Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1

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1 Reserch Arbidopsis downy mildew effector HRxL106 suppresses plnt immunity by binding to RADICAL-INDUCED CELL DEATH1 Lennrt Wirthmueller 1,2, Shut Asi 1, Ghnsym Rllplli 1, Jn Sklenr 1, Georgin Fbro 1, De Sung Kim 1, Ruth Lintermnn 2, Pinj Jspers 3, Michel Wrzczek 3, Jkko Kngsj rvi 3, Dniel McLen 1,FrnkL.H.Menke 1, Mrk J. Bnfield 4 nd Jonthn D. G. Jones 1 1 The Sinsbury Lbortory, Norwich Reserch Prk, Norwich, NR4 7UH, UK; 2 Dhlem Centre of Plnt Sciences, Deprtment of Plnt Physiology nd Biochemistry, Freie Universit t Berlin, K onigin-luise-strße 12 16, Berlin, Germny; 3 Division of Plnt Biology, Deprtment of Biosciences, University of Helsinki, FIN Helsinki, Finlnd; 4 Deprtment of Biologicl Chemistry, John Innes Centre, Norwich Reserch Prk, Norwich, NR4 7UH, UK Authors for correspondence: Lennrt Wirthmueller Tel: +49 (0) Emil: lennrt.wirthmueller@fu-berlin.de Jonthn D. G. Jones Tel: +44 (0) Emil: jonthn.jones@tsl.c.uk Received: 16 April 2018 Accepted: 9 My 2018 doi: /nph Key words: Arbidopsis thlin, Hyloperonospor rbidopsidis, oomycete pthogen, pthogen effector, plnt innte immunity, RADICAL-INDUCED CELL DEATH1, slicylic cid (SA). Summry The oomycete pthogen Hyloperonospor rbidopsidis (Hp) cuses downy mildew disese on Arbidopsis. To colonize its host, Hp trnsloctes effector proteins tht suppress plnt immunity into infected host cells. Here, we investigte the relevnce of the interction between one of these effectors, HRxL106, nd Arbidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pthogen infection ssys s well s moleculr nd biochemicl nlyses to test the hypothesis tht HRxL106 mnipultes RCD1 to ttenute trnscriptionl ctivtion of defense genes. We report tht HRxL106 suppresses trnscriptionl ctivtion of slicylic cid (SA)-induced defense genes nd lters plnt growth responses to light. HRxL106-medited suppression of immunity is bolished in RCD1 loss-of-function mutnts. We report tht RCD1-type proteins re phosphorylted, nd we identified Mut9-like kinses (MLKs), which function s phosphoregultory nodes t the level of photoreceptors, s RCD1-intercting proteins. An mlk1,3,4 triple mutnt exhibits stronger SA-induced defense mrker gene expression compred with wild-type plnts, suggesting tht MLKs lso ffect trnscriptionl regultion of SA signling. Bsed on the combined evidence, we hypothesize tht nucler RCD1/MLK complexes ct s signling nodes tht integrte informtion from environmentl cues nd pthogen sensors, nd tht the Arbidopsis downy mildew pthogen trgets RCD1 to prevent ctivtion of plnt immunity. Introduction Plnts rely on their innte immune system to distinguish beneficil microbes from hrmful pthogens or commensl bcteri. While plnt innte immunity fends off the mjority of ttempted infections, specilized pthogens cn subvert host defenses with effector proteins tht re trnslocted into host cells. Mny pthogen effectors interfere with cellulr processes tht re essentil for innte immunity, such s formtion of cell wll ppositions, secretion of ntimicrobil compounds, production of rective oxygen species (ROS), or trnscriptionl ctivtion of defense genes (DebRoy et l., 2004; Nomur et l., 2006; Bozkurt et l., 2011; Anderson et l., 2012; Gngdhrn et l., 2013; Asi et l., 2014). Bcteril pthogens hve evolved specilized secretion systems to deliver effectors into host cells (Deng et l., 2017). Likewise, the fungl rice blst pthogen Mgnporthe oryze employs specilized secretion pthwy to These uthors contributed eqully to this work. deliver host-cell-trgeted effectors into host-derived comprtment nmed the biotrophic interfcil complex (Khng et l., 2010; Girldo et l., 2013). How other filmentous plnt pthogens, such s oomycetes, trnslocte effectors into plnt cells remins poorly understood (Petre & Kmoun, 2014; Lo Presti & Khmnn, 2017). Like fungl pthogens, oomycetes elborte hustori, bulbous feeding structures tht induce the formtion of specilized extr-hustoril membrne, within the plsm membrne of infected host cells (Lo Presti & Khmnn, 2017). Notbly, hustori re lso sites of trgeted effector secretion (Whisson et l., 2007; Gilroy et l., 2011; Liu et l., 2014b; Wng et l., 2017). Most host-cell-trgeted oomycete effectors crry combintion of signl peptide nd conserved mino cid motif RXLR (where X represents ny mino cid). The RXLR motif is required for effector trnsloction into the host cell, nd there is evidence tht it functions s n internl sorting signl (Grouffud et l., 2008; Wwr et l., 2017). The effector protein domins downstrem of signl peptide nd RXLR motif 232 This is n open ccess rticle under the terms of the Cretive Commons Attribution License, which permits use, distribution nd reproduction in ny medium, provided the originl work is properly cited.

