Arabidopsis Endoplasmic Reticulum-Localized UBAC2Proteins Interact with PAMP-INDUCED COILED-COIL toRegulate Pathogen-Induced Callose Deposition andPlant Immunity

Zhe Wang,a,1 Xifeng Li,a,b,1 Xiaoting Wang,a,c Nana Liu,a,d Binjie Xu,a,e Qi Peng,a,f Zhifu Guo,a,g Baofang Fan,a

Cheng Zhu,h and Zhixiang Chena,h,2

a Department of Botany and Plant Pathology, 915 W. State Street, Purdue University, West Lafayette, Indiana 47907-2054bDepartment of Horticulture, Zhejiang University, Hangzhou, Zhejiang 310029, ChinacNational Center for Soybean Improvement, Nanjing Agricultural University, Nanjing, Jiangsu, 210095 ChinadCollege of Science, China Agricultural University, Beijing 100193, P.R. Chinae Triticeae Research Institute, Sichuan Agricultural University, Wenjiang, Sichuan 611130, Chinaf Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, ChinagCollege of Biosciences and Biotechnology, Shenyang Agricultural University, Shenyang, Liaoning 110866, ChinahCollege of Life Sciences, China Jiliang University, Hangzhou 310018, China

ORCID IDs: 0000-0001-9847-296X (Z.W.); 0000-0002-5933-3622 (X.L.); 0000-0002-5050-2397 (X.W.); 0000-0001-9745-8525(N.L.); 0000-0003-1349-7258 (B.X.); 0000-0002-3593-2153 (Q.P.); 0000-0003-4358-7836 (Z.G.); 0000-0002-8125-3346 (B.F.);0000-0002-8056-4943 (C.Z.); 0000-0002-5472-4560 (Z.C.)

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is initiated upon PAMP recognition by patternrecognition receptors (PRR). PTI signals are transmitted through activation of mitogen-activated protein kinases (MAPKs),inducing signaling and defense processes such as reactive oxygen species (ROS) production and callose deposition. Here,we examine mutants for two Arabidopsis thaliana genes encoding homologs of UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2), a conserved endoplasmic reticulum (ER) protein implicated in ER protein qualitycontrol. The ubac2 mutants were hypersusceptible to a type III secretion-deficient strain of the bacterial pathogenPseudomonas syringae, indicating a PTI defect. The ubac2 mutants showed normal PRR biogenesis, MAPK activation,ROS burst, and PTI-associated gene expression. Pathogen- and PAMP-induced callose deposition, however, wascompromised in ubac2 mutants. UBAC2 proteins interact with the plant-specific long coiled-coil protein PAMP-INDUCEDCOILED COIL (PICC), and picc mutants were compromised in callose deposition and PTI. Compromised callose deposition inthe ubac2 and picc mutants was associated with reduced accumulation of the POWDERY MILDEW RESISTANT 4 (PMR4)callose synthase, which is responsible for pathogen-induced callose synthesis. Constitutive overexpression of PMR4restored pathogen-induced callose synthesis and PTI in the ubac2 and picc mutants. These results uncover an ER pathwayinvolving the conserved UBAC2 and plant-specific PICC proteins that specifically regulate pathogen-induced callosedeposition in plant innate immunity.

INTRODUCTION

Plants have developed a complex immune system to protectthemselves from infection (Jones and Dangl, 2006). Upon rec-ognition of pathogen-associated molecular patterns (PAMPs),such as bacterial flagellin by pattern-recognition receptors (PRR),early plant defensemechanisms are triggered,which include a setof rapid responses such as a burst of reactive oxygen species(ROS), activation of mitogen-activated protein kinases (MAPKs),increased callose deposition, and defense gene expression

(Pitzschke et al., 2009). Pathogens can deliver effectors to plantcells to suppress PAMP-triggered immunity (PTI) but some of theeffectors may be recognized by plant resistance (R) proteins andactivate effector-triggered immunity (Jones and Dangl, 2006).Effector-triggered immunity is a strong defense response oftenmanifested as hypersensitive responses associated with rapidprogrammed cell death (Jones and Dangl, 2006) and increasedaccumulation of salicylic acid (SA) not only in local infected cellsbut also distant uninfected tissues to establish systemic acquiredresistance (Durrant and Dong, 2004).Many proteins involved in plant immune responses, including

the plasma membrane-localized PRRs and defense-relatedproteins, such as pathogenesis-related proteins, are membraneand secreted proteins that are synthesized on the endoplasmicreticulum (ER) and enter the secretory pathway to reach theplasmamembrane and extracellular destinations (Gu et al., 2017).These proteins are cotranslationally imported into the ER after

1 These authors contributed equally to this work.2 Address correspondence to zhixiang@purdue.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Zhixiang Chen (zhixiang@purdue.edu).www.plantcell.org/cgi/doi/10.1105/tpc.18.00334

The Plant Cell, Vol. 31: 153–171, January 2019, www.plantcell.org ã 2019 ASPB.

synthesis and go through an elaborate ER quality control (ERQC)system for proper folding and modification (Araki and Nagata,2011). Those terminally misfolded ER polypeptides are eliminatedthrough ER-associated degradation (ERAD) or autophagy.

Studies in yeast and mammals have shown several systems inERQC. First, the ER relies on the luminal binding immunoglobulinprotein, one of the most abundant ER chaperones that functiontogether with the Hsp40 family proteins as cochaperones andserve multiple roles in the ER, ranging from productive folding toERAD (Araki and Nagata, 2011). Another important maturationstep of ER proteins in the ER is the formation of disulfide bondscatalyzed by protein disulfide isomerases and other thiol oxi-doreductases that promote protein function and stability (Arakiand Nagata, 2011).

The best studied system of ERQC is the so-called calnexin/calreticulin (CNX/CRT) cycle (Araki and Nagata, 2011). In thissystem, polypeptides entering the ER are first modified by at-tachment of a preformed oligosaccharide to Asn (N) side chains inAsn-Xxx-Ser/Thr sequons, which is subsequently modified togenerate a monoglucosylated oligosaccharide. These N-glycansare recognized by ER lectin-like chaperones CNX and CRT topromote their proper folding.After release fromCNX/CRTafter thetrimming of their innermost Glc residue by glucosidase II, clientglycoproteins are delivered to UDP-Glc:glycoprotein glucosyl-transferase (UGGT), which senses the folding state of releasedglycoproteins. If the glycoproteins do not achieve the correctconformation, UGGT glucosylates the N-glycan again to be re-engaged by CNX/CRT. UGGT is inactive against correctly foldedproteins so they are released from the CNX/CRT cycle andtransported to their destinations (Araki and Nagata, 2011).

In Arabidopsis (Arabidopsis thaliana), a number of studieshave shown that specific ERQC components are required forbiogenesis of ELONGATION FACTOR-THERMO UNSTABLERECEPTOR (EFR), a PRR that recognizes bacterial ELONGATIONFACTOR-THERMO UNSTABLE (Li et al., 2009; Nekrasov et al.,2009; von Numers et al., 2010). These ERQC components in-clude CRT3, UGGT, a homolog of glucosidase II b-subunit,ERD2d (a homolog of yeast and mammalian HDEL receptorERD2 for retaining proteins in the ER lumen), SDF2 (a subunit ofthe Hsp40/ binding immunoglobulin protein complex), and STTa(a catalytic subunit of the oligosaccharyltransferase complex forcotranslational N-glycosylation; Li et al., 2009; Nekrasov et al.,2009; von Numers et al., 2010). Interestingly, the mutants forthese specific ERQC components have normal biogenesis andfunction of FLAGELLIN SENSITIVE2 (FLS2), which belongs tothe same subfamily of PRRs as EFR (Li et al., 2009; Nekrasovet al., 2009; von Numers et al., 2010). Arabidopsis ERQCcomponents UGGT and STT3a are also required for activation ofdefense responses in bak1-interacting receptor-like kinase 1,1(bir1-1) mutant plants (Zhang et al., 2015a). In rice (Oryza sativa),ERQC component SDF2 is copurified with the Xa21 immunereceptor and required for Xa21-mediated immunity (Park et al.,2013). In addition, upregulation of both the ER-associatedprotein folding/modification machinery and the secretorypathway are required for SA and NON-EXPRESSER OFPATHOGENESIS-RELATED GENES 1-mediated induction ofdefense genes during the establishment of systemic acquiredresistance (Wang et al., 2005).

