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TCP three-way handshake: linking developmental processes with plant immunity Jessica A. Lopez1, Yali Sun1, Peter B. Blair1, and M. Shahid Mukhtar1,2 1 2
Department of Biology, University of Alabama at Birmingham, AL 35294, USA Nutrition Obesity Research Center, University of Alabama at Birmingham, AL 35294, USA
The TCP gene family encodes plant-specific transcription factors involved in growth and development. Equally important are the interactions between TCP factors and other pathways extending far beyond development, as they have been found to regulate a variety of hormonal pathways and signaling cascades. Recent advances reveal that TCP factors are targets of pathogenic effectors and are likely to play a vital role in plant immunity. Our focus is on reviewing the involvement of TCP in known pathways and shedding light on other linkages in the nexus of plant immunity centered around TCP factors with an emphasis on the convergence of effectors, interconnected hormonal networks, utility of the circadian clock, and the potential mechanisms by which pathogen defense may occur. TCP transcription factors, highly connected proteins in plant cellular network Transcription factors (TFs) are essential components of cellular machinery that regulate the spatiotemporal expression of thousands of genes in response to both internal and external stimuli [1]. This ensures the fine-tuned cellular functions of an organism during cellular proliferation, normal growth and development, and various disease states. The TCP gene family was discovered in 1999 and named after the first three characterized members, TEOSINTE BRANCHED1 in maize (Zea mays), CYCLOIDEA in snapdragon (Antirrhinum majus), and PCF (proliferating cell nuclear antigen factor) in rice (Oryza sativa) [2]. In the past quarter-century, the advances in genome and transcriptome sequencing shaped the TCP family into a complex network of genes encompassing 24 members in Arabidopsis (Arabidopsis thaliana) [3–5]. These plant-specific factors encode a 59-amino acid basic helix–loop–helix (bHLH) motif that allows for the classification of TCP TF family members into two groups, known as class I (or PCF or TCP-P) and class II (or TCP-C) [4]. TCP class II is further subdivided into the CIN and CYC/TB1 subclades (Figure 1A). The TCP class I is distinctive as its members Corresponding author: Mukhtar, M.S. ([email protected]). Keywords: TCP factors; pathogen effectors; SAP11; SRFR1; hormonal network; circadian clock; transcriptional control; miR319. 1360-1385/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2015.01.005
possess a four-amino acid deletion within the TCP domain in addition to the presence of other subclade-specific conserved signatures (Figure 1B). Another hallmark difference is the presence of an R domain (an 18–20 residue arginine-rich motif) and an ECE motif (a glutamic acid– cysteine–glutamic acid stretch), both of unknown biological functions, in subsets of TCP class II [4,5]. It is likely that TCP genes appeared in the Streptophyta lineage before the divergence of Zygnemophyta, between 350 and 800 Mya [3,6]. It is also believed that the division of the TCP family resulted from an ancient duplication event before the divergence of Zygnemophyta [6]. Genes of the CIN-like clade are involved in lateral organ development such as CINCINNATA (CIN) in the snapdragon, and the CYC/TB1 clade’s gene control axillary meristem development, such as TB1 in maize and its orthologs in Arabidopsis [7]. It is plausible that the CIN clade is more ancient than the CYC/ TB1 clade because primitive plants such as Selaginella and Physcomitrella have neither flowers nor branches and possess TCP–CIN homologs in their genomes [3,4]. Reconstructing the phylogeny of TCP genes in basal eudicots [8] could yield a clearer framework for understanding the functional evolution of these genes. It is possible that this framework could play a crucial role in linking evolution of TCP factors to their cellular functions. TCP family members have demonstrated involvement in a wide range of biological processes throughout the entire life span of plants. This includes the coordination of cell proliferation [9–12], differentiation of several morphological traits including shoot apical meristem and leaf development [13–15], biosynthesis of phytohormones [16], as well as the regulation of circadian clock rhythms [17]. These findings have positioned TCP factors as central regulatory nodes in diverse signaling pathways. Given that cellular functions are highly coordinated, finely orchestrated interactions of numerous proteins and biomolecules, the emerging question is whether topological properties of TCP proteins play an essential role in this biological network. Recent large-scale protein–protein interaction studies have revealed a TCP-centered module composed of 419 interactions among five TCP proteins (TCP13, TCP14, TCP15, TCP19, and TCP21) interacting with 236 other Arabidopsis proteins (Figure 1C) [18]. The vast majority of TCP interactors are enriched in diverse GO categories signifying the importance of TCP factors in a Trends in Plant Science xx (2015) 1–8
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specific interactors. Here, we will review the mechanisms of established TCP interactions and their role as effector targets via NLR (nucleotide-binding domain leucine-rich repeat protein) and TIR (Toll/interleukin-1 receptor)– NLR-mediated effector-triggered immunity. Further, we will examine the ramifications, both direct and indirect, of various TCP factors on hormonal signaling to recognize virulence perturbation. Additionally, we will use the circadian clock as a model regulatory network rendering various connections to immunity via TCP regulation. Lastly, we will unveil how TCP family members regulate downstream target genes and the prominence of miRNAs (miRs) in gene expression, which delineate further immunity linkages via TCP factors.
TCP6, TCP7, TCP8, TCP9, TCP11, TCP14, TCP15, TCP16, TCP19, TCP20, TCP21, TCP22, and TCP23
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Pathways Auxin
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Figure 1. TCP transcription factor family, its classification, and position in the protein–protein interaction network. (A) A phylogenetic representation of class I (TCP-P) and class II (TCP-C), along with their respective subclades and TCP members. (B) Schematic representation of TCP domain structure. Sequence alignment of one member of each subclade is shown. Basic, helix I, loop, and helix II motifs within the TCP domain are marked. Red dashes on yellow background represent the lack of amino acids in TCP class I. Conserved residues among all the subclades are highlighted in purple. (C) A subnetwork encompassing the TCP protein in Arabidopsis interactome-1 is displayed using Cytoscape software. Members of TCP proteins in the module are labeled. In row 1, functional annotation clustering of TCP interactors is revealed, which was performed using DAVID Bioinformatics Resources 6.7. Key proteins with known functions are extracted (row 2) including AFB2 (AUXIN SIGNALING F-BOX 2), PRR2 (PSEUDORESPONSE REGULATOR 2), GRX480 (GLUTAREDOXIN FAMILY MEMBER ALSO KNOWN AS ROXY19), CAND1 (CANDIDATE G-PROTEIN-COUPLED RECEPTOR 1), ERF12 (ERF DOMAIN PROTEIN 12), DWA1 (DDB1-BINDING WD40 PROTEIN ALSO KNOWN AS DWD), HAT2 (HOMEOBOX-LEUCINE ZIPPER GENE), SWEET16 (NODULIN MTN3 FAMILY PROTEIN), PIA1 (PHYTOCHROME-INTERACTING ANKYRIN-REPEAT PROTEIN 1), CML41 (CALMODULIN-LIKE 41), MOS1 (MODIFIER OF SNC1), and ACX1 (ACYL-COA OXIDASE 1). Pathways relevant to the known TCP interactors are classified in row 3.
