when BIK1 is inactive (Liang et al., 2016). Upon activation of BIK1 (i.e., in response to PTI elicitation), the XLG2/3-AGB1-AGG1/2 inhibitory complex dissociates, releasing unblocked BIK1 for UPS mediated-degradation (Liang et al., 2016; Wang et al., 2018).

Given that the proteasome exists within the cytosol, nucleus, and vacuole, it is a reasonable assumption that free, soluble, proteins are targeted to the proteasome via simple diffusion processes. However, this is not the case of PM-associated proteins, which are typically anchored through a variety of mechanisms, including transmembrane domains, post-translational modification, as well as via association with PM-resident components. In this regard, PM-associated immune signaling components, such as FLS2, BAK1, SERK1, CERK1, LYK5, PERP1, and SlCf-4, all require endocytosis-based mechanisms as a means to regulate recycling and or degradation (Claus et al., 2018). In a general sense, the constitutive endocytosis of membrane components serves as a recycling mechanism to ensure that immunity is maintained in signaling-competent state. As a mechanism describing the naïve and activated recycling of immune receptors, the example of flg22-triggered FLS2 endocytosis is one of the best characterize models (Robatzek et al., 2006; Mbengue et al., 2016). As observed, following flg22 elicitation, the majority of FLS2 (GFP-tagged FLS2) was internalized from the PM to cytosolic vesicles within 20–40 min following flg22 treatment. As an illustration of immune attenuation and the regulation of PTI, during this same time, de novo synthesized FLS2 was not replenished at the PM. Instead, a marked induction in FLS2 transcription was observed, indicating that PRR endocytosis is an approach of immune regulation, in support of UPS, to maintain the equilibrium of immune signaling.

As a final example, the negative regulation of pro-immune transcription represents a key component of the defense signaling network attenuation. Just as immune activation requires the induction of TF-mediated gene expression, so does immune attenuation. Among the best example(s) of this process is illustrated by the activity of the plant-specific family of WRKY transcription factors, known for their broad roles in signaling processes associated with both abiotic and biotic stress (Tsuda and Somssich, 2015; Hussain et al., 2019). For example, Arabidopsis WRKY18 and WRKY40 are rapidly induced following P. syringae DC3000 and G. orontii perception, yet they function as synergistic negative regulators of resistance in response to both pathogens (Xu et al., 2006; Pandey et al., 2010). Using a series of ChIP-seq and RNA-seq approaches, it was further revealed that WRKY18 and WRKY40 possess broad transcriptional regulatory (presumably inhibitory) functions over defense genes during the early activation of PTI (Birkenbihl et al., 2017). Taken together, these studies indicate that certain “WRKY sub-regulatory networks” may serve as a mechanism to prevent over-induction of immunity, through balancing the in/activation of transcription following pathogen perception. Related to this hypothesis, Moore and colleagues (Moore et al., 2011) provide a similar network perspective, proposing a transcription pulse model to describe transcription cascading in plant immunity. In short, this posits that the expression of TFs in different temporal nodules display consecutive cyclical bursts, with sharp up- and down-regulated oscillations over the course of the lifecycle, a process regulated in part by UPS-mediated degradation of transcription activators in the nucleus. Indeed, such a mechanism is required for the degradation of activated NPR1, as well as the enrichment of pro-immune TF inhibitors, including MYC2-induced JAZ expression.

IV. Distal immune signaling

A. Distal immune signaling varies by pattern and pathway

In parallel to the activation of local immune signaling, plants also employ long-distance signaling as a mechanism to prime defense activation in advance of pathogen proliferation. This strategy, to “nip it in the bud”, functions to halt pathogen spread via the mobilization of a core, evolutionarily conserved, class of highly specific signaling molecules. Once mobilized, these signals activate defense responses in distal uninfected cells and tissues, which reduces secondary pathogen invasion, proliferation, and disease (see Figure 4). As a consequence, noninfected cells are primed to enter a pro-immune status. This process, referred to as systemic acquired resistance (SAR; (Durrant and Dong, 2004; Shine et al., 2019)), provides protection against a broad range of pathogens, including bacteria, fungi, and viruses.

Following pathogen perception, a broad spectrum of distal immune signaling is activated, which can be categorized into two basic forms. The first, the electrical wave, mediated by self-feedback ion fluxes that resemble the neural transmission networks found in animals (Leybaert and Sanderson, 2012). In brief, this type of signal travels along the charged PM and requires the operation of a regulated channel transport between the apoplast, cytosol, endoplasmic reticulum, and tonoplast. While a detailed mechanism of intercellular transmission of the electric wave is not fully defined, it is hypothesized that Ca²⁺ influx plays a dominant role and that plasmodesmata (PD) is significantly involved in the intercellular transmission of electric wave (see Figure 4, (C); Choi et al., 2017). Indeed, evidence in support of this is described in a recent study indicating that blockage of PD inhibits Ca²⁺ waves through mesophyll cells, yet not the vasculature (Toyota et al., 2018). Based on the robustness and speed of this signal, it is reasonable to hypothesize that the Ca²⁺ wave represents the first phase of long-distance signaling in response to biotic stress perception.

