Briefings in Functional Genomics Advance Access published April 2, 2015 Briefings in Functional Genomics, 2015, 1–8 doi: 10.1093/bfgp/elv011 Review paper

Functional studies of transcription factors involved in plant defenses in the genomics era Eunyoung Seo and Doil Choi Corresponding author. Doil Choi, Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Tel.: þ82 2 880 4572; Fax: þ82 2 873 2056; E-mail: [email protected]

Abstract Plant transcription factors (TFs) play roles in diverse biological processes including defense responses to pathogens. Here, we provide an overview of recent studies of plant TFs with regard to defense responses. TFs play roles in plant innate immunity by regulating genes related to pathogen-associated molecular pattern-triggered immunity, effector-triggered immunity, hormone signaling pathways and phytoalexin synthesis. Currently, genome-wide phylogenetic and transcriptomic analyses are as important as functional analyses in the study of plant TFs. The integration of genomics information with the knowledge obtained from functional studies provides new insights into the regulation of plant defense mechanisms as well as engineering crops with improved resistance to invading pathogens. Key words: plant transcription factor; genome-wide analysis; plant defense response; transcriptional regulation; plant disease resistance

Introduction Plants encounter diverse pathogens and pests including viruses, bacteria, fungi, nematodes and insects. Because they are sessile, plants have developed elaborate defense mechanisms against these organisms [1]. Briefly, when pathogens attack plants, the plants recognize conserved patterns associated with the pathogens (pathogen-associated molecular patterns, PAMPs) at the pathogen cell surface and trigger basic immune responses (PAMP-triggered immunity, PTI). Some pathogens secrete effectors into the host cells to suppress PTI, and the plants having corresponding resistance (R) proteins directly or indirectly recognize the effectors and activate immune responses (effector-triggered immunity, ETI) such as the hypersensitive response (HR) [2, 3]. In PTI and ETI, signal transduction and the fine-tuning of gene expression are required to regulate these defense mechanisms [4–6]. Transcription factors (TFs) are DNA-binding proteins that play roles in the modulation of gene expression by binding to specific DNA sequences called cis-elements in the gene

promoters. TFs act as transcriptional activators or repressors and are involved in large-scale biological phenomena, including growth and development [7–10]. The roles of TFs in plant defense have been elucidated by a large number of researchers. Loss-offunction studies using T-DNA insertion or virus-induced gene silencing and gain-of-function studies using overexpression have been adopted to investigate the functions of TFs. Along with rapid developments in sequencing technology, great progress in genomics has been made over the past 10 years. To date, the genome sequencing of >80 plants from algae to eudicots has been completed, which allows analysis of whole TF families [11]. On average, plant genomes contain 10003000 TFs or transcriptional regulators, which account for 5–15% of all the encoded proteins [12, 13]. In addition, transcriptome profiling of plant responses to pathogen infection has provided a genome-wide view of the expression of TF genes that might be involved in plant defense systems [14–16]. In this review, we focused on recent studies of five TF families that play roles in plant defense mechanisms: WRKY, APETALA2/ethylene responsive factor (AP2/ERF), basic-domain

Eunyoung Seo is a PhD candidate at Seoul National University. She worked on the pepper genome project and was in charge of transcription factor analysis. She is working on genome-wide evolution of resistance genes in Solanaceae plants. Doil Choi is a professor at Seoul National University. He is an expert in molecular plant–microbe interactions, and he directed the pepper genome project. Currently, he and his groups are working on nonhost resistance and evolution of resistance genes in Solanaceae plants. C The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected] V

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Table 1. Numbers of five families of transcription factors in plant genomes TFs

Dicot a

At AP2/ERF 146 bHLH 153 bZIP 74 NAC 113 WRKY 72

Monocot a

Sl 167 161 70 101 81

b

a

a

Ca Gm Mt 123 355 127 134 350 122 58 151 58 106 182 72 73 182 73

a

Gr 268 225 115 153 120

a

Pt 210 202 97 170 102

a

Bd 141 141 85 100 82

a

Os 158 156 96 141 102

Moss a

a

Sb Zm 181 214 170 202 95 131 127 134 96 125

Ppa 157 108 45 33 36

At: Arabidopsis thaliana, Sl: Solanum lycopersicum, Ca: Capsicum annuum, Gm: Glycine max, Mt: Medicago truncatula, Gr: Gossypium raimondii, Pt: Populus trichocarpa, Bd: Brachypodium distachyon, Os: Oryza sativa, Sb: Sorghum bicolor, Zm: Zea mays, Pp: Physcomitrella patens. a

Data were obtained from [12].

b

Data were obtained from [17].

leucine-zipper (bZIP), basic helix-loop-helix (bHLH) and NAM/ ATAF/CUC (NAC). Those families comprise a large portion of the TF protein repertoire in plants (Table 1). We discuss the known functions related to defense and genome-wide expression in each family. This information on defense-related TFs enhances our understanding of the roles of TFs in global gene expression networks during plant defense against pathogens. Furthermore, it provides insight into the regulation of defense mechanisms that can be used to improve disease resistance in crop plants.