wide spectrum of cellular processes. In addition to several interactors involved in biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), ethylene, abscisic acid (ABA), and auxin, proteins involved in circadian rhythms regulation, phytochrome-mediated light responses, calcium homeostasis, and even sugar transport have been identified as direct TCP interaction partners [7] (Figure 1C). It has only recently emerged that these seemingly independent physiological processes are implicated in the plant immune response [19,20]. Thus, members of the TCP family constitute highly connected nodes or hubs in a plant cellular network, asserting their essential roles at intersections of various signaling pathways, and control vital plant immune processes through recruitment of 2
TCP proteins as pathogen effector targets Although the diversity of pathogens is immense, various attackers across kingdoms utilize a common infectious strategy that causes global disruption of host responses. Specifically, specialized pathogens employ virulence factors (effectors) to attack cellular hubs in host biological networks, thereby hijacking cellular machinery in a manner conducive to pathogen proliferation [21,22] and to induce effector-triggered susceptibility (ETS). Conversely, host immune receptors including NLRs or PRRs (patternrecognition receptors) can recognize effectors and pathogen-associated molecular patterns (PAMPs) to prompt effector-triggered and PAMP-triggered immunity (ETI and PTI, respectively) [21,23,24]. Strikingly, plant–pathogen interaction network-1 (PPIN-1) and PPIN-2 revealed that multiple members of TCP factors (TCP13, TCP14, TCP15, TCP19, and TCP21) are targeted by effectors from three pathogens that span the eukaryote–eubacteria divergence, represented by the bacterium Pseudomonas syringae (Psy), the oomycete Hyaloperonospora arabidopsidis (Hpa), and the true fungus Golovinomyces orontii (Gor) (Figure 2) [25,26]. Plants lacking TCP13, TCP14, and TCP19 exhibited enhanced disease susceptibility phenotypes [25]; TCP21 remains to be tested in this aspect. This discovery opens a new avenue of research focused on further characterization of the TCP factors and expansion of their functions to plant immunity. Another question pertinent to these findings is whether TCP proteins are virulence targets of the effectors and, if so, what are the mechanisms whereby pathogen effectors target TCP proteins to induce ETS. A recent report showed that overexpression of tomato (Solanum lycopersicum) TCP14-2 exhibited enhanced immunity to Phytophthora capsici, a phenotype that can be reversed upon coexpression of CRN12_997, a P. capsici effector that physically interacts with SlTCP14-2 [27]. This lends additional support for the concept of effector–host hub interactions, which can be conserved in divergent host–microbe systems. However, it remains to be discovered how TCP proteins participate in plant disease resistance. Intriguingly, another study identified six TCP class I proteins (TCP8, TCP14, TCP15, TCP20, TCP22, and TCP23) interacting with SRFR1 (SUPPRESSOR OF rps4-RLD1) (Figure 2) [28], two of which are also effector targets in PPIN-1 [25]. SRFR1 encodes a tetratricopeptide repeat (TPR) protein conserved in both plants and animals and was previously demonstrated to
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Figure 2. Proposed model of involvement of TCP proteins in the plant immune system. TCP factors–SRFR1 (SUPPRESSOR OF rps4-RLD1)–EDS1 (ENHANCED DISEASE SUSCEPTIBILITY)–SRFR1-mediated activation of NLR (NUCLEOTIDEBINDING DOMAIN LEUCINE-RICH REPEAT PROTEINS) and induction of ETI (EFFECTOR-TRIGGERED IMMUNITY; manifested in the form of cell death) are illustrated. TIR (TOLL/INTERLEUKIN-1 RECEPTOR)–NLR, RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE 4), RPS6, and SNC1 (SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1) physically interact with EDS1 in the nucleus. Interactions of two pathogen effectors, AvrRps4 (red star) and HopA1 (blue star), with EDS1 are illuminated. SRFR1 interactions with either EDS1 or TCP proteins are shown. It is important to note that all of these proteins may not be part of the same complex. TCP proteins marked with a blue star designate TCP factors as effector(s) targets in PPIN-1 (PLANT–PATHOGEN INTERACTIONS NETWORK-1). The green star indicates TCP factors that are affected as a result of manipulating the jasmonic acid (JA) pathway. The red line with bar illustrates that MOS1 (MODIFIER OF SNC1) is a genetic suppressor of SNC1. TCP8, TCP14, and TCP15 may trigger ETI via TIR– NLR and CC (COILED-COIL)–NLR. The functions of TCP13, TCP19, and TCP21 in ETI or ETS (EFFECTOR-TRIGGERED SUSCEPTIBILITY) are not known.
act as a negative regulator of ETI [29–33]. As an adaptor protein, SRFR1 forms complexes in both the cytoplasm and the nucleus with several immune regulators including SGT1a (SUPPRESSOR OF THE G2 ALLELE OF SKP1 A; a cochaperone of the resistance proteins), SGT1b, EDS1 (ENHANCED DISEASE SUSCEPTIBILITY; a positive regulator of immunity), and TIR–NLR including RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE 4) and RPS6 [34–36]. It is worth noting that two different effectors, AvrRps4 and HopA1, target EDS1 and disrupt SRFR1–EDS1–NLR complexes leading to AvrRps4- or HopA1-triggered immunity (Figure 2) [36]. It was proposed that SRFR1 and SGT1b may modulate differential levels of NLR protein accumulation in response to the presence of diverse effectors [34]. It was established that tcp8-1 tcp14-5 tcp15-3 triple mutant plants display compromised ETI that can be triggered by four sequence unrelated effectors, that is, AvrRps4, HopA1, AvrRpm1, and AvrRpt2. These results suggest that TCP8, TCP14, and TCP15 positively regulate ETI-mediated resistance by the TIR–NLR, RPS4 and RPS6, and the CC (COILED-COIL)–NLR, RPS2, and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA 1) [28]. This is significantly different from EDS1/SRFR1-mediated ETI, which is controlled by only TIR–NLR. Exposure of TCP8, TCP14, and TCP15 to a range of effectors affirms the likelihood that these TFs determine an immune set point by modulating gene expression, which could provide an explanation for why they are targeted by pathogen effectors [28]. This is significantly
different from EDS1/SRFR1-mediated ETI, which is controlled by only TIR–NLR. Since the tcp8-1 tcp14-5 tcp15-3 mutants and Col-0 plants were equally susceptible to DC3000, it appears that TCP proteins are not general plant immune regulators but merely involved in CC– NLR and TIR–NLR-mediated ETI. This also suggests that the TCP proteins constitute host virulence targets, and some NLRs may have evolved to guard TCP factors from an effector’s attack allowing ETI to be triggered [25] (Figure 2). The first clue to the identity of TCP-related NLRs may originate from the interaction of TCP15 with MOS1 (MODIFIER OF SNC1), a BAT2 domain-containing protein [37]. MOS1 regulates SNC1 (SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1, a TIR–NLR)-mediated resistance responses by fine-tuning SNC1 expression. It is likely that MOS1, TCP15, and SNC1 form complexes in the nucleus. Furthermore, how SRFR1 maintains TCPrelated NLR protein accumulation has yet to be determined. Since only a subset of effector target TCP factors has been demonstrated to interact with SRFR1, we expect additional negative regulator adaptor proteins to form different nuclear complexes with NLRs. Future studies should shed more light on the mysteries surrounding effectors–TCP factors–NLR complexes. TCP-mediated hormonal crosstalk homeostasis Phytohormones are signal molecules produced within a plant that control nearly all stages of growth and development by regulating gene expression that in turn translates into appropriate morphological and/or physiological responses [20]. TCP factors are involved in a diverse spectrum of both mutualistic and antagonistic biological interactions. For instance, the antagonistic relationship between the two classes of TCP factors, TCP class I and TCP class II, was shown to balance contrasting activities of TCP class I members promoting cell proliferation in leaves and TCP–CIN factors, negative regulators of leaf growth, and positive regulators of senescence [10,38]. This contributes to fine-tuning of phytohormone production and ensuring normal leaf and plant morphology [39]. However, it is expected that the pathogen effectors manipulate hormonal crosstalk to their advantage. Indeed, it was demonstrated that SAP11, a secreted protein from Aster Yellows phytoplasma strain Witches’ Broom (AY–WB), interacts with and destabilizes all eight members of TCP–CIN, but does not interact with any related TCP members belonging to TCP class I family (Figure 3A) [40–43]. These biochemical data were further substantiated by genetic studies, revealing that SAP11 transgenics and octuple tcp–cin mutants, which exhibit reduced protein levels of all TCP–CIN, both display deeply lobed and highly crinkled leaves phenotypes [41]. This action of SAP11-mediated destabilization is biologically relevant, because SAP11 transgenics and AY–WB-infected plants produce less JA in response to wounding, leading to increased fecundity of AY–WB insect vector leafhopper (Macrosteles quadrilineatus) [41]. These findings spark questions into how TCP–CIN and TCP class I regulate JA biosynthesis under diverse environmental conditions. Selected members of TCP–CIN were shown to positively influence LOX2 (LIPOXYGENASE 2) gene expression and JA biosynthesis, whereas TCP4 binds directly 3
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Figure 3. Schematic representation of a model depicting effector-mediated manipulation of hormonal pathways. (A) SAP11 (a secreted protein from Aster Yellows phytoplasma strain Witches’ Broom)-mediated destabilization of TCP–CIN (CINCINNATA) members are illustrated. Interaction of SAP11 with TCP–CIN members leads to the disappearance of TCP proteins (shown in broken pink oval) and less jasmonic acid (JA) production. (B) TCP4, a CIN–TCP, and TCP9 and TCP20, two TCP class I members, directly bind onto the promoter of LOX2 (LIPOXYGENASE 2) and regulate LOX2 gene expression and JA synthesis antagonistically. Effectors are proposed to interfere with LOX2-mediated JA signaling. (C) TCP–CIN members, in complex with AS1 (ASYMMETRIC LEAVES 1) and AS2, directly bind onto the promoter of the KNOX gene and repress its expression. KNOX protein is shown to be an effector target in PPIN-1 (PLANT– PATHOGEN INTERACTION NETWORK-1). Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively.