2 Phytologist Reserch 233 re diverse nd constitute the prt of the effector tht mnipultes cellulr processes in the host cell (Frnceschetti et l., 2017). Plnts respond to infection by biotrophic pthogens with elevted biosynthesis of the defense hormone slicylic cid (SA), nd elevted SA levels led to thioredoxin-ctlyzed reduction of disulfide-linked oligomeric complexes of the NONEXPRESSOR OF PATHOGENESIS-RELATED GENE 1 (NPR1) protein (Mou et l., 2003; Td et l., 2008). Monomeric NPR1 trnsloctes to the nucleus, where it functions s trnscriptionl co-ctivtor nd is indispensble for SA responsiveness of mny SA-induced genes (Wng et l., 2006). Some biotrophic plnt pthogen effectors ctively suppress SA ccumultion nd/or SA signling. The mize smut fungus Ustilgo mydis produces host-cell-trgeted chorismte dismutse tht my suppress SAmedited immunity by diverting the SA-precursor chorismte into the phenylpropnoid pthwy (Djmei et l., 2011). The oomycete pthogen Phytophthor soje nd the fungl pthogen Verticillium dhlie ttenute SA signl trnsduction by delivery of isochorimtses into host cells (Liu et l., 2014b). The hosttrgeted effector Pi04314 from the lte blight pthogen Phytophthor infestns trgets severl nucler-loclized phosphtses nd ttenutes the trnscriptionl response to SA nd methyl jsmonte (MeJA) (Boevink et l., 2016). The Arbidopsis downy mildew pthogen Hyloperonospor rbidopsidis (Hp) lso suppresses trnscriptionl upregultion of the SA mrker gene PATHOGENESIS-RELATED GENE 1 (PR1) in infected host cells (Cillud et l., 2013). At lest two Hp effector proteins interfere with SA signling when expressed s trnsgenes in Arbidopsis. Effector HRxL44 ppers to ttenute SA signl trnsduction by trgeting the MEDIATOR subunit Med19 for protesoml degrdtion (Cillud et l., 2013), while effector HRxL62 interferes with SA signling by n unknown mechnism (Asi et l., 2014). Light perception nd signling lso influence the trnscriptionl response to SA (Genoud et l., 2002; de Wit et l., 2013; Gngpp et l., 2016). Simulted shde conditions, for exmple, suppress trnscript chnges induced by exogenous ppliction of SA or MeJA, thereby ttenuting plnt defense towrd biotrophic nd necrotrophic pthogens (Izguirre et l., 2006; Cerrudo et l., 2012; de Wit et l., 2013). This is remrkble given tht SA- nd jsmonic cid (JA)-responsive gene networks re ntgonisticlly regulted in response to infection by pthogens with either biotrophic or necrotrophic mode of infection (Pieterse et l., 2012; Crls et l., 2015). Arbidopsis RADICAL-INDUCED CELL DEATH1 (RCD1) hs been proposed to ct s positive regultor of SA signling. Loss of RCD1 function does not lter SA levels, but trnscript levels of mny NPR1 trget genes re lower in rcd1 mutnts compred with wild-type plnts (Ahlfors et l., 2004; Brosche et l., 2014). RCD1 ws initilly identified in screen for ozonesensitive Arbidopsis mutnts. The mutnt is impired in restricting progrmmed cell deth under sublethl ozone concentrtions (Overmyer et l., 2000). In ddition, rcd1 mutnts show pleiotropic phenotypes tht include smller rosette size nd ltered lef shpe, s well s prtil loss of picl dominnce nd n ltered root system rchitecture (Ahlfors et l., 2004; Teoti & Lmb, 2009). Loss of RCD1 function enhnces sensitivity to poplstic ROS nd slt stress but increses tolernce to chloroplstic ROS, nd this correltes with ltered trnscription of genes tht re responsive to ROS, bscisic cid, JA, ethylene, nd SA (Ahlfors et l., 2004; Overmyer et l., 2005; Ktiyr-Agrwl et l., 2006; Brosche et l., 2014). RCD1 is the founding member of plnt-specific protein fmily chrcterized by centrl domin with sequence similrity to the ctlytic domin of Poly-(ADP-ribose)-polymerses (PARPs) (Lmb et l., 2012). In contrst to cnonicl PARPs tht covlently modify trget proteins by ADP-ribosyltion, Arbidopsis RCD1 does not show PARP ctivity in vitro when expressed s GST fusion (Jspers et l., 2010). However, n RCD1 homologue from whet shows PARP ctivity when expressed in Escherichi coli, suggesting tht some RCD1-type proteins my be enzymticlly ctive (Liu et l., 2014). In ddition to the centrl PARP domin, RCD1 nd its prlogue SIMILAR TO RCD ONE1 (SRO1) hve n N-terminl WWE domin nd C-terminl RST domin. RCD1 nd sequence-relted proteins loclize to the plnt cell nucleus nd bind severl trnscription fctors vi their RST domins, possibly explining why loss of RCD1 function ffects plnt development nd severl stress signling pthwys (Ktiyr-Agrwl et l., 2006; Jspers et l., 2009; You et l., 2014). Accordingly, RCD1 might influence SA signl trnsduction by intercting with trnscription fctors tht control expression of defense genes. RCD1 intercts with the Hp effector HRxL106 in yesttwo-hybrid (Y2H) ssy, nd this effector renders Arbidopsis more susceptible to biotrophic pthogens when expressed s trnsgene (Fbro et l., 2011; Mukhtr et l., 2011). In plnt cells, HRxL106 is ctively trnsported into the nucleus, indictive of nucler virulence-promoting ctivity of the effector (Wirthmueller et l., 2015). Here, we report tht HRxL106, when expressed s trnsgene, ffects both SA signling nd light-regulted developmentl processes. We identify RCD1 s likely virulence trget of HRxL106 nd report tht RCD1 intercts with Mut9-like kinses (MLKs) tht, in ddition to their previously chrcterized function in light signl trnsduction, lso influence the trnscriptionl response to SA. Mterils nd Methods Plnts nd growth conditions For hypocotyl growth ssys, Arbidopsis seeds were sown on Murshige Skoog medium (Duchef #M0255) contining 0.1 g l 1 myoinositol nd 8 g l 1 Bctogr, strtified for 48 h t 4 C in the drk. Germintion ws induced by 6 h white light stimulus. The pltes were plced in long-dy (12 h : 12 h, light : drk) conditions t 21 C nd fluence rte of 12 lmol m 2 s 1 white light. Hypocotyl length ws determined on dy 5 using IMAGEJ softwre. Growth conditions for Nicotin benthmin nd ll other Arbidopsis experiments were s in Segonzc et l. (2011) nd Fbro et l. (2011). The nd rcd1-3 mutnts hve been described (Plm et l., 2005; Go et l., 2009; Jspers et l., 2009; Yng et l., 2017). The mlk1,2,3

3 234 Reserch Phytologist nd mlk1,3,4 triple mutnts hve been described (Hung et l., 2016). Genertion of trnsgenic Arbidopsis lines Trnsgenic Arbidopsis plnts expressing yellow fluorescent protein (YFP)-tgged HRxL106 hve been described (Wirthmueller et l., 2015). To generte trnsgenic lines expressing 3xHA- StrepII (HS):HRxL106, RFP:HRxL106, RFP:NLS: HRxL106DC, nd RFP:HRxL106-Cterm58 (ll Hp Emoy2) we used the following previously described pentr plsmids: pentr4-hrxl106, M followed by HRxL106 mino cids I 25 S 285 (Fbro et l., 2011), pentr/d-topo-sv40nls: HRxL106DC, sequence APKKKRKV followed by HRxL106 mino cids I 25 G 229, nd pentr/d-topo-hrxl106- Cterm58, HRxL106 mino cids G 228 S 285 (Wirthmueller et l., 2015). Plsmids pxcsg-hs nd ph7wgr2 were recombined with the forementioned pentr plsmids using Gtewy LR clonse II to generte HS- nd red fluorescent protein (RFP)-tgged versions of the HRxL106 constructs respectively. Trnsgenic HS-tgged HRxL106 lines were generted by trnsforming or 35S Pro :NPR1:GFP plnts with Agrobcterium tumefciens GV3101::pMP90RK crrying pxcsg-hs: HRxL106 constructs using florl dip (Logemnn et l., 2006). RFP-tgged HRxL106 lines were generted by trnsforming with A. tumefciens GV3101::pMP90 crrying the corresponding ph7wgr2 plsmids. lines expressing