The Plant Journal (2014) 78, 978–989

doi: 10.1111/tpj.12527

The Arabidopsis immune adaptor SRFR1 interacts with TCP transcription factors that redundantly contribute to effector-triggered immunity Sang Hee Kim1,2, Geon Hui Son1,2,3, Saikat Bhattacharjee1,2,†, Hye Jin Kim3, Ji Chul Nam1,2,‡, Phuong Dung T. Nguyen1,2, Jong Chan Hong1,3 and Walter Gassmann1,2,* 1 Division of Plant Sciences, University of Missouri, Columbia, MO 65211-7310, USA, 2 C.S. Bond Life Sciences Center and Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211-7310, USA, and 3 Division of Applied Life Science (BK21 Plus Program) and Department of Biochemistry, Gyeongsang National University, Jinju, 660-701 Korea Received 19 August 2013; revised 20 March 2014; accepted 24 March 2014; published online 1 April 2014. *For correspondence (e-mail [email protected]). † Present address: Regional Centre for Biotechnology, Gurgaon 122016, India. ‡ Present address: Department of Biology, Texas State University, San Marcos, TX 78666, USA.

SUMMARY The plant immune system must be tightly controlled both positively and negatively to maintain normal plant growth and health. We previously identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator specifically of effector-triggered immunity. SRFR1 is localized in both a cytoplasmic microsomal compartment and in the nucleus. Its TPR domain has sequence similarity to TPR domains of transcriptional repressors in other organisms, suggesting that SRFR1 may negatively regulate effector-triggered immunity via transcriptional control. We show here that excluding SRFR1 from the nucleus prevented complementation of the srfr1 phenotype. To identify transcription factors that interact with SRFR1, we screened an Arabidopsis transcription factor prey library by yeast two-hybrid assay and isolated six class I members of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor family. Specific interactions were verified in planta. Although single or double T-DNA mutant tcp8, tcp14 or tcp15 lines were not more susceptible to bacteria expressing AvrRps4, the triple tcp8 tcp14 tcp15 mutant displayed decreased effector-triggered immunity mediated by the resistance genes RPS2, RPS4, RPS6 and RPM1. In addition, expression of PATHOGENESIS-RELATED PROTEIN2 was attenuated in srfr1-4 tcp8-1 tcp14-5 tcp15-3 plants compared to srfr1-4 plants. To date, TCP transcription factors have been implicated mostly in developmental processes. Our data indicate that one function of a subset of TCP proteins is to regulate defense gene expression in antagonism to SRFR1, and suggest a mechanism for an intimate connection between plant development and immunity. Keywords: Arabidopsis thaliana, Pseudomonas syringae, effector-triggered immunity, SRFR1, TCP transcription factors, transcriptional regulation, yeast two-hybrid assay.

INTRODUCTION Plants must fine-tune immune responses to prevent deleterious developmental phenotypes, poor growth and reduced fertility (McDowell and Simon, 2006; Todesco et al., 2010). Most prominently, effector-triggered immunity (ETI) frequently, but not necessarily, results in localized programmed cell death called the hypersensitive response (HR) (Goodman and Novacky, 1994; Greenberg and Yao, 2004; Chisholm et al., 2006). The long-term negative effects of uncontained HR or low-level constitutive defenses may represent direct results of the toxicity of 978

defense compounds such as reactive oxygen species or secondary metabolites. Alternatively, plants may allocate resources towards development or defense. Whether this allocation occurs by default because of limiting resources or by a regulated process is an open question. Using RLD, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) in a suppressor screen for mutants with enhanced resistance to DC3000 (avrRps4) but not to virulent DC3000 (Kwon et al., 2004). The Arabidopsis accession RLD is naturally susceptible to the bacterial pathogen Pseudomonas © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd

TCPs interact with SRFR1 and regulate immunity 979 syringae pv. tomato strain DC3000 (DC3000) expressing the effector avrRps4 (Hinsch and Staskawicz, 1996). SRFR1 encodes a tetratricopeptide (TPR) protein, and proteins with similarity to SRFR1 across the whole sequence, including the C-terminal non-TPR domain, are found in other organisms (Kwon et al., 2009). To date, none of these SRFR1-like proteins have been assigned a function. Recently, SRFR1 was identified as an adaptor protein in cytoplasmic microsomal and nuclear protein complexes containing the defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and resistance proteins of the Toll/interleukin-1 receptor–nucleotide binding–leucine-rich repeat (TNL) class (Bhattacharjee et al., 2011; Heidrich et al., 2011). SRFR1 orthologs appear to be absent in Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. Nevertheless, the SRFR1 TPR domain shares significant sequence similarity with the TPR domains of the transcriptional repressors Ssn6 of S. cerevisiae and OGT1 of C. elegans, suggesting that SRFR1 may also function as a negative transcriptional regulator in ETI by interacting with transcription factors (Kwon et al., 2009). Transcription factors control a significant proportion of the defense response output by positively or negatively regulating the defense transcriptome. Well-studied examples of the roles of transcription factors include the involvement of WRKY transcription factors in basal resistance to biotrophs or necrotrophs, as well as ETI (Eulgem and Somssich, 2007; Chi et al., 2013), and of TGA transcription factors in regulation of systemic acquired resistance (Fu and Dong, 2013). TCP proteins are among the more recently recognized plant-specific transcription factor families (Mart
ın-Trillo and Cubas, 2010). The TCP protein family derives its name from the first characterized members, TEOSINTE BRANCHED1 (TB1) in maize (Zea mays), CYCLOIDEA (CYC) in snapdragon (Antirrhinum majus) and PCF in rice (Oryza sativa), and to date its members have mainly been implicated in plant developmental pathways (Cubas et al., 1999; Kosugi and Ohashi, 2002; Navaud et al., 2007). The TCP family may be divided into two subfamilies, PCF (TCP-P or class I) and TB1/CYC (TCP-C or class II), based on the structure of the TCP-specific basic helix-loop-helix (b-HLH) DNA-binding domain (Cubas et al., 1999; Navaud et al., 2007). There are 24 members of the TCP protein family in Arabidopsis (Mart
ın-Trillo and Cubas, 2010). Most characterized TCPs in Arabidopsis belong to class II, with functions in shoot lateral organ formation (TCP3) (Koyama et al., 2007) and JA biosynthesis and leaf senescence (TCP2, TCP3, TCP4, TCP10 and TCP24, whose expression is regulated by the microRNA miR319) (Schommer et al., 2008). Characterized functions of Arabidopsis class I TCPs include pollen development (TCP16) (Takeda et al., 2006), seed germination (TCP14) (Tatematsu et al., 2008), and control of the cell cycle, cell expansion and plant shape (TCP20)