onto the promoter of LOX2 (Figure 3B) [38]. Interestingly, it has been found that two TCP class I members, TCP9 and TCP20, have been found to bind to LOX2 promoter and exert a contrasting effect of downregulating the JA pathway (Figure 3B) [11]. This antagonistic relationship between TCP–CIN and TCP class I members, in JA biosynthesis, provides an opportunity for diverse pathogens to directly or indirectly target TCP factors, thereby tipping the hormonal balance for their benefits. In conjunction with the JA and other signaling pathways, serving as another important linkage in the search for connections with TCP factors and plant pathogen effectors is the protein known as TOPLESS (TPL; an effector target in PPIN-1) [25,44]. TPL is a universal corepressor that orchestrates the crosstalk between JA and other hormone signaling pathways [18,45]. A recent TPL-specific interactome study revealed that both members of TCP– CIN (TCP2, TCP3, and TCP4) and TCP class I (TCP8 and TCP14) interact with TPL [44]. TCP14 and TCP15, both members of TCP class I and effector targets, were shown to function in cell proliferation and in several plant hormone signaling pathways [46]. TCP14 interacts with RGA (REPRESSOR OF GA1–3) and GAI (GIBBERELLIN INSENSITIVE), the two most abundant DELLAs, and are involved in the regulation of GA-responsive gene expression [47]. Another finding suggests that TCP14 and TCP15 4
participate in the cytokinin (CK) responses through interaction with O-linked N-acetyl glucosamine transferase SPINDLY [48]. Expansion of the nexus continues from JA to SA, auxin, CK, GA, and ABA with the involvement of TPL–TCP factors, TCP–effectors, and TPL–effectors crosstalk. Another central player in hormonal homeostasis and the plant developmental process is BP (BREVIPEDICELLUS; a Knotted1-like homeobox or KNOX family member), an effector target in PPIN-1 [49]. TCP–CIN members, in complex with other TFs, AS1 (ASYMMETRIC LEAVES 1) and AS2, directly bind onto the BP promoter and regulate its expression [50]. Interestingly, a study determined that a TCP class I family member, TCP7, can bind to the promoter of STM (SHOOT MERISTEMLESS), a KNOX gene (Figure 3C) [10,49]. A tcp8 tcp22 mutant that loses the function of two TCP class I genes showed altered leaf shape, and this phenotype is further pronounced in triple mutants (as1 tcp8 tcp22) [10,49]. The possible participation of both TCP class I and TCP–CIN factors in the regulation of AS1, AS2, and KNOX genes could serve as another mechanism by which TCP factors can play an active role in pathogen defense. Taken together, it is conceivable that pathogen effectors target TCPmediated complexes both directly and indirectly to manipulate hormonal signaling network with the aim of causing virulence. Role of TCP proteins in circadian clock and plant defense Serving as a resonance between the internal timekeeper and environmental cues, the circadian clock regulatory network is crucial to enhancing fitness and survival of plants, and has been known to play a key role in plant physiology. Recently, strides have been made to develop a notion that the circadian clock is linked to plant immunity [51,52], but the key question remains whether TCP factors play significant roles in circadian clock regulation during plant defense. TCP21, also known as CHE (CCA1 HIKING EXPEDITION), is an effector target in PPIN-1 and its discovery seems to fit the bill as a missing piece in the circadian clock puzzle of Arabidopsis [17]. The molecular circuit of plant circadian rhythms consists of transcriptional feedback loops with a variety of positive and negative factors. The alterations to the expression of any one of its components can cause drastic arrhythmia and acute misregulation of the clock [53,54]. This includes eveningphased pseudoresponse regulator TOC1 (TIMING OF CAB EXPRESSION 1) and the two morning-expressed MYB TFs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL) [53,54]. In the reciprocal regulation between morning and evening clock players, CHE physically interacts with TOC1 and binds the CCA1 promoter to repress its gene expression [17]. While plants lacking functional CHE have not been tested for their response to immune stresses, cca1 and lhy mutants displayed increased susceptibility to both bacterial and oomycete pathogens [51,52]. Another direct link between the circadian clock, in conjunction with CCA1 and LHY, and defense regulation is established through a CCA1 downstream target gene, GRP7 (GLYCINE-RICH PROTEIN 7) [51]. GRP7 is an RNA-binding protein that
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Review associates with the transcripts of several biologically important genes including the mRNAs of two key pattern recognition immune receptors FLS2 (FLAGELLIN-SENSITIVE 2) and EFR (EF-TU RECEPTOR) [55]. Consistent with the wide-spectrum RNA-binding activities of GRP7, it has been implicated in the regulation of stomatal movement, stress responsiveness, floral transition, and the circadian clock [53]. Interestingly, a Psy effector HopU1 blocks GRP7-mediated association with the transcripts of immune receptors, resulting in disease susceptibility [55,56]. These findings expand the complexity of the TCP-mediated gene regulatory network in the circadian clock and plant defense. Since disruptions to circadian rhythms occur primarily at the RNA level, this could serve as a suitable platform for further investigations of involvement of TCP genes in plant defenses. In a study focused on a direct role of CHE in plant immunity, CHE was shown to bind the promoter and positively regulate expression of ICS1 (ISOCHORISMATE SYNTHASE 1), a major SA biosynthetic gene [53]. This provides unequivocal evidence that CHE is a key convergent node between plant defense and the circadian clock. It is worth mentioning that mutations in CHE did not result in severe alterations in phase, amplitude, or period of the circadian clock [17]. This suggests that additional members of the TCP family may regulate CCA1 and TOC1, directly effecting regulatory clock functions in addition to CHE. Thus, it is imperative to investigate other members involved in regulating TOC1, CCA1, and other clock components. This inference is supported by a TCP family-wide transcript abundance study that concluded that nearly all TCP factors display distinct diurnal expression patterns [57]. In addition, selected members of both TCP class I and TCP class II were found to physically interact with different components of the core circadian clock, such as CCA1, LHY, PRR1, PRR3, and PRR5 [57]. Notably, tcp11 and tcp15 mutants exhibited altered transcript profiles of several core clock components [57]. Taken together, TCP family members can be positioned as the convergent nodes between the circadian clock and plant immune systems. Transcriptional control of TCP proteins in the plant immune system Given that TCP factors participate in the regulation of a wide spectrum of fundamental cellular processes, such as growth, proliferation, differentiation, hormonal homeostasis, and diverse signaling pathways involving intricate transcriptional networks [58], it is important to unveil how TCP family members regulate downstream target genes during pathogen infection. To date, no genome-wide transcriptome studies involving the roles of TCP proteins in plant disease resistance have been conducted. In the absence of this knowledge, however, the extrapolation of previous findings pertinent to the roles of TCP factors in phytohormone crosstalk might provide initial clues in discovering TCP-mediated downstream target genes involved in the plant immune system and yet further strengthen the developmental linkages to immunity. Domain structure and DNA-binding analyses previously revealed that both TCP classes can recognize distinct but overlapping regulatory elements [2,5,58]. Specifically,
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Figure 4. Transcriptional and post-transcriptional regulation of TCP factors. (A) TCP class I members are shown to bind GGNCCCAC consensus sequences and site II element. TCP class II members are illustrated to bind GTGGNCCC consensus sequence and site II element. Different categories of target genes for the respective TCP classes are illumined to the right. (B) The influence of miR319 on selected members of the TCP–CIN (CINCINNATA) subfamily is emphasized. TCP3 and TCP4 factors regulate miR164 and miR396, respectively. NAC (NAC DOMAIN CONTAINING PROTEIN) and GRF (GROWTH REGULATING FACTOR) are targets of miR164 and miR396, respectively. Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively.