RCD1 mino cids s C-terminl fusion to green fluorescent protein (GFP) were generted by recombining corresponding pentr clone with pk7wgf2 (Krimi et l., 2002); the construct ws trnsformed into plnts s erlier. Site-directed mutnts of RCD1 were generted using the QuikChnge method (Agilent, Snt Clr, CA, USA). pentr/d-topo plsmids crrying the mutted RCD1 vrints in fusion with 2499 bp of the RCD1 promoter sequence were recombined with pgwb13 (Nkgw et l., 2007) to generte trnsltionl fusion with triple HA-tg t the C-terminus of the protein. The constructs were trnsformed into the mutnt. Hp infection nd quntifiction Mutnts nd trnsgenic lines were tested for ltered susceptibility to Hp Noco2 either in dult leves of 6-wk-old plnts (Fig. 1) or in cotyledons of 10-d-old seedlings grown on soil. For both types of experiments, plnts were spryed with suspension of spores ml 1. The plnts were plced in high (> 90%) humidity under plstic dome. Sporultion on seedlings ws scored t 5 d post infection, nd sporultion on dult plnts ws quntified 7 8 d post infection. For the dult lef ssy, 20 leves per genotype were stined with trypn blue. Following destining with chlorl hydrte solution, conidiophores on 20 lef res of 1cm 2 were counted using light microscope. For the seedling ssy, seedlings per genotype were incubted in 0.02% (w/v) Uvitex 2B (Polysciences, Hirschberg n der Bergstrsse, Germny) solution, then destined in wter for 2 min, mounted on Styrofom rck, nd imged through Leic UV filter (Leic # ) using Leic M165 FC fluorescent stereomicroscope connected to n EL6000 lser source. Only conidiophores on the upper side of the cotyledons were counted. SA tretment For SA tretment, 4-wk-old Arbidopsis plnts were spryed with solution contining 0.1 mm SA nd 0.01% Silwet L-77 1 h fter dwn (09:00 h). Rosette leves were hrvested 8 h lter. Results HRxL106-expressing Arbidopsis plnts exhibit ttenuted light nd defense signling To chrcterize HRxL106-intercting proteins from Arbidopsis we generted trnsgenic lines expressing HRxL106 with n N-terminl YFP or 3xHA-StrepII (HS) epitope tg under control of the 35S promoter. As previously reported for trnsgenic plnts expressing untgged HRxL106 (Fbro et l., 2011), these lines re hyper-susceptible to infection by the comptible Hp isolte Noco2 (Fig. 1; Supporting Informtion Tble S1). Notbly, lines expressing HRxL106 show phenotype reminiscent of plnts grown under shde; specificlly, longer hypocotyls nd elongted petioles (Fig. 1b). Differences in hypocotyl length between wild-type plnts nd trnsgenic lines were more pronounced when we grew seedlings under lower fluence rte of white light (12 lmol m 2 s 1 ) (Fig. 1c,d; Tble S2). Under these conditions, HRxL106-expressing seedlings were indistinguishble from the phyb-9 mutnt tht shows constitutive shde voidnce (Reed et l., 1993). Lines expressing control constructs YFP nd HS did not differ from wild-type plnts in hypocotyl length (Fig. 1c,d). By contrst, differences in hypocotyl length between HRxL106-expressing trnsgenic lines nd wild-type plnts were much smller when we grew seedlings in drkness (Fig. 1d; Tble S2). This suggests tht, in ddition to suppression of plnt immunity, HRxL106 lso ffects signl trnsduction between photoreceptors nd light-regulted elongtion growth. Effector HRxL106 suppresses SA signl trnsduction but not SA levels As phyb mutnts show n ttenuted trnscriptionl response to SA (Genoud et l., 2002; de Wit et l., 2013), nd given tht suppression of SA signl trnsduction would be conceivble virulence mechnism for n effector from biotrophic pthogen, we tested SA-induced upregultion of the defense mrker gene PR1 in plnts nd two trnsgenic lines, one expressing YFP: HRxL106 nd the other HS:HRxL106. SA induced PR1 mrna levels in plnts but not in the npr1-1 mutnt (Co et l., 1994) (Fig. 2; Tble S3). By contrst, PR1 expression levels in SA-treted HRxL106 trnsgenic lines were comprble to those in mock-treted plnts, suggesting tht HRxL106 ffects either endogenous SA levels or SA signl trnsduction

4 Phytologist Reserch 235 () Hp Noco2 (b) (c) Sporngiophores per lef YFP #4 YFP #6 YFP:HRxL106 #13 HS #1 #3 HS:HRxL106 #1 YFP:HRxL106 YFP #6 HS #3 phyb-9 Hypocotyl length (cm) (d) White light 12 μmol m - 2 s 1 Drkness Hypocotyl length (cm) YFP #6 HS #6 phyb-9 YFP #6 HS #6 phyb-9 Fig. 1 () Sporultion of the virulent Hyloperonospor rbidopsidis isolte Noco2 on 6-wk-old Arbidopsis thlin plnts of the indicted genotypes quntified by the number of conidiophores per lef. The results shown re representtive of two independent biologicl experiments, n = 20, error brs show plus/minus SEM, nd sterisks indicte differences from (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S1 for source dt nd sttistics. (b) Constitutive expression of HRxL106 induces phenotypes tht re reminiscent of the shde voidnce syndrome in Arbidopsis thlin. The top pnel shows 10-d-old seedlings of nd representtive 35S Pro :YFP:HRxL106 line. Bottom pnel shows 4-wk-old plnts from both genotypes grown under short dy condition nd fluence rte of c. 120 lmol m 2 s 1. (c) Five-dy-old seedlings of the indicted genotypes germinted under lower fluence rte of c.12lmol m 2 s 1. (d) Quntifiction of seedling hypocotyl length of the indicted genotypes grown s in (c) or in drkness. The results shown re representtive of three independent biologicl experiments, n = 30, horizontl brs denote medin, nd sterisks indicte men vlues different from (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Tble S2 for source dt nd sttistics. (Fig. 2). Quntifiction of unconjugted SA levels in leves 24 h fter infiltrtion of Pseudomons syringe pv. tomto (Pst) DC3000 reveled tht SA concentrtions in HRxL106- expressing lines were comprble to (one-wy ANOVA; P = 0.648) (Fig. 2b; Tble S4). These results suggest tht HRxL106 does not substntilly lter SA levels but nevertheless strongly ttenutes SA-induced trnscriptionl regultion of the SA mrker gene PR1. Effector HRxL106 ttenutes NPR1-dependent defense ctivtion HRxL106 is ctively trnsported into nuclei of plnt cells (Wirthmueller et l., 2015). Given tht NPR1 is n importnt nucler signl integrtor of the SA pthwy, we tested whether HRxL106 ffects NPR1 locliztion or protein levels. When plnts expressing 35S Pro :NPR1:GFP in n npr1-1 mutnt bckground (Kinkem et l., 2000) re grown under short dy conditions the plnts show signs of constitutive defense ctivtion, including severe stunting, development of micro lesions, nd elevted PR1 expression (Fig. 3,b; Tble S5; Love et l., 2012). We trnsformed the 35S Pro :NPR1:GFP line with the 35S Pro :HS:HRxL106 construct nd found tht expression of HRxL106 completely suppressed the stunting of the 35S Pro : NPR1:GFP line in 12 out of 14 independent trnsgenic lines (Fig. 3). HRxL106 lso reverted the constitutive PR1 expression of the 35S Pro :NPR1:GFP line (Fig. 3b). This suppression ws not due to lower NPR1:GFP protein levels, s shown by the western blot in Fig. 3c. Consistent with constitutively ctivted defense, we observed nucler locliztion of NPR1:GFP in gurd cells of plnts grown under short dy condition even without exogenous SA ppliction (Fig. 3d). NPR1:GFP lso loclized to nuclei in double trnsgenic lines co-expressing HS:HRxL106 (Fig. 3d). Tken together, these results show tht HRxL106 does not ttenute SA signl trnsduction by ltering protein levels or locliztion of NPR1. As HRxL106 suppresses

