BREAKTHROUGH REPORT

Spatiotemporal Monitoring of Pseudomonas syringaeEffectors via Type III Secretion Using Split FluorescentProtein FragmentsOPEN

Eunsook Park,a,1 Hye-Young Lee,a,b Jongchan Woo,a Doil Choi,b,c and Savithramma P. Dinesh-Kumara,1

a Department of Plant Biology and the Genome Center, College of Biological Science, University of California, Davis, California 95616bDepartment of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul, Koreac Plant Genomics and Breeding Institute, Seoul National University, Seoul, Korea

ORCID IDs: 0000-0003-2984-3039 (E.P.); 0000-0003-3797-889X (H.-Y.L.); 0000-0002-2994-6444 (J.W.); 0000-0002-4366-3627(D.C.); 0000-0001-5738-316X (S.P.D.-K.)

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type IIIsecretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellularcompartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP preventseffector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder greenfluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS intoplant cells. In this system, effectors are fused to 11th b-strand of super-folder GFP (sfGFP11), and when delivered into plant cellsexpressing sfGFP1-10 b-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number ofArabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localizationof sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringaeeffectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT systemdescribed here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

INTRODUCTION

A number of gram-negative bacteria are pathogenic to animalsand plants. These bacteria are taxonomically diverse and infecta range of hosts. However, irrespective of the host they infect,most pathogenic bacteria use the type III secretion system (T3SS)to take advantage of the host cells (He et al., 2004). The T3SSmachinery is afilamentoussupramolecular structure thatprovidesa channel through which type III effector (T3E) proteins are se-creted into the host cells to manipulate host defense responsesagainst bacteria (Büttner, 2016). The first T3SS-associated fila-mentous structure was discovered in Pseudomonas syringae,a plant pathogen with a broad host range including several im-portant crop species (Jin and He, 2001; Xin and He, 2013; Galánet al., 2014; Büttner, 2016). Our current understanding of detailedmolecular and biochemical properties of the T3SS machinerycome from studies of human pathogenic bacteria such asSalmonella and Yersinia (Galán et al., 2014).

T3Es are important for the pathogenicity of the bacteria and theyplay an important role in suppressing the first line of plant defenseresponses (Xin and He, 2013; Büttner, 2016). Several studies havedemonstrated that the effector proteins from pathogenic bacteriaare targeted to specific subcellular compartments in cells, wherethey alter the physiological properties of the cell and suppress innateimmunity (AlfanoandCollmer,2004;KayandBonas,2009;Choietal.,2013; Aung et al., 2017). Thus, the spatiotemporal localization of theeffectors is crucial for their function inside the host cell. However, thesubcellular dynamics of effectors directly delivered frombacteria intohost cells are largely unknown because of experimental difficulties,such as small amount of effector proteins secreted from the bacteriaand incompatibility of fluorescent tags with the T3SS machinery(Galán, 2009). In plants,most of the localization studies of T3Es havebeen performed in Agrobacterium tumefaciens-mediated transientexpression systems using constitutive promoters (Block and Alfano,2011; Macho, 2016). However, several biochemical studies revealedbacterial effectors undergo posttranslocational modification forproper targeting in the host cells (Boucrot et al., 2003; Reinicke et al.,2005; Patel et al., 2009; Fernández-Álvarez et al., 2012). Therefore,Agrobacterium-mediated expressions may not reflect dynamic lo-calization of respective effectors originally secreted by T3SS.Monitoring of T3E directly secreted from Salmonella has been

described using the self-assembling split green fluorescentprotein (GFP) system (Van Engelenburg and Palmer, 2010). GFPis a b-barrel protein with 11 b strands. This structure can be splitinto two fragments, 1-10 b strands (GFP1-10) and the 11th b

1 Address correspondence to spdineshkumar@ucdavis.edu or espark@ucdavis.edu.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Savithramma P.Dinesh-Kumar (spdineshkumar@ucdavis.edu) and Eunsook Park(espark@ucdavis.edu).OPENArticles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.17.00047

The Plant Cell, Vol. 29: 1571–1584, July 2017, www.plantcell.org ã 2017 ASPB.

strand with 13 amino acids (GFP11) (Cabantous et al., 2005;Supplemental Figure 1A). GFP1-10 is nonfluorescent becausethe conserved E222 residue in the GFP11 strand is critical forchromophorematuration (Barondeauetal.,2003).OnceGFP1-10andGFP11 exist in close proximity, the two fragments assemble a barrelstructure and then emit fluorescence. The original split GFP system(Ghosh et al., 2000) has been further engineered to improve proteinfolding kinetics and solubility to enhance fluorescence intensity(Cabantousetal., 2005;Pédelacqetal., 2006;Cabantousetal., 2013).For better protein folding efficiency, super-folder GFP (sfGFP) wasengineered for split GFP system (Pédelacq et al., 2006). However, thesolubility of the sfGFP1-10 fragment is poor, resulting in lower fluo-rescence intensity (Cabantous et al., 2005). Therefore, a variant ofsfGFP1-10 was engineered called GFP1-10OPT that significantlyimprovessolubilityandfluorescence intensity (Cabantousetal., 2005;Cabantous et al., 2013). In addition, recently, single amino acid mu-tants of sfGFP1-10OPT were generated that show emission spectrashifted to yellow or cyan color (Kamiyama et al., 2016).

Although the self-assembling split GFP systemhas been recentlyusedasatool tostudysubcellular localizationofmammalianproteins(Kamiyama et al., 2016; Leonetti et al., 2016) and Salmonella T3Elocalization (Van Engelenburg and Palmer, 2010) and to visualizeAgrobacteriumVirE2delivered throughT4SS intoplant cells (Li et al.,2014b), it has not been robustly optimized for plant biology researchincludingT3Edelivery fromplant bacterial pathogens. In addition, anattempt to use the system to visualize fungal pathogen Ustilagomaydis effectors in themaize (Zeamays) pathosystem has not beensuccessful (Tanaka et al., 2015). In this study, we optimized the splitGFP system based on the improved sfGFP1-10OPT to monitorsubcellular localization of T3Es delivered from P. syringae into plantcells. To facilitate localization studies of T3Es to different subcellularcompartments in the plant cells, we generated a set of transgenicArabidopsis thaliana plants that express sfGFP1-10OPT in varioussubcellular compartments. Furthermore, theuseof sfYFP/CFP1-10OPT

andthesplitsfCherrysystemwillallowstudies involvingdynamicandcomplex protein interactions at subcellular compartments. Finally,we provide a comprehensive toolkit to express effector proteinstagged toeithersfGFP11ora tandemrepeatofsfGFP11 inP.syringaeto examine secretion of the T3Es of interest. The seeds of varioustransgenicArabidopsis linesand theplasmids toexpressT3Escanbeobtained fromtheABRC(https://abrc.osu.edu/) andAddgene (https://www.addgene.org) (see Supplemental Table 1 for accession num-bers). The optimized split GFP system will facilitate investigation ofdynamics of effector secretion as well as bona fide localization ofeffectors delivered from bacteria into the host cells. The transgeniclineswe have developedwill also be useful for subcellular localizationstudies of Arabidopsis proteins that will overcome potential pertur-bationontraffickingto thetargetsubcellularcompartmentwhenfusedto full-length fluorescent proteins.