Callose is a b-(1,3)-D-glucan polymer normally found in sieveplates, pollen tubes, microsporocytes, cell plates, and plasmo-desmata (Ellinger andVoigt, 2014). In addition, pathogen infectioncan trigger callose deposition (Ellinger and Voigt, 2014; Voigt,2016). In the interactions between plants and filamentouspathogens, the spores of the pathogens first germinate on the leafsurface and develop infection pegs to invade the epidermal cells.This invasion can induce a range of plant defense responsesincluding the formation of local cell wall appositions (papilla) at thesites of pathogen attack (Collins et al., 2003; Assaad et al., 2004;Schulze-Lefert, 2004). Callose is amajor component of the papilla(Ellinger and Voigt, 2014). In Arabidopsis, callose deposition isalso triggered by PAMPs such as fungal cell wall componentchitosan and bacterial flagellin epitope flg22 from Pseudomonassyringae (Gómez-Gómez et al., 1999; Iriti and Faoro, 2009). It hasbeen proposed that callose polymers can reinforce the cell wallstructure at the site of infection to restrict the ingression ofpathogen-secreted cell wall-degrading enzymes (Stone andClarke, 1992). The proposed role of callose deposition in plantimmune responses is supported by its inhibition by defense-suppressing virulence effectors (Hauck et al., 2003).In Arabidopsis, pathogen- and PAMP-induced callose de-

position is dependent on the PMR4 callose synthase (Jacobset al., 2003; Nishimura et al., 2003). The Arabidopsis pmr4mutantsupports20-foldmoregrowth thanwild-typeplantsofastrainofP.syringae that is deficient in the type III secretion system (Kim et al.,2005). Overexpression of PMR4 in transgenic Arabidopsis plantsconfers complete restriction to penetration by powdery mildewfungal pathogen (Ellinger et al., 2013), further supporting thecritical role of callose deposition in plant disease resistance. In-triguingly, mutation of PMR4 also results in increased resistanceto powdery mildew fungal pathogens in association with hyper-activation of SA signaling (Nishimura et al., 2003). This findingindicates an unexpected role of PMR4-dependent callose de-position in suppression of SA-mediated defense responses.Therefore, the roles of PMR4-dependent callose deposition inplant defense against pathogen invasion appear to be quitecomplex.We have only very limited information about the regulatory

mechanisms for pathogen-induced, PMR4-dependent callosedeposition. In uninfected PMR4-overexpression lines, no sig-nificant increase in the callose synthase activity or callose de-position was observed (Ellinger et al., 2013). In yeast, activationand translocation of callose synthases involveGTPases (Qadotaet al., 1996;Calonge et al., 2003). In Arabidopsis, PMR4 interactswith GTPase RabA4c (Ellinger et al., 2014). Similar to PMR4overexpression, RabA4c overexpression leads to completepenetration resistance to the virulent powdery mildew fungalpathogen in aPMR4-dependentmanner (Ellinger et al., 2014). Bycontrast, overexpression of a dominant-negative form of Ra-bA4c fails to increase callose deposition or penetration re-sistance (Ellinger et al., 2014). These results indicate that theRabA4c GTPase plays a critical role in the activation andtranslocation of PMR4 during pathogen-induced callose de-position. As a transmembrane protein synthesized on the ER,PMR4 is likely subjected to additional regulatorymechanisms forits biogenesis, trafficking, and activation to ensure biosynthesisof callose in a timely manner.

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In this study, we report that two Arabidopsis genes encodingclose homologs of UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2), a conserved ER protein im-plicated in ERQC, play a critical role in PTI. This is based on thehyper-susceptibility of the ubac2 mutants to a type III secretionsystem-deficient strain of the bacterial pathogen P. syringae.Despite the compromised PTI, the ubac2mutants were normal inthe biogenesis of PRRs, activation of MAPKs, production of ROSand PTI-associated gene expression. The evolutionarily con-served UBAC2 proteins interact with the plant-specific longcoiled-coil protein PAMP-INDUCEDCOILEDCOIL (PICC), whosemutants were also compromised in PTI but normal in associatedsignaling and defense gene expression. Both the ubac2 and piccmutants, however, were compromised in pathogen- and PAMP-induced callose deposition. Compromised callose deposition inthe ubac2 and picc mutants was associated with reduced ac-cumulation of the PMR4 calllose synthase, which is responsiblefor pathogen-induced callose synthesis. Constitutive over-expression of PMR4 restored pathogen/PAMP-induced cal-lose synthesis and PTI in both the ubac2 and picc mutants.Based on these results, we propose that UBAC2 and PICC arecomponents of an ERQC pathway that plays a critical role inPMR4 accumulation and callose deposition in plant innateimmunity.

RESULTS

The ubac2a ubac2b Double Mutants Are Defective in PTI

We have recently reported that the mutants for the two closeArabidopsis homologs of UBAC2 are compromised in heat tol-erance and resistance to the necrotrophic pathogen Botrytiscinerea (Zhou et al., 2018). To further analyze the roles of theevolutionarily conservedproteins,weanalyzed theubac2mutantsfor response to a virulent strain of the bacterial pathogen P. sy-ringae DC3000 (PstDC3000). The ubac2a and ubac2b single anddouble mutants responded normally to the bacterial strain basedon bacterial growth, which was similar to that in wild-type plants(Figure 1A). Thus, the ubac2mutants have normal basal immunityagainst the virulent bacterial pathogen.

We then compared the ubac2 mutants with wild type for re-sponse to the PstDC3000 hrcCmutant strain deficient in the typeIII secretion system. As shown in Figure 1B, after infiltration witha suspension of the mutant bacterial strain, there was an;5-foldincrease in the bacterial population in the ubac2a and ubac2bsingle mutant and ;20- to 30-fold increase in the two ubac2aubac2b (ubac2a/2b) double mutants compared with that inwild type.

Since the two ubac2a/2b double mutants contain the sameubac2b mutation, we performed molecular complementationby transforming one of the ubac2a/2b double mutants witha UBAC2b gene under control of the Cauliflower mosaic virus(CaMV) 35S promoter. Resistance of the ubac2a/2b doublemutant plants toPstDC3000 hrcCwas restored by transformationof the mutant with the UBAC2b gene (Figure 1B). Thus, the twoclose UBAC2 genes have a critical and partially redundant rolein PTI.

To further analyze the role of the UBAC2 genes in PTI, wecompared the wild type and ubac2a/2b double mutants for in-duction of disease resistance by the bacterial PAMP flagellin-derived flg22. Wild-type and ubac2a/2b double mutant plantswere pretreated by leaf infiltration with water or 1mM flg22. After24 h, leaveswere infectedwith 105 cfu/mLPstDC3000. Bacterialgrowth was assessed 40 h after infection. As shown in Figure 2,flg22 treatment led to increased resistance toPstDC3000 inwild-type plants as indicated by more than 40-fold reduction inbacterial growth when compared with those from water-pretreated wild-type plants. By contrast, flg22 treatment ofthe ubac2a/2b double mutants led to only ;8-fold reduction inthegrowthofPstDC3000 (Figure 2). These results confirmed thatthe ubac2a/2b double mutant plants were compromised inPAMP-induced immunity.

Figure 1. The ubac2 Mutants Are Compromised in PTI.

(A) Growth of virulent PstDC3000.(B) Growth of type III secretion-deficient PstDC3000 hrcC. Three fullyexpanded leaves from each wild-type (WT) and ubac2 mutant plant wereinfiltrated with a suspension of PstDC3000 or PstDC3000 hrcC (OD600 =0.0002 in 10 mM MgCl2). Leaf samples were taken at 0 and 4 d post-inoculation (dpi) to determine bacterial colony-forming units (cfu). Meansand SE were calculated from 10 plants for each treatment. According toDuncan’smultiple range test (P=0.01),meansof cfu do not differ if they areindicated with the same letter. The experiment was repeated more thanthree times with similar results.

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The ubac2a/2b Double Mutants Are Normal in Biogenesisand Signaling of PRRs

To determine the molecular and cellular basis of the defectivePTI in the ubac2a/2b double mutants, we investigated thebiogenesis and signaling of FLS2 and EFR, two PRRs thatrecognize the PAMPs flg22 and elf18, respectively, leading toactivation of PTI (Gómez-Gómez and Boller, 2000; Zipfel et al.,2006). To compare biogenesis of FLS2 and EFR, we preparedprotoplasts from leaf mesophyll cells of wild-type and theubac2a/2b double mutant plants and transfected them withplasmid DNA of the FLS2- and EFR-YFP (yellow florescentprotein) constructs under control of the CaMV 35S promoter.As shown in Figure 3A, we observed similar levels and patternsof the YFP fluorescence signals in wild type and ubac2a/2bmutant protoplasts transfected with the FLS2- and EFR-YFPconstructs. These results did not support a major reductionin the biogenesis of the two PRRS from disruption of theUBAC2 genes.

Consistent with the normal biogenesis of the PRRs, we ob-served that the ubac2a/2b double mutants were normal in theactivation of MAPK3, MAPK6, and MAPK4 in responses to flg22and elf18 treatment (Figure 3B). Likewise, the ubac2a/2b doublemutant plants were normal in flg22- and elf18-triggered oxidativeburst (Figure 3C) and induction of two PTI-associated genes(WRKY33 and CYP81F2; Figure 3D). These results indicate thatthe biogenesis and signaling of FLS2 and EFR were not altered inthe ubac2a/2b doublemutants. This conclusion is consistent withthe observation that both flg22 and elf18 inhibited the growth ofwild-type and ubac2a/2b mutant seedlings to similar extents(Supplemental Figure 1).

Defects in Pathogen/PAMP-Induced Callose Depositionin ubac2a/2b

Despite the normal biogenesis and signaling of FLS2 andEFR, theubac2a/2b double mutants were defective in pathogen-inducedcallose deposition. As shown in Figure 4, at 16 h after inoculationwith PstDC3000 hrcC, there was a more than 30-fold induction incallose deposits in the inoculated leaves of wild-type plants butonly a 5- to 6-fold induction in the ubac2a/2bdoublemutants. Theubac2a/2b double mutants were also compromised in flg22- andelf18-inducedcallosedeposition (Figures 4Cand4D). At 16 hafterinfiltration with 1 mM flg22 or elf18, callose deposition in the in-filtrated leaf sampleswere inducedbymore than20-fold in thewildtype over water-infiltrated control leaves (Figures 4C and 4D). Bycontrast, onlya3- to4-fold inductionwasobserved in the leavesofthe ubac2a/2b double mutants after infiltrated with the sameconcentration of flg22 and elf18 (Figures 4C and 4D).