The second class of immune signal that has been described is broadly classified as messenger molecules, including hormones (e.g., SA, JA), RNA (Kehr and Kragler, 2018; Huang et al., 2019), proteins, and peptides (Segonzac and Monaghan, 2019). These signal molecules, which transmits by themselves, are distinguished from electric waves that stimulates membrane potential without transporting molecules to distal cells. As such, the long-distance messengers transmit signals with high specificity, robustness, and durability, at the expense of speed. Moreover, each of these characteristics determines their biological function to induce and maintain the second phase of distal immunity, when massive pathogen inhibitory molecules are synthesized.

To facilitate the activation and spread of distal signals, there are two transmission pathways in plant tissues - symplastic and apoplastic (Conde et al., 2011; Notaguchi and Okamoto, 2015; Canales et al., 2018). In the symplastic pathway, immune activation signals move within the symplast, the space on the interior of the plasma membrane, comprised of the plant cytosol and endomembrane system, an intercellular network connected by plasmodesmata (PD). In the case of the apoplastic pathway, the signal moves beyond the fringe of the PM, within the apoplast - the space between the PM and the cell wall - wherein solutes diffuse freely. However, intercellular signaling within local parenchyma (mesophyll) has limited speed (Toyota et al., 2018), potentially because the cell wall impedes the diffusion efficiency of signaling molecules within the apoplast. In addition, PD have reduced ion pools and small apertures, both of which diminish electrical signal transmission.

To accelerate the speed of distal immune signaling transmission, plants have evolved the use of the vascular system, particularly the phloem, for immune system activity. Among these signaling conduits, sieve tubes, the conducting cells in phloem, are wire-like cylinders that are joined in tandem with shared cytosol (symplast) and interconnected by multiple sieve poles. As subcellular structures required for intercellular communication, sieve poles originate from PD during development of the phloem, and are distinguished by a diameter of approximately 10–25-fold greater than PD themselves (Heo et al., 2014). Hence, within sieve tubes, electrical signals can be transmitted along the PM of the symplast with extremely minor reductions in speed due to reduced gaps between cells (see Figure 4, (D)). Similar to electrical waves, messenger molecules can diffuse at high speeds inside the sieve tube without physical impediment of the cell wall (De Schepper et al., 2013). As a general result, systemic signals usually travel as fast as 100–1000 mm/s across the vasculature (Choi et al., 2016; Choi et al., 2017), a speed that surpasses that of the spreading pathogen. Interestingly, the fastest speed recorded for vasculature mediated transmission in plants was the cold-shock signal following ice touch in Aloe vera, which was recorded at 132 m/s (Volkov et al., 2007), comparable to myelinated neurons.

B. Calcium: the vanguard of long distance immune signaling

Unlike neural exon networks in animals where ion influx is modulated by voltage-gated channels, current evidence supports that the plant Ca²⁺ electrical wave is driven by PM-associated ligand-gated channels (Leybaert and Sanderson, 2012). Once pathogen invasion is perceived locally, systemic signaling is initiated by the activation of PRR-associated signaling complexes and followed by ROS accumulation and Ca²⁺ influx. While a detailed mechanism is not yet fully described, current knowledge supports a model whereby ROS generation and Ca²⁺ influxes are partially interdependent, and function synergistically in support of immune signaling amplification (see Figure 4, (B)). Data in support of this mechanism includes the observation that the rbohd mutant is capable of inducing Ca²⁺ influx, yet lacks the sustained (i.e., second burst) signaling response (Ranf et al., 2011). Vice versa, in the absence of Ca²⁺, RBOHD can still be phosphorylated by BIK1 but not CPKs (Kadota et al., 2014), which results in dampened activity of RBOHD in the local (Beneloujaephajri et al., 2013; Miller et al., 2009). Therefore, the initial Ca²⁺ influx cannot be fully attributed to the activation of a hypothetical Ca²⁺ channel activated by H₂O₂, either directly or indirectly. Instead, the most likely mechanism is that Ca²⁺ influx is initiated by an unknown PRR signaling component which directly activates Ca²⁺-channels, a process in functions in parallel with H₂O₂-triggered Ca²⁺ influx (Yuan et al., 2017). Interestingly, channels gated by Ca²⁺-derived signals, as a positive-feedback loop, may be responsible the majority of PAMP-triggered Ca²⁺ influx, such as CNGC2/4 (cyclic nucleotide-gated channel 2/4), the calmodulin (CaM)-gated channels activation by flg22 elicitation (Tian et al., 2019).

For distal Ca²⁺ signal transmission, however, activation of channels strictly requires certain patterns of “loop-feedback”, because there is no activated PRR complex to “ignite the calcium spark”. In this process, H₂O₂ is presumably a significant signaling mediator (Miller et al., 2009). While the detailed mechanism linking H₂O₂ and Ca²⁺ remains largely unknown, one potential signaling pathway is via HPCA1, an H₂O₂-activated receptor-like kinase that contributes to the activation Ca²⁺ channels (Wu et al., 2020). As described in a classical model describing distal Ca²⁺ signaling, H₂O₂ generated by RBOHD defuses to adjacent region and activates Ca²⁺ influx, which next leads to the activation of specific CPKs and RBOHD (by CPK). In such a mechanism, the Ca²⁺ signal is transmitted the distal cells and tissues, thus forming a Ca²⁺ electric wave (see Figure 4). This model is supported by evidence demonstrating that the rbohd mutant has a severely dampened Ca²⁺ wave in the root-shoot transmission (Evans et al., 2016). Related to this, vacuolar ion channel TPC1 (two-pore channel 1) is identified as an essential gate regulating Ca²⁺ cytosolic influx from tonoplast during distal Ca²⁺ signaling (Choi et al., 2014b). Since it is dually-gated by both Ca²⁺ ligand and voltage indispensably (Guo et al., 2016), it may function to amplify Ca²⁺ influx following the initial influx mediated by H₂O₂.