RESULTS

Self-Assembling Split Super-Folder Fluorescent ProteinSystem to Visualize Proteins in Plant Cells

To develop subcellular protein visualization system in plantcells to monitor bacterial effectors using sfGFP1-10OPT andsfGFP11 tag (Supplemental Figure 1A), we first evaluated the

system using Agrobacterium-mediated transient assays inNicotiana benthamiana plants (Wroblewski et al., 2005). Forthis, an expression vector with sfGFP1-10OPT under thecontrol of an Arabidopsis UBIQUITIN10 (UBQ10) promoter(Grefen et al., 2010) and a NOS terminator was generated(Supplemental Figure 1B). In a separate expression vector,mCherry was fused to the sfGFP11 to confirm reconstitution ofsfGFP (Supplemental Figure 1B). Agrobacteria containingsfGFP1-10OPT and mCherry-sfGFP11 constructs were co-infiltrated into 4-week-old N. benthamiana leaves. Two dayspostinfiltration, the tissue was observed by confocal laserscanningmicroscopy (seeMethods fordetails). In tissueexpressingsfGFP1-10OPT and mCherry-sfGFP11, we observed GFP signal inthecytoplasm(Figure1A,panel3).However, therewasnodetectablesignal in the tissues infiltrated only with sfGFP1-10OPT or mCherry-sfGFP11 (Figure 1A, panels 1 and 2; mCherry localization was notimaged.). Tandem repeats of sfGFP11 can amplify the fluorescencesignal in mammalian cells (Kamiyama et al., 2016). Therefore, wetested tandem repeat of sfGFP11 (2xsfGFP11) with sfGFP1-10OPT

and observed increased fluorescence signal in the N. benthamianatissue (Figure 1B). These results indicate that self-assembling splitsfGFPOPT systemcould be used for localization and labeling studiesin the plant cells.To facilitate multicolor imaging in plant cells, we tested com-

plementation of sfGFP11 with sfYFP1-10OPT and sfCFP1-10OPT

describedbyKamiyamaet al. (2016). Expression of sfCFP1-10OPT

with mCherry-sfGFP11 reconstituted the cyan fluorescencesignal in the cytoplasm of N. benthamiana cells (Figure 1C, panel1). Coexpression of sfYFP1-10OPT fused to a signal peptide at theN terminus and an endoplasmic reticulum (ER) retention signal attheC terminus,withmCherry-sfGFP11 targeted to theERshoweda yellow fluorescent signal in the ER (Figure 1C, panel 2; mCherrylocalization was not imaged.). Next, we tested if the split sfCherry(Kamiyamaetal., 2016) is functional in theplantcells.Coinfiltrationof ER-targeted sfCherry1-10 with ER-targeted sfCherry11 fusedto b-glucuronidase (GUS) exhibited fluorescence signal in the ER(Figure 1C, panel 3). Similar to sfGFP1-10OPT (Figure 1A, panel 1),expressionofsfCFP1-10OPTaloneandsfYFP1-10OPTorsfCherry1-10alone targeted to the ER showed no detectable fluorescence(Supplemental Figure 2, panels 1–3). In addition, coexpression ofsfGFP1-10OPT with sfCherry11 targeted to the ER failed to re-constitute fluorescence signal (Supplemental Figure 2, panel 4),confirming that the reconstitution of fluorescence protein occursonly when a specific FP1-10 coexpressed with a corresponding11thb strand. These results indicate that the self-assembling splitsuper-folder fluorescent protein system is a versatile system formulticolor subcellular localization studies in plant cells.

Self-Assembling Split sfGFPOPT System to Visualize ProteinsTargeted to Various Subcellular Compartments

Bacterial effectors are known to target different subcellularcompartments in the plant cells including the cytoplasm, nucleus,ER, and other organelles to alter the physiological state of plantcells to dampenplant immune responses (BlockandAlfano, 2011;Macho, 2016). To visualize and to understand the effects of ef-fectors targeted to the plasma membrane, nucleus, and variousplant organelles, we generated various sfGFP1-10OPT constructs

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(Supplemental Figure 1B). Plasma membrane, Golgi, ER, perox-isomes,mitochondria, and plastid targeting sequences used herehave been previously shown to target FPs to the correspondingorganelle in transgenic Arabidopsis (Nelson et al., 2007). To targetsfGFP1-10OPT to the nucleus, the nuclear localization signal se-quence (Kalderon et al., 1984) was fused to the N terminus ofsfGFP1-10OPT. Since sfGFP1-10OPT does not fluoresce withoutthe sfGFP11 strand, we generated organelle-targeted sfGFP11strand fused to mCherry for better protein folding. Proper or-ganelle targeting of mCherry-sfGFP11 constructs was confirmedby transient expression in N. benthamiana leaves (SupplementalFigure 3).

Transient expressionof sfGFP1-10OPT fused todifferent targetingsequences with sfGFP11 targeted to the same subcellular com-partment in N. benthamiana leaves, reconstituted the sfGFPOPT

signal in respective subcellular compartments (Figure 2). We alsotested whether sfGFP11 targeted to a specific organelle can re-constitute the sfGFP signal at the respective organelle whencoexpressed with cytosolic sfGFP1-10OPT. The mCherry-sfGFP11targeted to the plasma membrane, nucleus, and peroxisome suc-cessfully reconstituted the fluorescence at the respective organellewhen coexpressed with cytosolic sfGFP1-10OPT (Figure 3). In thecaseof thenucleus, it ispossible that thesfGFP1-10thatdiffuses intothe nucleus could be reconstituted with nuclear-targeted sfGFP11.However,mCherry-sfGFP11 targeted toGolgi, ER, andplastid failed

to reconstitute the sfGFP signal when coexpressed with cytosolicsfGFP1-10OPT (Supplemental Figure 4A). Expression of mitochon-dria-targetedmCherry-sfGFP11 with cytosolic sfGFP1-10OPT led tosfGFP signal in the cytosol and in the nucleus but not in the mito-chondria (Supplemental Figure 4B). These results indicate that onlyplasma membrane and peroxisome-targeted sfGFP11 can re-constitute with cytosolic sfGFP1-10OPT. Therefore, it is better totarget both sfGFP11 and sfGFP1-10OPT to the appropriate organelleto visualize reconstituted sfGFP fluorescence signal in respectiveorganelle in plant cells.To facilitate visualization and subcellular studies of bacterial

effectors andplantproteins,wegenerated transgenicArabidopsisCol-0 plants expressing sfGFP1-10OPT targeted to the cytoplasm,plasma membrane, nucleus, plastid, mitochondria, peroxisome,ER, andGolgi (seeMethods for details). To validate these transgeniclines, we expressed sfGFP11 targeted to different subcellular com-partments in the corresponding organelle-targeted sfGFP1-10OPT

transgenic plants by the fast Agrobacterium-mediated seedlingtransformation (FAST) method (Li et al., 2009). We observed the re-constitution of sfGFP signal in all the corresponding subcellularcompartments (Supplemental Figure 5). These transgenic lineswill bea valuable tool to study subcellular localization not only ofP. syringaeeffector proteins delivered from bacteria but also of endogenousArabidopsisproteins. Theseedsof these transgenicArabidopsis linescan be obtained from the ABRC (https://abrc.osu.edu/).

Figure 1. Complementation of Split Fluorescent Protein in Plant Cells.