the consensus-binding site of TCP class I is defined by the sequence GTGGGNCC, whereas TCP class II proteins show a preference for the promoter element GTGGNCCC (Figure 4A) [5,59]. Similarly, another common cis element, named site II (TGGGCY) (Figure 4A), has been demonstrated to interact with several members of both class I and class II TCP factors [57]. Thus, both classes contrast by maintaining distinctive binding motifs, but they do possess a common binding element allowing for parallel activation and repression. This is likely to contribute to fine-tuning the expression of target genes. By the complex functionality achieved by this powerful multilayer mechanism, cooperative and competitive interplay between both TCP classes, under diverse physiological conditions including plant–pathogen interactions, is established. This raises a question as to how TCP members can incorporate a multitude of transcription signals under such diverse conditions. In fact, it has been shown that TCP family members can homodimerize, and preferentially heterodimerize, in a complex formation required for DNA binding [11]. A family-wide Y2H analysis resulted in the discovery of 64 detected dimer combinations, seven homodimers and 57 heterodimers [12], and indicated the presence of high redundancy among various TCP family members. This study also revealed that class-specific TCP 5
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Review members preferentially interact with each other, although interclass interactions do exist [12]. Thus, the answer lies, at least in part, in the physical, combinatorial protein– protein interactions of TCP factors that may generate a large enough address space to form a complex TCP-centered transcriptional regulatory network that controls a wide variety of target genes. In support of this model, several developmental-related transcriptomic studies using plants lacking functional TCP factors or overexpressing modified forms of TCP factors, such as transcriptional activator domain (VP16) or repressor domain (EAR) fusions, have been conducted to identify TCP member-specific target genes [58]. With significant crosstalk between developmental processes and plant immune systems, several noteworthy examples arise. Some of the most momentous examples related to hormonal signaling include TCP3, a controller of flavonoid biosynthesis and auxin-responsive genes [60]; TCP20, a regulator of cell expansion, cell division, and cell differentiation [61]; TCP4, a governor of genes required in cell proliferation, growth, senescence, and JA signaling pathway [38]; TCP1, an operator in brassinosteroid biosynthesis gene regulation [16]; TCP15, a controller of endoreduplication and auxin-responsive genes [62]; and TCP9 and TCP20, influential in the gene expression of JA signaling (Figure 4A) [11]. Evidently, exploring these TCP factors warrants significant considerations to uncovering their roles in plant disease resistance. In addition, a detailed list of predicted TCP target genes has been compiled recently [58], from which inquiry is ignited to determine how many of these genes are downstream role players in plant immunity. These candidate genes are only the starting point to unraveling the additional interactions required in plant immunity. Additional genome-wide transcriptomic studies, under diverse pathogens on single, double, and higher order combinatorial tcp mutant plants, in the presence of pathogen and pathogen mimicking stimuli, are needed to discover the roles of TCP family members in plant immunity. Still, an unresolved key question concerns the regulation of TCP TFs under diverse environmental conditions. While significant research efforts are needed to understand the molecular underpinnings of the TCP-centered regulatory network, miR-mediated regulation of selected TCP–CIN members has been demonstrated. miRs are a class of short, endogenous, noncoding RNAs that range in size from 19 to 25 nucleotides and have been shown to play essential roles in post-transcriptional regulation of gene expression in plants [63,64]. Evidence has shown that the expression of TCP–CIN members were downregulated in the jaw-D (JAGGED AND WAVY) mutant, an activation tag line that overexpresses miR319 and displays crinkly leaf phenotypes [65], while suppressor of jaw-D (SOJ) mutants with nearly wild type leaves have changes in the miR319-binding site of TCP4 [66], collectively advocated that miR319 post-transcriptionally controls TCP2, TCP3, TCP4, TCP10, and TCP24 (Figure 4B). Recently, the roles of miR319 have been expanded beyond leaf development to salt and drought tolerance in creeping bentgrass (Agrostis stolonifera) [13], cold tolerance in rice (Oryza sativa) [15], and sugarcane (Saccharum officinarum) [67]. Given that miR319 expression is induced by a variety of abiotic stresses and the existence of crosstalk between 6
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diverse environmental stimuli, it is highly plausible that miR319 and its cognate TCP–CIN targets play a role in plant immunity. Target gene analyses discovered the control of miR164 and miR396 by TCP3 and TCP4, respectively, suggesting a crosstalk between miR319 and other miRs [7,68]. Interestingly, a novel NAC TF from the NAM subfamily of wheat, a target gene of wheat miR164, has been found to negatively regulate resistance of wheat to stripe rust (Figure 4B) [69]. Collectively, we anticipate the existence of an orchestrated transcriptional interplay between miR319 and TCP–CIN genes under diverse physiological conditions. These findings bring about several pressing questions, such as (i) what TF(s) control miR319 expression under biotic and abiotic stresses? And (ii) how do miR319 and other yet-to-be identified TFs participate in a combinatorial and complex regulation of this subgroup of TCP factors? Concluding remarks The emergent roles of TCP factors, expanding from a 1D role player in growth and development into a multifaceted factor engaging in a variety of intraspecies and interspecies interactions, have created a strong conceptual framework for further immunological developments. The advancements made in our understanding of TCP gene regulation have significantly broadened in recent years with linkages to multiple pathogen effector studies, simultaneously strengthening the argument for the role of TCP in plant immunity. From impeccable timing in the regulation of growth and development via the circadian clock, to a cascade of hormonal signaling, to a nexus of transcriptional regulators and miRs, TCP factors are continuing to prove their universal competence and vital importance to cellular functions. This discussion does not cease here. Rather, it paves the way for new avenues of research and highlights critical questions pertaining to the diverse roles that TCP factors play in the nexus. Future research needs to focus on gaining a deep understanding of how diverse pathogen effectors target TCP factors to provoke virulence. It is imperative to identify what effector molecules mechanistically perturb TCP4–LOX2 and TCP9/TCP20–LOX2 complexes to influence the JA signaling pathway. Another essential question in need of addressing is how pathogen effectors manipulate CHE complexes to alter the circadian clock for the virulence outcome. Does the answer lie in decreased binding capacities of CHE to the ICS1 promoter or impaired interactions between CHE and its additional binding partners including other TCP factors? In addition, it is equally important to gain deep insights into the roles of TCP factors, in particular TCP–CIN members, TCP8 and TCP14, in connection with TPL, a driving force of the recent breakthroughs connecting plant development with its immune system. A further challenge is to obtain a mechanistic understanding of how TCP proteins participate in plant immune system signaling. Determining which NLRs guard these high-value TCP targets to trigger ETI is unknown and remains an attractive pursuit for future studies. Rightfully, this model provides that NLR–TCP factor associations might provide a robust transcriptional reprogramming upon effector recognition. Additional global-scale interactome studies involving effectors of diverse pathogens will prove advantageous
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Review in identifying saturated suites of TCP-focused modules. A combination of various next-generation transcriptomics approaches, such as mRNA-seq, miRNA-seq, and ChIPseq, will be essential to gain insights into in vivo regulatory processes, further shedding light on the interrelated nature of TFs and various miRs at the interface of plants and their pathogens. While many answers are still to be discovered, it is with great certainty that TCP research is conceivably the spearhead to a multitude of immune-related discoveries. Employing such studies may have a latent function of identifying targets of novel pathogen effectors and their functions in conferring host resistance. Through isolating pairwise interactions of TCP proteins with effectors and unraveling direct downstream transcriptional targets, we will gain a more comprehensive understanding of genetic pathways affected by these evolutionarily ancient proteins. Results from continued analyses of miRs and TCP factors are also likely to influence research with agronomical interests in an effort to develop disease-resistant crops. Acknowledgments The authors apologize to colleagues whose work we were unable to cite owing to space constraints. M.S.M. was supported through funds from the Department of Biology, The University of Alabama at Birmingham.