(Tremousaygue et al., 2003; Li et al., 2005; Herve
et al., 2009). Interestingly, an antagonistic regulation of JA biosynthesis by class I TCP20 and TCP9 and the miR319-regulated class II TCPs was observed recently (Danisman et al., 2012). A first indication that TCP transcription factors may play a role in plant defense responses to biotrophic pathogens was the finding that multiple pathogen effectors from diverse pathogen classes interact with TCP14 in yeast (Mukhtar et al., 2011). As a major function of the yeast protein Ssn6 is to repress expression of target genes via interaction with DNA-binding transcription factors, we screened an Arabidopsis transcription factor yeast two-hybrid library using SRFR1 as bait. We isolated several class I TCP transcription factors, and verified that SRFR1 interacts with these in planta. In addition, a triple tcp8 tcp14 tcp15 mutant was found to be more susceptible to DC3000 (avrRps4), and the defense marker PATHOGENESIS-RELATED PROTEIN2 (PR2) accumulated less in the srfr1-4 tcp8-1 tcp14-5 tcp15-3 quadruple mutant compared with the srfr1-4 mutant. We therefore propose that SRFR1 functions as a transcriptional repressor of plant defense genes and fine-tunes the Arabidopsis defense response by interacting with TCP proteins in the nucleus. RESULTS Nuclear localization of SRFR1 is required for complementation of srfr1-1 plants In previous studies, SRFR1 constructs N-terminally tagged with GFP and driven by the CaMV 35S promoter, or with the hemagglutinin (HA) epitope and driven by the native SRFR1 promoter, were found to complement srfr1 mutants in stable transgenic lines (Kwon et al., 2009; Kim et al., 2010). To examine the relationship between SRFR1 localization and function, we fused one of four cellular localization tags to the N-terminus of the HA tag in an HA-SRFR1 construct driven by the CaMV 35S promoter. The four localization tags consisted of the monopartite nuclear localization signal (NLS) derived from the SV40 T-antigen with the amino acid sequence PKKKRKV (Wu et al., 1996), the nuclear exclusion signal (NES) LPPLERLTLD derived from the HIV-1 Rev protein (Henderson and Percipalle, 1997), plus a mutated NLS (nls; PKNKRKV) (Wu et al., 1996) and a mutated NES (nes; LPPLERATAD) (Johnson et al., 1999) as controls. These constructs were used to transform Arabidopsis accession RLD carrying the srfr1-1 allele (Kwon et al., 2004). For each construct, three to five independent homozygous transgenic lines were tested for resistance or susceptibility to DC3000 (avrRps4) in disease assays and for SRFR1 expression by protein blotting. In repeated disease assays, a difference was discernible between NES lines, which showed resistance levels similar to srfr1-1, and NLS,

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

980 Sang Hee Kim et al. nls and nes lines, which on average were as diseased as wild-type RLD (Figure S1a,b and Table S1). We chose a representative line for each construct with comparable levels of total SRFR1 protein (Figure S1c) for detailed analysis. First, we examined SRFR1 levels in nuclear fractions and cytoplasmic microsomal or soluble fractions. As shown in Figure 1(a), the amount of nuclear NES–HA–SRFR1 protein was below the detection limit. Despite the nuclear localization tag, NLS–HA–SRFR1 was detected in the microsomal fraction at reduced but similar levels to control nls– HA–SRFR1 and nes–HA–SRFR1 proteins, preventing conclusions about the relative importance of the microsomal sub-pool of SRFR1 in this analysis. None of the fusion proteins localized to the cytoplasmic soluble fraction to detectable levels (Figure S2), consistent with previous localization of HA–SRFR1 and GFP–SRFR1 to nuclear and cytoplasmic microsomal fractions but not soluble fractions in Nicotiana benthamiana and Arabidopsis (Kwon et al., 2009; Kim et al., 2010; Bhattacharjee et al., 2011). Next, these transgenic lines were compared to untransformed Col-0, RLD and srfr1-1 in terms of in planta growth of DC3000 or DC3000 (avrRps4). All plant lines were equally susceptible to DC3000, whereas Col-0 and srfr1-1, respectively, supported approximately 200- and 30-fold less growth of DC3000 (avrRps4) compared with susceptible RLD (Figure 1b). Lines expressing the nls-HA-SRFR1 or nes-HA-SRFR1 constructs were as susceptible to DC3000 (avrRps4) as RLD, indicating that addition of amino acids to the N-terminus of HA–SRFR1 does not interfere with its function. Of the remaining two transgenic lines tested, only plants expressing the NLS-HA-SRFR1 construct were as susceptible as RLD, whereas NES-HA-SRFR1 plants were as resistant to DC3000 (avrRps4) as srfr1-1 (Figure 1b), indicating that this construct does not complement the mutant. We therefore conclude that a sub-pool of nuclear SRFR1 is required for its function in suppressing resistance to DC3000 (avrRps4) in RLD plants. Identification of SRFR1-interacting transcription factors To determine whether SRFR1 interacts with any nuclearlocalized transcription factors, we performed a yeast twohybrid screen using a pooled Arabidopsis transcription factor cDNA library (Son et al., 2012) as prey and Arabidopsis SRFR1 as the bait. Screening the prey library of over 1400 transcriptional regulators identified 18 positive clones representing nine distinct putative interactors. Re-testing by co-transformation with bait and prey vectors eliminated all but one possible interactor: At3g27010 (TCP20). Using individual co-transformations and higher stringency, we then re-screened the TCP prey sub-library representing all predicted TCP family members except TCP6 and TCP18, which could not be amplified. In repeated tests, yeast growth was not only weakly rescued with the TCP20 prey, but also with five additional TCP prey: TCP8, TCP14, TCP15, TCP22 and

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Figure 1. Nuclear localization of SRFR1 is required for full susceptibility of RLD to bacteria expressing AvrRps4. (a) Protein blot of nuclear and cytoplasmic microsomal fraction from four transgenic lines expressing HA–SRFR1 fused to the cellular localization tags NLS, NES, nls or nes. Organelle markers were used to determine fraction enrichment [anti-histone H3 (aH3), nucleus; anti-V-ATPase (aV-A), cytoplasmic microsomal; anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (aG)]. The nuclear fraction is fivefold concentrated compared with the microsomal fraction. The molecular mass values for protein standards (kDa) are shown on the left. The assay was repeated three times with similar results. (b) In planta bacterial growth of virulent DC3000 and DC3000 (avrRps4) on day 0 and day 3 after inoculation of the indicated plant lines with bacteria. Plants were inoculated with a bacterial suspension at a density of 5 9 104 colony-forming units per ml. Values are means  SD from triplicate leaf samples. Different letters above the bars indicate significant differences as determined by Student’s t test (P < 0.05). This experiment was repeated three times with similar results.