(A) N. benthamiana cells transiently expressing both sfGFP1-10OPT and mCherry-sfGFP11 showed sfGFP signal in the cytosol (panel 3). sfGFP signalwas not observed in cells expressing sfGFP1-10OPT (panel 1) or sfGFP11-mCherry (panel 2) alone. Magenta, chlorophyll autofluorescence.(B) Expression of mCherry-2xsfGFP11 with sfGFP1-10OPT resulted in brighter sfGFP signal. Magenta, chlorophyll autofluorescence.(C) Expression of sfCFP1-10OPT with mCherry-sfGFP11 in the cytoplasm reconstituted sfCFP fluorescence in the cytosol (panel 1). Expression ofER-targeted sfYFP1-10OPT and ER-sfCherry1-10 with ER-targeted mCherry-sfGFP11 and GUS-sfCherry11, respectively, reconstituted sfYFP (panel 2)and sfCherry (panel 3) fluorescence signal in the ER. Bars = 40 mm.

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Figure 2. Complementation of sfGFP1-10OPT and mCherry-sfGFP11 Targeted to Subcellular Compartments in N. benthamiana.

Coinfiltration of agrobacteria containing cytoplasmic (CYTO), PM, nucleus (NU), plastid (PT), mitochondria (MT), peroxisomes (PX), ER, and Golgi (GO)-targetedsfGFP1-10OPT and the other agrobacteria containing the same subcellular sites or organelle targeted mCherry-sfGFP11 reconstituted sfGFP signal in thecorresponding subcellular sites or organelles. sfGFP signal is pseudocolored to green, while mCherry is shown in magenta. Top panels show sfGFPimages overlapped with differential interference contrast (DIC) images (gray background) for cell architecture. Bottom panels are merged images ofsfGFP and mCherry. Bars = 40 mm.

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Development of sfGFP11 Tag System for Live Cell Imagingof P. syringae Effectors Delivered through T3SS

To visualize effector delivery through the T3SS using the splitsfGFPOPT system, we designed two different infection strategies(Figure 4A). First, a transgenic lineexpressingsfGFP1-10OPT in thecytoplasm was infected by P. syringae expressing effector fusedto sfGFP11 tag. This will be useful to observe the localization ofeffectors, whose subcellular localization is unknown (Figure 4A,left panel). The first subcellular compartment that T3SS effectorsencounter in the plant cell will be the cytoplasm. Thus, sfGFP11fused to an effector protein can reconstitute with cytosolicsfGFP1-10OPT. However, if effector proteins are targeted to themitochondria, plastid, or endomembrane system, cytosolicsfGFP1-10OPT could interfere with proper targeting of effectorproteins. In this case, transgenic plants expressing sfGFP1-10OPT

targeted to subcellular compartments will give a better signal atthe compartment if an effector fused to sfGFP11 translocates intothe corresponding compartment (Figure 4A, right panel). This isbecause the probability of reconstitution at the respective com-partment is much higher in the targeted sfGFP1-10OPT transgenicplants than in the cytosolic sfGFP1-10OPT plants. In addition, if theeffector proteins are processed in plant cells and consequentlychange their subcellular localization, these two strategies willbe complementary to study spatial and temporal dynamics ofeffectors in plant cells.

To monitor effectors delivered through the T3SS using the splitsfGFPOPT system,weengineeredGateway-compatible vectors toexpress an effector fused to the sfGFP11 tag in bacteria (Figure 4B).For this, the promoter of the P. syringae effector gene AvrRpm1

(Upadhyaya et al., 2014) followed by a Gateway cassette, sfGFP11,HA epitope tag, and AvrRpm1 terminator was introduced into thebroad-host-rangevectorspBBR1MCS-2and-5 (Kovachetal.,1995)(Figure4B). Todeliver effectors fromotherpathogens thatdonotuseT3SS, we engineered vectors with the AvrRpm1 promoter followedbyaT3SSsignal peptide (Upadhyayaet al., 2014). Sinceavery smallamount of the effectors might be delivered from bacteria, we gen-erated vectors containing 2xsfGFP11 to improve the fluorescencesignal (Figure 4B).To use these vectors to deliver effectors into N. benthamiana

and Arabidopsis plants, we initially tested six P. syringae pv to-mato DC3000 (Pst DC3000) strains, which carry the deletion ofdifferent effector genes (Kvitko et al., 2009). Pst CUCPB5500strain inwhich18effectors aredeleted (Kvitkoet al., 2009) showedrelatively reduced or no cell death comparedwith the other strainswhen inoculated into N. benthamiana leaves at 1 3 107 colony-forming units (cfu)/mL (Supplemental Figure 6).While amajority ofthe effector genes were deleted in PstCUCPB5500, the effectorsremaining in this strain are known to suppress pathogen-associatedmolecular pattern-triggered plant immunity (PTI) (Cunnac et al.,2011). Since PTI could inhibit bacterial survival in early time pointof their infection, suppressing PTI inPstCUCPB5500 strainmightallow us to observe the delivery of the effector tagged witha sfGFP11 or 2xsfGFP11 tag into cells of Arabidopsis andN. benthamiana during early time points of infection.To verify protein expression in our new vector system, we

transformed Pst CUCPB5500 strain with a construct containingmCherry fused to HA and sfGFP11 tags under the control ofthe AvrRpm1 promoter and AvrRpm1 T3SS signal peptide(pAvrRPM1:T3SSsp:mCherry-HA-sfGFP11). We infiltrated Pst

Figure 3. Plasma Membrane, Peroxisome, and Nuclear-Targeted mCherry-sfGFP11 Can Complement with sfGFP1-10OPT Targeted to Cytoplasm in N.benthamiana.

Expression of cytosolic sfGFP1-10OPT with PM, peroxisome (PX), or nucleus (NU) targeted mCherry-sfGFP11 (top panels, magenta) and reconstitutedsfGFP signal (green, middle panels) at the corresponding site or organelles. Fluorescence images merged to differential interference contrast to presentplant cell shape (bottom panels). Bars = 40 mm.

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Figure 4. Split Fluorescent Protein System to Monitor Delivery of Functional P. syringae T3SS Effectors into Plant Cells.

(A) Schematics of T3E detection system using split sfGFP. sfGFP11-tagged T3Es translocate via T3SS into host cells and complement with cytoplasmicsfGFP1-10OPT and then the effector-tagged sfGFP is targeted to the specific subcellular site or organelle directed by the effector (left). Alternatively, the

1576 The Plant Cell

CUCPB5500with pAvrRpm1:T3SSsp:mCherry-HA-sfGFP11 intothe transgenic Arabidopsis plants expressing cytosolic sfGFP1-10OPT.WeobservedmCherry spots on the infected leaves that arepresumably bacteria expressing mCherry (Figure 4C, left panel,magenta spots). As expected, therewasnodetectableGFPsignalin the infected cells, suggesting that mCherry was not secretedinto plant cells through T3SS. These results suggest that theAvrRpm1 promoter used in this construct is active inside bacteria.

