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. 2017 Jun 14;29(7):1571–1584. doi: 10.1105/tpc.17.00047

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN]

Eunsook Park a,1, Hye-Young Lee a,b, Jongchan Woo a, Doil Choi b,c, Savithramma P Dinesh-Kumar a,1
PMCID: PMC5559745  PMID: 28619883

Using the split GFP system to monitor type III effectors secreted from bacteria into plant cells.

Abstract

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments 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 prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described 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 animals and plants. These bacteria are taxonomically diverse and infect a 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 T3SS machinery is a filamentous supramolecular structure that provides a channel through which type III effector (T3E) proteins are secreted into the host cells to manipulate host defense responses against bacteria (Büttner, 2016). The first T3SS-associated filamentous structure was discovered in Pseudomonas syringae, a plant pathogen with a broad host range including several important crop species (Jin and He, 2001; Xin and He, 2013; Galán et al., 2014; Büttner, 2016). Our current understanding of detailed molecular and biochemical properties of the T3SS machinery come from studies of human pathogenic bacteria such as Salmonella and Yersinia (Galán et al., 2014).

T3Es are important for the pathogenicity of the bacteria and they play an important role in suppressing the first line of plant defense responses (Xin and He, 2013; Büttner, 2016). Several studies have demonstrated that the effector proteins from pathogenic bacteria are targeted to specific subcellular compartments in cells, where they alter the physiological properties of the cell and suppress innate immunity (Alfano and Collmer, 2004; Kay and Bonas, 2009; Choi et al., 2013; Aung et al., 2017). Thus, the spatiotemporal localization of the effectors is crucial for their function inside the host cell. However, the subcellular dynamics of effectors directly delivered from bacteria into host cells are largely unknown because of experimental difficulties, such as small amount of effector proteins secreted from the bacteria and incompatibility of fluorescent tags with the T3SS machinery (Galán, 2009). In plants, most of the localization studies of T3Es have been performed in Agrobacterium tumefaciens-mediated transient expression systems using constitutive promoters (Block and Alfano, 2011; Macho, 2016). However, several biochemical studies revealed bacterial effectors undergo posttranslocational modification for proper 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 localization of respective effectors originally secreted by T3SS.

Monitoring of T3E directly secreted from Salmonella has been described using the self-assembling split green fluorescent protein (GFP) system (Van Engelenburg and Palmer, 2010). GFP is a β-barrel protein with 11 β strands. This structure can be split into two fragments, 1-10 β strands (GFP1-10) and the 11th β strand with 13 amino acids (GFP11) (Cabantous et al., 2005; Supplemental Figure 1A). GFP1-10 is nonfluorescent because the conserved E222 residue in the GFP11 strand is critical for chromophore maturation (Barondeau et al., 2003). Once GFP1-10 and GFP11 exist in close proximity, the two fragments assemble a barrel structure and then emit fluorescence. The original split GFP system (Ghosh et al., 2000) has been further engineered to improve protein folding kinetics and solubility to enhance fluorescence intensity (Cabantous et al., 2005; Pédelacq et al., 2006; Cabantous et al., 2013). For better protein folding efficiency, super-folder GFP (sfGFP) was engineered for split GFP system (Pédelacq et al., 2006). However, the solubility of the sfGFP1-10 fragment is poor, resulting in lower fluorescence intensity (Cabantous et al., 2005). Therefore, a variant of sfGFP1-10 was engineered called GFP1-10OPT that significantly improves solubility and fluorescence intensity (Cabantous et al., 2005; Cabantous et al., 2013). In addition, recently, single amino acid mutants of sfGFP1-10OPT were generated that show emission spectra shifted to yellow or cyan color (Kamiyama et al., 2016).