5 236 Reserch Phytologist () SA-induced PR1 expression (b) SA levels Reltive PR1 trnscript SA (μg g 1 FW) Non-treted Mock P. syringe M SA M SA M SA M SA npr1-1 sid2-1 sid2-1 sid2-1 Fig. 2 () Hyloperonospor rbidopsidis effector HRxL106 suppresses slicylic cid (SA)-induced PATHOGENESIS-RELATED GENE 1 (PR1) expression in Arbidopsis thlin. Four-week-old plnts of the indicted genotypes were spryed with 0.1 mm SA or mock (M) solution nd PR1 expression levels were nlyzed by quntittive rel-time PCR 8 h lter. PR1 expression levels were normlized by ELONGATION FACTOR 1 ALPHA (EF1) expression. The plot shows the men of PR1/EF1 expression from three independent biologicl experiments. Error brs show plus/minus SEM; sterisk indictes men vlue different from mock tretment (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S3 for source dt nd sttistics. (b) Quntifiction of free SA levels in the indicted genotypes under nontreted conditions nd 24 h fter infiltrtion with 10 8 CFU ml 1 of Pseudomons syringe pv. tomto DC3000 or 10 mm mgnesium chloride mock solution. Red, green, nd blue represent dt from three independent biologicl experiments. Dots of the sme color represent technicl replictes. See Tble S4 for source dt nd sttistics. () NPR1:GFP NPR1:GFP (b) Reltive PR1 trnscript Bsl PR1 expression (c) #1 #3 HS:HRxL106 in NPR1:GFP NPR1:GFP #1 #3 HS:HRxL106 NPR1:GFP (d) 0 NPR1:GFP NPR1:GFP HS:HRxL106 NPR1:GFP #1 HS:HRxL106 NPR1:GFP #1 HS:HRxL106 NPR1:GFP #3 α-gfp CBB Fig. 3 Hyloperonospor rbidopsidis effector HRxL106 suppresses constitutive defense signling induced by NPR1:GFP overexpression in Arbidopsis thlin under short dy conditions. () Morphology of 5-wk-old nd 35S Pro :NPR1:GFP plnts (top row) nd two independent double trnsgenic 35S Pro : NPR1:GFP lines co-expressing 35S Pro :HS:HRxL106 (bottom row). The inset shows spontneous lesions forming in 35S Pro :NPR1:GFP plnts. (b) Bsl PATHOGENESIS-RELATED GENE 1 (PR1) expression in the lines shown in () s determined by quntittive rel-time PCR. PR1 expression levels were normlized by ELONGATION FACTOR 1 ALPHA (EF1) expression. The plot shows the men of PR1/EF1 expression from three independent biologicl experiments. Error brs show SEM, sterisk indictes men vlue different from (one-wy ANOVA; Tukey-Krmer post-hoc test, P < 0.05). See Tble S5 for source dt nd sttistics. (c) Western blot showing ccumultion of NPR1:GFP protein in the lines shown in (). The Western blot is representtive of three independent biologicl experiments. (d) Representtive (n > 10) confocl microscopy imges showing nucler ccumultion of NPR1:GFP protein in short dy conditions in 35S Pro :NPR1:GFP plnts nd plnts co-expressing 35S Pro :HS:HRxL106. The signl in is uto-fluorescence from stomt.

6 Phytologist Reserch 237 constitutive PR1 gene expression induced by the 35S Pro :NPR1: GFP trnsgene, the effector must either ct downstrem of nucler NPR1 signling or disrupt the nucler trnsctivtor function of NPR1 itself. HRxL106-overexpressing lines show prtil trnscription profile overlp with the rdicl-induced cell deth1-1 mutnt Similr to HRxL106-expressing lines, rcd1 mutnts show lower expression levels of PR1, s well s other SA mrker genes (Brosche et l., 2014). As the two proteins interct in Y2H, RCD1 could be virulence trget of HRxL106. We performed trnscriptome profiling experiment to chrcterize defense gene expression of representtive HS:HRxL106-expressing line nd the mutnt in more detil (Methods S1). We profiled complementry DNA from nontreted plnts s well s complementry DNA prepred from leves tht were infiltrted with Pst DC3000 or mgnesium chloride mock control 24 h erlier. As shown in Fig. S1, the mutnt showed prtil trnscriptionl overlp with the HRxL106-expressing line, prticulrly for repressed genes under nontreted conditions. A functionl clssifiction of this gene set using Gene Ontology terms reveled n overrepresenttion of SA-responsive defense genes (Tble S6). Fig. 4 shows the expression levels of 22 SA/NPR1-regulted genes (Wng et l., 2006) nd the JA mrker genes PDF1.2 nd VSP2 in the HRxL106-expressing line nd. In nontreted nd mock-treted plnts, SA mrker genes were repressed compred to. Expression levels of the two JA mrker genes were either repressed or not different from wild-type, nd this pttern is reminiscent of shde-grown plnts (de Wit et l., 2013). At 24 h fter infection with Pst DC3000, expression levels of SA mrker genes in were similr to, while 16 out of 22 SA mrker genes remined repressed in the HRxL106- expressing line (Fig. 4; Tble S6). Therefore, the enhnced susceptibility medited by ectopic expression of HRxL106 is likely due to its repressive effect on trnscription of SA-responsive defense genes. Loss of RCD1 function results in comprbly low expression level of SA mrker genes before pthogen chllenge. However, defense genes in re still trnscriptionlly induced upon bcteril infection, resulting in defense trnscriptome tht is more similr to wild-type plnts 24 h fter infection (Fig. 4; Tble S6). These results suggest tht, if RCD1 is virulence trget of HRxL106, the mnipultive effect of HRxL106 is not mimicked by complete loss of RCD1 function. HRxL106 intercts with RCD1 nd SRO1 proteins nd RCD1 quntittively contributes to SA signl trnsduction To test for interction between HRxL106 nd RCD1 in Arbidopsis, we trnsformed trnsgenic line in which the rcd1-3 muttion is complemented by expression of n RCD1 Pro :RCD1: HA construct (Jspers et l., 2009), with YFP:HRxL106 nd selected double trnsgenic lines. When we immunoprecipitted YFP:HRxL106 from these plnts, RCD1:HA co-purified with HRxL106, whilst cross-recting bnd detected by the -HA ARD3 At5g03350 NIMIN1 WRKY54 WRKY38 XTH10 PR1 LHT7 At3g28510 GRXS13 UGT76B1 PCC1 At3g29240 WRKY18 WRKY70 CPFTSY At5g45000 MLO2 At5g24210 At1g02360 LURP1 At2g27660 PDF1.2 VSP2 NT -6 mock -4 Pst ntibody did not (Fig. 5). Therefore, YFP:HRxL106 intercts with functionl epitope-tgged RCD1:HA protein in Arbidopsis. RCD1 nd its prlogue SRO1 show unequl genetic redundncy with respect to plnt development nd responses to biotic stress with RCD1 mking stronger contribution (Jspers et l., 2009; Teoti & Lmb, 2009). Trnsiently expressed, GFP-tgged versions of RCD1 nd SRO1 co-immunoprecipitted HS: HRxL106, wheres YFP control did not (Fig. 5b). This suggests tht HRxL106 intercts with both RCD1 nd SRO1 in plnt cells. The prtil redundncy between RCD1 nd SRO1 prompted us to test whether SRO1 lso contributes to trnscriptionl regultion of NPR1 trget genes. Overll, the nd sro1-1 muttions hd weker effect on SA-induced PR1 expression thn the YFP:HRxL106 trnsgene (Fig. 5c). PR1 levels in but not in sro1-1 showed significnt reduction compred with those in (one-wy ANOVA, Tukey Krmer post hoc test; P < 0.05) (Fig. 5c; Tble S7). Therefore, RCD1-2 NT log2 fold chnge mock Pst Fig. 4 Expression levels of slicylic cid (SA)-responsive defense genes nd the jsmonic cid (JA) mrker genes PDF1.2 nd VSP2 in Arbidopsis thlin HS:HRxL106 line #2 nd the mutnt. NT, nontreted plnts; mock, 24 h fter infiltrtion of 10 mm mgnesium chloride; Pst, 24 h fter infiltrtion with CFU ml 1 Pseudomons syringe pv. tomto DC3000. Blue nd yellow colors denote the level of down- nd upregultion respectively in comprison with expression levels in. See Supporting Informtion Tble S6 for source dt nd flse discovery rtes.