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58 Uberti-Manassero, N.G. et al. (2013) TCP transcription factors: architectures of plant form. Biomol. Concepts 4, 111–127 59 Aggarwal, P. et al. (2010) Identification of specific DNA binding residues in the TCP family of transcription factors in Arabidopsis. Plant Cell 22, 1174–1189 60 Li, S. and Zachgo, S. (2013) TCP3 interacts with R2R3–MYB proteins, promotes flavonoid biosynthesis and negatively regulates the auxin response in Arabidopsis thaliana. Plant J. 76, 901–913 61 Herve, C. et al. (2009) In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development. Plant Physiol. 149, 1462–1477 62 Uberti-Manassero, N.G. et al. (2012) The class I protein AtTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins. J. Exp. Bot. 63, 809–823 63 Schommer, C. et al. (2014) Repression of cell proliferation by miR319-regulated TCP4. Mol. Plant. Published online July 22, 2014. http://dx.doi.org/10.1093/mp/ssu084 64 Nag, A. et al. (2009) miR319a targeting of TCP4 is critical for petal growth and development in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 106, 22534–22539 65 Palatnik, J.F. et al. (2003) Control of leaf morphogenesis by microRNAs. Nature 425, 257–263 66 Palatnik, J.F. et al. (2007) Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319. Dev. Cell 13, 115–125 67 Thiebaut, F. et al. (2012) Regulation of miR319 during cold stress in sugarcane. Plant Cell Environ. 35, 502–512 68 Koyama, T. et al. (2010) TCP transcription factors regulate the activities of ASYMMETRIC LEAVES1 and miR164, as well as the auxin response, during differentiation of leaves in Arabidopsis. Plant Cell 22, 3574–3588 69 Feng, H. et al. (2014) The target gene of tae-miR164, a novel NAC transcription factor from the NAM subfamily, negatively regulates resistance of wheat to stripe rust. Mol. Plant Pathol. 15, 284–296
Review
TCP three-way handshake: linking developmental processes with plant immunity Jessica A. Lopez1, Yali Sun1, Peter B. Blair1, and M. Shahid Mukhtar1,2 1 2
Department of Biology, University of Alabama at Birmingham, AL 35294, USA Nutrition Obesity Research Center, University of Alabama at Birmingham, AL 35294, USA
The TCP gene family encodes plant-specific transcription factors involved in growth and development. Equally important are the interactions between TCP factors and other pathways extending far beyond development, as they have been found to regulate a variety of hormonal pathways and signaling cascades. Recent advances reveal that TCP factors are targets of pathogenic effectors and are likely to play a vital role in plant immunity. Our focus is on reviewing the involvement of TCP in known pathways and shedding light on other linkages in the nexus of plant immunity centered around TCP factors with an emphasis on the convergence of effectors, interconnected hormonal networks, utility of the circadian clock, and the potential mechanisms by which pathogen defense may occur. TCP transcription factors, highly connected proteins in plant cellular network Transcription factors (TFs) are essential components of cellular machinery that regulate the spatiotemporal expression of thousands of genes in response to both internal and external stimuli [1]. This ensures the fine-tuned cellular functions of an organism during cellular proliferation, normal growth and development, and various disease states. The TCP gene family was discovered in 1999 and named after the first three characterized members, TEOSINTE BRANCHED1 in maize (Zea mays), CYCLOIDEA in snapdragon (Antirrhinum majus), and PCF (proliferating cell nuclear antigen factor) in rice (Oryza sativa) [2]. In the past quarter-century, the advances in genome and transcriptome sequencing shaped the TCP family into a complex network of genes encompassing 24 members in Arabidopsis (Arabidopsis thaliana) [3–5]. These plant-specific factors encode a 59-amino acid basic helix–loop–helix (bHLH) motif that allows for the classification of TCP TF family members into two groups, known as class I (or PCF or TCP-P) and class II (or TCP-C) [4]. TCP class II is further subdivided into the CIN and CYC/TB1 subclades (Figure 1A). The TCP class I is distinctive as its members Corresponding author: Mukhtar, M.S. ([email protected]). Keywords: TCP factors; pathogen effectors; SAP11; SRFR1; hormonal network; circadian clock; transcriptional control; miR319. 1360-1385/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2015.01.005
possess a four-amino acid deletion within the TCP domain in addition to the presence of other subclade-specific conserved signatures (Figure 1B). Another hallmark difference is the presence of an R domain (an 18–20 residue arginine-rich motif) and an ECE motif (a glutamic acid– cysteine–glutamic acid stretch), both of unknown biological functions, in subsets of TCP class II [4,5]. It is likely that TCP genes appeared in the Streptophyta lineage before the divergence of Zygnemophyta, between 350 and 800 Mya [3,6]. It is also believed that the division of the TCP family resulted from an ancient duplication event before the divergence of Zygnemophyta [6]. Genes of the CIN-like clade are involved in lateral organ development such as CINCINNATA (CIN) in the snapdragon, and the CYC/TB1 clade’s gene control axillary meristem development, such as TB1 in maize and its orthologs in Arabidopsis [7]. It is plausible that the CIN clade is more ancient than the CYC/ TB1 clade because primitive plants such as Selaginella and Physcomitrella have neither flowers nor branches and possess TCP–CIN homologs in their genomes [3,4]. Reconstructing the phylogeny of TCP genes in basal eudicots [8] could yield a clearer framework for understanding the functional evolution of these genes. It is possible that this framework could play a crucial role in linking evolution of TCP factors to their cellular functions. TCP family members have demonstrated involvement in a wide range of biological processes throughout the entire life span of plants. This includes the coordination of cell proliferation [9–12], differentiation of several morphological traits including shoot apical meristem and leaf development [13–15], biosynthesis of phytohormones [16], as well as the regulation of circadian clock rhythms [17]. These findings have positioned TCP factors as central regulatory nodes in diverse signaling pathways. Given that cellular functions are highly coordinated, finely orchestrated interactions of numerous proteins and biomolecules, the emerging question is whether topological properties of TCP proteins play an essential role in this biological network. Recent large-scale protein–protein interaction studies have revealed a TCP-centered module composed of 419 interactions among five TCP proteins (TCP13, TCP14, TCP15, TCP19, and TCP21) interacting with 236 other Arabidopsis proteins (Figure 1C) [18]. The vast majority of TCP interactors are enriched in diverse GO categories signifying the importance of TCP factors in a Trends in Plant Science xx (2015) 1–8
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(A)
specific interactors. Here, we will review the mechanisms of established TCP interactions and their role as effector targets via NLR (nucleotide-binding domain leucine-rich repeat protein) and TIR (Toll/interleukin-1 receptor)– NLR-mediated effector-triggered immunity. Further, we will examine the ramifications, both direct and indirect, of various TCP factors on hormonal signaling to recognize virulence perturbation. Additionally, we will use the circadian clock as a model regulatory network rendering various connections to immunity via TCP regulation. Lastly, we will unveil how TCP family members regulate downstream target genes and the prominence of miRNAs (miRs) in gene expression, which delineate further immunity linkages via TCP factors.
TCP6, TCP7, TCP8, TCP9, TCP11, TCP14, TCP15, TCP16, TCP19, TCP20, TCP21, TCP22, and TCP23
Class I (TCP-P)
TCP gene family
TCP2, TCP3, TCP4, TCP5, TCP10, TCP13, TCP17, and TCP24
CIN subclade Class II (TCP-C)
TCP1, TCP12, and TCP18
CYC/TB1 subclade
(B)
TCP domain
Helix I
Basic
Loop
Helix II
TCP14 (Class I) TCP4 (CIN) TCP1 (CYC)
(C) TCP-centered module in arabidopsis interactome-1
Funconal annotaon clustering of TCPs’ interactors Photosynthesis
RNA binding
Abioc/hormone
TCP14
Ubiquin
Transcripon Growth
Kinase
Immunity
Key proteins with known funcons AFB2
PRR2
GRX480
CAND1
ERF12
DWA1
HAT2
SWEET16
PIA1
CML41
MOS1
ACX1
TCP21 TCP15 TCP19
Key:
TCP13
Nodes: 241 Edges: 411
TCP protein TCP interactor
Pathways Auxin
SA
JA
Sugar
Calcium
Light
ABA
Ethylene
Ubiquin Circadian
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Figure 1. TCP transcription factor family, its classification, and position in the protein–protein interaction network. (A) A phylogenetic representation of class I (TCP-P) and class II (TCP-C), along with their respective subclades and TCP members. (B) Schematic representation of TCP domain structure. Sequence alignment of one member of each subclade is shown. Basic, helix I, loop, and helix II motifs within the TCP domain are marked. Red dashes on yellow background represent the lack of amino acids in TCP class I. Conserved residues among all the subclades are highlighted in purple. (C) A subnetwork encompassing the TCP protein in Arabidopsis interactome-1 is displayed using Cytoscape software. Members of TCP proteins in the module are labeled. In row 1, functional annotation clustering of TCP interactors is revealed, which was performed using DAVID Bioinformatics Resources 6.7. Key proteins with known functions are extracted (row 2) including AFB2 (AUXIN SIGNALING F-BOX 2), PRR2 (PSEUDORESPONSE REGULATOR 2), GRX480 (GLUTAREDOXIN FAMILY MEMBER ALSO KNOWN AS ROXY19), CAND1 (CANDIDATE G-PROTEIN-COUPLED RECEPTOR 1), ERF12 (ERF DOMAIN PROTEIN 12), DWA1 (DDB1-BINDING WD40 PROTEIN ALSO KNOWN AS DWD), HAT2 (HOMEOBOX-LEUCINE ZIPPER GENE), SWEET16 (NODULIN MTN3 FAMILY PROTEIN), PIA1 (PHYTOCHROME-INTERACTING ANKYRIN-REPEAT PROTEIN 1), CML41 (CALMODULIN-LIKE 41), MOS1 (MODIFIER OF SNC1), and ACX1 (ACYL-COA OXIDASE 1). Pathways relevant to the known TCP interactors are classified in row 3.