A Critical Role of the UBA Domain of UBAC2 Protein in PTI

UBAC2 proteins contain a conserved ubiquitin-associated (UBA)domain at their C terminus (Christianson et al., 2011; Zhou et al.,2018). To determine the role of the UBA domain of ArabidopsisUBAC2 proteins in plant PTI, we generated a mutant UBAC2a inwhich twoconserved residues in itsUBAdomain (M262andL288)required for ubiquitin binding were changed to alanine residues.These changes in UBA domains have been previously shown toabolish ubiquitin binding (Kozlov et al., 2007). Genes encodingwild-type UBAC2a andmutant UBAC2aM262A/L288A proteins werefusedwith amyc-tagandcloned into aplant transformation vectorunder control of the CaMV 35S promoter. The constructs werethen transformed into the ubac2a/2b double-mutant plants,and transgenic lines overexpressing the UBAC2 transgeneswere selected based on RT-qPCR and protein blotting. Theselines were tested for PTI based on the reduced growth ofPstDC3000 hrcC (Figure 5A) and flg22-induced callose deposi-tion (Figure 5B). Transformation of the ubac2a/2b double mutantwith the wild-type UBAC2a gene completely restored PTI(Figure 5A) and flg22-induced callose deposition (Figure 5B). Thecomplete restoration of the ubac2a/2b double mutant by thewild-type UBAC2a gene probably resulted from its over-expression, which apparently offset the loss of theUBAC2b genein the ubac2a/2b double mutant. By contrast, transformation withthegeneencoding themutantUBAC2aM262A/L288Aprotein failed torestore PTI (Figure 5A) or flg22-induced callose deposition in theubac2a/2b double mutant (Figure 5B). These results indicate thatthe C-terminal UBA domain of the UBAC2 proteins is critical fortheir role in PTI and PAMP-induced callose deposition.

Overexpression of PMR4 Rescued the ubac2a/2bDouble Mutants

In Arabidopsis, the PMR4 callose synthase is responsible forpathogen-induced callose deposition (Jacobs et al., 2003;Nishimura et al., 2003). As a transmembrane protein, PMR4 issynthesized on the ER and transported to the plasma membranefor synthesis of callose, which is deposited at the plasma mem-brane andcell wall interface in response topathogen infection and

Figure 2. The ubac2 Mutant Plants Are Compromised in flg22-InducedResistance.

Three fully expanded leaves from each wild-type (WT) and ubac2a/2bdouble-mutant plant were pretreated by leaf infiltration with water or 1 mMflg22. After 24 h, leaves are infected by infiltration with virulent PstDC3000(105 cfu/mL). Bacterial growth was assessed 40 h after infection. Meansand SE were calculated from 10 plants for each treatment. According toDuncan’smultiple range test (P=0.01),meansof colony-formingunits (cfu)do not differ if they are indicated with the same letter. The experiment wasrepeated three times with similar results.

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wound stresses. Since the ubac2 mutants are compromised inboth PTI and pathogen/PAMP-induced callose deposition, wetestedwhether thesephenotypesof theubac2a/2bmutants couldbe rescued by PMR4 overexpression. The full-length PMR4coding sequence was cloned behind the CaMV 35S promoter ina plant transformation vector and transformed into the ubac2a/2bdouble-mutant plants. Three independent transgenic ubac2a/2bmutant lines with a 6- to 10-fold increase in the PMR4 transcriptlevel based on RT-qPCR analysis were compared with the wildtype and ubac2a/2b for both PTI and pathogen-induced callosedeposition. As shown in Figure 6A, overexpression of PMR4completely restored the PTI of the ubac2a/2b double mutantbased on the reduction in the growth of PstDC300 hrcC in the

transgenic PMR4 overexpression lines. Despite the constitutivePMR4 overexpression, callose deposition was not significantlyelevated inmock-inoculated transgenicplantsbutwas restored towild-type levels after pathogen infection (Figure 6B). These resultsimplicated PMR4 in the mutant phenotypes of compromised PTIand callose deposition in the ubac2a/2b mutants.

Interaction of UBAC2 with PICC

To gain insights into the action of UBAC2 proteins in PTI, we triedto identify UBAC2-interacting proteins using Gal4-based yeasttwo-hybrid screenswithUBAC2aasabait.After screening53106

Figure 3. The ubac2a/2b Double-Mutant Plants Are Normal in Biogenesis and Signaling of PRRs.

(A)Accumulationandsubcellular localization of FLS2-YFPandEFR-YFP.Leaf protoplastswereprepared fromwild-type (WT) andubac2a/2bmutant plantsand transfected with the FLS2-YFP and EFR-YFP plasmid DNA. The levels and subcellular localization of the YFP signals in transfected protoplasts wereobserved 12 h later by confocal fluorescence microscopy. The bright field images are also shown. Bar, 10 mm.(B)MAPkinase activation. Two-week-old Arabidopsis seedlingswere treatedwith 100 nM flg22 or elf18 for indicatedmin. The activatedMAP kinasesweredetected using an anti-p42/44 MAPK monoclonal antibody. Rubisco large subunit proteins stained with Ponceau S are shown as loading control.(C)Oxidativeburst. Leaf discs from3- to4-week-oldArabidopsis plantswere treatedwith1mMelf18orflg22, andROS levels at indicated timesof treatmentweremeasured in relative luminescence units (RLU). Results are shownasmeans and SE calculated from12 leaf discs for each treatment at each timepoint.(D) Induction ofWRKY33andCYP81F2. Two-week-old Arabidopsis seedlingswere treatedwith 100 nM flg22or elf18 for 30min, and the transcript levels ofthePTImarker genesweredeterminedbyRT-qPCRusinggene-specificprimers.MeansandSEwerecalculated from three replicates. Theexperimentswererepeated twice with similar results.

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independent transformants of an Arabidopsis complementaryDNA (cDNA) prey library, we isolated 10 positive clones based onboth prototrophy for His and LacZ reporter gene expressionthrough assays of b-galactosidase activity. Two positive clonesidentified from the screens encode the PICC protein (At2g32240),a plant-specific large protein (147 kD) that consists of a longcoiled-coil domainwith apredicted transmembrane domain at theC terminus (Venkatakrishnan et al., 2013). Like UBAC2 proteins,PICC is locatedat theER (Venkatakrishnanetal., 2013). Toconfirmwhether the interactionsbetweenPICCandUBAC2a in yeast cellsare specific, we performed two-hybrid assays using PICC-LIKE(PICL; At1g05320), a paralog of PICC that is also localized in theER (Venkatakrishnanet al., 2013), as acontrol. PICC, but notPICL,gave positive results of interaction with UBAC2a (Figure 7A).To determine whether UBAC2 and PICC interact in plant cells,

we performed bimolecular fluorescence complementation(BiFC) in Agrobacterium-infiltrated Nicotiana benthamiana.We fused Arabidopsis UBAC2a to the C-terminal yellowfluorescent protein (YFP) fragment (UBAC2a-C-YFP) andcoexpressed it with PICC-N-YFP in N. benthamiana leaves.BiFC signals were detected in transformed cells as a reticulumnetwork pattern characteristic of that of the ER (Figure 7B).Control experiments in which UBAC2a-C-YFP was coex-pressed with unfused N-YFP or unfused C-YFP was coex-pressed with PICC-N-YFP did not show fluorescence(Figure 7B). When PICL was fused to the N-terminal YFP (PICL-N-YFP) and coexpressed with UBAC2a-C-YFP, no BIFC signalwas observed either (Figure 7B). Similar results with UBAC2bwere also obtained for interaction with PICC in plant cells(Supplemental Figure 2). Thus, Arabidopsis UBAC2 and PICCcan interact and form complexes at the ER.

Compromised PTI and Pathogen/PAMP-Induced CalloseDeposition in picc

The role of PICC and PICL in plant immunity has been previouslycharacterized (Venkatakrishnan et al., 2013). PICC expressionwas induced by the bacterial PAMP flg22 (Venkatakrishnan et al.,2013). Like ubac2 mutants, picc mutants showed a normal re-sponse to the virulent strain of PstDC3000 but were highlycompromised in resistance to PstDC3000 hrcC (Venkatakrishnanet al., 2013). By contrast, the picl mutant plants responded nor-mally to PstDC3000 hrcC (Venkatakrishnan et al., 2013). Fur-thermore, the piccmutants were normal in flg22-induced burst ofROS and induction of PTI-associated genes despite com-promised PTI (Venkatakrishnan et al., 2013). There was nodifference either in pathogen-induced expression of MYB51(Venkatakrishnan et al., 2013), which encodes a PAMP-inducedtranscription factor essential for induced callose deposition atthe sites of pathogen attack (Clay et al., 2009).The normal induction of MYB51 in the picc mutants does not

support a role of PICC in the transcriptional regulation ofpathogen-induced callose deposition. However, given its locali-zation in the ER and physical interaction with UBAC2 proteins,PICC could function with its interacting UBAC2 proteins in theposttranscriptional regulation of pathogen-induced callose de-position. To test these possibilities, we directly compared twopicc knockout mutants with the ubac2a/2b double mutants

Figure 4. The ubac2a/2bDoubleMutants AreCompromised in Pathogen-and PAMP-Induced Callose Deposition.