Although the self-assembling split GFP system has been recently used as a tool to study subcellular localization of mammalian proteins (Kamiyama et al., 2016; Leonetti et al., 2016) and Salmonella T3E localization (Van Engelenburg and Palmer, 2010) and to visualize Agrobacterium VirE2 delivered through T4SS into plant cells (Li et al., 2014b), it has not been robustly optimized for plant biology research including T3E delivery from plant bacterial pathogens. In addition, an attempt to use the system to visualize fungal pathogen Ustilago maydis effectors in the maize (Zea mays) pathosystem has not been successful (Tanaka et al., 2015). In this study, we optimized the split GFP system based on the improved sfGFP1-10OPT to monitor subcellular localization of T3Es delivered from P. syringae into plant cells. To facilitate localization studies of T3Es to different subcellular compartments in the plant cells, we generated a set of transgenic Arabidopsis thaliana plants that express sfGFP1-10OPT in various subcellular compartments. Furthermore, the use of sfYFP/CFP1-10OPT and the split sfCherry system will allow studies involving dynamic and complex protein interactions at subcellular compartments. Finally, we provide a comprehensive toolkit to express effector proteins tagged to either sfGFP11 or a tandem repeat of sfGFP11 in P. syringae to examine secretion of the T3Es of interest. The seeds of various transgenic Arabidopsis lines and the plasmids to express T3Es can be obtained from the ABRC (https://abrc.osu.edu/) and Addgene (https://www.addgene.org) (see Supplemental Table 1 for accession numbers). The optimized split GFP system will facilitate investigation of dynamics of effector secretion as well as bona fide localization of effectors delivered from bacteria into the host cells. The transgenic lines we have developed will also be useful for subcellular localization studies of Arabidopsis proteins that will overcome potential perturbation on trafficking to the target subcellular compartment when fused to full-length fluorescent proteins.

RESULTS

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

To develop subcellular protein visualization system in plant cells to monitor bacterial effectors using sfGFP1-10OPT and sfGFP11 tag (Supplemental Figure 1A), we first evaluated the system using Agrobacterium-mediated transient assays in Nicotiana benthamiana plants (Wroblewski et al., 2005). For this, an expression vector with sfGFP1-10OPT under the control 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 of sfGFP (Supplemental Figure 1B). Agrobacteria containing sfGFP1-10OPT and mCherry-sfGFP11 constructs were coinfiltrated into 4-week-old N. benthamiana leaves. Two days postinfiltration, the tissue was observed by confocal laser scanning microscopy (see Methods for details). In tissue expressing sfGFP1-10OPT and mCherry-sfGFP11, we observed GFP signal in the cytoplasm (Figure 1A, panel 3). However, there was no detectable signal in the tissues infiltrated only with sfGFP1-10OPT or mCherry-sfGFP11 (Figure 1A, panels 1 and 2; mCherry localization was not imaged.). Tandem repeats of sfGFP11 can amplify the fluorescence signal in mammalian cells (Kamiyama et al., 2016). Therefore, we tested tandem repeat of sfGFP11 (2xsfGFP11) with sfGFP1-10OPT and observed increased fluorescence signal in the N. benthamiana tissue (Figure 1B). These results indicate that self-assembling split sfGFPOPT system could be used for localization and labeling studies in the plant cells.

Figure 1.

Figure 1.

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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 signal was 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 of ER-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 μm.

To facilitate multicolor imaging in plant cells, we tested complementation of sfGFP11 with sfYFP1-10OPT and sfCFP1-10OPT described by Kamiyama et al. (2016). Expression of sfCFP1-10OPT with mCherry-sfGFP11 reconstituted the cyan fluorescence signal in the cytoplasm of N. benthamiana cells (Figure 1C, panel 1). Coexpression of sfYFP1-10OPT fused to a signal peptide at the N terminus and an endoplasmic reticulum (ER) retention signal at the C terminus, with mCherry-sfGFP11 targeted to the ER showed a yellow fluorescent signal in the ER (Figure 1C, panel 2; mCherry localization was not imaged.). Next, we tested if the split sfCherry (Kamiyama et al., 2016) is functional in the plant cells. Coinfiltration of ER-targeted sfCherry1-10 with ER-targeted sfCherry11 fused to β-glucuronidase (GUS) exhibited fluorescence signal in the ER (Figure 1C, panel 3). Similar to sfGFP1-10OPT (Figure 1A, panel 1), expression of sfCFP1-10OPT alone and sfYFP1-10OPT or sfCherry1-10 alone targeted to the ER showed no detectable fluorescence (Supplemental Figure 2, panels 1–3). In addition, coexpression of sfGFP1-10OPT with sfCherry11 targeted to the ER failed to reconstitute fluorescence signal (Supplemental Figure 2, panel 4), confirming that the reconstitution of fluorescence protein occurs only when a specific FP1-10 coexpressed with a corresponding 11th β strand. These results indicate that the self-assembling split super-folder fluorescent protein system is a versatile system for multicolor subcellular localization studies in plant cells.