wide spectrum of cellular processes. In addition to several interactors involved in biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), ethylene, abscisic acid (ABA), and auxin, proteins involved in circadian rhythms regulation, phytochrome-mediated light responses, calcium homeostasis, and even sugar transport have been identified as direct TCP interaction partners [7] (Figure 1C). It has only recently emerged that these seemingly independent physiological processes are implicated in the plant immune response [19,20]. Thus, members of the TCP family constitute highly connected nodes or hubs in a plant cellular network, asserting their essential roles at intersections of various signaling pathways, and control vital plant immune processes through recruitment of 2
TCP proteins as pathogen effector targets Although the diversity of pathogens is immense, various attackers across kingdoms utilize a common infectious strategy that causes global disruption of host responses. Specifically, specialized pathogens employ virulence factors (effectors) to attack cellular hubs in host biological networks, thereby hijacking cellular machinery in a manner conducive to pathogen proliferation [21,22] and to induce effector-triggered susceptibility (ETS). Conversely, host immune receptors including NLRs or PRRs (patternrecognition receptors) can recognize effectors and pathogen-associated molecular patterns (PAMPs) to prompt effector-triggered and PAMP-triggered immunity (ETI and PTI, respectively) [21,23,24]. Strikingly, plant–pathogen interaction network-1 (PPIN-1) and PPIN-2 revealed that multiple members of TCP factors (TCP13, TCP14, TCP15, TCP19, and TCP21) are targeted by effectors from three pathogens that span the eukaryote–eubacteria divergence, represented by the bacterium Pseudomonas syringae (Psy), the oomycete Hyaloperonospora arabidopsidis (Hpa), and the true fungus Golovinomyces orontii (Gor) (Figure 2) [25,26]. Plants lacking TCP13, TCP14, and TCP19 exhibited enhanced disease susceptibility phenotypes [25]; TCP21 remains to be tested in this aspect. This discovery opens a new avenue of research focused on further characterization of the TCP factors and expansion of their functions to plant immunity. Another question pertinent to these findings is whether TCP proteins are virulence targets of the effectors and, if so, what are the mechanisms whereby pathogen effectors target TCP proteins to induce ETS. A recent report showed that overexpression of tomato (Solanum lycopersicum) TCP14-2 exhibited enhanced immunity to Phytophthora capsici, a phenotype that can be reversed upon coexpression of CRN12_997, a P. capsici effector that physically interacts with SlTCP14-2 [27]. This lends additional support for the concept of effector–host hub interactions, which can be conserved in divergent host–microbe systems. However, it remains to be discovered how TCP proteins participate in plant disease resistance. Intriguingly, another study identified six TCP class I proteins (TCP8, TCP14, TCP15, TCP20, TCP22, and TCP23) interacting with SRFR1 (SUPPRESSOR OF rps4-RLD1) (Figure 2) [28], two of which are also effector targets in PPIN-1 [25]. SRFR1 encodes a tetratricopeptide repeat (TPR) protein conserved in both plants and animals and was previously demonstrated to
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RPS4
Nucleus
TCP15
SNC1
TIR-NLR
CC-NLR
TCP13
TCP14
MOS1
TCP21
EDS1
1 FR SR
TCP19
RPS6
TCP8
? ETS/ETI ?
Target genes ETI
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Figure 2. Proposed model of involvement of TCP proteins in the plant immune system. TCP factors–SRFR1 (SUPPRESSOR OF rps4-RLD1)–EDS1 (ENHANCED DISEASE SUSCEPTIBILITY)–SRFR1-mediated activation of NLR (NUCLEOTIDEBINDING DOMAIN LEUCINE-RICH REPEAT PROTEINS) and induction of ETI (EFFECTOR-TRIGGERED IMMUNITY; manifested in the form of cell death) are illustrated. TIR (TOLL/INTERLEUKIN-1 RECEPTOR)–NLR, RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE 4), RPS6, and SNC1 (SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1) physically interact with EDS1 in the nucleus. Interactions of two pathogen effectors, AvrRps4 (red star) and HopA1 (blue star), with EDS1 are illuminated. SRFR1 interactions with either EDS1 or TCP proteins are shown. It is important to note that all of these proteins may not be part of the same complex. TCP proteins marked with a blue star designate TCP factors as effector(s) targets in PPIN-1 (PLANT–PATHOGEN INTERACTIONS NETWORK-1). The green star indicates TCP factors that are affected as a result of manipulating the jasmonic acid (JA) pathway. The red line with bar illustrates that MOS1 (MODIFIER OF SNC1) is a genetic suppressor of SNC1. TCP8, TCP14, and TCP15 may trigger ETI via TIR– NLR and CC (COILED-COIL)–NLR. The functions of TCP13, TCP19, and TCP21 in ETI or ETS (EFFECTOR-TRIGGERED SUSCEPTIBILITY) are not known.
act as a negative regulator of ETI [29–33]. As an adaptor protein, SRFR1 forms complexes in both the cytoplasm and the nucleus with several immune regulators including SGT1a (SUPPRESSOR OF THE G2 ALLELE OF SKP1 A; a cochaperone of the resistance proteins), SGT1b, EDS1 (ENHANCED DISEASE SUSCEPTIBILITY; a positive regulator of immunity), and TIR–NLR including RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE 4) and RPS6 [34–36]. It is worth noting that two different effectors, AvrRps4 and HopA1, target EDS1 and disrupt SRFR1–EDS1–NLR complexes leading to AvrRps4- or HopA1-triggered immunity (Figure 2) [36]. It was proposed that SRFR1 and SGT1b may modulate differential levels of NLR protein accumulation in response to the presence of diverse effectors [34]. It was established that tcp8-1 tcp14-5 tcp15-3 triple mutant plants display compromised ETI that can be triggered by four sequence unrelated effectors, that is, AvrRps4, HopA1, AvrRpm1, and AvrRpt2. These results suggest that TCP8, TCP14, and TCP15 positively regulate ETI-mediated resistance by the TIR–NLR, RPS4 and RPS6, and the CC (COILED-COIL)–NLR, RPS2, and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA 1) [28]. This is significantly different from EDS1/SRFR1-mediated ETI, which is controlled by only TIR–NLR. Exposure of TCP8, TCP14, and TCP15 to a range of effectors affirms the likelihood that these TFs determine an immune set point by modulating gene expression, which could provide an explanation for why they are targeted by pathogen effectors [28]. This is significantly
different from EDS1/SRFR1-mediated ETI, which is controlled by only TIR–NLR. Since the tcp8-1 tcp14-5 tcp15-3 mutants and Col-0 plants were equally susceptible to DC3000, it appears that TCP proteins are not general plant immune regulators but merely involved in CC– NLR and TIR–NLR-mediated ETI. This also suggests that the TCP proteins constitute host virulence targets, and some NLRs may have evolved to guard TCP factors from an effector’s attack allowing ETI to be triggered [25] (Figure 2). The first clue to the identity of TCP-related NLRs may originate from the interaction of TCP15 with MOS1 (MODIFIER OF SNC1), a BAT2 domain-containing protein [37]. MOS1 regulates SNC1 (SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1, a TIR–NLR)-mediated resistance responses by fine-tuning SNC1 expression. It is likely that MOS1, TCP15, and SNC1 form complexes in the nucleus. Furthermore, how SRFR1 maintains TCPrelated NLR protein accumulation has yet to be determined. Since only a subset of effector target TCP factors has been demonstrated to interact with SRFR1, we expect additional negative regulator adaptor proteins to form different nuclear complexes with NLRs. Future studies should shed more light on the mysteries surrounding effectors–TCP factors–NLR complexes. TCP-mediated hormonal crosstalk homeostasis Phytohormones are signal molecules produced within a plant that control nearly all stages of growth and development by regulating gene expression that in turn translates into appropriate morphological and/or physiological responses [20]. TCP factors are involved in a diverse spectrum of both mutualistic and antagonistic biological interactions. For instance, the antagonistic relationship between the two classes of TCP factors, TCP class I and TCP class II, was shown to balance contrasting activities of TCP class I members promoting cell proliferation in leaves and TCP–CIN factors, negative regulators of leaf growth, and positive regulators of senescence [10,38]. This contributes to fine-tuning of phytohormone production and ensuring normal leaf and plant morphology [39]. However, it is expected that the pathogen effectors manipulate hormonal crosstalk to their advantage. Indeed, it was demonstrated that SAP11, a secreted protein from Aster Yellows phytoplasma strain Witches’ Broom (AY–WB), interacts with and destabilizes all eight members of TCP–CIN, but does not interact with any related TCP members belonging to TCP class I family (Figure 3A) [40–43]. These biochemical data were further substantiated by genetic studies, revealing that SAP11 transgenics and octuple tcp–cin mutants, which exhibit reduced protein levels of all TCP–CIN, both display deeply lobed and highly crinkled leaves phenotypes [41]. This action of SAP11-mediated destabilization is biologically relevant, because SAP11 transgenics and AY–WB-infected plants produce less JA in response to wounding, leading to increased fecundity of AY–WB insect vector leafhopper (Macrosteles quadrilineatus) [41]. These findings spark questions into how TCP–CIN and TCP class I regulate JA biosynthesis under diverse environmental conditions. Selected members of TCP–CIN were shown to positively influence LOX2 (LIPOXYGENASE 2) gene expression and JA biosynthesis, whereas TCP4 binds directly 3
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Figure 3. Schematic representation of a model depicting effector-mediated manipulation of hormonal pathways. (A) SAP11 (a secreted protein from Aster Yellows phytoplasma strain Witches’ Broom)-mediated destabilization of TCP–CIN (CINCINNATA) members are illustrated. Interaction of SAP11 with TCP–CIN members leads to the disappearance of TCP proteins (shown in broken pink oval) and less jasmonic acid (JA) production. (B) TCP4, a CIN–TCP, and TCP9 and TCP20, two TCP class I members, directly bind onto the promoter of LOX2 (LIPOXYGENASE 2) and regulate LOX2 gene expression and JA synthesis antagonistically. Effectors are proposed to interfere with LOX2-mediated JA signaling. (C) TCP–CIN members, in complex with AS1 (ASYMMETRIC LEAVES 1) and AS2, directly bind onto the promoter of the KNOX gene and repress its expression. KNOX protein is shown to be an effector target in PPIN-1 (PLANT– PATHOGEN INTERACTION NETWORK-1). Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively.