To monitor effectors delivered through this system in plantcells, we generated constructs with AvrB, AvrRps4, and AvrRps4variants fused to HA tag and sfGFP11. It has been shown thatP. syringaepvpisieffectorAvrRps4 iscleaved inside theplantcellsbetween Gly-133 and Gly-134, resulting in generation of the Nterminus (1–133 amino acids; AvrRps4N) and theC terminus (134–221 amino acids; AvrRps4C) fragments (Sohn et al., 2009). Thus,we created Pst CUCPB5500 strain with full-length AvrRps4(pAvrRpm1:AvrRps4-HA-2xsfGFP11), N terminus of AvrRps4(pAvr Rpm1:AvrRps4N-HA-2xsfGFP11), and C terminus ofAvrRps4 (pAvrRpm1:T3SSsp:AvrRps4C-HA-2xsfGFP11). In addi-tion, we created a cleavage-resistant AvrRps4RL mutant describedby Sohn et al. (2009) (pAvrRpm1:AvrRps4RL-HA-2xsfGFP11) andfull-length AvrB (pAvrRpm1: AvrB-HA-2xsfGFP11). We first tested ifexpressionof effector fused to sfGFP11 tagaffects bacterial growth.Growth of Pst CUCPB5500 containing effectors with sfGFP11 tagas well as those with effectors without a tag showed no growthdifference in the King’s Bmedia (Supplemental Figure 7A) as well asin the hrp-derepressing minimal medium supplemented with fruc-tose (Supplemental Figure 7B), indicating that the sfGFP11 fusionhas no effect on bacterial growth.

To further confirm effectors’ functionality when fused to sfGFP11tag, we monitored bacterial growth on the CYTO-sfGFP1-10OPT

transgenic Arabidopsis plants. As controls, we used effectors in thesame expression cassette without any tag. AvrB and AvrRps4 ef-fectorsare recognizedbyRPM1andRPS4NLRs (nucleotidebindingdomainsand leucine-rich repeats), respectively, inArabidopsisCol-0

ecotype and induce immunity-related cell death and defense re-sponses resulting in reduced bacterial growth. Furthermore, theAvrRps4C is sufficient to induce immunity-related cell death inArabidopsisCol-0 (Heidrichetal.,2011).Tomonitorbacterialgrowth,13105 cfu/mLofPstCUCPB5500 andPstCUCPB5500 containingAvrB, AvrRps4, or AvrRps4C with or without sfGFP11 fusion wereinoculated onto 3-week-old transgenic Arabidopsis plants ex-pressing sfGFP1-10OPT. For cell death, 1 3 107 cfu/mL of cell ofbacteria was cocultivated with 2-week-old transgenic Arabidopsisplants expressing sfGFP1-10OPT and cell death was monitored bytrypan blue staining (Koch and Slusarenko, 1990) 24 h after in-oculation (Ishiga et al., 2011). Bacterial growth was significantlyreduced in plants infected with Pst CUCPB5500 expressing AvrB,AvrRps4, or AvrRps4C fused to 2xsfGFP11 compared with plantsinfectedwithPstCUCPB5500 alone orPstCUCPB5500 expressingmCherry-HA-2xsfGFP11 (Figure 4D). In addition, there was no dif-ference in bacterial growth of PstCUCPB5500 expressing effectorsfused to sfGFP11 tag compared with effectors without any tag(Figure 4D). Similarly, the cell death induced by effectors withand without sfGFP11 tag fusion shows statistically no differ-ence (Figure 4E). Together, these results indicate that thesfGFP11 tagged effectors are functional inside the plants cells.

Self-Assembling Split sfGFPOPT System Reveals DynamicLocalization of the AvrB Effector in Infected Plant Cells

To further validate our split sfGFPOPT system in planta, we testedthe localization of AvrB fused to sfGFP11 delivered from PstCUCPB5500 in N. benthamiana and Arabidopsis plants. AvrB isaT3E fromP. syringaepvglycinea, known to localize at theplasmamembrane (Tamaki et al., 1991). We infiltrated Pst CUCPB5500containing AvrRPM1pro:AvrB-HA-2xsfGFP11 into the leaves oftransgenic Arabidopsis plants expressing cytoplasmic CYTO-sfGFP1-10OPT. At 3 h postinfiltration (hpi), we observed recon-stituted sfGFP signal as small foci at the plasma membrane of

Figure 4. (continued).

sfGFP11-tagged T3E delivered via T3SS localizes to the specific subcellular site or organelle and reconstitutes with sfGFP1-10OPT targeted to thesubcellular site or organelle (right).(B) Gateway-compatible T3E delivery vectors based on the broad host range pBBR background. T3Es could be cloned into the Gateway cassette. Theexpression of T3Es will be under the control of AvrRpm1 T3E promoter (pAvrRpm1). The expressed T3E will be in-frame with HA epitope tag and sfGFP11(HA-11) or tandem sfGFP11 (HA-2x11). To express non-T3SS effectors from other pathogens, vectors with signal peptide of AvrRpm1 T3E (sp) weregenerated.(C) mCherry-HA-2xsfGFP11 is expressed in P. syringae. Infiltration of 1 3 106 cells mL21 of Pst CUCPB5500 expressing mCherry-HA-2xsfGFP11 intoArabidopsis Col-0 transgenic plants expressing cytoplasmic sfGFP1-10OPT showed mCherry fluorescence spots at the leaf epidermis surface 3 h afterinfiltration (right panel; magenta). No sfGFP signal was observed. Pst CUCPB5500 expressing AvrB without sfGFP11 fusion showed no detectablefluorescence signals (left panel). Bars = 40 mm.(D)Effectors fused tosfGFP11 tagdonot interferewitheffector function.GrowthofPstCUCPB5500,PstCUCPB5500expressingmCherry-HA-2xsfGFP11,or Pst CUCPB5500 expressing AvrB, AvrRps4, and AvrRps4C with or without 2xsfGFP11 in CYTO-sfGFP1-10OPT transgenic Arabidopsis leaves wasmonitored 4 d after infection with 13 105 cells mL21. Four leaves from four plants were infected for each strain. Experiments were repeated three times.Graph showsaverageof Log[cfu/cm2], and error bars indicate SE of themean. Letter codes indicate statistical differences analyzedbyone-wayANOVAwithTukey’s multiple comparisons in Prism7.0.(E) Infection with 13 107 cells mL21 of Pst CUCPB5500 with effectors fused to sfGFP11 tag or no tag showed increased immune related cell death at theinfiltrated sites of the CYTO-sfGFP1-10OPT transgenic Arabidopsis plants compared with Pst CUCPB5500 alone or Pst CUCPB5500 with mCherry-HA-2xsfGFP infected plants. Scale bar = 1mm. Trypan blue stains dead cells. Bars in the graph represent the average number of dead cells observed in panels1 to 7. Error bars indicate SE of the mean. Letters at the top of bars indicate statistically significant differences by Dunnett’s multiple comparison (P < 0.05).Experiments were repeated two times with eight biological replicates.

Type III Secretion Compatible Split GFP System 1577

epidermal cells (Figure 5A).Counterstainingwithpropidium iodide(PI) showed the foci of sfGFP fluorescence at the periphery of thePI-stainedcellwall (Figure5B). To further confirm these results,weinfiltrated Pst CUCPB5500 expressing AvrB-HA-2xsfGFP11 intoone-half of the leavesof transgenic Arabidopsis plants expressingplasmamembrane (PM)-targeted PM-sfGFP1-10OPT. At 3 hpi, weobserved the reconstituted sfGFP signal at the plasmamembrane(Figure 5C, right panel), while no signalwasdetectable at theotherhalf of the leaves of the same plants where PstCUCPB5500 ex-pressing AvrB without tag were infiltrated (Figure 5C, left panel).