(A)Callose deposition triggered byPstDC3000 hrcC (hrcC ). Five-week oldArabidopsisplantswere infiltratedwith 10mMMg2Cl (mock) or 53107cfu/mL PstDC3000 hrcC. Callose deposition was assayed at 16 h after in-filtration. Bar, 20 mm.(B) Callose deposits triggered by PstDC3000 hrcC (hrcC ).(C) Callose deposits triggered by 1 mM flg22 for 16 h.(D) Callose deposits triggered by 1 mM elf18 for 16 h. Means and SE ofcallosedepositspermicroscopicfield (1253100mm)werecalculated frommore than 10microscopic fields per treatment per genotype. According toDuncan’s multiple range test (P = 0.01), means of callose deposits do notdiffer if they are indicated with the same letter. The experiments wererepeated at least three times with similar results.

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for phenotypes in disease resistance and in pathogen- andPAMP-induced callose deposition. As previously reported(Venkatakrishnan et al., 2013), thepiccmutantswere normal inresponse toPstDC3000 but highly compromised in resistanceto PstDC3000 hrcC based on the growth of the bacterialstrains, just as found with the ubac2a/2bmutants (Figures 8Aand 8B). Furthermore, flg22-induced resistance to the virulentPstDC3000 was compromised to similar extents in the uba-c2a/2b and picc mutants (Figure 8C).

Callose deposition following infection by PstDC3000 hrcC ortreatmentwith flg22or elf18was also reduced in thepiccmutants,again to similar extents as in the ubac2a/2b double mutants

(Figure 9). Thus, compromised PTI was associated with reductionin pathogen-induced callose deposition in both the ubac2a/2band picc mutants. Importantly, the compromised phenotypes ofthe picc mutants in PTI and pathogen/PAMP-induced callosedeposition could also be rescued by overexpression of PMR4, asin the ubac2 double mutants (Figures 8 and 9).

Figure 6. Overexpression of PMR4 Restores PTI and Pathogen-InducedCallose Deposition in the ubac2a/2b Double Mutant.

(A) Growth of type III secretion-deficient PstDC3000 hrcC. The wild type(WT), ubac2a/2b mutant, and ubac2a/2b mutant overexpressing PMR4were infiltrated with PstDC3000 hrcC (OD600 = 0.0002 in 10 mM MgCl2).Leaf samples were taken at 0 and 4 d post-inoculation (dpi) to determinebacterial colony-forming units (cfu). Means and SE were calculated from 10plants for each treatment. According to Duncan’s multiple range test (P =0.01), means of cfu do not differ if they are indicated with the same letter.(B) Pathogen-induced callose deposition. Five-week-old Arabidopsisplants were infiltrated with 10 mM Mg2Cl (mock) or 5 3 107 cfu/mLPstDC3000 hrcC (hrcC ). Callose deposition was assayed at 16 h afterinfiltration. Means and SE of callose deposits per microscopic field (1253

100 mm) were calculated from more than 10 microscopic fields pertreatment per genotype. According to Duncan’s multiple range test (P =0.01), means of callose deposits do not differ if they are indicated with thesame letter. Three independent overexpression lines (L1 to L3) were usedfor the analysis. The experiments were repeated twice with similar results.

Figure 5. The UBA Domain of UBAC2a Is Required for Its Role in PTI andPAMP-Induced Callose Deposition.

(A) Growth of type III secretion-deficient PstDC3000 hrcC. The wild type(WT), ubac2 mutant, and indicated transgenic lines were infiltrated witha suspension of PstDC3000 hrcC (OD600 = 0.0002 in 10 mM MgCl2). Leafsamples were taken at 0 and 4 d post-inoculation (dpi) to determinebacterial colony-forming units (cfu). Means and SEwere calculated from 10plants for each treatment. According to Duncan’s multiple range test (P =0.01), means of cfu do not differ if they are indicated with the same letter.(B) Flg22-induced callose deposition. Arabidopsis plants were infiltratedwith water or 1 mM flg22. Callose deposition was assayed at 16 h afterinfiltration. Means and SE of callose deposits per microscopic field (1253

100 mm) were calculated from more than 10 microscopic fields pertreatment per genotype. According to Duncan’s multiple range test (P =0.01), means of callose deposits do not differ if they are indicated with thesame letter. The experiment was repeated twice with similar results.

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Mutual Effects between UBAC2 and PICC

Given their colocalization in the ER, their physical interaction andthe strikingly similar phenotypes of their respective mutants, it islikely that UBAC2 and PICC proteins act in the same pathwayeither sequentially or coordinately in the ER. As homologs ofa highly conservedprotein implicated inERQCandERAD,UBAC2

proteins might regulate the synthesis and stability of PICC or viceversa. To test this possibility, we first determined the effect of thedisruption of theUBAC2 genes on the accumulation of PICC. Thefull-length PICC coding sequence was cloned, tagged withmCherry at its N terminus, and placed in a plant transformationvector. The resulting mCherry-PICC fusion gene is functionalbased on its ability to restore PTI and pathogen-induced callosedeposition when expressed in the picc mutant plants (Sup-plemental Figure 3).ThemCherry-PICC expression construct was transformed into

wild-type and ubac2a/2b double mutant plants, and the trans-genic lines were examined for the levels of the mCherry-PICCsignals. Preliminary survey of the transgenic mCherry-PICC linesin wild-type and ubac2a/2b mutant backgrounds failed to reveala clear pattern of difference in the levels of mCherry-PICC signalsbetween the two genotypes. However, because the mCherry-PICC transgene under control of the CaMV 35S promoter wasintegrated into different genome sites in different transgenic lines,the variability of the transgene expression was substantial evenamong the lines in the same genetic background, making thecomparison difficult and unreliable.To avoid this problem, we generated transgenic lines that

contained the same mCherry-PICC transgene integrated at thesame genome site but differed in the zygosity of the ubac2a/2bmutations. First, we introduced themCherry-PICC transgene intothe ubac2a/2b double mutant and obtained single-copy homo-zygous lines expressing the mCherry-PICC transgene. Twotransgenic mCherry-PICC lines in the ubac2a/2b mutant back-ground were then selected and crossed with both the wildtype and ubac2a/2b double mutant to generate heterozygousUBAC2a/2b+/2 and homozygous ubac2a/2b2/2 progeny, re-spectively, that contained the same mCherry-PICC transgene.The two types of F1 progeny for each of the two lines were thencompared for the levels of mCherry-PICC signals. As shown inFigure 10A, the levels of mCherry-PICC signals were similar in theheterozygous UBAC2a/2b+/2 and homozygous ubac2a/2b2/2

progeny. This result indicated that the disruption ofUBAC2 geneshad little effects on the accumulation of PICC proteins.Furthermore, overexpression ofPICCdid not rescue themutant

phenotypes of the ubac2a/2bmutant in PTI (Figure 10B) or flg22-induced callose deposition (Figure 10C). Using the same ap-proach, we found that the mutation of PICC did not affect theaccumulation of UBAC2a (Supplemental Figure 4A) and over-expression of UBAC2 did not restore PTI or PAMP-induced cal-lose deposition in the piccmutant (Supplemental Figures 4B and4C). Thus, UBAC2 and PICC proteins did not affect each other’saccumulation, and both proteins were required for PTI- andPAMP-induced callose deposition.

Reduced Accumulation of PMR4 in ubac2a/2b andpicc Mutants

Based on the phenotypes of the ubac2a/2b and picc mutants inboth PTI and pathogen/PAMP-induced callose deposition andtheir rescue by overexpression of PMR4 callose synthase, weexamined the transcript levels of PMR4 and the accumulation ofthe callose synthase protein in the mutants. Analysis with RT-qPCR revealed, however, that the transcript levels of PMR4

Figure 7. UBAC2a interacts with PICC.

(A) Interaction of UBAC2a and PICCproteins in yeast cells. TheGal4 DNA-binding domain vector (bait) with or without (2) fused UBAC2a gene werecotransformed with the activation domain vector (prey) with or without (2)fused PICC or PICL gene into yeast cells, and the transformed cells weregrown in the selection medium with (+) or without (2) histidine (His).(B) BiFC analysis of UBAC2a interaction with PICC. BiFC fluorescencewas observed in the transformed N. benthamiana leaf epidermal cellsfrom complementation of the N-terminal half of the YFP fused with PICC(PICC-N-YFP) by the C-terminal half of the YFP fused with UBAC2a(UBAC2a-C-YFP). No fluorescencewas observedwhen PICC-N-YFPwasco-expressedwithunfusedC-YFPorwhenunfusedN-YFPorPICL-N-YFPwas co-expressed with UBAC2a-C-YFP. YFP epifluorescence, bright-field, and emerged images of the same cells are shown. Bar, 10 mm.