onto the promoter of LOX2 (Figure 3B) [38]. Interestingly, it has been found that two TCP class I members, TCP9 and TCP20, have been found to bind to LOX2 promoter and exert a contrasting effect of downregulating the JA pathway (Figure 3B) [11]. This antagonistic relationship between TCP–CIN and TCP class I members, in JA biosynthesis, provides an opportunity for diverse pathogens to directly or indirectly target TCP factors, thereby tipping the hormonal balance for their benefits. In conjunction with the JA and other signaling pathways, serving as another important linkage in the search for connections with TCP factors and plant pathogen effectors is the protein known as TOPLESS (TPL; an effector target in PPIN-1) [25,44]. TPL is a universal corepressor that orchestrates the crosstalk between JA and other hormone signaling pathways [18,45]. A recent TPL-specific interactome study revealed that both members of TCP– CIN (TCP2, TCP3, and TCP4) and TCP class I (TCP8 and TCP14) interact with TPL [44]. TCP14 and TCP15, both members of TCP class I and effector targets, were shown to function in cell proliferation and in several plant hormone signaling pathways [46]. TCP14 interacts with RGA (REPRESSOR OF GA1–3) and GAI (GIBBERELLIN INSENSITIVE), the two most abundant DELLAs, and are involved in the regulation of GA-responsive gene expression [47]. Another finding suggests that TCP14 and TCP15 4
participate in the cytokinin (CK) responses through interaction with O-linked N-acetyl glucosamine transferase SPINDLY [48]. Expansion of the nexus continues from JA to SA, auxin, CK, GA, and ABA with the involvement of TPL–TCP factors, TCP–effectors, and TPL–effectors crosstalk. Another central player in hormonal homeostasis and the plant developmental process is BP (BREVIPEDICELLUS; a Knotted1-like homeobox or KNOX family member), an effector target in PPIN-1 [49]. TCP–CIN members, in complex with other TFs, AS1 (ASYMMETRIC LEAVES 1) and AS2, directly bind onto the BP promoter and regulate its expression [50]. Interestingly, a study determined that a TCP class I family member, TCP7, can bind to the promoter of STM (SHOOT MERISTEMLESS), a KNOX gene (Figure 3C) [10,49]. A tcp8 tcp22 mutant that loses the function of two TCP class I genes showed altered leaf shape, and this phenotype is further pronounced in triple mutants (as1 tcp8 tcp22) [10,49]. The possible participation of both TCP class I and TCP–CIN factors in the regulation of AS1, AS2, and KNOX genes could serve as another mechanism by which TCP factors can play an active role in pathogen defense. Taken together, it is conceivable that pathogen effectors target TCPmediated complexes both directly and indirectly to manipulate hormonal signaling network with the aim of causing virulence. Role of TCP proteins in circadian clock and plant defense Serving as a resonance between the internal timekeeper and environmental cues, the circadian clock regulatory network is crucial to enhancing fitness and survival of plants, and has been known to play a key role in plant physiology. Recently, strides have been made to develop a notion that the circadian clock is linked to plant immunity [51,52], but the key question remains whether TCP factors play significant roles in circadian clock regulation during plant defense. TCP21, also known as CHE (CCA1 HIKING EXPEDITION), is an effector target in PPIN-1 and its discovery seems to fit the bill as a missing piece in the circadian clock puzzle of Arabidopsis [17]. The molecular circuit of plant circadian rhythms consists of transcriptional feedback loops with a variety of positive and negative factors. The alterations to the expression of any one of its components can cause drastic arrhythmia and acute misregulation of the clock [53,54]. This includes eveningphased pseudoresponse regulator TOC1 (TIMING OF CAB EXPRESSION 1) and the two morning-expressed MYB TFs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL) [53,54]. In the reciprocal regulation between morning and evening clock players, CHE physically interacts with TOC1 and binds the CCA1 promoter to repress its gene expression [17]. While plants lacking functional CHE have not been tested for their response to immune stresses, cca1 and lhy mutants displayed increased susceptibility to both bacterial and oomycete pathogens [51,52]. Another direct link between the circadian clock, in conjunction with CCA1 and LHY, and defense regulation is established through a CCA1 downstream target gene, GRP7 (GLYCINE-RICH PROTEIN 7) [51]. GRP7 is an RNA-binding protein that
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Review associates with the transcripts of several biologically important genes including the mRNAs of two key pattern recognition immune receptors FLS2 (FLAGELLIN-SENSITIVE 2) and EFR (EF-TU RECEPTOR) [55]. Consistent with the wide-spectrum RNA-binding activities of GRP7, it has been implicated in the regulation of stomatal movement, stress responsiveness, floral transition, and the circadian clock [53]. Interestingly, a Psy effector HopU1 blocks GRP7-mediated association with the transcripts of immune receptors, resulting in disease susceptibility [55,56]. These findings expand the complexity of the TCP-mediated gene regulatory network in the circadian clock and plant defense. Since disruptions to circadian rhythms occur primarily at the RNA level, this could serve as a suitable platform for further investigations of involvement of TCP genes in plant defenses. In a study focused on a direct role of CHE in plant immunity, CHE was shown to bind the promoter and positively regulate expression of ICS1 (ISOCHORISMATE SYNTHASE 1), a major SA biosynthetic gene [53]. This provides unequivocal evidence that CHE is a key convergent node between plant defense and the circadian clock. It is worth mentioning that mutations in CHE did not result in severe alterations in phase, amplitude, or period of the circadian clock [17]. This suggests that additional members of the TCP family may regulate CCA1 and TOC1, directly effecting regulatory clock functions in addition to CHE. Thus, it is imperative to investigate other members involved in regulating TOC1, CCA1, and other clock components. This inference is supported by a TCP family-wide transcript abundance study that concluded that nearly all TCP factors display distinct diurnal expression patterns [57]. In addition, selected members of both TCP class I and TCP class II were found to physically interact with different components of the core circadian clock, such as CCA1, LHY, PRR1, PRR3, and PRR5 [57]. Notably, tcp11 and tcp15 mutants exhibited altered transcript profiles of several core clock components [57]. Taken together, TCP family members can be positioned as the convergent nodes between the circadian clock and plant immune systems. Transcriptional control of TCP proteins in the plant immune system Given that TCP factors participate in the regulation of a wide spectrum of fundamental cellular processes, such as growth, proliferation, differentiation, hormonal homeostasis, and diverse signaling pathways involving intricate transcriptional networks [58], it is important to unveil how TCP family members regulate downstream target genes during pathogen infection. To date, no genome-wide transcriptome studies involving the roles of TCP proteins in plant disease resistance have been conducted. In the absence of this knowledge, however, the extrapolation of previous findings pertinent to the roles of TCP factors in phytohormone crosstalk might provide initial clues in discovering TCP-mediated downstream target genes involved in the plant immune system and yet further strengthen the developmental linkages to immunity. Domain structure and DNA-binding analyses previously revealed that both TCP classes can recognize distinct but overlapping regulatory elements [2,5,58]. Specifically,
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Figure 4. Transcriptional and post-transcriptional regulation of TCP factors. (A) TCP class I members are shown to bind GGNCCCAC consensus sequences and site II element. TCP class II members are illustrated to bind GTGGNCCC consensus sequence and site II element. Different categories of target genes for the respective TCP classes are illumined to the right. (B) The influence of miR319 on selected members of the TCP–CIN (CINCINNATA) subfamily is emphasized. TCP3 and TCP4 factors regulate miR164 and miR396, respectively. NAC (NAC DOMAIN CONTAINING PROTEIN) and GRF (GROWTH REGULATING FACTOR) are targets of miR164 and miR396, respectively. Positive and negative regulatory actions are indicated by arrows and lines with bars, respectively.