Self-Assembling Split sfGFPOPT System to Visualize Proteins Targeted to Various Subcellular Compartments

Bacterial effectors are known to target different subcellular compartments in the plant cells including the cytoplasm, nucleus, ER, and other organelles to alter the physiological state of plant cells to dampen plant immune responses (Block and Alfano, 2011; Macho, 2016). To visualize and to understand the effects of effectors targeted to the plasma membrane, nucleus, and various plant organelles, we generated various sfGFP1-10OPT constructs (Supplemental Figure 1B). Plasma membrane, Golgi, ER, peroxisomes, mitochondria, and plastid targeting sequences used here have been previously shown to target FPs to the corresponding organelle in transgenic Arabidopsis (Nelson et al., 2007). To target sfGFP1-10OPT to the nucleus, the nuclear localization signal sequence (Kalderon et al., 1984) was fused to the N terminus of sfGFP1-10OPT. Since sfGFP1-10OPT does not fluoresce without the sfGFP11 strand, we generated organelle-targeted sfGFP11 strand fused to mCherry for better protein folding. Proper organelle targeting of mCherry-sfGFP11 constructs was confirmed by transient expression in N. benthamiana leaves (Supplemental Figure 3).

Transient expression of sfGFP1-10OPT fused to different targeting sequences with sfGFP11 targeted to the same subcellular compartment in N. benthamiana leaves, reconstituted the sfGFPOPT signal in respective subcellular compartments (Figure 2). We also tested whether sfGFP11 targeted to a specific organelle can reconstitute the sfGFP signal at the respective organelle when coexpressed with cytosolic sfGFP1-10OPT. The mCherry-sfGFP11 targeted to the plasma membrane, nucleus, and peroxisome successfully reconstituted the fluorescence at the respective organelle when coexpressed with cytosolic sfGFP1-10OPT (Figure 3). In the case of the nucleus, it is possible that the sfGFP1-10 that diffuses into the nucleus could be reconstituted with nuclear-targeted sfGFP11. However, mCherry-sfGFP11 targeted to Golgi, ER, and plastid failed to reconstitute the sfGFP signal when coexpressed with cytosolic sfGFP1-10OPT (Supplemental Figure 4A). Expression of mitochondria-targeted mCherry-sfGFP11 with cytosolic sfGFP1-10OPT led to sfGFP signal in the cytosol and in the nucleus but not in the mitochondria (Supplemental Figure 4B). These results indicate that only plasma membrane and peroxisome-targeted sfGFP11 can reconstitute with cytosolic sfGFP1-10OPT. Therefore, it is better to target both sfGFP11 and sfGFP1-10OPT to the appropriate organelle to visualize reconstituted sfGFP fluorescence signal in respective organelle in plant cells.

Figure 2.

Figure 2.

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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)-targeted sfGFP1-10OPT and the other agrobacteria containing the same subcellular sites or organelle targeted mCherry-sfGFP11 reconstituted sfGFP signal in the corresponding subcellular sites or organelles. sfGFP signal is pseudocolored to green, while mCherry is shown in magenta. Top panels show sfGFP images overlapped with differential interference contrast (DIC) images (gray background) for cell architecture. Bottom panels are merged images of sfGFP and mCherry. Bars = 40 μm.

Figure 3.

Figure 3.

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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 reconstituted sfGFP signal (green, middle panels) at the corresponding site or organelles. Fluorescence images merged to differential interference contrast to present plant cell shape (bottom panels). Bars = 40 μm.

To facilitate visualization and subcellular studies of bacterial effectors and plant proteins, we generated transgenic Arabidopsis Col-0 plants expressing sfGFP1-10OPT targeted to the cytoplasm, plasma membrane, nucleus, plastid, mitochondria, peroxisome, ER, and Golgi (see Methods for details). To validate these transgenic lines, we expressed sfGFP11 targeted to different subcellular compartments in the corresponding organelle-targeted sfGFP1-10OPT transgenic plants by the fast Agrobacterium-mediated seedling transformation (FAST) method (Li et al., 2009). We observed the reconstitution of sfGFP signal in all the corresponding subcellular compartments (Supplemental Figure 5). These transgenic lines will be a valuable tool to study subcellular localization not only of P. syringae effector proteins delivered from bacteria but also of endogenous Arabidopsis proteins. The seeds of these transgenic Arabidopsis lines can be obtained from the ABRC (https://abrc.osu.edu/).