TCP23. TCP8, TCP14 and TCP15 interacted most strongly with SRFR1, resulting in selective yeast growth at 1 mM 3amino-1,2,4-triazole (3-AT) (Figure 2a). The b-galactosidase activity assay further confirmed interactions of SRFR1 with specific TCPs in yeast (Figure 2b). Phylogenetic analysis of TCP proteins revealed that only a subset of class I TCP transcription factors

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

TCPs interact with SRFR1 and regulate immunity 981

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Figure 2. Interaction of SRFR1 with six members of the TCP transcription factor family in yeast. (a) Full-length SRFR1 was used as bait with the prey constructs indicated in the first two columns. (+), pDEST32-HY5 and pDEST22-STO (positive control); (–), pDEST32-SRFR1 and pEXP-AD502 (negative control). The background for TCP proteins that interact strongly with SRFR1 is shaded dark gray, while that for TCP proteins that interact more weakly with SRFR1 is shaded light gray. The three panels on the right indicate yeast growth on selective media. –TL, medium lacking Trp and Leu (selection for prey and bait vectors); –TLH, medium lacking Trp, Leu and His (selection for bait and prey interaction). 3-AT was added at a concentration of 0.5 or 1 mM to inhibit low-level constitutive expression of the HIS3 reporter. (b) Phylogenetic tree of 24 Arabidopsis full-length TCP protein sequences. The scale bar indicates the evolutionary distance as the mean number of amino acid substitutions per site. Specific b-galactosidase (b-gal) activity levels were determined to quantify SRFR1 interactions with TCP transcription factors, and are shown on the right, with pEXP-AD502 (vector) as a negative control. In the replicate shown, the b-gal activity level for the positive control comprising pDEST32HY5 and pDEST22-STO was 135.9  6.1 Miller units. Plate and liquid yeast two-hybrid assays were repeated twice with similar results.

interacted with SRFR1. Interactions with class II TCPs were not observed (Figure 2a,b). We further analyzed whether the SRFR1–TCP interaction was domain-specific. SRFR1 consists of an N-terminal TPR domain (SRFR11–567) and a conserved C-terminal domain of unknown function (SRFR1568–1052) (Figure S3a) (Kwon et al., 2009). For TCP8, the interaction with SRFR11–567 was as strong as with fulllength SRFR1, whereas the interaction with SRFR1568–1052 was weaker (Figure S3b,c). A similar pattern was observed with TCP14 and TCP15, although TCP15 interacted with SRFR1568–1052 almost as well as with full-length SRFR1 and SRFR11-567. Conversely, when the TCP8 prey was divided into an N-terminal fragment containing the TCP domain (TCP81–200) and a C-terminal domain (TCP8201–401), interactions with full-length SRFR1 were lost completely (Figure S3d). Both TCP8 fragments accumulated when transiently expressed in N. benthamiana (Figure S3e). The interaction between SRFR1 and TCPs was therefore strongest between the SRFR1 TPR domain and full-length TCP8. We also specifically screened WRKY and TGA prey sublibraries containing 59 WRKY and nine TGA family members, respectively, using SRFR1 because of the known functions of WRKY and TGA transcription factors in defense response regulation (Eulgem and Somssich, 2007; Chi et al., 2013). However, no SRFR1 interactors were identified among these two transcription factor families. Because of the specificity of interactions, we mainly

focused on the strong SRFR1 interactors TCP8, TCP14 and TCP15 for further study. Subcellular localization of TCP interactors Computational prediction of protein localizations (Horton et al., 2007) indicated that TCP8, TCP14 and TCP15 are likely to be nuclear proteins. To verify the subcellular localization of these TCPs, we used Agrobacterium tumefaciens-mediated transient expression of N-terminally GFPtagged TCPs in N. benthamiana leaf cells. TCP3, a class II TCP protein that did not interact with SRFR1 in yeast, was included for comparative purposes. As shown in Figure S4a, the control GFP was uniformly distributed throughout the nucleus and cytoplasm. GFP–TCP8 and GFP–TCP14 were exclusively targeted to the nucleus, while GFP–TCP15 and GFP–TCP3 were found in both the nucleus and cytoplasm (Figure S4a). Magnification of confocal images indicated that GFP–TCP14 in particular localized to distinct sub-nuclear bodies, whereas GFP–TCP15 gave a diffuse nucleoplasmic signal (Figure S4b). Protein blots confirmed that the GFP–TCP fusion proteins, including GFP–TCP15 and GFP–TCP3, were full-length (Figure S4c). SRFR1 interacts with TCP8, TCP14 and TCP15 in planta As a first step to verify the interactions of SRFR1 with TCP8, TCP14 and TCP15 in planta, we performed bimolecular fluorescence complementation (BiFC), allowing