7 238 Reserch Phytologist RCD1:HA RCD1:HA YFP:HRxL106 #1 #3 Soluble protein RCD1:HA RCD1:HA YFP:HRxL106 () (b) (c) #1 #3 IP α-gfp α-gfp α-ha (RCD1) α-ha (ns) CBB YFP YFP RCD1:GFP SRO1:GFP + HS:HRxL106 Soluble protein IP α-gfp RCD1:GFP SRO1:GFP < RCD1 / SRO1 α-gfp < free YFP/GFP α-ha CBB Reltive PR1 trnscript b SA-induced PR1 expression M SA M SA M SA M SA sro1-1 b M npr1-1 SA Fig. 5 Hyloperonospor rbidopsidis effector HRxL106 intercts with Arbidopsis thlin RADICAL-INDUCED CELL DEATH1 (RCD1) nd SIMILAR TO RCD ONE1 (SRO1) in plnt cells nd RCD1 contributes to slicylic cid (SA)-induced PATHOGENESIS-RELATED GENE 1 (PR1) expression. () Functionl RCD1:HA protein co-immunoprecipittes with YFP:HRxL106 in Arbidopsis. YFP:HRxL106 ws immunoprecipitted from double trnsgenic lines expressing RCD1:HA, proteins were resolved by sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS-PAGE), trnsferred onto polyvinylidene fluoride (PVDF) membrne nd probed with the indicted ntibodies. ns, nonspecific bnd detected by the -HA ntibody. CBB, Coomssie brillint blue stin. This result is representtive of three independent biologicl experiments. (b) HS:HRxL106 co-immunoprecipittes with GFP-tgged vrints of RCD1 nd SRO1 following trnsient expression in Nicotin benthmin. GFP-tgged proteins, or YFP s control, were immunoprecipitted, proteins were resolved by SDS-PAGE, trnsferred onto PVDF membrne, nd probed with the indicted ntibodies. Co-immunoprecipittion of HRxL106 with RCD1 nd SRO1 is bsed on three nd two independent biologicl experiments respectively. (c) SA-induced PR1 gene expression in, YFP: HRxL106 line #12,, sro1-1, nd npr1-1 mutnts. Four-week-old plnts were spryed with 0.1 mm SA or mock (M) solution nd PR1 expression levels were nlyzed by quntittive rel-time PCR 8 h lter. PR1 expression levels were normlized by ELONGATION FACTOR 1 ALPHA (EF1) expression. The plot shows the men of PR1/EF1 expression from five independent biologicl experiments. Error brs show plus/minus SEM; letters indicte differences between genotypes/tretments (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S7 for source dt nd sttistics. quntittively contributes to SA-induced PR1 expression. Owing to the requirement of either RCD1 or SRO1 for norml embryogenesis (Teoti & Lmb, 2009), we were not ble to test n rcd1 sro1 double mutnt for SA-induced PR1 expression. The C-terminl 58 mino cids of HRxL106 re required for RCD1 binding nd ttenution of light nd defense signling To test whether HRxL106 binding to RCD1 correltes with its defense-suppressing ctivities, we generted mutnt vrint of HRxL106 tht does not bind to RCD1. We used the Y2H system to compre RCD1 binding to full-length HRxL106, n HRxL106 vrint lcking the 56 C-terminl mino cids (HRxL106DC), with RCD1 binding to the C-terminl 58 mino cids lone (HRxL106-Cterm58). In contrst to Mukhtr et l. (2011), we did not detect interction between the two full-length proteins by Y2H under our conditions. However, we found tht the HRxL106 C-terminus intercts with RCD1 (Fig. 6). Next, we tested which domins of RCD1 re required for this interction. As shown in Fig. 6(), the HRxL106 C-terminus intercted with frgment spnning the WWE nd PARP domins but did not bind to the isolted WWE, PARP, or RST domins. Notbly, the RCD1 WWE PARP construct lso showed interction with full-length HRxL106 protein (Fig. 6). This suggests tht HRxL106 specificlly binds to the RCD1 WWE PARP domins vi its C-terminl region. We next tested whether the HRxL106 C-terminl 58 mino cids re necessry for ltered light nd SA signling in Arbidopsis. We trnsformed n RFP:NLS:HRxL106DC construct lcking the C-terminus of the effector into. Becuse the HRxL106DC construct lso lcks the effector s NLS, this fusion protein crries n SV40 NLS to ensure efficient nucler import (Wirthmueller et l., 2015). As controls, we generted trnsgenic RFP:HRxL106 lines nd lines expressing RFP fused to the 58 C-terminl mino cids of HRxL106 (RFP: HRxL106-Cterm58). All constructs were under control of the 35S promoter, nd we confirmed expression of the RFP fusion proteins by western blot (Fig. 6b). In comprison with, trnsgenic lines expressing RFP-tgged HRxL106 developed longer petioles nd reduced lef re (Fig. 6c). By contrst, RFP:NLS:HRxL106DC lines were indistinguishble from wildtype plnts. RFP-HRxL106-Cterm58 lines resembled trnsgenics expressing full-length HRxL106, suggesting tht the C-terminus of the effector is required nd sufficient for ttenution of light signling (Fig. 6c). We then tested resistnce to Hp Noco2 in these lines. In contrst to RFP:HRxL106, the truncted RFP:NLS:HRxL106DC protein filed to suppress defense (Fig. 6d; Tble S8). Trnsgenic lines expressing RFP:HRxL106- Cterm58 were more susceptible to Hp Noco2 thn were, but less so thn lines expressing the full-length effector. Therefore, the C-terminl 58 mino cids of HRxL106 re required to ttenute defense signling, nd the sme prt of the effector protein lters plnt growth responses to light. Similr to RFP:

8 Phytologist Reserch 239 () pdest32 HRxL106 HRxL106-Cterm58 HRxL106ΔC pdest22 RCD1 WWE WWE-PARP PARP PARP-RST WWE PARP RST SD -WLH (b) kd RFP RFP:HRxL106 #2 RFP:HRxL106 #6 RFP:NLS:HRxL106ΔC #2 RFP:NLS:HRxL106ΔC #24 RFP:HRxL106-Cterm58 #2 RFP:HRxL106-Cterm58 #14 α-rfp (long exp.) α-rfp (short exp.) (c) RFP:HRxL106 #2 #6 #2 #24 #2 #14 CBB RFP:NLS:HRxL106ΔC RFP:HRxL106-Cterm58 (d) Hp Noco2 Hp Noco2 (e) SA-induced PR1 expression Sporngiophores per cotyledon pir Sporngiophores/cotyledon pir Reltive PR1 trnscript M SA M SA M SA M SA M SA M SA M SA M SA #2 #6 #2 #24 #2 #14 RFP:HRxL106 RFP:NLS: HRxL106ΔC RFP: HRxL106- Cterm58 RFP:HRxL106 #2 RFP:HRxL106 #6 RFP:NLS: HRxL106ΔC #2 RFP:NLS: HRxL106ΔC #24 RFP:HRxL106- Cterm58 #2 RFP:HRxL106- Cterm58 #14 npr1-1 Fig. 6 The C-terminl 58 mino cids of Hyloperonospor rbidopsidis (Hp) effector HRxL106 re required for binding to Arbidopsis thlin RADICAL-INDUCED CELL DEATH1 (RCD1) nd ffect light-regulted plnt growth nd defense signling. () Protein protein interctions between RCD1, HRxL106, nd the indicted deletion constructs in yest-two-hybrid ssy. pdest32 nd pdest22 re empty bit nd prey vectors respectively. HRxL106DC is deletion construct lcking HRxL106 s 56 C-terminl mino cids. HRxL106-Cterm58 is n N-terminl deletion construct consisting only of the 58 C-terminl mino cids. (b) Western blot showing protein levels of RFP-tgged HRxL106, NLS:HRxL106DC, nd HRxL106-Cterm58 in selected stble trnsgenic Arbidopsis lines. Asterisks indicte the expected migrtion bsed on the moleculr weight of the fusion