To determine the localization in N. benthamiana plants, we firstexpressed CYTO-sfGFP1-10OPT by Agrobacterium-mediated tran-sient expression for 2 d. Then, Pst CUCPB5500 containingAvrRPM1pro:AvrB-HA-2xsfGFP11 was infiltrated into the sameleaves.At 3hpi,weobserved the reconstitutedsfGFP11signal at theplasmamembrane in the epidermal andmesophyll cells (Figure 5D).Interestingly, at 6 hpi of Pst CUCPB5500 containing AvrRPM1pro:AvrB-HA-2xsfGFP11 into the leaves of N. benthamiana plantsexpressing CYTO-sfGFP1-10OPT, we observed vesicular locali-zation of sfGFP signal comparedwith 3 hpi, whichwas primarily inthe plasma membrane (cf. Figures 5D and 5E). These resultssuggest that AvrB effector may undergo trafficking in response toplant immune responses.

Visualization of Subcellular Localization of P. syringaeEffector AvrRps4 Using the Split sfGFPOPT System

AvrRps4 effector has been shown to localize to the cytoplasm,nucleus, and plastid in Agrobacterium-mediated transient ex-pression system in N. benthamiana plants (Bhattacharjee et al.,2011; Heidrich et al., 2011; Li et al., 2014a) and in Arabidopsistransgenic plants overexpressing AvrRps4 under the control ofan inducible promoter (Bhattacharjee et al., 2011; Heidrich et al.,2011; Li et al., 2014a). To determine the localization of AvrRps4whendelivered frombacteria, we generated full-length AvrRps4,AvrRps4 R112L noncleavable mutant (AvrRps4RL), AvrRps4N,and AvrRps4C fused to the HA-2xsfGFP11 tag under the controlof AvrRpm1 promoter. Since AvrRps4C lacks T3SS signal peptide,we fused AvrRpm1 T3SS signal peptide at the N terminus. Uponinfiltration of Pst CUCPB5500 with AvrRps4-HA-sfGFP11 intoN. benthamiana leaves expressing CYTO-sfGFP1-10OPT, we oc-casionally observed a few cells showing reconstituted sfGFP signalin the cytoplasm 3 hpi (Supplemental Figure 8A). At 6 hpi, the cy-tosolic sfGFP signal was more frequently detected (Figure 6A, leftpanel). We also observed bright sfGFP signal in the nucleus at 6 hpi(Figure 6A, middle panel). Interestingly, we observed small vesiclesthatare localized incytosoloccasionally (Figure6A, rightpanel,whitearrows; Supplemental Figure 8).

To further confirm these results, we infiltrated Pst CUCPB5500with AvrRps4-HA-2xsfGFP11 into leaves of transgenic Arabi-dopsis Col-0 plants expressing sfGFP1-10OPT in the cytoplasmand in the nucleus. We observed reconstituted sfGFP signal 6 hpiin the cytoplasm (Figure 6B; left panel; Supplemental Figures 9Aand 10A) and a weak signal in the nucleus (Figure 6B; right panel,white arrows; Supplemental Figure 10A). To confirm that theobserved fluorescence was generated by reconstituted sfGFP bycytosolic sfGFP1-10OPT and secretedAvrRps4-HA-2xsfGFP11 inthe infected cells, Arabidopsis Col-0 plants were infected with

Pst CUCPB5500 expressing AvrRps4-HA-2xsfGFP11. We ob-served no fluorescence in these cells (Supplemental Figure 9B).Similarly, transgenic Arabidopsis Col-0 plants expressingsfGFP1-10OPT with Pst CUCPB5500 expressing AvrRps4without any tag failed to showafluorescent signal (SupplementalFigure 9C). Similar to AvrRps4-HA-2xsfGFP11, at 6 hpi,AvrRps4RL mutant was observed in the cytoplasm and in thenucleus (Figure 6C; Supplemental Figure 10B). We were unableto detect the reconstituted sfGFP signal at 6 hpi with AvrRps4N

in the cytoplasm or in the nucleus (data not shown). However,at 24 hpi, occasionally we could detect the reconstitutedsfGFP signal at small foci in the cytoplasm (Figure 6D, left panel,white arrow). Previously, AvrRps4N has been shown to localizeto chloroplasts when overexpressed under an inducible pro-moter in Arabidopsis transgenic plants (Li et al., 2014a).Therefore, we tested AvrRps4N-HA-2XsfGFP11 in plastid-targetedPT-sfGFP1-10OPT expressing transgenic Arabidopsis plants.However,wecouldnotdetect anysfGFPsignalsat3, 6, and24hpi(Figure 6D, right panel for 24 hpi; data for 3 and 6 hpi are notshown). The AvrRps4C fused to the T3SS signal peptide ofAvrRpm1 was successfully delivered into the plant cell and thereconstituted sfGFP signal was visible 6 hpi in the cytoplasm(Figure 6E, left panel). In addition, strong expression in the nu-cleus was detected (Figure 6E, right panel). Nuclear localization ofreconstituted sfGFP was confirmed by PI staining (SupplementalFigures 9D and 9E). Together, our sfGFP11-T3SS delivery systemcombinedwith sfGFP1-10OPT targeted to subcellular compartmentsin the host cells successfully demonstrated temporal and spatialinformation of P. syringae effector delivery through the T3SS.

DISCUSSION

Microbial pathogens deliver effector proteins into host cells tointerfere with various cellular functions to cause diseases. Thelocation and the timing of delivery of these effectors determinesuccessful pathogenesis. Thus, a reliable method to monitor thespatiotemporal dynamics of effectors in host cells is necessary tounderstand the function of these effectors. Translocation of theXanthomonas bacterial pathogen effector AvrBs2 through theT3SS has been validated using the adenylate cyclase (Cya) do-mainof theBordetellapertussiscyclolysin fused to theAvrBs2 thatallows measurement of cAMP production after translocation(Casper-Lindley et al., 2002). In animal cells, an effector fused tob-lactamase that cleaves a fluorescence resonance energytransfer (FRET)-based sensorwas developed tomonitor a changein FRET signal (Enninga and Rosenshine, 2009). However, thesesystems fail to provide spatial information of effectors in the in-fected cells. The self-assembling split sfGFPOPT system de-scribed in this study allows direct, real-time visualization ofeffectors delivered through the T3SS in plant cells. In addition,Arabidopsis transgenic lines with a series of organelle targetedsfGFP1-10OPT lines will be useful to detect effector localization ina variety of organelles and subcellular sites. Furthermore, T3SS-compatible broad-host-range Gateway vectors that we de-veloped provide a convenient and efficient route for cloningmultiple T3SS effectors.Using the self-assembling split sfGFPOPT system, we demon-

strate the delivery and visualization of P. syringae effector AvrB in

1578 The Plant Cell

Figure 5. AvrB Effector Tagged with sfGFP11 Delivered through P. syringae Is Detectable in Arabidopsis and N. benthamiana Plants.