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

982 Sang Hee Kim et al. determination of the subcellular localization of interacting protein complexes in living cells (Citovsky et al., 2006; Ohad et al., 2007). SRFR1 was fused to the C-terminus of Venus (an enhanced YFP version) (nVenus–SRFR1), and TCP3, TCP8, TCP14 and TCP15 were fused to the C-terminus of CFP (cCFP–TCP). As shown in Figure 3(a), SRFR1 interacted with TCP8, TCP14 and TCP15 in the nucleus. Moreover, SRFR1–TCP8 and SRFR1–TCP14 complexes were detected in sub-nuclear bodies, whereas SRFR1– TCP15 showed a diffuse nucleoplasmic localization (Figure 3a). Identical localization patterns were obtained when the BiFC tags were swapped, except for nVenus– TCP14 and cCFP–SRFR1, which also showed cytoplasmic localization (Figure S5). Consistent with the yeast twohybrid data, no YFP fluorescence was detected for SRFR1 and TCP3 with either BiFC tag combination (Figures 3a and S5). The SRFR1–TCP interactions were also confirmed by nuclear co-immunoprecipitation assays of transiently expressed Myc–TCP8, Myc–TCP14, Myc–TCP15 or Myc– TCP3 in HA–SRFR1-expressing stably transgenic N. benthamiana (Kim et al., 2010). We included TCP21, a class I TCP that did not interact with SRFR1 in yeast, to test for additional interactions. The negative control protein GUS and TCP3 did not interact with SRFR1, whereas TCP8, TCP14, TCP15 and TCP21 did form complexes with SRFR1 (Figure 3b). In addition to SRFR1–TCP interactions, we detected TCP8, TCP14 and TCP15 homodimers in the nucleus, and TCP3 homodimers in the cytoplasm (Figure S6). Interestingly, all four TCPs also interacted with each other to form heterodimers in the nucleus, with dimer combinations that included TCP8 being most strongly associated with

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sub-nuclear bodies (Figure S6). This suggests that heterodimerization of TCPs occurs even between class I TCPs (TCP8, TCP14 and TCP15) and class II TCPs (TCP3). To verify this, we performed nuclear isolation and co-immunoprecipitation assays of transiently expressed Myc–TCP3 with HA–TCP3, HA–TCP8, HA–TCP14 or HA–TCP15 in N. benthamiana (Figure 4). Consistent with the BiFC results, the amount of TCP3–TCP3 complexes in the nuclear fraction was below the detection limit, but TCP8, TCP14 and TCP15 were found in a nuclear complex with TCP3 in these transient over-expression assays. Together, the yeast two-hybrid, BiFC and co-immunoprecipitation assays indicate that SRFR1 interacts specifically with class I TCPs. In addition, these interactions were most robust with TCP8, TCP14 and TCP15. However, additional interactions of class I TCPs with SRFR1 may occur in planta, perhaps via heterodimerization with TCP8, TCP14 or TCP15 if this also occurs at native expression levels. Partial loss of resistance of tcp8 tcp14 tcp15 plants to DC3000 (avrRps4) To examine whether SRFR1-interacting TCPs positively or negatively regulate AvrRps4-triggered immunity, we first generated TCP8 over-expression lines of Col-0 using an N-terminally Myc-tagged TCP8 construct whose expression was driven by the CaMV 35S promoter. Protein blots confirmed that Myc–TCP8 was stably over-expressed (Figure S7a). However, two independent lines over-expressing Myc-tagged TCP8 did not display altered bacterial growth after inoculation with virulent DC3000 or DC3000(avrRps4) (Figure S7b). Figure 3. Interaction of SRFR1 with TCP proteins in planta. (a) BiFC indicates that SRFR1 and TCP8, TCP14 and TCP15, but not TCP3, interact in the nucleus in N. benthamiana leaf epidermal cells. Scale bar = 10 lm. (b) SRFR1 interacts with TCP8, TCP14, TCP15 and TCP21, but not with TCP3 or GUS, in the nuclear fraction. Stable transgenic N. benthamiana plants expressing HA–SRFR1 were infiltrated with Agrobacterium containing binary vectors for transient expression of Myc-tagged TCPs or GUS. Input nuclear extracts are shown in the top two panels. Antibodies used to detect immunoprecipitates (IP) by immunoblotting (IB) are indicated on the right. Molecular mass values for protein standards (kDa) are shown on the left. Asterisks indicate the expected sizes of full-length Myc-tagged proteins.

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TCPs interact with SRFR1 and regulate immunity 983

Figure 5. Redundant functions of TCP8, TCP14 and TCP15 in AvrRps4-triggered immunity. In planta bacterial growth was measured in Col-0 or tcp8-1 tcp14-5 tcp15-3 (t8 t14 t15) plants on day 0, day 2 and day 4 after inoculation with DC3000 or DC3000 (avrRps4). Plants were inoculated with a bacterial suspension at a density of 5 9 104 colony-forming units per ml. Values are mean colonyforming units cm 2 leaf tissue  SD. Asterisks indicate that growth of DC3000 (avrRps4) on days 2 and 4 was significantly different between Col-0 and tcp8-1 tcp14-5 tcp15-3, as determined by a two-tailed Student’s t test (P < 0.01). This experiment was repeated three times with similar results. Figure 4. TCPs heterodimerize across classes in the nucleus. Nuclear co-immunoprecipitation of TCP3 with TCP8, TCP14 and TCP15, but not with TCP3 itself or GUS, transiently expressed in N. benthamiana. Input nuclear extracts are shown in the top two panels. Antibodies used to detect immunoprecipitates (IP) by immunoblotting (IB) are indicated on the right. Molecular mass values of protein standards (kDa) are shown on the left.

is likely that higher-order tcp knockout mutants would show even stronger susceptibility to DC3000 (avrRps4). Susceptibility of the tcp8 tcp14 tcp15 triple mutant is not specific to DC3000 (avrRps4)

As a second step to identify the function of TCPs in ETI, we analyzed resistance in tcp8, tcp14 and tcp15 T-DNA knockout lines. Because of likely redundancy, we also generated tcp8 tcp14 tcp15 triple mutant lines and corresponding double mutant lines using T-DNA knockout lines of TCP8 (tcp8-1, SAIL_656_F11) (Sessions et al., 2002), TCP14 (tcp14-5, GK-611C04-021892) (Mukhtar et al., 2011; Kleinboelting et al., 2012) and TCP15 (tcp15-3, SALK_011491) (Alonso et al., 2003; Kieffer et al., 2011). RT-PCR suggested that tcp15-3 is a transcriptional knockout allele of TCP15, while low levels of tcp8 and tcp14 transcript were detected from the tcp8-1 and tcp14-5 alleles (Figure S8). Because the T-DNAs were inserted within the open reading frames of TCP8 and TCP14, these transcripts are unlikely to encode functional protein. In planta bacterial growth assays showed that the growth of DC3000 (avrRps4) in single tcp mutants was similar to growth in wild-type Col-0, and all plant lines were fully susceptible to virulent DC3000 (Figure S9). As shown in Figure 5, by day 4, triple mutant lines supported approximately 30-fold more growth of DC3000 (avrRps4) than wild-type Col-0. The corresponding double mutants showed intermediate growth in repeated experiments that usually was statistically not significantly different from wild-type (Figure S9). We conclude that TCP8, TCP14 and TCP15 contribute redundantly to DC3000 (avrRps4) immunity. Because these TCPs and SRFR1 interacted with additional TCPs such as TCP20, TCP22 and TCP23 (Figure 2), it