proteins. (c) Visul phenotype of trnsgenic Arbidopsis lines expressing RFP-tgged HRxL106, HRxL106DC, or HRxL106-Cterm58. (d) Resistnce to the virulent Hp isolte Noco2 in 10-d-old seedlings of nd trnsgenics expressing RFP-tgged HRxL106, HRxL106DC, or HRxL106-Cterm58. The plots show the number of conidiophores per cotyledon pir. Dt from three independent biologicl experiments were pooled, horizontl brs show medin, verticl box height represents interqurtile rnge (IQR), nd whisker rnge is 1.59 IQR. Circles represent dt points beyond 1.59 IQR. Asterisks indicte men vlues different from (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S8 for source dt nd sttistics. (e) Slicylic cid (SA)-induced PATHOGENESIS-RELATED GENE 1 (PR1) gene expression in, nd trnsgenics expressing RFP-tgged HRxL106, NLS: HRxL106DC or HRxL106-Cterm58. Four-wk-old plnts were spryed with 0.1 mm SA or mock (M) solution nd PR1 expression levels were nlyzed by qrt-pcr 8 h lter. PR1 expression levels were normlized by ELONGATION FACTOR 1 ALPHA (EF1) expression. The plot shows the men of PR1/EF1 expression from three independent biologicl experiments. Error brs show plus/minus SEM; sterisks indicte men vlues different from mock tretment (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S9 for source dt nd sttistics. HRxL106 lines, trnsgenics expressing the HRxL106 C- terminus responded with lower PR1 trnscript levels thn wildtype plnts to SA sprying, wheres RFP:NLS:HRxL106DC lines responded like wild-type (Fig. 6e; Tble S9). RCD1 is dispensble for resistnce to Hp but required for HRxL106-medited suppression of defense To test whether RCD1 contributes to resistnce to Hp, we infected the mutnt with Hp Noco2. The mutnt showed enhnced resistnce compred with (Fig. 7; Tble S10), which is consistent with previous lrge-scle Hp phenotyping report (Weßling et l., 2014). This suggests tht the lower level of defense gene expression before pthogen chllenge in does not compromise resistnce ginst Hp. Therefore, if RCD1 is virulence trget of HRxL106, inhibition of RCD1 s function(s) or signling is unlikely to be responsible for the enhnced susceptibility induced by HRxL106. We considered tht HRxL106 my mnipulte RCD1 in wy tht is not mimicked by complete loss of RCD1 function; for exmple, by

9 240 Reserch Phytologist converting RCD1 into trnscriptionl co-repressor. We compred susceptibility to Hp Noco2 in the YFP:HRxL106 line nd trnsgenic line expressing the sme construct in n mutnt bckground. Although the YFP:HRxL106 fusion protein ccumulted to similr levels in both trnsgenic lines (Fig. 7b), the muttion completely suppressed the enhnced susceptibility induced by YFP:HRxL106. Reintroduction of functionl RCD1 by crossing restored HRxL106 function (Fig. 7; Tble S10). Therefore, functionl RCD1 protein is essentil for suppression of defense by HRxL106. Loss of RCD1 function lso ttenuted the extent of petiole elongtion in the YFP:HRxL106 bckground (Fig. 7c). A crystl structure of RCD1 s PARP domin suggests tht RCD1-type proteins do not function s cnonicl ADPribosyl trnsferses Our finding tht suppression of defense by HRxL106 is lrgely dependent on RCD1 nd tht the effector binds to RCD1 s WWE PARP domins prompted us to further investigte the moleculr function(s) of RCD1. We resoned tht if RCD1 hd PARP or relted trnsferse ctivity, HRxL106 might mnipulte this enzymtic function. We solved crystl structure of the RCD1 PARP domin by X-ry crystllogrphy (PDB ID 5NGO; Tble S11; Methods S1). The RCD1 PARP domin dopts fold tht is overll similr to mmmlin PARP domins (Fig. 8). However, the RCD1 PARP domin structure confirmed tht the mino cid trid H-Y-E, constituting the ctive site of mmmlin nd plnt PARPs, is not conserved in RCD1 (Kleine et l., 2008; Jspers et l., 2010) (Fig. 8b). Nonconservtion of the His nd Tyr residues criticl for NAD + binding in cnonicl ADP-ribosyl-trnsferses suggests tht the RCD1 PARP domin does not bind NAD + nd therefore is likely to lck cnonicl PARP ctivity. Consistent with this inference, the RCD1 PARP domin is not stbilized by 6(5H)- phennthridinone, n inhibitor of mmmlin PARPs (Whlberg et l., 2012), t elevted tempertures (Fig. 8c; Tble S12). Conceivbly, the cleft of RCD1 tht corresponds to the ctlytic center of ctive PARPs hs evolved to bind other smll compounds. To test whether this region of the protein is required for the biologicl function of RCD1, we designed three RCD1 mutnt vrints with single mino cid exchnges in the cleft region. When expressed under trnscriptionl control of 2.5 kb of the ntive RCD1 promoter, ll constructs complemented the developmentl phenotype (Fig. 8d) nd the enhnced prqut tolernce of the mutnt (Fig. 8e; Tble S13; Ahlfors et l., 2004). One trnsgenic line expressing RCD1 D421A did not complement the oxidtive stress phenotype of (Fig. 8e) nd only prtilly complemented the developmentl phenotype of (compre lines B nd B in Fig. 8d). However, n independent trnsgenic line (A) expressing the sme construct fully complemented both phenotypes, suggesting tht differences in expression levels might ccount for prtil complementtion in line B. Overll, our results suggest tht the integrity of the Sporngiophores per cotyledon pir () (b) (c) Hp Noco2 b c b d cd kd YFP:HRxL106 #5 α-gfp CBB sro1-1 YFP:HRxL106 #3 YFP:HRxL106 #5 YFP:HRxL106 #5 #55 #56 YFP:HRxL106 RCD1 (BC) Fig. 7 Hyloperonospor rbidopsidis (Hp) HRxL106-medited suppression of immunity in Arbidopsis thlin is bolished in RADICAL-INDUCED CELL DEATH1 (RCD1) loss-of-function mutnts. () Resistnce to the virulent Hp isolte Noco2 in 10-d-old seedlings of,, the YFP: HRxL106 line #12, trnsgenic lines expressing YFP:HRxL106 to comprble levels in the bckground (#5), nd two descendnt lines of #5 in which RCD1 hs been reintroduced by bckcrossing to (#55 nd #56). The plots show the number of conidiophores per cotyledon pir. Dt from five independent biologicl experiments were pooled, horizontl brs show medin, verticl box height represents interqurtile rnge (IQR), nd whisker rnge is 1.59 IQR. Circles represent dt points beyond 1.59 IQR. Letters indicte differences between men vlues (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Supporting Informtion Tble S10 for source dt nd sttistics. (b) Western blot showing protein levels of YFP:HRxL106 in line #12 () nd line #5 (). CBB, Coomssie brillint blue stin. (c) Visul phenotype of 4-wk-old plnts of, sro1-1,, nd lines expressing YFP:HRxL106 in either or bckgrounds. The YFP:HRxL106 fusion protein ws not detectble by western blot in line YFP: HRxL106 #3.