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

To visualize effector delivery through the T3SS using the split sfGFPOPT system, we designed two different infection strategies (Figure 4A). First, a transgenic line expressing sfGFP1-10OPT in the cytoplasm was infected by P. syringae expressing effector fused to sfGFP11 tag. This will be useful to observe the localization of effectors, whose subcellular localization is unknown (Figure 4A, left panel). The first subcellular compartment that T3SS effectors encounter in the plant cell will be the cytoplasm. Thus, sfGFP11 fused to an effector protein can reconstitute with cytosolic sfGFP1-10OPT. However, if effector proteins are targeted to the mitochondria, plastid, or endomembrane system, cytosolic sfGFP1-10OPT could interfere with proper targeting of effector proteins. In this case, transgenic plants expressing sfGFP1-10OPT targeted to subcellular compartments will give a better signal at the compartment if an effector fused to sfGFP11 translocates into the corresponding compartment (Figure 4A, right panel). This is because the probability of reconstitution at the respective compartment is much higher in the targeted sfGFP1-10OPT transgenic plants than in the cytosolic sfGFP1-10OPT plants. In addition, if the effector proteins are processed in plant cells and consequently change their subcellular localization, these two strategies will be complementary to study spatial and temporal dynamics of effectors in plant cells.

Figure 4.

Figure 4.

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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 cytoplasmic sfGFP1-10OPT and then the effector-tagged sfGFP is targeted to the specific subcellular site or organelle directed by the effector (left). Alternatively, the sfGFP11-tagged T3E delivered via T3SS localizes to the specific subcellular site or organelle and reconstitutes with sfGFP1-10OPT targeted to the subcellular 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. The expression 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) were generated.

(C) mCherry-HA-2xsfGFP11 is expressed in P. syringae. Infiltration of 1 × 106 cells mL−1 of Pst CUCPB5500 expressing mCherry-HA-2xsfGFP11 into Arabidopsis Col-0 transgenic plants expressing cytoplasmic sfGFP1-10OPT showed mCherry fluorescence spots at the leaf epidermis surface 3 h after infiltration (right panel; magenta). No sfGFP signal was observed. Pst CUCPB5500 expressing AvrB without sfGFP11 fusion showed no detectable fluorescence signals (left panel). Bars = 40 μm.

(D) Effectors fused to sfGFP11 tag do not interfere with effector function. Growth of Pst CUCPB5500, Pst CUCPB5500 expressing mCherry-HA-2xsfGFP11, or Pst CUCPB5500 expressing AvrB, AvrRps4, and AvrRps4C with or without 2xsfGFP11 in CYTO-sfGFP1-10OPT transgenic Arabidopsis leaves was monitored 4 d after infection with 1 × 105 cells mL−1. Four leaves from four plants were infected for each strain. Experiments were repeated three times. Graph shows average of Log[cfu/cm2], and error bars indicate se of the mean. Letter codes indicate statistical differences analyzed by one-way ANOVA with Tukey’s multiple comparisons in Prism7.0.

(E) Infection with 1 × 107 cells mL−1 of Pst CUCPB5500 with effectors fused to sfGFP11 tag or no tag showed increased immune related cell death at the infiltrated 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 = 1 mm. Trypan blue stains dead cells. Bars in the graph represent the average number of dead cells observed in panels 1 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.

To monitor effectors delivered through the T3SS using the split sfGFPOPT system, we engineered Gateway-compatible vectors to express 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 the broad-host-range vectors pBBR1MCS-2 and -5 (Kovach et al., 1995) (Figure 4B). To deliver effectors from other pathogens that do not use T3SS, we engineered vectors with the AvrRpm1 promoter followed by a T3SS signal peptide (Upadhyaya et al., 2014). Since a very small amount of the effectors might be delivered from bacteria, we generated vectors containing 2xsfGFP11 to improve the fluorescence signal (Figure 4B).

To use these vectors to deliver effectors into N. benthamiana and Arabidopsis plants, we initially tested six P. syringae pv tomato DC3000 (Pst DC3000) strains, which carry the deletion of different effector genes (Kvitko et al., 2009). Pst CUCPB5500 strain in which 18 effectors are deleted (Kvitko et al., 2009) showed relatively reduced or no cell death compared with the other strains when inoculated into N. benthamiana leaves at 1 × 107 colony-forming units (cfu)/mL (Supplemental Figure 6). While a majority of the effector genes were deleted in Pst CUCPB5500, the effectors remaining in this strain are known to suppress pathogen-associated molecular pattern-triggered plant immunity (PTI) (Cunnac et al., 2011). Since PTI could inhibit bacterial survival in early time point of their infection, suppressing PTI in Pst CUCPB5500 strain might allow us to observe the delivery of the effector tagged with a sfGFP11 or 2xsfGFP11 tag into cells of Arabidopsis and N. benthamiana during early time points of infection.