To date, direct effects of mutations in SRFR1 have only been observed with resistance specificities that require EDS1 (Kwon et al., 2004; Kim et al., 2009b). In contrast, it was recently shown that TCP14 is an interaction target of multiple effectors from two pathogens (Mukhtar et al., 2011). To assess the specificity of the enhanced susceptibility phenotype of the triple mutant against isogenic pathogen strains, we measured the growth of bacteria expressing the effectors HopA1, which is recognized by the TNL protein RPS6 (Kim et al., 2009b), and AvrRpt2 and AvrRpm1, which are recognized by the coiled-coil– nucleotide binding–leucine-rich repeat (CNL) proteins RPS2 (Bent et al., 1994; Mindrinos et al., 1994) and RPM1 (Grant et al., 1995), respectively, and compared these to growth of virulent DC3000 and DC3000 (avrRps4). As shown in Figure S10, although enhanced growth of DC3000 (avrRps4) was most pronounced, the triple mutant showed enhanced susceptibility to bacteria expressing the other effectors as well. Mean growth differences of similar proportions were observed in three additional replicates, although this difference was not statistically significant in one case each for DC3000 (avrRps4), DC3000 (hopA1) and DC3000 (avrRpm1) due to variability of bacterial growth. In contrast, wild-type and triple mutant plants were equally susceptible to virulent DC3000 (Figure S10a). In addition, triple mutant plants were not more susceptible to the type III secretion-deficient strain DC3000 (hrcC-) (Figure S10b), indicating that these plants are not compromised in

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

984 Sang Hee Kim et al. pathogen-associated molecular pattern-triggered immunity and basal resistance in general. These data suggest that TCP8, TCP14 and TCP15, and probably additional TCP family members, positively contribute to ETI to a diverse set of effectors that signal through resistance proteins of both the TNL and CNL classes. Genetic interaction between SRFR1 and TCP genes As independent support that the physical interactions between SRFR1 and TCPs observed in heterologous systems influence Arabidopsis defense phenotypes, we analyzed the tcp8-1 tcp14-5 tcp15-3 srfr1-4 quadruple mutant line. The srfr1-4 T-DNA allele induces marked autoimmune responses such as stunted growth that are environmentally conditioned and complicate phenotypic analyses (Kim et al., 2010; Li et al., 2010). When plants were grown under short-day conditions at 24°C, both srfr1-4 and quadruple mutant plants were significantly smaller than Col-0 and triple mutant plants, and were not significantly different from each other (Figure S11a). When grown under long-day conditions at 22°C, the srfr1-4 phenotype was more severe, and a marginal rescue of growth in the quadruple mutant was measured in two out of three experiments (Figure S11b). This indicated that rescue of srfr1-4 plant growth inhibition, if any, by mutations in TCP8, TCP14 and TCP15 may be masked by the presence of additional TCPs. As a more sensitive assay, we determined protein levels of PR2, a marker for plant defense activation whose mRNA levels are up-regulated in srfr1 mutants (Dong et al., 1991; Cordelier et al., 2003; Kim et al., 2009a). Plants were grown under short-day conditions at 24°C to minimize confounding effects of altered growth. In all three tests, we observed lower PR2 expression in the quadruple mutant compared to srfr1-4 (Figures 6 and S12). This showed that mutations in TCP8, TCP14 and TCP15 do indeed lower the constitutive defense phenotype of srfr1-4 plants, indicating that SRFR1 and TCP genes interact genetically. DISCUSSION Genetically, SRFR1 functions as a negative regulator of AvrRps4-triggered immunity because mutations in srfr1 were recessive, and these mutants were fully susceptible

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Figure 6. High constitutive PR2 expression in srfr1-4 plants is attenuated by mutations in TCP8, TCP14 and TCP15. Protein blots of total protein isolated from Col-0, the tcp8-1 tcp14-5 tcp15-3 (t8 t14 t15) triple mutant, the tcp8-1 tcp14-5 tcp15-3 srfr1-4 (t8 t14 t15 s1) quadruple mutant, and srfr1-4 plants. GAPDH is shown as a loading control.

to virulent DC3000, with specifically enhanced resistance to DC3000 (avrRps4) (Kwon et al., 2004, 2009). The known repertoire of SRFR1 regulatory targets was subsequently expanded to additional TNL resistance proteins (Kim et al., 2009b, 2010), and specificity for the TNL class of resistance proteins is most likely explained by the physical association of SRFR1 with both microsomal and nuclear protein complexes containing the immune regulator EDS1 (Bhattacharjee et al., 2011). In addition to SRFR1 and EDS1, a subpool of the resistance proteins RPS4 and SNC1 as well as the effector AvrRps4 are localized to the nucleus; such localization is required for gene expression changes accompanying resistance (Wirthmueller et al., 2007; Garc
ıa et al., 2010; Zhu et al., 2010; Heidrich et al., 2011). Indeed, the nucleus is emerging as a central node in ETI mediated by several resistance proteins (Maekawa et al., 2011; Bhattacharjee et al., 2013). In addition, expression of several defense genes is slightly up-regulated in srfr1-1 and srfr1-2 plants (Kim et al., 2009a), indicating that SRFR1 may have a broader role in the nucleus in addition to its interactions with components of ETI. SRFR1 is active in the nucleus and interacts with TCP transcription factors Here we show that NES–SRFR1 was excluded from the nucleus and its expression did not complement a srfr1 mutant, indicating that a proportion of SRFR1 must be localized in the nucleus for wild-type regulation of plant immunity by SRFR1. The TPR domain of SRFR1 shows sequence similarity to that of the transcriptional regulator Ssn6 in S. cerevisiae (Kwon et al., 2009). Ssn6 physically binds to the WD40 protein Tup1, and the Ssn6–Tup1 complex recognizes DNA-binding transcription factors to form a general transcriptional repressor complex (Smith and Johnson, 2000). Based on this model, we speculated that SRFR1 may interact with transcription factors to repress expression of defense-related genes, and screened an Arabidopsis transcription factor-specific library using yeast two-hybrid assay to identify transcription factors that interact with SRFR1. Interestingly, screening for SRFR1-interacting proteins in yeast did not result in isolation of well-known immunerelated transcription factors such as WRKYs or TGAs. Instead, we found that SRFR1 interacts with several members of the TCP transcription factor family, most robustly with TCP8, TCP14 and TCP15. These all belong to the class I sub-family of TCPs, for which few functions have yet been described in Arabidopsis. TCP14 was recently proposed to be a virulence target of two distinct biotrophic pathogens, and a tcp14 mutant was more susceptible to the Hyaloperonospora parasitica isolates Emwa1 and Emoy2 (Mukhtar et al., 2011). Interactions of SRFR1 with the three main TCPs in a yeast two-hybrid system were confirmed by BiFC and