In addition to H₂O₂, glutamate has been identified as another key messenger molecule for Ca²⁺-mediated long-distance signaling. It is demonstrated that the distal Ca²⁺ wave, naturally triggered by mechanical damage, requires glutamate receptor like (GRL) family proteins GRL3.3/3.6, and simultaneously, generates wave-like apoplastic glutamate accumulation (Toyota et al., 2018). Because, plant GRLs are broadly classified as amino acid gated ion channels (Forde and Roberts, 2014), it is reasonable to hypothesize that GRLs (such as GRL3.3/3.6 in this case) and an unknown glutamate release mechanism establish a ROS-like loop-feedback system to deliver Ca²⁺ distant signal. However, its relationship with H₂O₂-Ca²⁺ loop is uncertain: they may work independently but synergistically, or dramatically in tandem.

While our understanding of the function and mechanisms underpinning the transmission of Ca²⁺ waves is growing, we are still just scratching the surface in terms of the downstream signaling components following the arrival of Ca²⁺ waves in distal cells/tissues. For example, numerous Ca²⁺-signaling cascades associated with calmodulin (CaM), CaM-like (CML), CPKs, calcineurin B-like protein (CBL)-interacting protein kinase (CIPK), and Ca²⁺/calmodulin-dependent protein kinase (CCaMK) suggest a requirement for both transcriptional-dependent and independent defense responses (Marcec et al., 2019). This includes but is not limited to regulation of TF activity (Bredow and Monaghan, 2019), induction of SA synthesis (Guerra et al., 2020; Wang et al., 2009; Wang et al., 2011), and cross-talk with second messengers (e.g., ROS and NO) (Marcec et al., 2019). However, a critical question remains: how does plant distinguish specific type Ca²⁺ signals with diverse output, while using the shared Ca²⁺-mediated mechanism? As a developing foundation which illustrates this complexity, the “Ca²⁺ signature model” offers a plausible hypothesis to describe signal origination and specificity (Yuan et al., 2017; Marcec et al., 2019). In brief, it is hypothesized that distal Ca²⁺ signals may differ in terms of oscillation dynamics, thus supporting a mechanism whereby Ca²⁺ signals possess an “identity” that is unique to their downstream effect(s). In support of this model, the pattern of the Ca²⁺ wave contributing to several immune processes has already been identified using mathematical approaches (Lenzoni et al., 2018; Liu et al., 2020).

C. Raging defense hormones

As a classic example of long-distance mobile signaling molecules in plants, hormones are not only critical regulators of growth and development, but also play integral roles in stress signaling, including in response to environmental (i.e., abiotic), bacterial, fungal, viral, and insect stimuli. Of the numerous host-derived defense signaling molecules associated with plant immunity, SA and JA are arguably the 2 best characterized hormones required for defense activation in response to a range of pests and pathogens. In simplest terms, SA biosynthesis and activity is triggered following biotrophic pathogen (e.g., bacteria) invasion, while JA is essential for the activation of defense to necrotrophic pathogens and pests. While current models describing defense signaling in plants often bifurcate based on SA- and/or JA-dependent modes of signaling, there is a growing body of literature that describes roles for additional plant hormones in both abiotic and biotic signaling. Indeed, the ethylene (ET), abscisic acid (ABA), brassinosteroids (BR), cytokinins (CKs), auxin (AUX), gibberellins (GAs) and strigolactones (SLs) also contribute to the regulation of plant immunity. However, for sake of brevity, we primarily focus on SA and JA, and thus, point the reader to several recent reviews that cover the role(s) of additional plant hormones in response to pathogen and pest perception (e.g., (Berens et al., 2017; Burger and Chory, 2019)).