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were normal in the ubac2a/2b and picc mutants before or afterpathogen infection.To examine the effects of themutations ofUBAC2 andPICC on

the accumulation of PMR4, we generated a PMR4-GFP con-struct andplaced it in a plant transformation vector under controlof thePMR4native promoter.However, we failed to reproduciblydetect the PMR4-GFP signals even in the wild-type backgroundafter protoplast transfection or plant transformation with orwithout pathogen infection or PAMP treatment, due to very lowlevels of PMR4-GFP under its native promoter. Therefore, weplaced the PMR4-GFP construct under control of theCaMV 35Spromoter and introduced it into Arabidopsis plants. For reliablecomparison of the transgene expression between the wild typeandubac2a/2b,wefirst generated transgenic lines in theubac2a/2b mutant backgrounds and two independent single-copytransgenic lines expressing different levels of PMR4-GFPtranscripts were selected. One of these transgenic lines (line 1)contained about a 5-fold increase in the transcript level ofPMR4,while the other line (line 2) contained an;3.5-fold increase in thetranscript level of PMR4 based on RT-qPCR analysis. The twolines were each crossed to both the wild type and ubac2a/2bmutant. For each line, the crosses generated both heterozygousUBAC2a/2b+/2 and homozygous ubac2a/2b2/2 progeny thatcontained the same PMR4-GFP transgene and similar levels ofPMR4 transcripts based on RT-qPCR (Supplemental Figure 5),which were then compared for the levels of the PMR4-GFPsignals.For both lines, PMR4-GFP fluorescent signals with a pattern of

plasma membrane localization were observed in the heterozy-gous UBAC2a/2b+/2 progeny (Figure 11A). PMR4-GFP signals inthe homozygous ubac2a/2b2/2 progeny were weaker than in theheterozygous UBAC2a/2b+/2 progeny (Figure 11A). Despite thereduced levels of PMR4-GFP signals in the homozygous ubac2a/2b2/2 progeny generated from line 1, flg22-induced callose de-position was almost fully restored (Figure 11B). For line 2, whichcontained a lower level of the PMR4-GFP transcript, flg22-induced callose deposition was only partially restored in thehomozygous ubac2a/2b2/2 progeny (Figure 11B).Using the same approach, we examined the role of PICC in the

accumulation of PMR4. Two independent transgenic PMR4-GFP lines in the picc mutant background with an ;4-fold in-crease in the PMR4 transcript level were crossed with both thewild type and the picc mutant. Comparison of the PMR4 tran-scripts and PMR4-GFP signals were then made between theheterozygous PICC/picc and homozygous picc/picc linescontaining the same PMR4-GFP transgene and similar levels ofPMR4 transcripts. As found in the ubac2a/2bmutant, disruptionof PICC reduced the accumulation of PMR4-GFP, but not thePMR4 transcripts (Figure 12A; Supplemental Figure 5). Fur-thermore, the PMR4-GFP transgene only partially restoredflg22-induced callose deposition in the homozygous picc/piccprogeny (Figure 12B). Thus, the mutation of PICC also reducedthe accumulation of PMR4-GFP.We also tried to determine the accumulation of PMR4-GFP in

the ubac2 and piccmutants with immunoblotting using anti-GFPantibodies. After repeated tries, however, we failed to reliablydetect the PMR4-GFP proteins using a number of anti-GFP an-tibodies. Comparison of the transgenic PMR4-GFP lines with

Figure 8. Compromised PTI of the picc Mutants and the Restoration byPMR4 Overexpression.

(A) and (B) Growth of virulent PstDC3000 and type III secretion-deficientPstDC3000 hrcC, respectively. Wild-type (WT), ubac2, picc, and piccmutant plants overexpressing PMR4 were infiltrated with a suspension ofPstDC3000 or PstDC3000 hrcC (OD600 = 0.0002 in 10 mM MgCl2). Leafsamples were taken at 0 and 4 d post-inoculation (dpi) to determinebacterial colony-forming units (cfu).(C) flg2-induced resistance to PstDC3000. Wild-type (WT), ubac2, picc,and picc mutant plants overexpressing PMR4 were pretreated by leafinfiltration with water or 1 mM flg22. After 24 h, leaves were infected byinfiltration with virulent PstDC3000 (105 cfu/mL). Bacterial growth wasassessed 40 h after infection. Two independent PMR4 overexpressionlines (L1 and L2) were used for the analysis. Means and SE were calculatedfrom 10 plants for each treatment. According to Duncan’s multiple rangetest (P = 0.01), means of cfu do not differ if they are indicatedwith the sameletter. The experiments were repeated three times with similar results.

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transgenic plants expressing other GFP fusion proteins indicatedthat the PMR4-GFP fluorescent signals were very low even in thewild-type background.

To increase the sensitivity of detection of the PMR4 proteins byimmunoblotting, we generated transgenic ubac2 and picc linesthat expressing PMR4 taggedwith four copies of theMyc epitopeat its C terminus (PMR4-4xMyc). Independent PMR4-4xMyctransgenic lines in the ubac2 and picc backgrounds were againcrossed with the wild type and their respective mutants to gen-erate transgenic lines that contained the same PMR4-4xMyctransgene integrated at the same genome site but differed inthe zygosity of the ubac2a/2b or picc mutations. The transcriptlevels of PMR4 were not significantly by the zygosity of theubac2a/2b or picc mutations in the progeny (Supplemental Fig-ure 5). On the other hand, immunoblotting showed that thePMR4-4xMyc protein levels were 4 to 6 times higher in the heterozygousUBAC2a/2b+/2 and PICC+/2 progeny than in their respectivehomozygous ubac2a/2b+/2 and picc+/2 progeny (Figure 13).These results support that mutations of UBAC2a/2b and PICCreduce PMR4 protein accumulation.

The Role of UBAC2 in PTI is Mediated through a PathwayThat Differs from the HRD3 and ATI3 Pathways

As a highly conserved protein, UBAC2was initially identified fromproteomic profiling of human ERAD complexes and has beenimplicated in mammalian ERAD through its action with the Hrd1and gp78 ubiquitin ligases (Christianson et al., 2012; Zhang et al.,2015b). In Arabidopsis, two mutated forms of the BRASSINOS-TEROID INSENSITIVE 1 (BRI1) brassinosteroid (BR) receptorkinases, bri1-5 and bri1-9, are retained in the ER by ERQC anddegraded by ERAD (Hong et al., 2008, 2009; Jin et al., 2009; Suet al., 2011, 2012).To determine the possible association of the roles of plant

UBAC2s in PTI and in ERAD,we first examinedwhethermutationsof theUBAC2genescould suppress themutantphenotypesof thebri1-5 and bri1-9 mutants by blocking ERAD of the bri1-5 andbri1-9mutant proteins. For this purpose,wegeneratedubac2a/2bbri1-5 and ubac2a/2b bri1-9 triple mutants but failed to observeany improved growth of themutant bri1mutants in the ubac2a/2bmutant background (Supplemental Figure 6). This result arguedagainst a role of theUBAC2proteins in the ERQCandERADof thebri1-5 and bri1-9 mutant proteins. We also found that a knockoutmutant for the Arabidopsis HRD3 protein required for ERAD of thebri1-5andbri1-9mutantproteinswasnormal inPTIandpathogen/PAMP-induced callose deposition (Supplemental Figure 7).We have recently reported that Arabidopsis UBAC2 proteins

interact with the ATI3 selective autophagy receptors and medi-ate a novel selective autophagy pathway in targeting spe-cific unknown ER proteins during plant responses to heat andnecrotrophic fungal pathogens (Zhou et al., 2018). To determinewhether the role of UBAC2 in plant PTI is mediated through theATI3 selective autophagy pathway, we determined the responseof the ati3a/3b/3c triple mutant to PstDC3000 hrcC but found thatthe mutant supported similar levels of the bacterial pathogen aswild-type plants (Supplemental Figure 6A). Furthermore, patho-gen- andPAMP-induced callose depositionwasnormal in the ati3triple mutant (Supplemental Figure 7B). These results indicatedthat UBAC2 proteins play a critical role in plant PTI and pathogen-inducedcallosedeposition throughamechanismdistinct from the

Figure 9. Compromised Callose Deposition of picc Mutants and theRestoration by PMR4 Overexpression.

(A) Callose deposition triggered by PstDC3000 hrcC. Five-week-oldArabidopsis plants were infiltrated with 10 mM Mg2Cl (mock) or 5 3 107

colony-formingunits (cfu)/mLPstDC3000hrcC (hrcC ).Callosedepositionswere assayed at 16 h after infiltration. Bar, 20 mm.(B) Callose deposits triggered by PstDC3000 hrcC (hrcC ).(C) Callose deposits triggered by 1 mM flg22 for 16 h.(D) Callose deposits triggered by 1 mM elf18 for 16 h. Means and SE ofcallosedepositspermicroscopicfield (1253100mm)werecalculated frommore than 10microscopic fields per treatment per genotype. According toDuncan’s multiple range test (P = 0.01), means of callose deposits do notdiffer if they are indicated with the same letter. The experiments wererepeated at least three times with similar results.

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previously characterized Hrd3 ERAD and ATI3 selective au-tophagy pathways.

DISCUSSION

In eukaryotic cells, about one-third of total cell proteins aresynthesized on ER-associated ribosomes and, therefore, the ERcontains a complex network of proteins that have evolved tomediate translocation, folding, modification, and degradation ofa large number of structurally diverse proteins (Pfeffer et al., 2017).Many proteins involved in plant immune responses, includingPRRs and extracellular defense-related proteins, are synthesizedon the ER, go through the ERQC, and ultimately are transported totheir destinations through the secretory pathway. As a result, theER has been increasingly recognized as a critical subcellularcompartment that regulates the plant immune system throughcontrolling the biogenesis and associated trafficking of importantcomponentsofplant immune responses (Liebrandetal., 2012).SAand NON-EXPRESSER OF PATHOGENESIS-RELATED GENES1-mediated defense gene expression during the establishmentof systemic acquired resistance, for example, is dependent onthe upregulation of both the ER-associated protein folding/modification machinery and the secretory pathway (Wang et al.,2005). In addition, specific components of the ERQC are requiredfor thebiogenesisand functionofanumberofplasma-membrane-localized PRRs from different plants including EFR from Arabi-dopsis (Li et al., 2009; Lu et al., 2009; Nekrasov et al., 2009; Saijoet al., 2009; vonNumersetal., 2010),Xa21andOsCERK1 fromrice(Chen et al., 2010; Park et al., 2013), IRK from N. benthamiana(Caplan et al., 2009), and Cf from tomato (Liebrand et al., 2012).In this study, we have discovered that two Arabidopsis

homologs of UBAC2, a conserved ER protein implicated in ERQCand ERAD, play a critical role in PTI, based on the highly com-promised responses of the ubac2a/2b double mutants toPstDC3000 hrcC (Figure 1B) and the defects in PAMP-inducedresistance to PstDC3000 (Figure 2). Unlike previously reportedmutants defective in the CNX/CRT cycle, the ubac2mutants havenormal biogenesis of FLS2 and EFR and associated signaling anddefense responses including MAPK activation, ROS generation,

Figure 10. Overexpression of PICC and the Effect on PTI and flg22-Induced Callose Deposition in the ubac2a/2b Mutant.