10 Phytologist Reserch 241 presumed NAD + binding cleft is not essentil for RCD1 s functions in plnt development nd signl trnsduction under oxidtive stress conditions. HRxL106 binds to the N-terminl domins of RCD1 nd SRO1 tht medite homo- nd heterodimeriztion Using the Y2H system, we further nrrowed down the HRxL106 binding site of RCD1 to n N-terminl frgment encompssing the WWE domin nd the linker region up to the beginning of the PARP domin (Fig. 9). Deletion of the linker region resulted in loss of interction with HRxL106, suggesting tht the WWE domin on its own is not sufficient for effector binding. The isolted PARP domin did not interct with HRxL106, irrespective of whether or not we included the linker region (Fig. 9). This suggests tht the WWE-linker region is required nd sufficient for binding to HRxL106. We found tht the RCD1 WWE-linker region intercts with itself nd the corresponding region of SRO1 in Y2H ssys, indictive of the formtion of homo- nd hetero-oligomers (Fig. 9b). We obtined comprble results for the corresponding prt of the SRO1 protein (Fig. 9c). These dt suggest tht the RCD1 nd SRO1 WWE-linker regions could medite formtion of RCD1/SRO1 oligomers. RCD1 s WWE domin forms protein complexes with MLKs Given tht RCD1 does not hve PARP ctivity, we further chrcterized RCD1 protein function(s) by screening for in plnt interctors of RCD1. Attempts to immuno-purify epitope-tgged RCD1 protein in mounts sufficient for LC MS/MS nlysis of co-purifying proteins from trnsient expression ssys in N. benthmin or stble Arbidopsis trnsgenics were not successful. We therefore resorted to screening for interctors of RCD1 s WWE-linker region following trnsient expression in N. benthmin, s this prt of the protein binds to HRxL106 nd is more stble (Fig. 9d). The predominnt interctors were severl importin- isoforms, full-length RCD1-type proteins nd protein kinses with sequence homology to csein I kinses (Fig. 9e; Tble S14). Identifiction of peptides from the PARP nd RST domins in pulldown experiments of the WWE-linker domin suggest tht this domin forms homo- nd heterooligomers with endogenous RCD1-type proteins in N. benthmin (Fig. 9; Fig S2). This is consistent with oligomer formtion of the WWE-linker regions in Y2H. A BLASTP serch of the co-purifying csein-i-relted kinses ginst the Arbidopsis protein dtbse (TAIR11) identified MLKs s likely orthologues (Fig. 9e). MLKs, lso described s PHOTOREGULATORY PROTEIN KINASES, re nucler-loclized Ser/Thr kinses tht phosphorylte the photoreceptor cryptochrome 2 (CRY2) nd PHYTOCHROME INTERACTING FACTOR 3 (PIF3) (Liu et l., 2017; Ni et l., 2017). In Arbidopsis nd Chlmydomons, phosphoryltion of histone H3 Thr3 (H3T3ph) is nother wellchrcterized MLK phosphoryltion site (Css-Mollno et l., 2008; Wng et l., 2015). We identified severl phosphopeptides from the RCD1 WWE-linker region (Fig. S3; Tble S15). While most of these phosphorylted peptides were locted in the GFP: WWE-linker bit protein from Arbidopsis, we lso detected two phosphopeptides from the WWE PARP linker region of copurifying N. benthmin RCD1 orthologue (Figs S3, S4; Tble S15), indicting tht RCD1-type proteins re phosphoproteins. We confirmed interctors identified in N. benthmin nd phosphoryltion of the RCD1 WWE-linker region in single experiment using stble trnsgenic Arbidopsis line expressing 35S Pro :GFP:WWE-linker protein (Tble S16; Figs S2, S3). Overll, our results show tht MLKs interct with the RCD1Nterminl domin in plnt cells, suggesting possible role of RCD1 nd sequence-relted proteins in influencing covlent modifictions of light-regultory components nd/or histone tils. MLKs hve been previously reported to ffect H3T3ph levels in response to osmotic nd slt stress, nd, like rcd1 mutnts, the mlk1,2 double mutnt is hypersensitive to sublethl concentrtions of sodium chloride (Ktiyr-Agrwl et l., 2006; Wng et l., 2015). As MLKs nd RCD1 form protein complexes in plnt cells, nd given tht SA mrker genes re expressed t lower levels in rcd1 mutnts, we sked whether MLKs lso ffect the trnscriptionl response to SA. We spryed mlk1,2,3 nd mlk1,3,4 triple mutnts with SA nd determined PR1 trnscript levels 8 h lter (Fig. 9f; Tble S17). While PR1 levels in the mlk1,2,3 triple mutnt did not differ from wild-type, the mlk1,3,4 triple mutnt consistently showed elevted PR1 trnscript levels in response to SA. However, under our growth conditions, the mlk1,3,4 triple mutnt did not show enhnced disese resistnce upon infection with the dpted Pst DC3000 strin (Fig. S5; Tble S18). Discussion Severl biotrophic pthogens evolved virulence mechnisms to counterct ctivtion of SA-dependent defense genes (Asi et l., 2014; Lewis et l., 2015). Aprt from enzymtic conversion of SA precursors (Djmei et l., 2011; Liu et l., 2014b), effectormedited ctivtion of JA signling ppers to be the min strtegy of biotrophic pthogens to ttenute SA-dependent defense (Zheng et l., 2012; Cillud et l., 2013; Yng et l., 2017). Here, we show tht ectopic expression of Hp effector HRxL106 suppresses both the expression of SA mrker genes nd the JA/ ethylene mrker gene PDF1.2 in noninfected plnts (Fig. 4; Tble S6). This suggests tht HRxL106 mnipultes SA signling vi mechnism tht does not rely on ctivtion of JA signling. The growth phenotype of HRxL106-expressing trnsgenic plnts is consistent with constitutive shde voidnce, nd conceivbly HRxL106 could suppress plnt immunity by mnipulting light signl trnsduction. However, we note tht n effect on temperture sensing, brssinosteroids (BRs), or uxin levels could lso underlie this phenotype. Severl genes tht re overexpressed in the HRxL106 trnsgenic line re lso ltered in expression in constitutive BR signling mutnts (Tble S6). However, unlike constitutive BR-signling mutnts or plnts with elevted BR levels tht develop longer hypocotyls thn wildtype plnts do in drkness (Choe et l., 2001; Jillis et l., 2011;