1. Salicylic acid: biosynthesis, regulation, and accumulation

Foundational work in our understanding of the regulation of SA-mediated pathogen defense was first provided by Wildermuth et al., who demonstrated that in Arabidopsis the majority (ca. 90%) of SA utilized for plant defense signaling is generated by isochorismate synthase 1 (ICS1) (Wildermuth et al., 2001). In the chloroplast, ICS1 converts chorismate into isochorismate - a product of the shikimate pathway and a common intermediate compound of primary and secondary metabolism. Next, isochorismate is exported from chloroplast by EDS5, a member of the multidrug and toxin extrusion (MATE) transporter family (Serrano et al., 2013). In cytosol, isochorismate is then converted into sochorismate-9-glutamate by the acyl-adenylate/thioester-forming protein PBS3, and finally transformed to SA spontaneously or catalyzed by EPS1, an isochorismate-9-glutamate pyruvoyl-glutamate lyase (Rekhter et al., 2019; Torrens-Spence et al., 2019). Similar to the biological significance of ICS1, PBS3 and EDS5 also play essential roles in SA signaling, as mutation of either of them results in significant reductions in SA accumulation and signaling associated with local and systemic defense activation. However, according to current knowledge, ICS1 is the rate-limiting enzyme and hence, the dominant regulatory site for pathogen-induced SA accumulation. Upon immune signaling activation, ICS1 expression is directly induced by pro-immune TF SARD1 and CBP60g, which activates SA biosynthesis (Wang et al., 2011; Sun et al., 2015). Subject to this mechanism, significant SA accumulation, as well as an increase in ICS1 expression can be detected 4 h after induction of ETI in local leaves (Wang et al., 2015; Liu et al., 2016), and 36 hpi in distal leaves following bacterial infection (Návarová et al., 2012).

2. Salicylic acid: signaling and defense activation

Pathogen-induced SA production and transport from the chloroplast leads to the activation of both local and distal defense signaling processes. In response to the accumulation of SA, the NPR (nonexpressor of pathogenesis related genes) family of proteins, including NPR1, NPR3, and NPR4, serve as SA co-receptors and dominantly regulates SA-mediated defense (Ding et al., 2018; Backer et al., 2019). In brief, NPR1 generally serves as an activator of SA signaling. When SA binds to the C-terminal transactivating domain of NPR1, it disassociates from the N-terminal autoinhibitory domain, which enables NPR1 to activate defense genes in a TF-like manner. On the other hand, NPR3/4 is hypothesized to serve as general negative regulators. At the absence of SA, NPR3/4 act as repressors that inhibit key pro-immune genes, such as WRKY70, SARD1 and TFs in TGA protein family. Upon pathogen perception, induced SA binds to NPR3/4 and inhibits their repressor activity, which acts in parallel with NPR1 to up-regulate defense genes in a stringent manner of control (Ding et al., 2018; Fu et al., 2012; Wu et al., 2012).

3. SA-mediated defense signaling and systemic acquired resistance

While PTI and ETI describe immune processes within infected regions, SAR imparts protection to distal and noninfected sites. As a function of distal immune signaling, and by definition, SAR describes the priming of systemic defense following local activation of immunity, whereby SA is hypothesized as one of the key messenger molecules to transmit the signal. As a first description of the molecular mechanisms underpinning SAR, the induction of a suite of genes (i.e., pathogenesis-related (PR) genes) following pathogen perception and the accumulation of SA provided a set of genetic markers whose expression patterns correlated with the onset of defense signaling, including in association with the activity of SA (Linthorst and Van Loon, 1991). Thanks to this early work, a body of knowledge describing the function and transmission of other SAR messengers has emerged, including the role of pipecolic acid (Pip) and its derivative N-hydroxy-pipecolic acid (NHP) (Hartmann and Zeier, 2018). As candidates for distal immune signal transmission, both Pip and NHP accumulate in systemic leaves as early as 24 h post-inoculation, suggesting a role for these 2 molecules in SAR and immunity. As evidence in support, mutation of critical biosynthetic enzymes required for NHP production, namely ALD1 (AGD2-like defense response protein 1) and SARD4 (SAR deficient 4), causes deficiency in Pip production, with concomitant reductions in SAR. (Hartmann and Zeier, 2018). Further studies demonstrated that mutation of FMO (flavin-containing monooxygenase), which catalyzes conversion of Pip into NHP, results in compromised SAR in plants challenged with P. syringae and oomycete pathogen Hyaloperonospora arabidopsidis (Hartmann et al., 2018b). Interesting, the fmo1 phenotype could be rescued by addition of exogenous NHP, but not Pip, further demonstrating the role for FMO in the SAR, as well as reinforcing the hypothesis that NHP may in fact be the bioactive signal.

4. Jasmonic acid: synthesis, perception, and signaling

Like SA, biosynthesis of JA also occurs within the chloroplast, and requires the conversion of galactolipids to 12-oxophytodienoic acid (OPDA), an intermediate step in the generation of JA by jasmonoyl isoleucine conjugate synthase1 (JAR1) (Ruan et al., 2019). As an inducer of JA biosynthesis, the polypeptide systemin plays an indispensable role in JA-mediating signaling following wounding, including damage resulting from insect herbivory and necrotrophic fungal pathogen infection (Campos et al., 2014). As an illustration of the speed of the JA-induced response, a rapid induction in JA marker genes (e.g., PDF1.2) has been observed as early as 15 min following wounding in both local and distal leaves (Manners et al., 1998). Interestingly, preceding the transcriptional activation of the JA response, increases in the levels of JA-Ile have been observed as early as 5 min post-elicitation, a response that is sustained up at ca. 6 h in local leaves following elicitation. In systemic leaves, JA-Ile levels increased rapidly (ca. 5 min post-elicitation) yet had levels that were substantially reduced as compared to local leaves. Likewise, systemic levels of JA-Ile were also diminished, with reductions as early as ~1 h post-elicitation (Schuman et al., 2018).