(A) Accumulation of mCherry-PICC. A PICC-mCherry construct was firstintroduced into the ubac2a-1/2b-1 double mutant, and two transgeniclines (L7 and L9) were selected for crossing to both the wild type (WT)and ubac2a/2b double mutant. For each transgenic line, the crossinggenerated two types of F1 progeny that contained the same mCherry-PICC transgene but differed in the zygosity for the UBAC2a and UBAC2bgenes: theheterozygousUBAC2a/2bubac2a/2b (UBAC2a/2b+/2) progenyand the homozygous ubac2a/2b ubac2a/2b (ubac2a/2b2/2) progeny. ThemCherry-PICC signals from the two types of progeny for each of the two

transgenic lines were observed by confocal fluorescence microscopy.

Bar, 10 mm.

(B) Growth of PstDC3000 hrcC. Wild-type (WT), ubac2a/2b, and ubac2/ubac2 mutant plants overexpressing mCherry-PICC were infiltrated withPstDC3000 hrcC (hrcC ). Leaf samples were collected at 0 and 4 dpi fordetermining bacterial colony-forming units (cfu). Means and SE were cal-culated from 10 plants for each treatment. According to Duncan’smultiplerange test (P = 0.01), means of cfu do not differ if they are indicatedwith thesame letter.(C) flg22-induced callose deposition. Wild-type (WT), ubac2a/2b, andubac2a/2bmutant plants overexpressingmCherry-PICCwere treatedwithwater or 1 mM flg22. After 16 h, callose deposits were determined. Meansand SE of callose deposits per microscopic field (125 3 100 mm) werecalculated from more than 10 microscopic fields per treatment per ge-notype. According to Duncan’s multiple range test (P = 0.01), means ofcallose deposits do not differ if they are indicated with the same letter. Theexperiments were repeated at least three times with similar results.

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and PTI-associated gene expression (Figure 3). The normal sig-naling and defense gene expression of the ubac2 mutants areconsistent with their normal sensitivity to flg22 and elf18 forseedling growth inhibition (Supplemental Figure 1). However, theubac2 mutants were compromised in pathogen- and PAMP-induced callose deposition (Figure 4). The UBAC2 proteins in-teract with the plant-specific PICC protein (Figure 7), which is alsolocalized in the ER and is important for PTI but dispensable for PTI-associated signaling and defense gene expression (Figure 8;Venkatakrishnanetal.,2013).Likeubac2mutants,compromisedPTIof the picc mutants was associated with defects in pathogen- andPAMP-induced callose deposition (Figure 9). Therefore, the evolu-tionarily conserved UBAC2 and plant-specific PICC proteins arecritical components of anERpathwaywith an important role in plantimmunity by regulating pathogen-induced callose deposition.

Thecompromisedphenotypesof theubac2andpiccmutants inPTI and pathogen/PAMP-induced callose deposition could be

rescued by overexpression of PMR4 (Figures 6 and 8). Theseresults suggest that disruption of UBAC2 and PICC genes mayreduce the accumulation of PMR4 callose synthase in the ubac2and piccmutants, leading to defects in pathogen/PAMP-inducedcallose deposition and compromised PTI. Indeed, by comparingfluorescent signals between heterozygous and homozygousmutants for the ubac2a/2b or picc mutations that expressed thesame PMR4-GFP or PMR-4xMyc transgene, we discovered thatdisruption of UBAC2 or PICC reduced the levels of plasma-membrane-localized PMR4 (Figures 11 to 13).Through comparison of transgenic ubac2 and picc lines ex-

pressing the PMR4-GFP or PMR4-4xMyc transgenes, it becamefurther apparent that UBAC2 and PICC modulate but not com-pletely control the accumulation of PMR4. In the transgenic ubac2and picc lines expressing a sufficient level of the PMR4-GFPtransgene, plasma-membrane-associated PMR4-GFP signalswere substantially reduced when compared with those in the

Figure 11. Disruption of UBAC2a and UBAC2b Reduced Accumulation of PMR4-GFP.

(A)Accumulation of PMR4-GFP. APMR4-GFP construct was first introduced into the ubac2a-1/2b-1 doublemutant and two independent transgenic lines(L1 and L2) with increased expression of PMR4 based on RT-qPCR andGFP signals from confocal fluorescencemicroscopy were selected for crossing toboth the wild type (WT) and ubac2a/2b double mutant to generate two types of progeny that contain the same PMR4-GFP gene for each line: the het-erozygousUBAC2/ubac2 (+/2) progeny and the homozygous ubac2/ubac2 (2/2) progeny. ThePMR4-GFP signals from the two types of progeny for eachof the two lines were compared by confocal fluorescence microscopy. Bar, 10 mm.(B) Flg22-induced callose deposition. Wild-type (WT), ubac2a/2b, and the ubac2/ubac2 homozygous progeny lines expressing PMR4-GFP at high or levellevelswere treatedwithwater or 1mMflg22.After 16h, callosedepositsweredetermined.Meansand SEof callosedeposits for eachmicroscope field (1253100mm)were calculated frommore than 10microscopic fields per treatment per genotype. According to Duncan’smultiple range test (P = 0.01), means ofcallose deposits do not differ if they are indicated with the same letter.

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heterozygous UBAC2a/2b+/2 or PICC/picc genetic backgroundbut were still detectable (Figures 11A and 12A).

Furthermore, flg22-induced callose deposition of these highexpressor lines in the homozygous ubac2 and picc mutantbackgroundswasalmost fully restored (Figures 11B).On theotherhand, in the relatively low expressor lines for the PMR4-GFPtransgene, flg22-induced callose deposition was only partiallyrestored in the homozygous ubac2a/2b2/2 or picc/picc mutantbackgrounds (Figures11Band12B).Thus,disruptionofUBAC2orPICC reduced but did not completely abolish the accumulation ofPMR4-GFP. As a result, when the PMR4-GFP transgene wasexpressed at relatively high levels, the reduced levels of the PMR4callose synthase in the ubac2a/2b mutant background were ap-parently still sufficient for PAMP-induced callose deposition (Fig-ures 11). On the other hand, when the expression levels of thePMR4-GFP transgene were low, functional UBAC2 and PICCproteinsplayacritical role inpromotingaccumulationofPMR4-GFP

for PAMP-induced, PMR4-dependent callose deposition (Figures11 and12). Based onboth the analysis ofPMR4 transcripts and thefailure todetectfluorescencesignalsofPMR4-GFPundercontrolofitsnativepromoter, theexpression levelsof thenativePMR4gene inArabidopsis are even lower than those PMR4-GFP low-expressorlines tested in this study. With such low expression levels of thenativePMR4gene, UBAC2andPICCwould be highly important forpositive modulation of the PMR4 protein levels for pathogen/PAMP-induced callose deposition and PTI.As a large transmembrane protein, PMR4 belongs to the family

of glucan synthases. Glucan synthases include plant cellulosesynthases, which are synthesized on the ER and go through theERQC system for proper folding and modification before beingassembled intocomplexes in theGolgi apparatus and transportedto the plasma membrane through the Golgi- and post-Golgitrafficking system (Bashline et al., 2014). In mammalian cells,the ER-localized UBAC2 interacts with UBXD8 in a protein

Figure 12. Disruption of PICC Reduced Accumulation of PMR4-GFP.

(A) Accumulation of PMR4-GFP. A PMR4-GFP construct was first introduced into the piccmutant and two independent transgenic lines (L1 and L2) withincreased expression of PMR4 based on RT-qPCR and GFP signals from confocal fluorescence microscopy were crossed to both the wild type (WT) andpicc mutant to generate two types of progeny that contain the same PMR4-GFP gene for each line: the heterozygous PICC/picc progeny and the ho-mozygous picc/picc progeny. The PMR4-GFP signals from the two types of progeny for each of the two lines were compared by confocal fluorescencemicroscopy. Bar, 10 mm.(B)Flg22-induced callose deposition.Wild-type (WT),picc, and thepicc/picc homozygous progeny lines expressingPMR4-GFP at high or level levelsweretreated with water or 1mM flg22. After 16 h, callose deposits were determined. Means and SE of callose deposits were calculated frommore than 10 1253100mmmicroscopic fields per treatment per genotype. According to Duncan’smultiple range test (P = 0.01), means of callose deposits do not differ if theyare indicated with the same letter.

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complex that also contains the ER-membrane-anchored gp78 E3ubiquitin ligase in the ERAD (Christianson et al., 2012). In Arabi-dopsis,UBAC2proteins also interactwith theselectiveautophagyreceptors ATI3s and target unknown ER proteins in plant stressresponses (Zhou et al., 2018). However, unlike previously char-acterized ERAD components from Arabidopsis, disruption ofUBAC2 genes did not block the ERAD of the bri1-9 and bri1-5mutant proteins (Supplemental Figure 6), two ER-retained mu-tated BR receptors (Su et al., 2011). On the other hand, loss-of-function mutation of Hrd3, a critical component of the Hrd1 E3ligase complex in ERAD, blocks the ERAD of bri1-9 and bri1-5 (Suet al., 2011) but did not affect plant PTI or pathogen-inducedcallose deposition (Supplemental Figure 7). Therefore, the role ofUBAC2 in the accumulation ofPMR4proteins is not dependent onHrd1/Hrd3-mediated ERAD.