(A)PstCUCPB5500containingAvrB-sfGFP11was infiltrated into leavesof transgenicArabidopsisexpressingCYTO-sfGFP1-10OPT.AprojectionofZslicesof infected cells 3 hpi showed reconstituted sfGFP signals (left panel, arrows).Middle panel corresponds tomagnified image corresponding towhite boxedarea in the left panel.Occasionally, small spot-like localizationwasobservedalong theplasmamembrane (right panel, arrows). Stronggreen fluorescenceatstomata indicates guard cell autofluorescence.(B) Cell wall was stained by PI (magenta) supports that the complemented sfGFP fluorescence is at the plasma membrane (green) in the Pst CUCPB5500containing AvrB-sfGFP11 infected Arabidopsis transgenic plants expressing sfGFP1-10OPT at 3 hpi. Fluorescence intensity of sfGFP (green) and PI(magenta) was compared by a line (dashed yellow line), showing sfGFP signal at the plasma membrane.(C) Infection of half leaf of Arabidopsis transgenic plants expressing plasmamembrane targeted sfGFP1-10OPT (PM-sfGFP1-10OPT) with PstCUCPB5500containing AvrB-2xsfGFP11 consistently showed complemented sfGFP fluorescence at the plasmamembrane at 3 hpi (right panel, arrows). The other halfleaf of the same plants infected with PstCUCPB5500 containing AvrBwithout any tag do not show detectable GFP fluorescence (left panel). Magenta, cellwall staining by PI.(D) Reconstituted sfGFP fluorescence was observed in N. benthamiana leaves transiently expressing cytosolic sfGFP1-10OPT (CYTO-sfGFP1-10OPT)infected with Pst CUCPB5500 expressing AvrB-sfGFP11. Images were captured 3 hpi.(E)Reconstituted sfGFP fluorescencewas observed as puncta structures frequently, 6 hpi withPstCUCPB5500, suggesting trafficking of AvrB containingmembranestructure. In (D)and (E), thefluorescence imagesweremerged todifferential interferencecontrast images toshowplantcell shape.Bars=20mm.Togenerate results shown in thisfigure, 13106cfu/mL21bacteria forArabidopsis and13107cfu/mL21bacteria forN.benthamianawereused for infection.

Type III Secretion Compatible Split GFP System 1579

Figure 6. AvrRps4 Effector and Its Variants Taggedwith sfGFP11Delivered throughP. syringaeAre Detectable in Arabidopsis andN. benthamianaPlants.

(A)ReconstitutedsfGFPsignalsweredetected in thecytoplasm (left panel), nucleus (middlepanel), andunidentifiedpunctatestructures (rightpanel), 6hpiofPst CUCPB5500 with AvrRps4-sfGFP11 in leaves of N. benthamiana transiently expressing sfGFP1-10OPT. Fluorescence images were merged to dif-ferential interference contrast images to show cell shape.(B)Weak reconstituted sfGFPsignalwasobserved in the cytoplasmofArabidopsis transgenic plants expressing sfGFP1-10OPT in the cytoplasm (left panel)and in the nucleus of Arabidopsis transgenic plants expressing sfGFP1-10OPT in the nucleus (right panel, white arrows), 3 hpi with Pst CUCPB5500expressing AvrRps4-2xsfGFP11. The original confocal imageswere cropped and the fluorescence intensity was digitally enhanced for better visualization.The original images are shown in Supplemental Figure 10A.(C)Strong reconstitutedsfGFPsignalwasobserved in thecytoplasmofArabidopsis transgenicplantsexpressingsfGFP1-10OPT in thecytoplasm (leftpanel)and in the nucleus of Arabidopsis transgenic plants expressing sfGFP1-10OPT in the nucleus (right panel, white arrows), 6 hpi with Pst CUCPB5500expressingnoncleavableAvrRps4RL-2xsfGFP11mutant. Imageswerecroppedand thefluorescence intensitywasdigitallyenhanced tobetter visualization.The original confocal images are shown in Supplemental Figure 10B.(D) Only faint sfGFP fluorescence in the cytoplasm was detected in Arabidopsis transgenic plants expressing sfGFP1-10OPT in the cytoplasm (left panel,whitearrow), andnosignal in theplastidswasdetected inArabidopsis transgenicplantsexpressingsfGFP1-10OPT in theplastids (rightpanel), 24hpiwithPstCUCPB5500 expressing AvrRps4N-2xsfGFP11. Fluorescence images were merged to differential interference contrast images to show cell shape.

1580 The Plant Cell

the plasmamembrane of the infected plant cells. Interestingly, theAvrB changes its localization from the plasma membrane to un-known vesicles at different time points after inoculation, sug-gesting a potential trafficking of AvrB. Further investigation willdetermine the precise molecular mode of AvrB trafficking andfunction during pathogenesis. Our imaging results using the splitsfGFPOPT also revealed that both cleavable and noncleavableP. syringae AvrRps4 effector are localized to the cytoplasm andnucleus. Interestingly, AvrRps4 is also localized to unknownvesicles that were not previously observed using transient ex-pression systems (Figure 6A). AvrRps4C has been detected inthe microsomal fraction extracted from the Arabidopsis trans-genic plants expressing AvrRps4C under an inducible promoter(Bhattacharjee et al., 2011; Heidrich et al., 2011), suggesting thatour system might support the previous biochemical data. Furthercharacterization of these vesicles might provide mechanism ofAvrRps4 processing inside the plant cells. Previously, in Arabi-dopsis transgenic plants expressing AvrRps4N or full-lengthAvrRps4 under an inducible promoter has been shown to localizeto plastids (Li et al., 2014a). However, both the full-length AvrRps4and AvrRps4N delivered from bacteria failed to reconstitutesfGFP fluorescence in transgenic Arabidopsis plants expressingsfGFP1-10OPT targeted to the plastids.

Our study showed that localization to some organelles could bedetected only when sfGFP1-10OPT targeted to the correspondingorganelles. Reconstitution of sfGFP in the chloroplast, mito-chondria, ER, and Golgi occurs only when both sfGFP11 andsfGFP1-10OPT are targeted to the same subcellular compartment.Several P. syringae effectors have been shown to target differentorganelles in plants cells to alter plant physiology and promotepathogenesis. The Pst DC3000 effector HopM1 has been bio-chemically shown to localize to the endomembrane compartmentin transgenic Arabidopsis plants (Nomura et al., 2012). HopM1interacts with Arabidopsis AtMIN7, which is involved in vesiculartrafficking. Expression and interaction of Pst DC3000 effectorHopD1 and Arabidopsis NTL9 protein in ER was shown byAgrobacterium-mediated transient expression in N. benthamiana(Block et al., 2014). Our various organelle-targeted sfGFP1-10OPT

transgenicArabidopsis lines should facilitate dynamic localizationstudies of effectors in real time.

Recently, the split sfGFP system was used to visualize effectortranslocation from fungal pathogen, U. maydis, without success(Tanaka et al., 2015). Lack of detection of the reconstituted GFPsignal in this study could be due to a strong background signal atthe infection site that masked the low complemented GFP signal(Tanaka et al., 2015). Since the original sfGFP1-10 (Cabantouset al., 2005) was used in the study, GFP complementation mighthaveoccurred lessefficiently due to theaggregationof sfGFP1-10(Cabantous et al., 2013). Here, we generated a plant expressionsystem using sfGFP1-10OPT, which is optimized to overcome theprotein aggregation issue (Cabantous et al., 2013). Our vector set

with the AvrRpm1 effector signal peptide should facilitate local-ization studies of fungal and other plant pathogen effectors.Despite the versatility of the split sfGFPOPT system, there are

some limitations to the study of effector localization and dy-namics. The observed reconstituted sfGFP fluorescent signal isrelatively weak. This could be due to the low level of expression ofeffectors when delivered directly from P. syringae compared withAgrobacterium-mediated transient overexpression. The signalcould be improved using sfYFP1-10OPT since YFP is brighter thanGFP (HeimandTsien, 1996). In addition,fluorescencesignal couldalso be increased by multimerizing sfGFP11 tag. In mammaliancells, seven repeats of sfGFP11 increased the signal significantlycompared with single sfGFP11 tag (Kamiyama et al., 2016). Weconfirmed that transient expression of sfGFP11 tandem repeatyielded a brighter signal than those of single sfGFP11 tag (Figure1B). However, effector fused to the tandemsfGFP11 tag deliveredfrom bacteria to the plant cells still showed weak reconstitutedsfGFPsignal, presumably due to lowexpressionof effectors. Itwillbe interesting to test in the future if four or seven repeat sfGFP11sfused to effector can be efficiently delivered into plant cellsto increase the fluorescence signal. In conclusion, the self-assembling split sfGFPOPT system described here will facilitatestudies of direct visualization of pathogen effectors delivered bybacterial pathogens at subcellular level.