5. JA perception

Once JA-Ile is synthesized and properly localized within the immune-activated cell, it is recognized by the receptor COI1 (coronatine insensitive 1; (Sheard et al., 2010)), together with the co-receptor JAZ (JA ZIM domain). As a complex, COI1 and JAZ mediate the downstream signaling of defense through a highly complex, yet elegant, series of events. In short, JAZ proteins are comprised of a N-terminal ZIM domain and a C-terminal Jas domain, which facilitate JA-Ile binding to the receptor. First, the COI1 protein forms a pocket which accommodates JA-Ile with high affinity. Following substrate recognition, the conserved degron motif at N-terminus of the Jas domain found in JAZ, forms a loop to trap JA-Ile into the COI1 pocket, with the C-terminal region of Jas provides a helical structure for COI1 docking (Sheard et al., 2010). Once fully docked, the perception of JA-Ile results in JAZ degradation, via the activity of the SCF^COI1 ubiquitin ligase and the 26S proteasome system, which initiates a series of transcription de-repression events, including the activation of MYC2, an essential TF responsible for the activation of JA-regulated genes (Withers et al., 2012). At the same time, MYC2 stimulates the expression of its downstream repressors (e.g., MTB1/2/3; Liu et al., 2019), which act as an elegant negative feedback loop to attenuate this signaling cascade. Interestingly, and as a mechanism illustrating the antagonistic relationship between JA and SA (noted above), biotrophic pathogens (i.e., SA-dependent), such as P. syringae, utilize secreted effectors that manipulate SA-mediated immunity via modulation of the JA signaling pathway. For instance, the P. syringae effector HopZ1a promotes JAZ1 degradation in a COI1-dependent manner through the activation of JA signaling, leading to a suppression of SA-mediated immunity via down-regulation of ICS1 (Gimenez-Ibanez et al., 2014). Similarly, additional recent studies have demonstrated pathogen effector-mediated manipulation of SA defense via targeting of JAZ-associated function and JA signaling processes (Jiang et al., 2013; Gimenez-Ibanez et al., 2016), illustrating the both the complexity and connectivity of SA and JA signaling in plants.

D. Danger, danger

In addition to a critical role for defense hormones as signaling molecules following pathogen perception and infection, plants also utilize the recognition of self-derived molecules to activate distal defense signaling. The compounds, damage associated molecular patterns (DAMPs), refer to a class of plant-derived signaling molecules that accumulate as a result of pathogen infection, cell injury, and/or the activation of death signaling (Hou et al., 2019). Similar in concept to SAR, DAMPs mediate immune priming in distal cells and tissues, and similar to the recognition of PAMPs, DAMP perception requires plasma membrane-localized receptors, whose activation leads to the initiation of similar signaling cascades (e.g., MAPK, Ca²⁺) and transcriptional reprograming.

Broadly, DAMPs encompass peptides, ATP, host-derived proteins released from damaged cells, and degraded cell wall polysaccharides. As one of the most diverse group of DAMPs, peptide signaling molecules have been well-explored, and in total, illustrates not only defense mechanisms that plants employ to defend against pathogen invasion, but also highlight the evolution and adaptation of pathogens to subvert host defense processes (Heil and Land, 2014; Hirakawa et al., 2017). Among the numerous peptide-based DAMPs identified in plants, the activity of systemin and PEP represent classic examples of wound-induced signaling molecules (Savatin et al., 2014). The first DAMP identified was systemin, an 18-amino acid peptide that accumulates in the apoplast in response to wounding and/or insect damage (Pearce et al., 1993). As a host-derived activator of defense responses, systemin is derived from prosyste-min, a ~200 amino acid precursor that accumulates in the cytosol following wound response activation. Not surprisingly, systemin perception is mediated by a plasma membrane associated receptor (Scheer and Ryan, 1999). Once perceived, receptor binding to systemin results in the rapid induction of JA biosynthesis, which as described above, activates systemic defense signaling in response to fungal pathogenesis and insect herbivory (Wang et al., 2018a; Zhang et al., 2020).

Pep1, another well characterized DAMP, is a 23 amino acid bioactive signaling molecule derived from a larger “propeptide” (i.e., 92 amino acid PROPEP1), whose accumulation leads to the activation of defense signaling, including the generation of H₂O₂ and regulation of JA-responsive genes (Huffaker et al., 2006). In Arabidopsis, it has been demonstrated that transformation of Pep1 from PROPEP1 in cytosol is catalyzed by the cysteine protease metacaspase 4 (MC4), which is activated by wound-induced Ca²⁺ influx (Hander et al., 2019). Once released, Pep1 binds the co-receptors PEPR1 and PEPR2 (PEPR1/2) of adjacent cells, which in turn leads to the activation of defense signaling (Krol et al., 2010; Yamaguchi et al., 2010). Not surprisingly, PEPR1 forms a complex with the LRR kinase BAK1, a critical component of many PRR complexes, further illustrating the connectivity among different immune signaling pathways (Yamada et al., 2016). Such similarity is also embodied in the dynamics of protein recycling. Like FLS2, the PERP1 complex is internalized for degradation in a clathrindependent manner ca. 20 min after exogenous application of Pep1; however, recycling of PERP1 and FLS2 utilize different trafficking pathways (Ortiz-Morea et al., 2016; Mbengue et al., 2016).