the consensus-binding site of TCP class I is defined by the sequence GTGGGNCC, whereas TCP class II proteins show a preference for the promoter element GTGGNCCC (Figure 4A) [5,59]. Similarly, another common cis element, named site II (TGGGCY) (Figure 4A), has been demonstrated to interact with several members of both class I and class II TCP factors [57]. Thus, both classes contrast by maintaining distinctive binding motifs, but they do possess a common binding element allowing for parallel activation and repression. This is likely to contribute to fine-tuning the expression of target genes. By the complex functionality achieved by this powerful multilayer mechanism, cooperative and competitive interplay between both TCP classes, under diverse physiological conditions including plant–pathogen interactions, is established. This raises a question as to how TCP members can incorporate a multitude of transcription signals under such diverse conditions. In fact, it has been shown that TCP family members can homodimerize, and preferentially heterodimerize, in a complex formation required for DNA binding [11]. A family-wide Y2H analysis resulted in the discovery of 64 detected dimer combinations, seven homodimers and 57 heterodimers [12], and indicated the presence of high redundancy among various TCP family members. This study also revealed that class-specific TCP 5
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Review members preferentially interact with each other, although interclass interactions do exist [12]. Thus, the answer lies, at least in part, in the physical, combinatorial protein– protein interactions of TCP factors that may generate a large enough address space to form a complex TCP-centered transcriptional regulatory network that controls a wide variety of target genes. In support of this model, several developmental-related transcriptomic studies using plants lacking functional TCP factors or overexpressing modified forms of TCP factors, such as transcriptional activator domain (VP16) or repressor domain (EAR) fusions, have been conducted to identify TCP member-specific target genes [58]. With significant crosstalk between developmental processes and plant immune systems, several noteworthy examples arise. Some of the most momentous examples related to hormonal signaling include TCP3, a controller of flavonoid biosynthesis and auxin-responsive genes [60]; TCP20, a regulator of cell expansion, cell division, and cell differentiation [61]; TCP4, a governor of genes required in cell proliferation, growth, senescence, and JA signaling pathway [38]; TCP1, an operator in brassinosteroid biosynthesis gene regulation [16]; TCP15, a controller of endoreduplication and auxin-responsive genes [62]; and TCP9 and TCP20, influential in the gene expression of JA signaling (Figure 4A) [11]. Evidently, exploring these TCP factors warrants significant considerations to uncovering their roles in plant disease resistance. In addition, a detailed list of predicted TCP target genes has been compiled recently [58], from which inquiry is ignited to determine how many of these genes are downstream role players in plant immunity. These candidate genes are only the starting point to unraveling the additional interactions required in plant immunity. Additional genome-wide transcriptomic studies, under diverse pathogens on single, double, and higher order combinatorial tcp mutant plants, in the presence of pathogen and pathogen mimicking stimuli, are needed to discover the roles of TCP family members in plant immunity. Still, an unresolved key question concerns the regulation of TCP TFs under diverse environmental conditions. While significant research efforts are needed to understand the molecular underpinnings of the TCP-centered regulatory network, miR-mediated regulation of selected TCP–CIN members has been demonstrated. miRs are a class of short, endogenous, noncoding RNAs that range in size from 19 to 25 nucleotides and have been shown to play essential roles in post-transcriptional regulation of gene expression in plants [63,64]. Evidence has shown that the expression of TCP–CIN members were downregulated in the jaw-D (JAGGED AND WAVY) mutant, an activation tag line that overexpresses miR319 and displays crinkly leaf phenotypes [65], while suppressor of jaw-D (SOJ) mutants with nearly wild type leaves have changes in the miR319-binding site of TCP4 [66], collectively advocated that miR319 post-transcriptionally controls TCP2, TCP3, TCP4, TCP10, and TCP24 (Figure 4B). Recently, the roles of miR319 have been expanded beyond leaf development to salt and drought tolerance in creeping bentgrass (Agrostis stolonifera) [13], cold tolerance in rice (Oryza sativa) [15], and sugarcane (Saccharum officinarum) [67]. Given that miR319 expression is induced by a variety of abiotic stresses and the existence of crosstalk between 6
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diverse environmental stimuli, it is highly plausible that miR319 and its cognate TCP–CIN targets play a role in plant immunity. Target gene analyses discovered the control of miR164 and miR396 by TCP3 and TCP4, respectively, suggesting a crosstalk between miR319 and other miRs [7,68]. Interestingly, a novel NAC TF from the NAM subfamily of wheat, a target gene of wheat miR164, has been found to negatively regulate resistance of wheat to stripe rust (Figure 4B) [69]. Collectively, we anticipate the existence of an orchestrated transcriptional interplay between miR319 and TCP–CIN genes under diverse physiological conditions. These findings bring about several pressing questions, such as (i) what TF(s) control miR319 expression under biotic and abiotic stresses? And (ii) how do miR319 and other yet-to-be identified TFs participate in a combinatorial and complex regulation of this subgroup of TCP factors? Concluding remarks The emergent roles of TCP factors, expanding from a 1D role player in growth and development into a multifaceted factor engaging in a variety of intraspecies and interspecies interactions, have created a strong conceptual framework for further immunological developments. The advancements made in our understanding of TCP gene regulation have significantly broadened in recent years with linkages to multiple pathogen effector studies, simultaneously strengthening the argument for the role of TCP in plant immunity. From impeccable timing in the regulation of growth and development via the circadian clock, to a cascade of hormonal signaling, to a nexus of transcriptional regulators and miRs, TCP factors are continuing to prove their universal competence and vital importance to cellular functions. This discussion does not cease here. Rather, it paves the way for new avenues of research and highlights critical questions pertaining to the diverse roles that TCP factors play in the nexus. Future research needs to focus on gaining a deep understanding of how diverse pathogen effectors target TCP factors to provoke virulence. It is imperative to identify what effector molecules mechanistically perturb TCP4–LOX2 and TCP9/TCP20–LOX2 complexes to influence the JA signaling pathway. Another essential question in need of addressing is how pathogen effectors manipulate CHE complexes to alter the circadian clock for the virulence outcome. Does the answer lie in decreased binding capacities of CHE to the ICS1 promoter or impaired interactions between CHE and its additional binding partners including other TCP factors? In addition, it is equally important to gain deep insights into the roles of TCP factors, in particular TCP–CIN members, TCP8 and TCP14, in connection with TPL, a driving force of the recent breakthroughs connecting plant development with its immune system. A further challenge is to obtain a mechanistic understanding of how TCP proteins participate in plant immune system signaling. Determining which NLRs guard these high-value TCP targets to trigger ETI is unknown and remains an attractive pursuit for future studies. Rightfully, this model provides that NLR–TCP factor associations might provide a robust transcriptional reprogramming upon effector recognition. Additional global-scale interactome studies involving effectors of diverse pathogens will prove advantageous
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Review in identifying saturated suites of TCP-focused modules. A combination of various next-generation transcriptomics approaches, such as mRNA-seq, miRNA-seq, and ChIPseq, will be essential to gain insights into in vivo regulatory processes, further shedding light on the interrelated nature of TFs and various miRs at the interface of plants and their pathogens. While many answers are still to be discovered, it is with great certainty that TCP research is conceivably the spearhead to a multitude of immune-related discoveries. Employing such studies may have a latent function of identifying targets of novel pathogen effectors and their functions in conferring host resistance. Through isolating pairwise interactions of TCP proteins with effectors and unraveling direct downstream transcriptional targets, we will gain a more comprehensive understanding of genetic pathways affected by these evolutionarily ancient proteins. Results from continued analyses of miRs and TCP factors are also likely to influence research with agronomical interests in an effort to develop disease-resistant crops. Acknowledgments The authors apologize to colleagues whose work we were unable to cite owing to space constraints. M.S.M. was supported through funds from the Department of Biology, The University of Alabama at Birmingham.
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