WRKY The WRKY TF family comprises a large number of members that are present in plant genomes, and few WRKY genes have been identified in non-photosynthetic eukaryotes [18, 19]. They have one or two WRKY domains consisting of 60 amino acids and are divided into three major groups depending on the number of WRKY domains and the type of zinc-finger motif [20]. The general cis-element bound by WRKY TFs is called the W-box, which has a consensus sequence of TTGACT/C [21]. However, alternative binding sites also have been identified [22, 23]. In recent years, the genome-wide identification and characterization of WRKY TFs have been performed in many plants including crop species [24–28]. The roles of WRKY TFs in defense have been extensively studied, mainly in Arabidopsis. WRKY TFs are involved in PAMP signaling downstream of mitogen-activated protein kinase (MAPK) cascades [29]. Plant MAPK cascades play essential signaling roles in multiple defense responses, especially in sensing PAMPs or pathogen effectors and in downstream signaling [30]. In Arabidopsis, WRKY33 plays a role in resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola [31]. A recent study revealed that WRKY33 is phosphorylated by MPK3/MPK6 in response to B. cinerea, and it plays roles in phytoalexin biosynthesis [32]. WRKY33 binds not only to the promoter of phytoalexin deficient 3, which catalyzes the final step in camalexin biosynthesis, but also to its own promoter, indicating a potential positive feedback regulatory loop. In addition, another study revealed that WRKY33 binds to the promoters of 1-aminocyclopropane-1-carboxylic acid synthase 2 (ACS2)/ACS6 in the ethylene biosynthetic pathway and activates the expression of ACS2/ACS6 in response to B. cinerea [33]. Genome-wide expression profiling of wrky33 loss-of-function mutants and wild-type plants following B. cinerea infection revealed differential transcriptional reprogramming, indicating unidentified targets and roles of WRKY33 in the defense

responses [34]. Nicotiana benthamiana WRKY8 (NbWRKY8), the closest homolog of WRKY33, is also phosphorylated by MAPKs, increasing expression of defense-related genes. In addition, NbWRKY8 silencing causes increased susceptibility to the oomycete Phytophthora infestans and the ascomycete fungus Colletotrichum orbiculare [35]. WRKY TFs interact with R proteins. Most R proteins belong to the nucleotide-binding/leucine-rich repeat (NB-LRR) family. In barley, mildew resistance locus A10 (MLA10), a coiled coil (CC)-NB-LRR protein conferring resistance to powdery mildew, interacts with Hordeum vulgare WRKY1 (HvWRKY1) and HvWRKY2 in the presence of the AVRA10 effector [36]. HvWRKY1 and HvWRKY2 repress the basal defense response against virulent powdery mildew fungus, Blumeria graminis. The interaction with MLA10 occurs following inoculation with B. graminis expressing AVRA10, and the defense becomes de-repressed. A recent study also revealed that rice panicle blast 1 (Pb1), a CCNB-LRR protein, interacts with Oryza sativa WRKY45 (OsWRKY45) in the nucleus and mediates blast resistance to the fungus Magnaporthe oryzae [37]. In addition, Arabidopsis WRKY52/RRS1 is a Toll/interleukin-1 receptor (TIR)-NB-LRR protein containing a WRKY domain that confers resistance against the bacterial pathogen Ralstonia solanacearum [38]. RRS1 acts with RPS4, a TIR-NB-LRR protein, for dual resistance against fungal and bacterial pathogens [39]. A recent study revealed crystal structures of the TIR domains of RPS4 and RRS1, and that their heterodimerization is required for effector recognition [40]. WRKY TFs also function in antiviral defense. It was reported that Arabidopsis WRKY8 is a negative and positive regulator of basal defense to Pseudomonas syringae and B. cinerea, respectively [41]. In recent work, mutations in Arabidopsis WRKY8 resulted in the accumulation of crucifer-infecting tobacco mosaic virus (TMV-cg) in systemically infected leaves [42]. WRKY8 induces a gene related to abscisic acid (ABA) signaling but represses genes involved in ethylene signaling by binding to the promoters of genes involved in both pathways. This work indicates that WRKY TFs play roles in mediating the long-distance movement of the viruses and the cross talk between the ABA and ethylene signaling pathways in antiviral defense. WRKY TFs play roles as repressors as well as activators of basal defense responses. The negative regulatory roles in defense have importance related to costs of resistance and balancing growth versus defense. The loss of WRKY11 in Arabidopsis conferred resistance to both avirulent and virulent P. syringae pv. tomato strains, and the resistance was more obvious in the wrky11/wrky17 double mutants [43]. In pepper, the silencing of Capsicum annuum WRKY1 (CaWRKY1) and CaWRKY58 reduced disease symptoms caused by the virulent bacteria Xanthomonas axonopodis pv. vesicatoria race 1 and R. solanacearum, respectively [44, 45]. OsWRKY62.1 in rice interacts with Xa21, a receptor-like kinase, and acts as a negative regulator in Xa21-mediated defense as well as in basal defense against Xanthomonas oryzae pv. oryzae [46]. In addition, a pair of alleles, OsWRKY45-1 and OsWRKY45-2, in different rice varieties showed opposite roles in resistance against X. oryzae pv. oryzae and X. oryzae pv. oryzicola [47]. These results indicate the negative regulatory roles of WRKY in defense. Transcriptome analyses revealed that many WRKY genes were induced following infection by pathogens. Microarray data from Arabidopsis following inoculation with B. cinerea revealed that WRKY TFs were rapidly induced within 18 h after infection [16]. In addition, eight WRKY genes were identified as transcriptional targets of nonexpressor of pathogenesis-related