Likewise, the mutant plants for the selective autophagy re-ceptors ATI3s were normal in plant PTI and pathogen-inducedcallosedeposition (Supplemental Figure 7), indicating that the roleof UBAC2s in plant PTI and pathogen-induced callose depositionis not mediated by the ATI3 selective autophagy pathway. In-terestingly, the functional C-terminal UBA domain of UBAC2s isrequired for their role in plant PTI and pathogen-induced callosedeposition (Figure 5). Thus, the action of UBAC2 proteins inpromoting accumulation of the PMR4 callose synthase may in-volveprotein ubiquitination,which is often associatedwithproteinturnover (Weissman, 1997; Lu and Hunter, 2009).Several possible mechanisms could account for the apparently

paradoxical roles of UBAC2 in ERQC/ERAD and in promotingPMR4 accumulation. First, UBAC2 and PICC-mediated path-way may target degradation of a negative regulator of PMR4

Figure 13. Disruption of and UBAC2 and PICC Reduced Accumulation of PMR4-4xMyc.

A PMR4-4xMyc construct was first introduced into the ubac2a/2b and picc mutants. Two independent transgenic lines (L1 and L2) from each mutantbackgroundwith increased expression of PMR4 based on RT-qPCRwere crossed to both the wild type (WT) and their respective mutants to generate twotypes of progeny for each line that contain the same PMR4-4xMyc gene but differ in the zygosity of the ubac2a/2b or piccmutations. The protein levels ofPMR4-4xMyc in the two types of progeny for each line were compared by immunoblotting using an anti-Myc monoclonal antibody. Rubisco large subunitproteins stained with Ponceau S as loading control and the migration positions of two molecular weight markers of 250 and 130 kD were also shown. Therelativedensitiesof thePMR4-mycproteinbands fromthe immunoblotsweremeasuredby ImageJagainst theRubisco loadingcontrol for each laneandareshown below the blots. The experiments were repeated twice with very similar results.

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accumulation to increase the accumulation of the callose syn-thase in plant cells. Second, misfolded proteins require promptremoval through refolding or degradation to prevent them frombindingnonspecifically tootherproteins to further increaseproteinmisfolding (Jackson and Hewitt, 2016). UBAC2/PICC-mediatedERQC could specifically target misfolded proteins in the ER toprevent their interference with the folding and modification ofPMR4 proteins, thereby promoting the accumulation and foldingof the callose synthase. Given the normal phenotype of the hrd3mutant plants in PTI and pathogen-induced callose synthase,UBAC2-mediated removal of potential negative regulators ofPMR4 biogenesis and accumulation would involve an ERmembrane-localized ligase other than the well-characterizedHRD1/HRD3 E3 complex in Arabidopsis. Alternatively, the inter-actingUBAC2andPICCproteins couldbe involved in theERQCofPMR4 for its biogenesis and trafficking through a mechanismdistinct from the established ERAD pathways. Studies in mam-malian cells have shown that theUBAC2 in theERalso plays a rolein cellular energy homeostasis through controlling cytoplasmiclipid droplet turnover (Suzuki et al., 2012; Wang and Lee, 2012;Olzmann et al., 2013). Polymorphisms in the UBAC2 gene havealso been associated with Behcet’s disease, a systemic in-flammatory disorder in human (Sawalha et al., 2011; Hou et al.,2012; Yamazoe et al., 2017). Given the diverse and complex rolesof UBAC2 proteins, it is likely that the evolutionarily conserved ERproteins participate in a range of cellular andmolecular processesthrough distinct mechanisms and pathways.

Callose was among the earliest components reported to beassociatedwith the formation of papillae, one of the first observedplant defenses that has been extensively analyzed overmore than150 years at the molecular and cellular levels (Voigt, 2014). Morerecent studies have revealed critical roles of callose deposition inplant immune responses to fungal, bacterial, and viral pathogens(Kim et al., 2005; Li et al., 2012; Ellinger et al., 2013, 2014; Voigt,2016). Our studies with Arabidopsis UBAC2 and PICC providefurther evidence for a critical role of pathogen-induced callosedeposition in plant innate immunity. Intriguingly, loss-of-functionmutations of PMR4 leads to hyperactivated SA responses(Nishimura et al., 2003). The inducible nature of PMR4-dependentcallose depositionmay also reflect potential detrimental effects ofconstitutive PMR4-dependent callose overproduction on plantgrowthanddevelopmentgiven theestablishedactionof callose toimportant plant cellular structures including cell wall and plas-modesmata. Therefore, PMR4-dependent callose depositionrequires tight regulation as an effective defense mechanismwithout causing harmful effects in plant growth and development.

Studies over the past decade have shown that regulation ofpathogen-induced callose deposition involve inducible tran-scription of the PMR4 gene (Jacobs et al., 2003), controlledtrafficking (Kulich et al., 2018), and activation of the PMR4 callosesynthase (Ellinger et al., 2014). In addition, the protein levels of thePMR4 callose synthase are subjected to regulation by the inter-acting UBAC2 and PICC proteins in the ER (Figures 11 and 12).Given the PAMP-induced nature of thePICC gene (Venkatakrishnanetal.,2013), theUBAC2/PICC-mediatedERmechanismcouldalsobe responsive topathogen infection for increasedaccumulationofPMR4during theactivationofplant immuneresponses.Therefore,as a critical defense mechanism, pathogen-induced callose

deposition is under regulation by a complex network of regulatorymechanisms. Further analysis of these regulatorymechanisms forthe synthesis, assembly, trafficking, andactivation of PMR4couldprovide new important insights into the molecular basis of theplant immune system.

METHODS

Arabidopsis Genotypes and Plant Growth Conditions

The Arabidopsis (Arabidopsis thaliana) mutants and wild-type plants usedin the study are all in the Col-0 background. The uba2a-1, uba2a-2, anduba2b-1 mutants have been previously described (Zhou et al., 2018).Homozygous picc1-1 (Salk_205507C), picc1-2 (WiscDsLox447A08), fls2(Salk_062054), efr1 (Salk_044334), mpk3 (Salk_151594), and mpk6(Salk_062471) mutants were identified by PCR using primers flanking theT-DNA insertions (Supplemental Table). The pmr4-1 mutant has beenpreviously described (Nishimura et al., 2003). Arabidopsis and Nicotianabenthamianaplantsweregrown ingrowthchambersat22°C,120mEm22s21

light (Philips T5 daylight white high fluorescent tubes) on a photoperiod of12-h light and 12-h dark.

Plasmid Construction and Generation of TransgenicArabidopsis Plants

The FLS2- and EFR-YFP constructs have been previously described(Häweker et al., 2010). For generation of myc-taggedUBAC2a genes, full-length UBAC2a coding sequence was PCR amplified using gene-specificprimers (59-agcgtcgacatgaacggcggtccctcc-39and59-agcttaattaagtgggactgtgcttcgagaagga-39). The gene for the UBAC2aM262A/L288A protein de-ficient inubiquitinbindingwasgeneratedbyoverlappingPCRaspreviouslydescribed with appropriate primer and confirmed by DNA sequencing.Bothwild-typeUBAC2aandmutantUBAC2aM262A/L288Ageneswere fusedwith amyc-tag in aplant transformation vector. For generation ofmCherry-tagged PICC gene, full-length PICC coding sequence was PCR amplifiedusing PICC-specific primers (59-agcggatccatggaagaagcaactcaagtaacg-39 and 59-atctctagattaatactttctcccaagaattataccg-39). The amplified PICCfragment was fused with anmCherry tag sequence behind theCauliflowermosaic virus (CaMV ) 35S promoter in a modified pCAMBIA1300 planttransformation vector. For generation of PMR4-GFP fusion construct, theN-terminal half of the PMR4 coding sequence was amplified using gene-specificprimers (59-agcgtcgacatgagcctccgccaccgca-39and59-ccgtctcgcctctagattca-39) and cloned between the CaMV 35S promoter and a GFPgene in a modified pCAMBIA1300 plant transformation vector. TheC-terminal half of the PMR4 coding sequence was then PCR-amplifiedusing gene-specific primers (59-acagcgtccctgtgaatcta-39 and 59-agcttaattaagacatcgccttttgatttcttc-39) and cloned between behind the N-terminalhalf of the PMR4 coding sequence in the plant transformation vector. Thefull-length PMR4 coding sequence was also fused with a 4xMyc tag ina plant transformation vector. The resulting constructs were introducedinto the Agrobacterium tumefaciens (strain GV3101) and transformed intoArabidopsis by floral-dip method (Clough and Bent, 1998).

Pathogen Inoculation

Bacterial inoculation by leaf infiltration was performed as previously de-scribed (Zhengetal., 2009). Inoculated leaveswerehomogenized in10mMMgCl2 and diluted before plating on King’s B Agar with appropriate anti-biotics. Colony-forming units were determined 2 to 3 days after bacteriagrowth at room temperature. For determining PAMP-induced diseaseresistance,Arabidopsis leaveswerefirst infiltratedwithwater or 1mMflg22,and PstDC3000 inoculation was performed 1 d after the pretreatment.