In addition to peptide-based elicitors of damage perception, other types of host-derived signaling molecules also function as elicitors of wound-induced defense signaling (Li et al., 2020). One of such DAMPs is extracellular adenosine 5-triphosphate (eATP), which is perceived by the lectin receptor kinase DORN1 (DOes not Respond to Nucleotides; (Choi et al., 2014a). At the downstream, the activation of DONR1 leads to many PTI-like signaling processes, which include Ca²⁺ influx, ROS/NO generation, MAPK phosphorylation, and transcriptional regulation of genes involved in SA and JA signaling (Jewell et al., 2019; Wang et al., 2018b). Similarly, and as a second example of nonpeptide derived elicitor, the co-factor NAD⁺ also plays an important role as a host-derived elicitor of immune signaling, which serves as indicator of cell damage in plant (Wang et al., 2017). Like eATP, NAD⁺ perception also results in the activation of PTI-like responses, yet those specific to SA-dependent defense processes. Not surprisingly, NAD⁺, together with its receptor LecRK-1.8, a PM-localized kinase and homolog of DORN1, is required for SAR (Luo et al., 2017).

V. The development of the plant immune system

A. Age related resistance and immune system development

The ontogenesis of an organism is an amazing biological process. Indeed, as organismal systems grow and mature, “new” signals are activated and “old” processes are attenuated; collectively, this dynamic signaling landscape highlights the concept of organismal development. In the context of an integrated system, we would argue that much of the plant immune system is invisible, because, as described above, the immune system is associated with and connected to nearly all processes within the living cell. Unlike the immune system of animals, which possesses a narrowly defined, yet highly differentiated immune surveillance network, the plant immune system is not specifically differentiated based on cell and/or tissue type (Handley et al., 2005). In this vein, the development and maturity of the plant immune system is virtually indistinguishable from the development and maturation of the organism itself.

For the sake of comparison, we propose two general perspectives to illustrate the development of the plant immune system. First, in temporal scales, the chronological age of a plant, as well as the developmental stage of growth, is positively corelated with the robustness of pathogen resistance (Hu and Yang, 2019). In this regard, the robustness of immune signaling is known to increase from early developmental stages to reproductive stages, after which time, the fitness/robustness of the immune system is reduced, presumably as a function of host senescence (Eichmann and Schafer, 2015; Haffner et al., 2015). During this process, discernable patterns of age-related immunity can be described as: (1) transitions in immune robustness at each developmental checkpoint in plant growth (Rusterucci et al., 2005); and (2) the accumulation of immunity as a feature of organ maturation (Ficke et al., 2002; Gadoury et al., 2003). Taken together, it is also tempting to hypothesize that the biological significance of age-related immunity may have its foundation in the energy tradeoff of immunity vs reproduction. If true, this would suggest a mechanism which reinforces the necessity to protect reproduction by investing energy to immunity during development. In support of this hypothesis, a recent ecogenetic study reveals that two strategies are adapted by Arabidopsis natural population to secure their reproduction: an extension of the vegetative stage for higher energy gain to invest a robust immune system, or a shortened vegetative stage for fast reproduction to avoid pathogen infection and death (Glander et al., 2018).

Second, and as a function of spatial scales, the robustness of immune system increases from early to late developed organs. To illustrate this concept, we present a simple disease symptom record when Arabidopsis is inoculated with P. syringae (Figure 5, (A)). As shown, an obvious trend emerges whereby late developed leaves have increasingly lower disease symptom development (i.e., enhanced resistance). Indeed, this phenomenon is further supported by several studies, including recent work which demonstrated that juvenile rosettes, adult rosettes, and cauline leaves from 8-week-old Arabidopsis plants have dramatically different levels of resistance against both P. syringae and S. sclerotiorum (Hu and Yang, 2019; Kus et al., 2002); a similar observation has also been observed using N. benthamiana and tobacco (Xu et al., 2018). At a mechanistic level, age-related enhancements in immunity in mature organs require the function of key defense hormone signaling components, as evidenced by compromised resistance in adult and cauline leaves in the hormone biosynthesis mutants sid1 (SA), jar1 (JA), and aba1 (abscisic acid) (Wilson et al., 2017). As a step to further describe this mechanism at a transcriptional level, we analyzed publicly available mRNA datasets derived from Arabidopsis leaves of different developmental stages and observed that mRNA expression profiles of immune-related genes show certain corelated patterns to the morphogenesis order of different leaf samples (Figure 5, (B)). In short, this correlation suggests the involvement of a shared upstream signaling nodule that regulates plant morphogenesis and age-dependent immunity in an integral and synergistic manner.

Figure 5.