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proteins 1 (NPR1), a key transcriptional coregulator in salicylic acid (SA)-dependent systemic acquired resistance (SAR) and a factor in the regulation of pathogenesis-related (PR) gene expression, using microarray experiment [48]. In grape, the expression profiles of 38 of 59 putative WRKY genes were altered during powdery mildew (Erysiphe necator) infection [27]. These works indicate that many WRKY TFs might play roles in defense mechanism. To elucidate the roles of the WRKY TF family members in plant defense, massive genome-wide data sets should be integrated with functional analyses based on gainand loss-of-function studies.

be manipulated by a pathogen. XopD, a type III effector from Xanthomonas euvesicatoria, targets and desumoylates SlERF4, preventing ethylene production and ethylene-related immunity [64]. The expression of a number of AP2/ERF TFs is induced or repressed by various biotic stresses as well as abiotic stresses including salt, drought and cold. For example, the expression levels of 53 AP2/ERF TFs were altered during B. cinerea infection [16]. A previous report indicated that most AP2/ERF TFs involved in disease resistance belong to the ERF group rather than to the DREB group [65]. Understanding the correlation between the functions and the phylogeny of AP2/ERF TFs is expected to provide insight into the roles of AP2/ERF TFs in defense responses.

AP2/ERF

NAC

The AP2/ERF family is one of the largest TF families. The existence of the AP2 domain is reported in bacteria, bacteriophage, a ciliate and plants [49]. AP2/ERF TFs have one or two AP2 domains consisting of 60 conserved amino acids, and they are divided into three groups: AP2, ERF and RAV [50]. The ERF group can be subdivided into the dehydration-responsive elementbinding proteins (DREBs) and the ERFs, each consisting of six subgroups [51]. They also can be subdivided into 12 subgroups based on phylogenetic relationships. Nomenclatures based on both classification systems are commonly used [52]. Many DREBs and ERFs interact with promoters called the C-repeat/ dehydration-responsive element (CCGAC) and the GCC box (GCCGCC), respectively. Genome-wide identification and phylogenetic analyses of the AP2/ERF TFs have been performed for many plant genomes including Arabidopsis, rice, soybean, grapevine and tomato [52–55]. A number of studies about the roles of AP2/ERF TFs in biotic and abiotic stress responses as well as in developmental processes have been reported. Most AP2/ERF TFs act as transcriptional activators, although some AP2/ERF TFs containing the ERF-associated amphiphilic repression (EAR) motif suppress gene expression [56]. AP2/ERF TFs are involved in defense responses against various pathogens. Gain- and loss-of-function experiments with tomato RAV (SlRAV) demonstrated their role in resistance to R. solanacearum [57]. SlRAV2 induces the expression of the SlERF5, increasing tolerance to bacterial wilt. In Arabidopsis, ERF5, ERF6 and RAP2.2 (RELATED TO AP2.2) act as positive regulators in resistance to the B. cinerea [58, 59]. Those defense responses were linked to hormones such as jasmonic acid (JA) and ethylene. In rice, OsERF3 containing an EAR motif plays positive roles in resistance against the chewing herbivore, Chilo suppressalis, influencing the expression of genes involved in the MAPK cascades and hormone biosynthesis [60]. Some reports also showed that AP2/ERF TFs can act as negative regulators. For example, the OsERF922 RNAi plants showed increased resistance to M. oryzae, increasing the expression of PR genes, phenylalanine ammonia lyase and a gene related to