METHODS

Plant Materials and Growth Conditions

Nicotiana benthamiana plants were grown in a controlled growth roomat ;24°C, 65% humidity, in 16/8-h light/dark photoperiod with light in-tensity of ;140 mE m22 s21 using fluorescent tubes. Six-week-oldplants were used for all experiments. Arabidopsis thaliana plants weregrown in a controlled environment growth chamber (Conviron) at 23°C,70% humidity, in 10/14-h light/dark photoperiod with light intensity of;100 mE m22 s21 using fluorescent tubes. Four-week-old plants wereused for Pst infection.

Plasmid Construction

Constructs generated in this study are listed in Supplemental Table 1.Briefly, the plant expression vector for sfGFP1-10OPT expression andvariants for expressing in subcellular compartments were constructed byinserting the UBQ10 promoter and corresponding fragments of sfGFP1-10OPT into the pCAMBIA1380 binary vector (GenBank accession numberAF234301). For targeting to plasma membrane, plastid, mitochondria,peroxisome, ER, andGolgi, targeting signalswere amplifiedbasedonNelsonet al. (2007). For nuclear-targeted sfGFP1-10OPT, the sfGFP1-10OPT wasfused to nuclear localization signal at theN terminus (Kalderon et al., 1984).mCherry-sfGFP11 and variants for the transient gene expression in N.benthamiana leaves were generated by replacing sfGFP1-10OPT withmCherry-sfGFP11. Sequence information of sfYFP1-10OPT, sfCFP1-10OPT,sfCherry, and sfCherry11 were obtained from Kamiyama et al. (2016) and

Figure 6. (continued).

(E) Reconstituted sfGFP signal was observed in the cytoplasm (right panel) and in the nucleus (left panel, white arrow) of transgenic Arabidopsis plantsexpressing cytosolic sfGFP1-10OPT, 6 hpi with Pst CUCPB5500 expressing AvrRpm1 signal peptide fused to AvrRps4C-2xsfGFP11. Occasionally, smallpunctate fluorescence structures were detected in the cytoplasm (right panel, yellow arrow). Fluorescence images weremerged to differential interferencecontrast images to show cell shape. Bars = 20 mm. To generate results shown in this figure, 1 3 106 CFU/mL21 bacteria was used for infection.

Type III Secretion Compatible Split GFP System 1581

constructed as described above. Plasmids were sequenced to confirm theidentity of various inserts.

For the expression vector of the bacterial T3E with sfGFP11, broad-host-range vector, pBBR1MCS-2 and -5 (Kovach et al., 1995) weremodified to introduceAvrRpm1promoter (Upadhyayaet al., 2014) followedby the Gateway cassette with AvrRpm1 terminator. The series of sfGFP11and linker were amplified using synthetic oligos and the correspondingfragments were introduced after the Gateway cassette in frame(Supplemental Figure 1B).

All primers used formaking plasmids are listed inSupplemental Table 2.The sequences of various plasmids have been deposited into theAddgene(stock numbers are in Supplemental Table 1).

Bacterial Strains and Growth Conditions

The bacterial strains used in this study are Pst CUCPB5500 (Kvitko et al.,2009), Agrobacterium tumefaciens GV3101, Escherichia coli DH10B, andSurvival 2 T1R (Thermo Fisher Scientific). Pseudomonas syringae wasgrown at 28°C in King’s B (KB) agar plates or KB liquid media (Sohn et al.,2009) or hrp-derepressing liquid media (Preiter et al. 2005). Agrobacteriaand E. coli were grown in Luria-Bertani (LB) agar plates or LB liquid mediawith shakingat28°Cor37°C, respectively. Antibioticswereusedat thefinalconcentrations of 100mg/mL rifampicin, 25mg/mLkanamycin,and50mg/mLgentamycin. Effector fused to sfGFP11 tagswas transformed intoP. syringaeCUCPB5500 using a standard electroporation.

Generation of Arabidopsis Transgenic Lines

Transgenic plants generated in this study are listed in Supplemental Table1. Briefly, constructs were transformed into Arabidopsis Col-0 plants byAgrobacterium-mediated transformation (Clough and Bent, 1998). Stabletransgenic plants were screened on 0.53 strength Murashige and Skoogmediumcontaining1%sucrose (pH5.8) andsolidifiedwith0.22%phytagel(Sigma-Aldrich) with hygromycin at a final concentration of 25 mg/mL. Alltransgenic lines seeds have been deposited to the ABRC and seed stocknumbers are in Supplemental Table 1.

N. benthamiana Transient Expression Assay

Agrobacterium-mediated transient expression in N. benthamiana leaveswas performed as described by Shamloul et al. (2014) with modifications.Briefly, agrobacteria containing corresponding constructs were infiltratedinto the leavesof 4-week-oldplants usinganeedlelesssyringe. For validationof theorganelle-targetedself-assemblingsplit sfXFP,agrobacteriacontainingthe given sfXFP1-10OPT and the other strain containing the correspondingmCherry fused to sfGFP11 were grown in LB liquid media overnight. Twostrains were then mixed at equal concentration to final OD600 to 0.5 and in-filtrated intoN.benthamiana leaves.Fluorescencesignals incellswere imagedbyconfocalmicroscopy (described below). Experimentswere repeated threetimes with three biological repeats for each experiment.

Arabidopsis Expression Assay

GFPcomplementationusing transgenicArabidopsisplantswasperformedas described by Li et al. (2009). Briefly, T2 transgenic plants expressingcytoplasmic, plasma membrane, nucleus, ER, and organelle targetedsfGFP1-10OPT were germinated on 0.53 Murashige and Skoog-0.22%phytagel (CaissonLabs) plate containing1%sucroseandpHadjusted to5.8.Three days prior to cocultivation, agrobacteria carrying the cytoplasmic,plasma membrane, nucleus, ER, and organelle targeted mCherry-sfGFP11were streaked on agar plate containing LB medium with appropriate anti-biotics. One day before cocultivation, a colony was inoculated in liquid LBmedia overnight. Bacteria cells were collected by centrifugation at 6000g for5 min and washed with a washing solution and resuspended in cocultivation

medium at the final density of OD600 to 0.5 (Li et al., 2009). Twenty seedlingswere cocultivated with agrobacteria cells in a plant growth chamber in thedark. Fluorescence signals were observed 40 h after cocultivation. Experi-ments were repeated three times independently.

P. syringae Infection to Visualize Effectors in Plant Cells

P.syringaecellscontainingvariousconstructsweregrownonKing’sBagarplates at 28°C for 2 d. A loopful of bacterial cells were inoculated in MGliquid media overnight and cells were resuspended in 10 mM MgCl2 andOD600 adjusted to 0.02 (1 3 107 cfu/mL) for N. benthamiana and to 0.002(1 3 106 cfu/mL) for Arabidopsis. To observe effectors in N. benthamianaleaves, agrobacteria containing sfGFP1-10OPT were infiltrated into two4-week-oldN. benthamiana leaves 2 d prior to P. syringae infection. Threeplants were included for each construct. For Arabidopsis, P. syringae wasinfiltrated into two4-week-old short-day (14 h light/10 hdark) grown leavesand four plants were included for each construct. At specific time points,two 2-cm2 leaf discs from the single plant were imaged under confocalmicroscope. For cell wall staining, 20mMPIwas infiltrated into the leaf discprior to observation. For nucleus staining, leaf discs were first submergedinto 0.1% paraformaldehyde for 5 min followed by washing with water.Then, 10 mM PI was infiltrated into the leaf disc 5 to 10 min before mi-croscopy. Experiments were repeated three times.