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

TCPs interact with SRFR1 and regulate immunity 985 co-immunoprecipitation assays in planta. In addition, we found that these three SRFR1-interacting TCP proteins form homo- and heterodimers in the nucleus, at least when transiently over-expressed. Members of several transcriptional regulator families are known to form homodimers and heterodimers to recognize core cis-acting elements. Rice PCF1 and PCF2, both class I TCPs, interact with themselves and each other for DNA binding in a yeast two-hybrid system (Kosugi and Ohashi, 1997). Recently, it was shown that Arabidopsis class I TCPs form extensive protein–protein interaction networks, with high preference for other class I TCP members (Danisman et al., 2013). In a matrix-based yeast two-hybrid analysis of all 24 TCP members, the six TCPs that interacted with SRFR1 in our analysis also interacted with each other. The weaker interactions of SRFR1 with additional class I TCPs, such as TCP20, TCP22 and TCP23, and heterodimerization of TCPs suggest that several TCP family members participate in protein complexes with SRFR1, perhaps explaining the fairly subtle defense phenotypes found even with tcp8 tcp14 tcp15 triple mutants and why over-expressing TCP8 alone did not result in a measurable change in resistance. Balancing development and immunity The founding members of the TCP transcription factor family, TB1, CYC and PCF, were identified based on their functions in plant development. TB1 proteins in maize and rice repress lateral branching (Doebley et al., 1995; Takeda et al., 2003; Doebley, 2004), and CYC in Antirrhinum majus functions in control of floral asymmetry (Luo et al., 1996, 1999), suggesting that the TCP class II sub-family plays a role in regulating plant architecture (Mart
ın-Trillo and Cubas, 2010). Rice PCF1 and PCF2, the first identified class I TCPs, affect cell growth and proliferation and bind to the site II motif (TGGGCC/T), a cis-acting element found in genes encoding proteins that are involved in the cell cycle, ribosomal protein synthesis and axillary bud outgrowth (Kosugi and Ohashi, 1997, 2002; Tremousaygue et al., 2003; Tatematsu et al., 2005). Because SRFR1 and TCP8, TCP14 and TCP15 form protein complexes that were proposed to function in transcriptional repression, we anticipated to observe similar changes in levels of plant resistance in srfr1 and tcp mutants. Instead, we found that tcp8 tcp14 tcp15 plants were more susceptible to DC3000 expressing various effectors. This suggests that TCP8, TCP14 and TCP15, and probably additional class I TCPs, are positive regulators of defense gene expression and immunity. The observation that single tcp13, tcp14 and tcp19 mutant plants were slightly compromised in ETI to Hyaloperonospora arabidopsidis also supports the conclusion that these TCPs are positive regulators of immunity (Mukhtar et al., 2011). A potentially direct connection between SRFR1–TCP complexes and regulation of resistance is suggested by the observation that auto-active snc1

and transcriptional co-repressors of the TOPLESS family interact to repress genes such as DND1 encoding proteins that down-regulate defenses (Zhu et al., 2010). While SNC1 has been shown to interact with SRFR1 and EDS1 (Kim et al., 2010; Bhattacharjee et al., 2011), a study of the TOPLESS interactome identified several TCPs, including TCP8 and TCP14, that interacted with TOPLESS family members (Causier et al., 2012). SRFR1, either alone or as part of a larger protein complex consisting of positive and negative regulators of ETI and broader defenses, may prevent promoter binding by TCPs by sequestering them away from promoters, or may regulate target gene expression by interacting with TCPs at these promoters. SRFR1 and TCPs may constitute a nexus that determines whether disease resistance or development occurs. The trade-off between pathogen resistance and plant development is a common observation (Todesco et al., 2010), and is probably achieved via multiple routes. An added level of complexity arises from our observation that TCPs heterodimerize when transiently over-expressed, perhaps even across TCP sub-families. Because class I and class II TCPs have been reported to have antagonistic effects on gene regulation (Danisman et al., 2012), it will be important to determine whether class I/class II heterodimers function as transcriptional activators or repressors. Also, activation or repression by particular TCPs is likely to be promoter-specific. In summary, the TCP family represents an interactive network of transcriptional regulators that, together with the adaptor protein SRFR1, is involved in fine-tuning plant immunity and perhaps development. Interestingly, TCP14 and TCP15 were recently found to interact with the O-linked N-acetyl transferase SPINDLY (SPY) and mediate cytokinin responses (Steiner et al., 2012). The N-terminal TPR domains of SRFR1, SPY and Ssn6 show significant sequence similarity to each other but the C-terminal domains are highly diverged, with the SRFR1 C-terminal domain not showing any discernible sequence similarity to proteins of known enzymatic function (Kwon et al., 2009). This raises the possibility that SRFR1 and SPY competitively bind TCPs to tip the balance towards activation or repression. A remaining open question is whether TCPs directly or indirectly regulate levels of immunity. Even though TCPs were found to interact with effectors from bacterial and oomycete pathogens in yeast, they are unlikely to be direct targets of AvrRps4. Mukhtar et al. (2011) included AvrRps4 from P. syringae pv. phaseolicola strain 1448a (AvrRps4Pph1448a) in their yeast two-hybrid analysis. The amino acid sequence of this protein is 95% identical to the original AvrRps4 from P. syringae pv. pisi strain 151 (Hinsch and Staskawicz, 1996) that was used in this study, and was recently shown to trigger RPS4/RRS1-dependent immunity (Sohn et al., 2012). However, AvrRps4Pph1448a was not one of the effectors that interacted with TCP14 in yeast