phytoalexin biosynthesis [61]. Recent studies have revealed the mode of action for AP2/ERF TFs. Like WRKY33, ERF6 was phosphorylated by MPK3/MPK6 in response to B. cinerea infection, and it activated defense-related genes such as PLANT DEFENSIN 1.1 (PDF1.1) and PDF1.2 [62]. AP2/ ERF TFs are also involved in the synthesis of secondary metabolites. The overexpression of Artemisia annua ORA (AaORA) increased the level of antimicrobial artemisinin as well as the expression of genes involved in artemisinin biosynthesis [63]. In addition, the overexpression of AaORA had enhanced resistance to B. cinerea, increasing expression of defense-related genes. On the other hand, a recent study demonstrated that an ERF could

The NAC family comprises a large plant-specific group of TFs. NAC TFs share a NAC domain consisting of about 150 amino acids at their N-terminus. The NAC domain has DNA-binding ability and is divided into five subdomains, A–E [66]. Typically, NAC TFs have a transcriptional regulatory (TR) domain in the C-terminal region, and some NAC TFs have a transmembrane domain within the TR domain. The genome-wide identification of NAC TFs has been performed in many plant genomes [67–71]. Phylogenetic analyses of NAC TFs in nine plant species revealed that the NAC TFs were divided into 21 subfamilies, some of which were specific to either monocots or eudicots [72]. NAC TFs play diverse roles in response to biotic and abiotic stresses and in growth and development. Recent studies revealed that the responses to biotic stresses are closely related to the responses to abiotic stresses and/or to hormone signaling. For example, cold signals enhanced the proteolytic activation of a plasma membrane-bound NAC TF, NTL6, in Arabidopsis [73]. The activated NTL6 directly binds to the promoters of PR genes and induces resistance to infection by pathogens. In addition, Arabidopsis ATAF1 seems to have multiple functions in responses to abiotic and biotic stresses [74]. Transcripts of ATAF1 were increased in response to drought, high salinity, ABA, methyl jasmonate, wounding and B. cinerea infection. The overexpression of ATAF1 caused not only increased tolerance to drought but also enhanced susceptibility to B. cinerea infection, indicating downstream signaling cross talk. In tomato, the most closely related NAC TFs (66% identity at the amino acid level), jasmonic acid 2 (JA2) and JA2-like (JA2L), play different roles in pathogen-induced stomatal closure and reopening [75]. The different roles were attributed to the regulation of ABA and SA signaling. Specifically, JA2 regulates ABA biosynthesis and ABA-mediated stomatal closure, whereas JA2L regulates SAmetabolism genes and JA/Coronatine (COR)-mediated stomatal reopening. These data indicate NAC TFs play roles in plant defense in a complex manner. Some NAC TFs can be targeted by various pathogens to enhance disease susceptibility and/or to act as negative regulators of the plant defense responses. A recent study demonstrated that an RxLR effector from P. infestans interacts with two ERassociated potato NAC TFs, Solanum tuberosum NTP1 (StNTP1) and StNTP2. This interaction prevented the movement of the TFs from the ER to the nucleus and suppressed defense responses [76]. In addition, HopD1, a type III effector from P. syringae, interacts with membrane-tethered NTL9 at the ER, suppressing ETI responses [77]. A similar situation was found in viral pathogenesis, in which the TMV replicase protein interacts with the NAC TF ATAF2, suppressing basal host defense [78]. A toxin from P. syringae also promotes virulence by mediating NAC TF genes. COR mimics jasmonic acid isoleucine, and it