Microscopy

Images were generated in a Zeiss 710 laser scanning confocal systemusing an Axio observer Z1 inverted microscope with 403/1.2 NAC-Apochromat water immersion objective or 603/0.8 NA C-Apochromatoil immersion objective (Carl Zeiss). About 2 to 15%of 488-nmargon laser,514-nm argon laser, 405-nm diode laser, and 561-nm HeNe1 laser wereused to detect sfGFP, sfYFP, sfCFP, and sfCherry excitation, respectively.Images were pseudocolored to green, yellow, and cyan for sfGFP, sfYFP,and sfCFP, respectively. Magenta corresponds to sfCherry and mCherry.In some images, chlorophyll autofluorescence was pseudocolored tomagenta. Images were resized and cropped using ImageJ (NIH).

Bacteria Growth Assay in Arabidopsis Leaves

Bacteria strains were streaked and grown on KB media plates with ap-propriate antibiotics for 2 d. A colony was resuspended in KB liquid mediawith shaking at 28°C overnight (Sohn et al., 2009). Bacteria were collectedby centrifugation and resuspended in 10 mMMgCl2 to a concentration of13105 cfu/mLcells. Cellswere infiltrated into leavesof 4-week-oldCYTO-sfGFP1-10OPT transgenic Arabidopsis plants. Bacterial growth was de-termined at 0 and 4 d postinfiltration. Two 2-cm2 leaf discs were collectedfrom one leaf and grounded with a mini beadbeater (Biospec). Colonieswere counted from a serial dilution of plant extracts after growing on KBagar plates at 28°C for 2 d. Four plants were used for one experiment withthree repeat experiments. Statistical analysis and graph generation wereperformed using Prism7 (GraphPad). Numbers of dead cells were com-pared by one-way ANOVA (Supplemental Table 3A) followed by Tukey’smultiple comparisons using a statistic module in Prism7.

Cell Death Assay

For cell death assay in Arabidopsis, seedling flood inoculation assay wasperformed (Ishiga et al., 2011). CYTO-sfGFP1-10OPT Arabidopsis trans-genicplantsweregrown in short-dayconditions for 2weeks, andseedlingswere inoculated in 0.025%Silwet L-77 solution containing 13 107 cfu/mLof Pst CUCPB5500 expressing corresponding effectors. At 24 hpi, leaveswere stained by heating for 10 min in lactophenol trypan blue solutionfollowed by boiling for 1 min and cooling for 30min (Koch and Slusarenko,1990). Leaves were destained in chloral hydrate solution and mounted in

1582 The Plant Cell

10% glycerol for imaging. Trypan blue-stained dead cells were photo-graphed using a stereomicroscope (3.43 digital zoom) and counted usinga cell counter plug-in in ImageJ (NIH). Eight leaves from four plants wereused for each condition and experiments were repeated twice with similarresults. Statistical analysis and graph generation were performed usingPrism7 (GraphPad). Numbers of dead cells were compared by one-wayANOVA (Supplemental Table 3B) followed by Dunnett’s multiple com-parisons using a statistic module in Prism7.

Accession Numbers

Sequence data from this study can be found at Addgene (https://www.addgene.org), andseedscanbeobtained fromtheABRC(https://abrc.osu.edu/). Accession and stock numbers are listed in Supplemental Table 1.

Supplemental Data

Supplemental Figure 1. Illustration of plasmids used in this study.

Supplemental Figure 2. No FP fluorescence in leaf tissue expressingonly FP1-10 or coexpressing sfCFP1-10OPT with sfCherry11.

Supplemental Figure 3. Appropriate subcellular compartment target-ing of mCherry-sfGFP11.

Supplemental Figure 4. GFP complementation fails to occur whencytosolic sfGFP1-10OPT coexpressed with some organelle-targetedmCherry-sfGFP11.

Supplemental Figure 5. Reconstitution of sfGFP fluorescence intransgenic Arabidopsis plants.

Supplemental Figure 6. Cell death assay of various Pst DC3000effector deletion mutants.

Supplemental Figure 7. Bacterial growth assay of Pst CUCPB5500with various effectors fused to sfGFP11.

Supplemental Figure 8. Reconstitution of sfGFP in N. benthamianaplants after delivery of AvrRps4-HA-sfGFP11 at different time points.

Supplemental Figure 9. Confirmation of AvrRps4-HA-2xsfGFP11 andAvrRps4C-HA-2xsfGFP11 localization by propidium iodide staining.

Supplemental Figure 10. The original confocal images shown inFigures 6B and 6C.

Supplemental Table 1. List of constructs and transgenic plants usedin this study.

Supplemental Table 2. List of primers used in this study.

Supplemental Table 3. Analysis of variance of bacterial growth andcell death in Arabidopsis displayed in Figures 4D and 4E.

Supplemental Table 4. Analysis of variance of bacterial growth in KBmedia and hp-derepressing media displayed in Supplemental Figure 7.

ACKNOWLEDGMENTS

We thank Andreas Nebenfuehr for the organelle targeting sequence con-taining plasmids that were used as templates for PCR, Bo Huang forsfCFP1-10OPT, sfYFP1-10OPT, sfCherry1-10, and sfCherry11 plasmids,and ShisongMa for AvrB entry vector.We thank Barry Chan for generatingER-sfYFP1-10OPT, sfCFP1-10OPT, GUS-sfCherry11 plasmids used in thisstudy and Susan Wu for screening Arabidopsis sfGFP1-10OPT transgenicplants.Wealso thankAlanCollmer for variousPstDC3000effectordeletionstrains. This work was supported by National Institutes of Health GM097587and National Science Foundation IOS-1354434 funds to S.P.D.-K. H.-Y.L.was supported through the basic Science Research Program of the National

Research Foundation of Korea (NRF) in the Ministry of Science, ICT, andFuture Planning (NRF-2015R1A2A1A01002327 to D.C.).

AUTHOR CONTRIBUTIONS

E.P. andS.P.D.-K.conceivedresearchplan.E.P.,H.-Y.L.,J.W.,andS.P.D.-K.designed experiments. E.P., H.-Y.L., J.W., and S.P.D.-K. constructed plas-mids.E.P. andH.-Y.L. generatedmicroscopy images for transient expressionand generated transgenic plants. E.P. and J.W. performed other experimentsand analyzed images. E.P., H.-Y.L., J.W., D.C., and S.P.D.-K. wrote themanuscript.

ReceivedJanuary17,2017; revisedMay30,2017;acceptedJune14,2017;published June 14, 2017.

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1584 The Plant Cell

DOI 10.1105/tpc.17.00047; originally published online June 14, 2017; 2017;29;1571-1584Plant Cell

Eunsook Park, Hye-Young Lee, Jongchan Woo, Doil Choi and Savithramma P. Dinesh-KumarFluorescent Protein Fragments

Effectors via Type III Secretion Using SplitPseudomonas syringaeSpatiotemporal Monitoring of

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