Phenotypic example of age-related immunity in Arabidopsis. (A) Disease symptom varies in simultaneously inoculated rosette leaves of 5-week-old Arabidopsis Col-0 following dip-inoculation with Pst DC3000 (10⁸ CFU/mL). While early-developed rosette leaves (red arrow) show severe disease symptoms (i.e., shrinking, chlorosis, and water-soaking), late-developed leaves (blue arrow) do not show disease symptoms in response to pathogen inoculation. (B) Immune-associated gene expression gradually changes among leaves in different development order. To illustrate this, we downloaded published RNA-microarray data (Winter et al., 2007) reflecting the transcriptome of Arabidopsis rosette leaves 2, 4, 6, 7, 8, 10, 12, and both healthy and senescent cauline leaves. To screen for immune-associated genes, we selected genes within 10 key immune-associated categories: immune, immunity, resistance, defense, biotic, chitin, fungus, flagellin, peptidoglycan, and bacterium. As an output of this analysis, we identified 3901 genes with potential roles related to plant immunity. Next, we used a Pearson filter (|r| > 0.5 and P-value ≤ 0.05) to select and categorize genes whose expression pattern are corelated to the development order of different samples and determined 2104 immune-associated genes that can be categorized into 4 groups, with differing but significant trends, during development of Arabidopsis. The average pattern of each group of these genes is presented. Bold, tinted line: average. Thin, dark line: average ± se.

B. Autologous genetic regulation controls immune system development

Similar to the development of any organ/system during ontogenesis, the development of the plant immune system is mediated by autologous transcriptional regulation. So far, studies focusing on a group of microRNA, miR156/157, have revealed a spatiotemporal regulatory network over the maturation process of plant immune system. At upstream, miR156/157 is regulated by the age-sensitive CDK8-MED12/13 mediator complex (Gillmor et al., 2014); at downstream, miR156/157 target TFs in the SPL (Squamosa-promoter binding protein-like) family and inhibit their expression (Preston and Hileman, 2013). Since SPLs directly and indirectly regulate expression of defense genes (see Figure 6), the miR156/157-SPLs signaling module plays central role in regulating the development of the plant immune system, thereby shaping age-related immunity (Zheng et al., 2019).

Figure 6.

The development of plant immune system is regulated by both autologous genes and commensal microbiota. Plant immune system maturation is correlated with the system development of the plant. In this process, miR156/157-SPLs plays a significant role in regulating the expression of genes functioning in immunity, including JAZ3, N, RPS4, ICS, and FLS2. As a central regulatory module of plant development, miR156/157-SPLs also play a key role in the synchronization of plant aging and organ morphogenesis. Additionally, the development of the plant immune system is indispensable to its commensal microbiota. While they do not necessarily cause disease, these microbes stimulate the development, maturation, and activity of the plant immune system, as well as the general development of the plant. To recruit a healthy microbiota, plants can selectively or nonselectively repel pathogens and attract beneficial microbes. Beneficial microbes can also inhibit the population growth of host associated pathogens, and as such, indirectly influence plant immunity.

For example, miR156-SPL9 contributes to resistance against P. syringae during early vegetative stages through the regulation of defense genes and ROS accumulation (Yin et al., 2019). As one of the best characterized mechanisms in behind, miR156-SPL9 controls FLS2 and basal defense through regulating miR172. In brief, miR172, promoted by SPL9 (and maybe SPL10/15 redundantly; Wu et al., 2009), inhibits two FLS2-repressive TFs, TOE1/2. Thus, while miR159 decreases in seedlings from day 2 to 6, the miR172 is upregulated for 7 folds, which eliminates TOE1/2 transcript by 65% and therefore increases FLS2 transcript by 7 folds (Zou et al., 2018), leading to the immune maturation of seedlings. Besides miR172, the miR156/157-SPLs module also regulates other components of immunity through the lifetime of plant, including facilitating JA signaling by stabilizing JAZ3 from UPS mediated degradation (Gaquerel and Stitz, 2017; Mao et al., 2017) and contributing to the ETI mediated by TIR-NB-LRR protein N and RPS4 (Padmanabhan et al., 2013).

Interestingly, SPLs are considered critical regulators of organ morphogenesis rather than having exclusive roles as defense genes (Ye et al., 2019). This is significant, as it offers a unique perspective to understand the relationship between plant development and immunity - instead of antagonistic signaling modules (in terms of energy tradeoff) or relatively independent processes, they are synergistic pathways subject to a common upstream signaling center (i.e., miR156/157-SPLs). In accordance with this perspective, many essential genes in charge of plant vegetative/reproductive morphogenesis at the downstream of miR156/157-SPLs can also regulate the defense genes simultaneously, such as LEAFY (Yamaguchi et al., 2009) and SOC1 (Lee and Lee, 2010), which contribute to PTI (Winter et al., 2011) and SA signaling (Wilson et al., 2017), respectively. Hence, plant morphogenesis and immune maturation are merely two sides of a coin named “development”.

Furthermore, this regulatory framework enables an advanced strategy to bypass certain antagonistic tradeoffs when confronting biotic stresses, by dynamic replacements of dominant signaling pathway at deferent developmental stages. For example, older plants possess dampened JA signaling-associated processes because a high level of SPL9 stabilizes JAZs. However, these plants also show robust resistance against insects, potentially due to an abundance of glucosinolates accumulated in leaves (Gaquerel and Stitz, 2017). As a result, such mechanism, which is subject to the JA/SA antagonism paradigm, would enable a high dynamic range to mobilize SA-mediated immunity, thus maintaining broad levels of resistance. The next step in advancing our understanding of these mechanisms is to reveal how plants utilize a dynamically developing immune system to overcome what is typically a “zero sum game” with respect to antagonistic tradeoffs.