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mediates three NAC TF genes (ANAC019, ANAC055 and ANAC072) directly through MYC2, repressing expression of isochorismate synthase 1 and SA biosynthesis [79]. In rice, 26 NAC TFs were upregulated following infection by ‘rice stripe virus’ or ‘rice tungro spherical virus’ [69]. Six of those genes were upregulated in both viral infections, indicating important roles of the genes in defense. A phylogenetic analysis of NAC TFs with known functions or expression profiles in response to certain stimuli revealed that sequence conservation among the NAC TFs somewhat matched the functions of the proteins [71]. These data will provide critical clues for the function of NAC TFs in plant defense responses.

bZIP bZIP TFs are found in all eukaryotes. They have a bZIP domain consisting of 60–80 amino acids, a DNA-binding basic region and a leucine zipper for homo- or hetero-dimerization. The number of bZIP TFs in most plants is more than twice that in other eukaryotes such as Drosophila, Caenorhabditis elegans and yeast, indicating their expansion and specific roles in the plant kingdom (Table 1) [80]. In Arabidopsis, bZIP TFs are divided into 10 groups, A–I and S, based on structural features and functional information [81]. Another study identified a total of 11 groups when bZIP TFs were classified according to their phylogenetic relationships in maize, Arabidopsis and rice [82]. bZIP TFs regulate diverse biological processes such as seed formation, floral development and responses to abiotic and biotic stress. The best-known bZIP TFs involved in plant defense belong to the TGA family of which the member, TGA1a in tobacco, was first cloned [83]. Seven of 10 TGAs in Arabidopsis interact with NPR1 and play roles in basal resistance and/or regulation of PR genes [84, 85]. A recent study revealed that some TGA TFs are involved in JA- and ethylene-dependent defense responses as well as in SA-dependent SAR resistance [86]. In addition, OsTGAP1 in rice is involved in diterpenoid phytoalexin synthesis [87]. Overexpression of OsTGAP1 enhances accumulation of momilactones, regulating expression of the clustered five momilactone biosynthetic genes. In addition to the TGA family, other bZIP TFs have also shown to be involved in plant defense. Arabidopsis bZIP10 (AtbZIP10) and lesions simulating disease resistance 1 (LSD1), plant-specific zinc-finger proteins, antagonistically control basal defense and the pathogen-induced HR. AtbZIP10 interacts with LSD1 and is retained outside the nucleus when it is not activated [88]. In pepper, CabZIP1 is induced by both compatible and incompatible Xanthomonas campestris pv. vesicatoria. The overexpression of CabZIP1 in Arabidopsis enhances resistance to P. syringae pv. tomato DC3000 [89]. Few studies have been conducted on genome-wide identification of pathogen-regulated bZIP genes. Differential gene expression analysis using microarrays in maize following four fungal infections identified some bZIP TFs that were up- or downregulated [82]. For better understanding of bZIP function in plant defense, further studies on pathogen-responsive genome-wide analyses as well as identification of interacting partners and downstream target genes are needed.

bHLH bHLH TFs are distributed among all eukaryotes. They all have a conserved bHLH domain of 60 amino acids that comprises a basic region for DNA binding and a helix-loop-helix region for protein–protein interaction. In animals, bHLH TFs mainly play

roles in cell differentiation and neurogenic and myogenic processes [90]. bHLH TFs in plants are involved in diverse biological processes as well. A recent genome-wide analysis of bHLH TFs from Arabidopsis, poplar, rice, moss and algae classified the bHLH TFs into 32 subfamilies [91]. There are few examples of bHLH TFs acting in plant defense against pathogens. One family that is known in plant defense is the MYC family. MYC TFs are closely related to JA signaling mediated by the coronatine insensitive 1 and jasmonate ZIM domain proteins [92]. Arabidopsis MYC2 (AtMYC2) is induced by JA and negatively and positively regulates pathogen- and wound-responsive genes, respectively [93]. A genome-wide comparison of transcriptional profiles between wild-type and myc2/jin1 mutant plants under JA treatment revealed that AtMYC2 modulates diverse gene expression in JA-mediated processes. In addition, AtMYC2 likely plays roles in JA-mediated tolerance to the herbivore Helicoverpa armigera [94]. In Nicotiana attenuata, NaMYC2 is involved in nicotine biosynthesis and regulates plant resistance against herbivores [95]. In addition to MYC2, MYC3 and MYC4 also play roles in JA signaling, and their functions additive to that of MYC2 [96]. Recently, jasmonate associated MYC2-like (JAM) 1, 2 and 3 were identified as targets of MYC2. Those targets negatively regulate other targets of MYC2, such as genes related to anthocyanin biosynthesis and defense against herbivores during JA responses [97].