11 242 Reserch Phytologist () (b) S375 D421 N428 H365 L333 RCD1 PARP domin HsPARP14 PARP domin (c) HsPARP1 PARP domin RCD1 PARP domin -d(fluorescence)/dt -d(fluorescence)/dt (d) Temperture (C) (e) Temperture (C) Tolernce to 1 μm prqut RCD1 line A RCD1 line B RCD1 H365Q line A RCD1 H365Q line B True leves (%) RCD1 S375W line A RCD1 S375W line B RCD1 D421A line A RCD1 D421A line B RCD1 D421A line B RCD1 (A) RCD1 (B) H365Q (A) H365Q (B) S375W (A) S375W (B) Fig. 8 Muttions in Arbidopsis thlin RADICAL-INDUCED CELL DEATH1 s (RCD1 s) puttive NAD + binding pocket do not compromise RCD1 function in development nd oxidtive stress signling. () Crystl structure of the RCD1 PARP domin (light blue, PDB code 5NGO) superimposed onto the structure of HsPARP14 (beige, PDB code 3SE2) (Whlberg et l., 2012). For X-ry dt collection, refinement, nd vlidtion sttistics, see Supporting Informtion Tble S11. (b) Structurl comprison of HsPARP14 s NAD + binding site (beige) with the corresponding re of the RCD1 PARP domin (light blue). RCD1 residues L333, H365, nd N428 tke the positions of the conserved H-Y-E trid in cnonicl PARPs. (c) Effect of the nonspecific PARP inhibitor 6(5H)-phennthridinone on the therml stbility of HsPARP1 s PARP domin (left pnel) nd the RCD1 PARP domin (right pnel). See Tble S12 for source dt from three independent experiments. (d) Morphologicl phenotypes of RCD1 site-directed mutnts with the indicted mino cid exchnges in the puttive NAD + binding site. Imges re representtive of > 10 plnts per line. RCD1 D421A line B showed both plnts tht fully complemented nd individuls tht prtilly complemented (sterisk) the developmentl phenotype of. (e) Tolernce of,, nd RCD1 site-directed mutnts to prqut. Seeds were sown on Murshige Skoog pltes contining 1 lm prqut nd the percentge of seedlings (n = 48) tht hd developed true leves fter 20 d ws determined. The plot shows the men vlue of two independent biologicl experiments. Error brs show plus/ minus SEM. See Tble S13 for source dt. D421A (A) D421A (B)

12 Phytologist Reserch 243 () WWE PARP RST (b) pdest32 pdest22 (c) pdest32 pdest22 pdest32 HRxL106 HRxL106ΔC SD -WLH 10 mm 3AT pdest22 WWE WWE PARP PARP WWE PARP WWE WWE PARP PARP WWE PARP RCD1 WWE RCD1 WWE RCD1 WWE RCD1 WWE SD -WLH 10 mm 3AT SD -WL RCD1 WWE SRO1 WWE RCD1 RCD1 WWE SRO1 WWE RCD1 SRO1 WWE SRO1 WWE SRO1 WWE SRO1 WWE SD -WLH 10 mm 3AT SD -WL SRO1 WWE RCD1 WWE SRO1 SRO1 WWE RCD1 WWE SRO1 (d) (e) (f) kd YFP GFP:WWE Accession number Cumultive spectr GFP:WWE/ YFP ctrl Best Arbidopsis mtch (% ID) Arbidopsis protein nme NbS g0015.1_SGN 630/0 AT1G (27) RCD1 NICBE_ _TGAC 547/2 AT3G (84) MLK2 NICBE_ _TGAC 535/0 AT2G (75) MLK3 NICBE_ _TGAC 463/2 AT3G (75) MLK4 Reltive PR1 trnscript SA-induced PR1 expression M SA M SA M SA mlk1,2,3 mlk1,3,4 Fig. 9 The N-terminl WWE-linker region of Arbidopsis thlin RADICAL-INDUCED CELL DEATH1 (RCD1) forms homo- nd hetero-oligomers nd intercts with Mut9-like kinses (MLKs). () Mpping protein protein interctions between HRxL106 nd the indicted RCD1 domin(s) in the yesttwo-hybrid ssy. The HRxL106DC deletion construct does not bind RCD1 nd is shown s control. (b, c) Formtion of homo- nd hetero-oligomers by RCD1 s (b) nd SIMILAR TO RCD ONE1 s (SRO1 s) (c) WWE-linker region in the yest-two-hybrid ssy., pdest22, or pdest32 vector control. (d) Representtive sodium dodecyl sulfte polycrylmide gel electrophoresis imge of trnsiently expressed YFP nd RCD1 GFP:WWE-linker proteins immuno-purified from Nicotin benthmin. The gel ws stined with colloidl blue Coomssie. The red sterisk indictes the expected migrtion of the GFP:WWE-linker fusion protein. (e) Selected proteins tht were enriched in immunoprecipittes of the GFP:WWE-linker fusion protein compred with the YFP control. For the full list, see Supporting Informtion Tble S14. (f) Slicylic cid (SA)-induced PATHOGENESIS-RELATED GENE 1 (PR1) gene expression in nd the mlk1,2,3 nd mlk1,3,4 triple mutnts. Four-week-old plnts were spryed with 0.1 mm SA or mock (M) solution nd PR1 expression levels were nlyzed by quntittive rel-time PCR 8 h lter. PR1 expression levels were normlized by ELONGATION FACTOR 1 ALPHA (EF1) expression. The plot shows the men of PR1/EF1 expression from three independent biologicl experiments. Error brs show plus/minus SEM; sterisk indictes men vlue different from SA tretment (one-wy ANOVA; Tukey Krmer post-hoc test, P < 0.05). See Tble S17 for source dt nd sttistics. Gou et l., 2012), HRxL106-expressing lines do not show this phenotype (Fig. 1d; Tble S2). Elevted tempertures promote uxin-medited hypocotyl elongtion in the light (Gry et l., 1998). By contrst, etiolted seedlings of uxin-overproducing lines develop shorter hypocotyls thn wild-type seedlings do (Zho et l., 2001; Nishimur et l., 2014). Despite trend for shorter hypocotyls in etiolted seedlings of HRxL106-expressing lines, the effect ws smll nd not sttisticlly significnt in most experiments (Fig. 1d; Tble S2). Notbly, PIF trnscription fctors ply dul role in light nd elevted-temperture signling (Koini et l., 2009; Leivr & Monte, 2014), nd recent results suggest tht phytochromes my ct s light nd temperture sensors (Jung et l., 2016; Legris et l., 2016). Given this pprent erly conversion of signling pthwys for light nd elevted temperture, dissecting how HRxL106 promotes plnt elongtion growth in response to environmentl signls requires more detiled nlysis. We mpped the defense-mnipulting ctivity of HRxL106 to short C-terminl prt of the effector, which is essentil for binding to RCD1 (Figs 6, 7). Notbly, the effect of HRxL106 on plnt growth responses to light is medited by the sme region of the effector (Fig. 6). In RCD1 loss of function mutnts, HRxL106-medited suppression of defense is bolished nd HRxL106-induced petiole elongtion is diminished (Fig. 7). These results suggest tht RCD1 integrtes both environmentl signls nd informtion from immune receptors, nd tht Hp exploits this function of RCD1 to ttenute plnt immunity. Notbly, the growth nd defense phenotypes of rcd1 null mutnts re opposite to those induced by ectopic expression of HRxL106 (Fig. 7). This indictes tht the effector mnipultes RCD1 in wy tht is not mimicked by RCD1 loss of function lleles. RCD1 is essentil for mintining the bsl expression levels of SA-inducible defense genes in noninfected plnts, but it is dispensble for trnscriptionl ctivtion of these defense genes upon pthogen infection (Fig. 4; Tble S6). One conceivble mode of HRxL106 function is tht effector binding to RCD1 converts the ltter into trnscriptionl co-repressor of defense genes (Fig. S6). However, this hypothesis cnnot esily be tested without better understnding of the moleculr function of both proteins.