C. The role of beneficial microbial associations on the plant immune system

Plants serve as host to numerous microorganisms originated from a variety of sources, including via aerosols, animals, rain, and soil (Müller et al., 2016). There are two primary interfaces for microbial interaction: above-ground (aka phyllosphere) and below-ground (aka rhizosphere; see Figure 1). In either case, the assembly and maintenance of the plant-associated microbiome is mediated in part by plant immune system itself, which involves phytoalexin, ETI, PTI, and other unknown approaches to shape the commensal microbiota selectively or unselectively (Hacquard et al., 2017; Teixeira et al., 2019; Vannier et al., 2019). In return, as introduced next, the commensal microbiota perform critical functions in facilitating the development of immune system and plant morphogenesis extensively, potentially due to their signaling cross-talk (see Figure 6).

In the broadest sense, the nonpathogenic components of commensals influence the plant immune system in two primary ways. First, microbial communities can stimulate the development and maturation of the immune system (Vannier et al., 2019). In an effort to identify the relationship(s) between microbiome function maturation of host immunity, a recent study utilized a germ-free (axenic) environment to evaluate immune system performance over the ontogenesis of the plant (Kremer, 2017; Kremer et al., 2018). As demonstrated, Arabidopsis grown in an axenic environment showed subtle, yet measurable, differences in shape and size as compared to plants in holoxenic (with a natural microbiome) environments. However, in terms of immune system maturation and performance, axenic plants showed significant deficiencies compared to holoxenic plants, because the axenic plants display an overall shut-down of defense gene expression and immune signaling processes (e.g., MAPK activity, ROS burst, and defense se hormone biosynthesis). Echoing this study, Dur an et al. also demonstrated that microbial community functions to enhance plant resistance against invading pathogens and identified certain components of protective microbes (Dur an et al., 2018). Collectively, these studies convincingly demonstrate that the development of immune system is indispensable to the engagement of plant-associated microbiome. Interestingly, while facilitating the development of plant immune system, commensal microbiome can also promote root morphogenesis in an auxin signaling-dependent manner (Zamioudis et al., 2013; Klikno and Kutschera, 2017). In agreement with current models that organ morphogenesis and immune system development are potentially coupled, as discussed above, these data supports the hypothesis that the plant-associated microbiome not only plays a critical role in promoting the development of plant immune system, but also contributes to additional aspects of whole plant ontogenesis.

A second process through which commensal microbes assist plant immunity is via inhibiting pathogen proliferation by direct microbe-microbe interaction(s) (Vannier et al., 2019). While a full mechanistic understanding is still lacking, significant insight has been gained through the identification and characterization of the role of secreted microbial anti-biotic compounds. For instance, a rhizosphere bacteria, Streptomyces sp. S4–7, was shown to secrete a thiopeptide that significantly inhibits the growth of F. oxysporum (a fungal pathogen causing Fusarium-wilt) during pathogenesis (Cha et al., 2016). In this study, mRNA-seq analysis revealed that the antimicrobial thiopeptide can impair RNA metabolism, cytoskeleton architecture, and cell wall biosynthesis in its targeted fungal pathogens. In a similar case, Pseudomonas piscium, isolated from wheat, can secrete an antimicrobial compound, phenazine-1-carboxamide, that suppresses F. graminearum via the inhibition of FgGcn5, a histone acetyltransferase, resulting in histone acetylation dysfunction (Chen et al., 2018). Another common mechanism that enhance immune system function and broad defense mechanisms is through the reduction of pathogenicity and abundance of potential pathogens. Recently, through a large-scale analysis of microcosms and greenhouse disease assays, it was shown that rhizosphere commensals with high diversity and a large niche overlap with R. solanacearum can suppress pathogen population growth and disease symptom development on tomato (Wei et al., 2015). In addition to these processes, several additional mechanisms have been described that have the potential to alleviate the threat of the pathogen over-taking the plant immune system, including nutrition interdependency, biofilm formation, endosymbiosis, quorum sensing, and predation (Hassani et al., 2018).

VI. Final thoughts

To date, numerous processes involved in plant-pathogen interactions have been identified, the analysis of which has greatly contributed to a better understanding of the molecular mechanisms underpinning plant immunity. However, while most research in this area has necessarily been guided by a reductionist approach, a full picture of plant immunity, including the connectivity of different immune processes, requires higher resolution-based approaches and a systemic perspective. Moreover, it requires a full assessment and integration of the temporal and spatial events to generate a complete picture. In this review, we focused on the presentation of an integrative and dynamic process, one that is mediated by local and distal signaling of immunity, as well as plant ontogenesis and its interaction with the environment. However, on the frontier of this field of study still stand many old and new questions: How do plants dynamically promote and attenuate immunity in a tradeoff? How do plant neuron-like transmission systems function? How do plants decode single (e.g., Ca²⁺) and combined signals for robust signaling that is specific to and appropriate for the nature of the stimulus? Can growth and immunity be integrated synergistically rather than antagonistically? These are just a few of the outstanding questions that remain on the battlefield of plant-pathogen interactions.