Others In addition to the five TF families described above, other TFs have been reported to play roles in plant defense. The CCCHtype zinc-finger protein in rice, C3H12, positively regulates resistance to X. oryzae pv. oryzae. The overexpression of C3H12 promotes the accumulation of JA and JA-related gene and partially enhances resistance to X. oryzae pv. oryzae [98]. Another recent study showed that the SQUAMOSA promoter binding protein-like (SPL) TF is involved in ETI. SPL TFs are known as regulators of flowering time and leaf and pollen development. In N. benthamiana, NbSPL6 interacts with TIR-NB-LRR-type N protein in the presence of p50-U1, a TMV replicase fragment that elicits the N-mediated defense response. The silencing of NbSPL6 demonstrated that NbSPL6 is crucial for N-mediated resistance to TMV [99]. In the case of the MYB TF family, MYB30 is relatively well known in plant defense responses. MYB30 is a positive regulator of the HR and resistance to bacteria. AtsPLA2a, a secreted phospholipase, is relocalized from golgi vesicles to the nucleus where it negatively regulates MYB30 and the defense responses [100]. A recent study also revealed that MYB30 is ubiquitinated and degraded by MYB30-interacting E3 ligase 1 in the absence of pathogens [101]. The negative regulation of MYB30 is attenuated in response to bacterial inoculation. In addition, a study of interactome network revealed that Arabidopsis TCP14 is a major hub targeted by two pathogen effectors [102].

Perspectives In plant defense responses, the roles of TFs are extremely important in the regulation and fine-tuning of defense-related gene expression and metabolite synthesis. To date, a number of TFs in certain families have been shown to induce or repress a variety of genes needed for defense responses such as the HR and phytoalexin synthesis. Recent studies also revealed some of the mechanisms of TF activation, such as phosphorylation or

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Figure 1. Scheme of functional studies to identify plant TFs involved in defense response in the genomics era

degradation. In addition, some TFs can be manipulated by pathogens to suppress defense responses. Advancement in sequencing technology has dramatically expanded genomic and transcriptomic data that are publically available. To elucidate the complex mechanisms and transcriptional reprogramming that function in defense responses, bioinformatic and experimental approaches should be integrated (Figure 1). All TF repertoires within plant genomes can be identified from in silico annotation data. The identified TFs can be classified into subgroups based on phylogenetic relationships or on the features of specific domains, providing useful information for comparative and evolutionary analyses among species in the plant kingdom. In addition, the targets of TFs can be predicted by the analysis of motifs in gene promoters. For example, a motif analysis was integrated into co-expression networks in Arabidopsis to identify gene expression modules regulated by a specific motif [103]. Transcriptome data from diverse pathogen infections provide global overviews of the transcriptional changes that occur in response to infection and also provide useful information for the prediction of TFs involved in defense responses. Proteome data such as that from protein microarrays might also be helpful in finding proteins that interact with and regulate TFs. Functional analyses based on experimental approaches are also essential for the identification of TF functions in defense responses. Gain- and loss-of-function studies have been conducted, and massive screening of genes of interest is also available along with the development of cloning methods.

Interactions between TFs and promoters can be confirmed by ChIP sequencing. Recently, a rapid in vivo screening system for interactions between TFs and promoters was developed using a reporter gene [103]. Using these approaches, the specific functions of candidate TFs will be validated, and transcriptional networks and/or specific target genes will be identified. The relationships between TFs and hormone signaling and phytoalexin synthesis can be elucidated. Ultimately, a better understanding of the target genes of TFs and of transcriptional regulation in plant immunity will be applied to genetic engineering and the breeding of improved crop resistance against plant pathogens.

Key points • Recent advances in genome sequencing allow the gen-

ome-wide identification and characterization of TFs from diverse plant genomes. • Expression of a number of TFs is induced or repressed by pathogen attack, and the expression of whole TF repertoires can be monitored using transcriptome analysis. • A number of TFs positively or negatively regulate defense-related gene expression following pathogen infection. • Functional analyses have revealed their targets, binding sites and modes of action.

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Acknowledgment The authors apologize to all colleagues whose work could not be discussed because of space limitations.

Funding This work was supported by grants from the National Research Foundation of the Korea Ministry of Education, Science and Technology [2010-0015105 to D.C., Global Ph.D Fellowship Program 2012-016405 to E.S.] and the Agricultural Genome Center, the Next Generation Biogreen 21 program, Rural Development Administration of the Korean government [PJ011275 to D.C.].

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