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

986 Sang Hee Kim et al. (Mukhtar et al., 2011). As we have shown an increased susceptibility of tcp8 tcp14 tcp15 plants to a range of effectors, we consider it more likely that these TCP transcription factors determine a general immune set-point by modulating gene expression, which in turn may explain why they are targeted by pathogen effectors. Interference with TCP functions by endogenous DC3000 effectors may also underlie the similar levels of susceptibility displayed by wild-type and tcp8 tcp14 tcp15 plants to virulent DC3000. Future research is required to determine which genes are directly regulated by TCP8, TCP14 and TCP15. EXPERIMENTAL PROCEDURES Yeast two-hybrid screening and phylogenetic analysis A full-length SRFR1 cDNA cloned into pDONR201 (Kwon et al., 2009) was recombined into the GATEWAY-compatible pDEST32 bait vector (Invitrogen, www.lifetechnologies.com). The prey library contained over 1400 Arabidopsis transcriptional regulator full-length cDNAs in the CEN-based GATEWAY-compatible pDEST22 low-copy prey vector (Invitrogen). The SRFR1 bait and the prey library were transformed into yeast strain PJ69-4a (MATa trp1-901 leu2-3,112 ura3-52 his3-200 Dgal4 Dgal80 LYS2::GAL1HIS3 GAL2-ADE2 met2::GAL7-lacZ) by a standard yeast transformation procedure (Clontech, www.clontech.com), and the transformation mixture was plated on SD drop-out media lacking Trp and Leu, lacking Trp, Leu and His but including 0.1, 0.5 or 1 mM 3-AT, or lacking Trp, Leu, Ade and His. Plates were kept at 30°C and examined 4 days later. Approximately 1.7 9 104 colonies were screened. The interaction between STO and HY5 (Jiang et al., 2012) was used as a positive control, and pDEST32-SRFR1 with empty pEXP-AD502 vector (Invitrogen) as a negative control. Plasmids were isolated from putative positive yeast colonies and sequenced. Pairs of potential interactors were directly cotransformed into PJ69-4a, and then screened on selective media containing varying concentrations of 3-AT to confirm interactions. In addition, protein interactions in yeast were quantified by a liquid culture assay using O-nitrophenyl-b-D-galactopyranoside in a MATCHMAKER two-hybrid system (Clontech) according to the manufacturer’s instructions. For phylogenetic analyses, full-length TCP protein sequences were aligned using the neighbor-joining method implemented in ClustalW2 (Larkin et al., 2007), and the tree was drawn using the MEGA5 program, with evolutionary distances calculated using the Poisson correction (Tamura et al., 2011).

Molecular cloning of plasmids To generate plant expression constructs for HA–SRFR1 fused to cellular localization tags, we used an HA-SRFR1 cDNA driven by the CaMV 35S promoter in vector pBA-HA (Kwon et al., 2009; Kim et al., 2010) as the template for overlap PCR. Coding sequences for NLS, NES, nls or nes, including a start codon, were contained in the primers listed in Table S2. The resulting PCR products were individually incorporated into pBA-HA-SRFR1 by replacing the unmodified EcoRV–XmaI fragment with the respective NLS, NES, nls or nes cassettes. The insertions were confirmed by sequencing. The coding regions of TCP8, TCP14, TCP15, TCP21 and TCP3 were re-amplified by PCR using first-strand cDNA from Arabidopsis (Col-0), and sub-cloned into the pDONR201 entry vector using

BP reactions (Invitrogen). The entry clones were recombined into GATEWAY-compatible destination vectors using the LR reaction (Invitrogen) for protein localization, BiFC and co-immunoprecipitation analyses. For over-expression of TCP8, the TCP8 entry clone was recombined into a GATEWAY-compatible binary vector derived from myc-pBA (Zhou et al., 2005). Primer sequences used for cloning are listed in Table S2.

Plant lines and generation of transgenic plants Arabidopsis plants were grown in an E-7/2 reach-in growth chamber (Controlled Environments Ltd, www.conviron.com) under an 8 h light/16 h dark cycle at 24°C, 70–80% relative humidity, and a light intensity of 140–180 lmol photons m 2 sec 1. T-DNA insertion lines for TCP8 (At1g58100), TCP14 (At3g47620) and TCP15 (At1g69690) were obtained from the Syngenta Arabidopsis Insertion Library (tcp8-1; SAIL_656_F11) (Sessions et al., 2002), the GABI-Kat collection (tcp14-5; GK-611C04-021892) (Mukhtar et al., 2011; Kleinboelting et al., 2012) and the Salk T-DNA Express collection (tcp15-3; SALK_011491) (Alonso et al., 2003; Kieffer et al., 2011). Double and triple homozygous mutants of tcp8-1, tcp14-5 and tcp15-3 were genotyped by PCR using the primer pairs listed in Table S2. To generate transgenic Arabidopsis containing pBA-Myc-TCP8 or pBA-NLS/NES/nls/nes-HA-SRFR1 constructs, plasmids were transferred into Agrobacterium strain C58C1 and transformed into wild-type Col-0 by floral dipping (Clough and Bent, 1998). Plants were screened on 20 lg ml 1 glufosinate ammonium.

In planta bacterial growth curve assay Pseudomonas syringae pv. tomato strain DC3000 containing the empty vector or expressing the indicated effectors was grown on Pseudomonas Agar F (Becton Dickinson, www.bd.com). In planta bacterial growth assays were performed by syringe infiltration of leaves of 4-week-old plants with bacterial suspensions of 5 9 104 colony-forming units per ml (OD600 = 0.00005). Leaf discs with a total area of 0.5 cm2 per sample were ground in 10 mM MgCl2, and solutions were plated in serial dilutions on selective medium in triplicate at the indicated time points.

Agrobacterium-mediated transient expression Transient expression constructs were electroporated into A. tumefaciens strain GV3101. Bacteria cultured overnight were harvested by centrifugation and re-suspended in 10 mM MgCl2 with 100 lM acetosyringone (Sigma-Aldrich, www.sigmaaldrich.com) to an OD600 of 0.3. The agrobacteria were was incubated for 2 h at room temperature and infiltrated into N. benthamiana leaves using a 1 ml needleless syringe. N. benthamiana plants were placed in an E-7/2 reach-in growth chamber (Controlled Environments Ltd) under a 16 h light/8 h dark cycle at 25°C, 70% relative humidity. Tissues were collected 2 days after infiltration for protein blotting and confocal microscopy.