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Preparation and Transfection of Leaf Protoplasts

Biogenesis of PRRs FLS2 and EFR was analyzed in Arabidopsis leafprotoplasts. Leaf protoplasts were prepared using the tape-Arabidopsis-sandwich procedure (Wu et al., 2009). Approximately 53 104 protoplastswere used in transfection using 20 mg FLS2-YFP and EFR-YFP plasmidDNA. YFP signals were observed with confocal fluorescence microscopyusing a Leica confocal microscope (excitation 514 nm, emission 525 to555 nm).

MAP Kinase Assay and Immunoblotting

Arabidopsis 14-d-old seedlings were treated with water, 100 nM elf18, orflg22. Proteinswere extracted from the treated seedlings and separated bySDS-PAGE. The separated proteins were blotted onto polyvinylidene di-fluoride membrane (Bio-Rad), and the activated MAP kinases were de-tected using an anti-p42/44 MAPK monoclonal antibody (Cell SignalingTechnology). Immunoblotting analysis of Myc-tagged UBAC2 and PMR4proteins in the transgenic plants was performed using an anti-mycmonoclonal antibody (Sigma).

ROS Assays

Twelve leaf discs (4-mm diameter) of four 3- to 4-week-old plants werecollected and floatedovernight on sterilewater in awhite 96-well plate. Theleaf discswere then treatedwith 1 mMelf18 or flg22 in a solution containing17 mg/mL (w/v) luminol (Sigma) and 10 mg/mL horseradish peroxidase(Sigma). Luminescence was captured using infinite M200 PROmultimodeplate reader (TECAN).

RT-qPCR

Arabidopsis seedlings were treated with water, 100 nM flg22, or elf18 for30 min. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen)followed by DNase treatment with Turbo DNA-free. RNA was quantifiedwith aNanodrop spectrophotometer (Thermo Fisher Scientific). cDNAwassynthesized from 2.5 mg total RNA using SuperScript III reverse tran-scriptase (Invitrogen). Transcript levels were determined by RT-qPCRusing gene-specific primers: WRKY33 (59-gctgctattgctggtcactcc-39 and59-ggtctcctcgtttggttcttcc-39), CYP81F2 (59-gcccgagaagtttatgcctgag-39and 59-caacgaacctaaagccaacaatacc-39), PMR4 (59-agctgtcggaatcgaagagg-39 and 59-ggacgacgaaggtctccaac-39) with ACTIN2 as an internalcontrol.

Seedling Growth Inhibition

Seedling growth inhibition assay was performed as described(Schwessinger et al., 2011). In brief, fresh harvested seeds were surfacesterilized, sown on 1/2 Murashige and Skoog plate, stratified for 2 d at 4°Cin dark. Five-day old Arabidopsis seedlings were transferred into liquid 1/2Murashige andSkoogmediumwith or without flg22 or elf18 and incubatedfor another 8 d before measuring seedling fresh weight. The weight of sixreplicates per treatment was measured for analysis.

Analysis of Callose Deposition

Analysis of pathogen/PAMP-inducedcallosedepositionwasperformedasprevious described with minor modification (Kulich et al., 2015). In brief,leavesof5-week-oldArabidopsiswere infiltratedwitheither 53107cfu/mLPstDC3000 hrcC, 1 mM flg22, or elf18, and leaf discs were taken 16 h afterthe infiltration. The leaf discs were cleared overnight by incubation in 1:3acetic acid/ethanol at room temperature and then washed in 150 mM K2

HPO4, pH 9.5. Cleared leaves were stained in a solution containing 0.01%aniline blue (Sigma-Aldrich) in 150 mMK2HPO4, pH 9.5, in dark for at least

30 min before being mounted in 50% glycerol. Pictures were taken withNikon eclipse E800 epifluorescence microscopy, and callose depositswere quantified from more than 10 microscopic fields (1253 100 mm) pertreatment per genotype.Callose depositswere counted using the “analyzeparticles” function of ImageJ.

Yeast Two-Hybrid Screen and Assays

Gal4-based yeast two-hybrid system was utilized for identification ofUBAC2a-interacting proteins, as described previously (Zhou et al., 2018).In brief, full-length UBAC2a coding sequence was PCR-amplified usinggene-specific primers (59-agcgaattcatgaacggcggtccctcc-39 and 59-agcgtcgacttagtttctgtcgaatcccatt-39) and cloned into pBD-GAL4 vector togenerate thebait vector. TheArabidopsisHybridZAP-2.1 two-hybridcDNAlibrary, which was prepared from Arabidopsis plants, has been describedpreviously (Zhou et al., 2018). The bait plasmid and the cDNA library wereused to transform yeast strain YRG-2. Yeast transformants were platedonto selection medium lacking Trp, Leu, and His and confirmed byb-galactosidase activity assays using o-nitrophenyl-b-D-galactopyrano-side as substrate.

BiFC Assays

The full-length UBAC2a, PICC, and PICL coding sequences were PCR-amplified using gene-specific primers (UBAC2a, 59-agcgagctcatgaacggcggtccctcc-39 and 59-agctctagagtgggactgtgcttcgaga-39; PICC, 59-agcggatccatggaagaagcaactcaagtaacg-39 and 59-atctctagaatactttctcccaagaattataccg-39; PICL, 59-agctctagaataatttttcccaacaatgatacc-39 and 59-agctctagatcaataatttttcccaacaatgatac-39) and cloned into pFGC-C-YFP orpFGC-N-YFP, as appropriate. The plasmids were introduced into A. tu-mefaciens (strain GV3101) and infiltration into Nicotiana benthamiana asdescribed previously (Zhou et al., 2018). BiFC fluorescent signals in in-fected tissues were analyzed at 24 h after infiltration with a Nikon A1 Rsiconfocal microscope with appropriate filter sets (excitation 514 nm,emission 524 to 555 nm).

Confocal Fluorescence Microscopy

Imaging of GFP and mCherry signals was performed with a Nikon A1 Rsiconfocal laser microscope with appropriate filter sets: GFP (excitation 488nm,emission500 to550nm)andmCherry (excitation561nm,emission570to 620 nm). Specific settings for the confocal microscopy include thefollowing: optical section, 2.92 mm; optical resolution, 0.4 mm; pixel dwelltime, 2.2ms; laser power, 5%; pinhole, 52.36mm;HVgain, 90 to 120; offsetbackground adjustment, 0.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibrary under the following accession numbers: AT3g56740 (UBAC2a),AT2g41160 (UBAC2b),AT2g32240 (PICC),AT1g05320 (PICL),AT4g03550(PMR4), AT1g18260 (Hrd3), AT4g26750 (LIP5), AT1g17780 (ATI3a),AT1g73130 (ATI3b), AT2g16575 (ATI3c), AT2g38470(WRKY33),AT5g57220 (CYP81F2), AT4g39400 (BRI1), AT5g46330 (FLS2), AT5g20480(EFR), AT3g18780 (ACTIN2).

Supplemental Data

Supplemental Figure 1. PAMP inhibition of Arabidopsis seedlinggrowth.

Supplemental Figure 2. BiFC analysis of UBAC2b interactionwith PICC.

168 The Plant Cell

Supplemental Figure 3. Effect of mCherry-PICC expression on PTIand pathogen-induced callose deposition in the picc mutant.

Supplemental Figure 4. Accumulation of UBAC2a and effect of itsoverexpression on PTI and flg22-induced callose deposition in thepicc mutant.

Supplemental Figure 5. Disruption of UBAC2 and PICC did not affectaccumulation of PMR4 transcripts.

Supplemental Figure 6. Mutations of UBAC2a and UBAC2b fail tosuppress bri1-5 or bri1-9 mutant phenotypes.

Supplemental Figure 7. Phenotypes of ati3, hrd3a, and ubac2mutants in PTI and callose deposition.

Supplemental Table. Primers for identification of Arabidopsismutants.

ACKNOWLEDGMENTS

We thank Dr. Justin Lee of Leibniz Institute of Plant Biochemistry and Dr.Antje von Schaewen of Westfalische Wilhelms University in Germany forthePXCSG-FLS2-YFPandPXCSG-EFR-YFP constructs andDr. IrisMeierof Ohio State University for the PICC-GFP and PICL-GFP constructs. Wethank Drs. Tesfaye Mengiste and Chao-Jan Liao for the help with theassays of ROS and Dr. Yun Zhou for the help with confocal fluorescencemicroscopy. We thank the Arabidopsis Resource Center at the Ohio StateUniversity for the Arabidopsis mutants. This work is supported by the U.S.National Science Foundation (grant no. IOS1456300 and IOS-1758767).

AUTHOR CONTRIBUTIONS

Z.C. conceived the research plan and designed the experiments; Z.W. andX.L performed the majority of the experiments; X.W., N.L., B.X., Q.P.,Z.G., and B.F. performed some of the experiments; Z.W., C.Z., andZ.C. analyzed the data; Z.W. and Z.C. wrote the article with contribu-tions by all authors.

Received April 26, 2018; revised November 26, 2018; accepted December27, 2018; published January 3, 2019.

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The Plant Cell 171

DOI 10.1105/tpc.18.00334; originally published online January 3, 2019; 2019;31;153-171Plant Cell

Zhu and Zhixiang ChenZhe Wang, Xifeng Li, Xiaoting Wang, Nana Liu, Binjie Xu, Qi Peng, Zhifu Guo, Baofang Fan, Cheng

COILED-COIL to Regulate Pathogen-Induced Callose Deposition and Plant ImmunityArabidopsis Endoplasmic Reticulum-Localized UBAC2 Proteins Interact with PAMP-INDUCED

 This information is current as of March 28, 2021

 

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References /content/31/1/153.full.html#ref-list-1

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