Bimolecular fluorescence complementation and confocal fluorescence microscopy BiFC was performed using A. tumefaciens-mediated transient expression in N. benthamiana. nVenus and cCFP fusions were used for SRFR1 and TCPs. RFP was co-expressed to identify the cell cytoplasm and nucleus. Using entry vector clones in pDONR201, the sequences were cloned into the nVenus and cCFP tag binary vectors (Bhattacharjee et al., 2011). Agroinfiltration was performed as described above. For co-infiltrations, each strain was adjusted to an OD600 of 0.3, and the strains were mixed.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 978–989

TCPs interact with SRFR1 and regulate immunity 987 Agrobacterium containing the Hc-Pro silencing suppressor was also co-infiltrated into N. benthamiana when SRFR1 constructs were used. Plant tissues were viewed under a Zeiss (www.zeiss. com) LSM 510 META NLO two-photon point-scanning confocal system mounted on an Axiovert 200M inverted microscope with a 409/1.2 C-Apochromat water immersion objective.

grants from the Rural Development Administration Next Generation Biogreen 21 Program (Systems & Synthetic Agrobiotech Center grant PJ00951402) and the Basic Science Research Program funded by the Ministry of Education (National Research Foundation grant 2013R1A1A2010131) (to J.C.H.).

SUPPORTING INFORMATION

Protein fractionation and co-immunoprecipitation analysis For transgenic Arabidopsis expressing localization-tagged HA– SRFR1, cellular fractionation was performed on plant tissue from liquid cultures based on a modified protocol adapted from Calikowski and Meier (2006) by Ying Wan and Scott Peck (Division of Biochemistry, University of Missouri, Columbia, MO). Briefly, tissue was processed in nuclei isolation buffer (13.8% hexylene glycol, 20 mM b-mercaptoethanol, 50 lM spermine, 125 lM spermidine, 20 mM KCl, 20 mM HEPES, pH 7.4) containing 0.3% Triton and protease and proteasome inhibitors (Sigma). Extracts were filtered and centrifuged at 1000 g for 10 min at 4°C to produce a nuclear pellet. The supernatant was further subjected to ultracentrifugation at 100 000 g to separate the soluble cytoplasmic fraction and the cytoplasmic microsomal fraction (pellet). The nuclear pellet from the previous centrifugation was separated on a Percoll gradient (Sigma) by centrifugation for 1.5 h at 4°C at 1000 g. The nuclear fraction located between the 30 and 60% layers of the Percoll gradient was recovered and washed twice at 4°C in buffer. Nuclei were lysed by sonication and centrifuged at 10 000 g for 15 min at 4°C to isolate nuclear protein (supernatant). Nuclear proteins from N. benthamiana were prepared as described by Palma et al. (2007). Nuclear lysates were centrifuged at 14 000 g for 10 min, and immunoprecipitation was performed as described previously (Bhattacharjee et al., 2011). Immunoblot assays were performed using the antibodies horseradish peroxidase-conjugated anti-Myc (Santa Cruz Biotechnology, www.scbt.com), horseradish peroxidase-conjugated anti-HA (Roche, www.roche.com), anti-GFP (Sigma-Aldrich) or anti-PR2 (Agrisera, www.agrisera.com). Detected proteins were visualized using an ECL Plus chemiluminescent kit (GE Healthcare, www. gehealthcare.com). The degree of enrichment after cellular fractionation was determined by immunoblot analyses using the antibodies anti-GAPDH (Genscript, www.genscript.com), anti-VATPase (Agrisera) and anti-histone H3 (Abcam, www.abcam.com).

TAIR accession numbers The TAIR accession numbers for the sequences referred to in this paper are At4g37460 (SRFR1), At5g45250 (RPS4), At5g46470 (RPS6), At1g53230 (TCP3), At1g58100 (TCP8), At3g47620 (TCP14), At1g69690 (TCP15), At3g27010 (TCP20), At5g08330 (TCP21), At1g72010 (TCP22) and At1g35560 (TCP23).

ACKNOWLEDGEMENTS We thank the Arabidopsis Biological Resource Center and Dan Riggs (Department of Cell & Systems Biology, University of Toronto at Scarborough, Canada) for providing tcp T-DNA insertion lines, Scott Peck and Ying Wan (Division of Biochemistry, University of Missouri, Columbia, MO) for advice on Arabidopsis protein fractionation, the University of Missouri DNA Core for sequencing services, and the University of Missouri Molecular Cytology Core for assistance with confocal fluorescence microscopy. This work was supported by Daniel F. Millikan Graduate Fellowships, Division of Plant Sciences (to P.D.T.N. and J.C.N.), US National Science Foundation Integrative Organismal Systems Program grants IOS-0715926 and IOS-1121114 (to W.G.), and

Additional Supporting Information may be found in the online version of this article. Figure S1. Evaluation of transgenic Arabidopsis lines expressing HA–SRFR1 tagged with NES, NLS, nes or nls sequences. Figure S2. Protein blot of soluble fractions from HA–SRFR1 expressing plants shown in Figure 1. Figure S3. Yeast two-hybrid interactions of SRFR1 and TCP8 fragments. Figure S4. Subcellular localization of TCP proteins. Figure S5. BiFC analysis of SRFR1–TCP interactions with reciprocal tags. Figure S6. TCP homo- and heterodimerizations analyzed by BiFC. Figure S7. TCP8 over-expressing plants do not show changes in bacterial resistance. Figure S8. Molecular characterization of the tcp8-1 tcp14-5 tcp15-3 triple T-DNA insertion line. Figure S9. Bacterial growth assays in single and double T-DNA insertion lines of TCP8, TCP14 and TCP15. Figure S10. Reduced ETI to DC3000 expressing avrRps4, avrRpm1, avrRpt2 and hopA1, but no increased susceptibility to the type III secretion-deficient strain DC3000 (hrcC-), in tcp8-1 tcp14-5 tcp15-3 mutant lines. Figure S11. Growth analysis of the tcp8-1 tcp14-5 tcp15-3 srfr1-4 quadruple mutant line. Figure S12. Replicate PR2 protein blots of plant lines shown in Figure 6. Table S1. Disease scores for plants shown in Figure S1b inoculated with virulent DC3000. Table S2. PCR primers used in this study.

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