To verify protein expression in our new vector system, we transformed Pst CUCPB5500 strain with a construct containing mCherry fused to HA and sfGFP11 tags under the control of the AvrRpm1 promoter and AvrRpm1 T3SS signal peptide (pAvrRPM1:T3SSsp:mCherry-HA-sfGFP11). We infiltrated Pst CUCPB5500 with pAvrRpm1:T3SSsp:mCherry-HA-sfGFP11 into the transgenic Arabidopsis plants expressing cytosolic sfGFP1-10OPT. We observed mCherry spots on the infected leaves that are presumably bacteria expressing mCherry (Figure 4C, left panel, magenta spots). As expected, there was no detectable GFP signal in the infected cells, suggesting that mCherry was not secreted into plant cells through T3SS. These results suggest that the AvrRpm1 promoter used in this construct is active inside bacteria.

To monitor effectors delivered through this system in plant cells, we generated constructs with AvrB, AvrRps4, and AvrRps4 variants fused to HA tag and sfGFP11. It has been shown that P. syringae pv pisi effector AvrRps4 is cleaved inside the plant cells between Gly-133 and Gly-134, resulting in generation of the N terminus (1–133 amino acids; AvrRps4N) and the C 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 of AvrRps4 (pAvrRpm1:T3SSsp:AvrRps4C-HA-2xsfGFP11). In addition, we created a cleavage-resistant AvrRps4RL mutant described by Sohn et al. (2009) (pAvrRpm1:AvrRps4RL-HA-2xsfGFP11) and full-length AvrB (pAvrRpm1: AvrB-HA-2xsfGFP11). We first tested if expression of effector fused to sfGFP11 tag affects bacterial growth. Growth of Pst CUCPB5500 containing effectors with sfGFP11 tag as well as those with effectors without a tag showed no growth difference in the King’s B media (Supplemental Figure 7A) as well as in the hrp-derepressing minimal medium supplemented with fructose (Supplemental Figure 7B), indicating that the sfGFP11 fusion has no effect on bacterial growth.

To further confirm effectors’ functionality when fused to sfGFP11 tag, we monitored bacterial growth on the CYTO-sfGFP1-10OPT transgenic Arabidopsis plants. As controls, we used effectors in the same expression cassette without any tag. AvrB and AvrRps4 effectors are recognized by RPM1 and RPS4 NLRs (nucleotide binding domains and leucine-rich repeats), respectively, in Arabidopsis Col-0 ecotype and induce immunity-related cell death and defense responses resulting in reduced bacterial growth. Furthermore, the AvrRps4C is sufficient to induce immunity-related cell death in Arabidopsis Col-0 (Heidrich et al., 2011). To monitor bacterial growth, 1 × 105 cfu/mL of Pst CUCPB5500 and Pst CUCPB5500 containing AvrB, AvrRps4, or AvrRps4C with or without sfGFP11 fusion were inoculated onto 3-week-old transgenic Arabidopsis plants expressing sfGFP1-10OPT. For cell death, 1 × 107 cfu/mL of cell of bacteria was cocultivated with 2-week-old transgenic Arabidopsis plants expressing sfGFP1-10OPT and cell death was monitored by trypan blue staining (Koch and Slusarenko, 1990) 24 h after inoculation (Ishiga et al., 2011). Bacterial growth was significantly reduced in plants infected with Pst CUCPB5500 expressing AvrB, AvrRps4, or AvrRps4C fused to 2xsfGFP11 compared with plants infected with Pst CUCPB5500 alone or Pst CUCPB5500 expressing mCherry-HA-2xsfGFP11 (Figure 4D). In addition, there was no difference in bacterial growth of Pst CUCPB5500 expressing effectors fused to sfGFP11 tag compared with effectors without any tag (Figure 4D). Similarly, the cell death induced by effectors with and without sfGFP11 tag fusion shows statistically no difference (Figure 4E). Together, these results indicate that the sfGFP11 tagged effectors are functional inside the plants cells.

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

To further validate our split sfGFPOPT system in planta, we tested the localization of AvrB fused to sfGFP11 delivered from Pst CUCPB5500 in N. benthamiana and Arabidopsis plants. AvrB is a T3E from P. syringae pv glycinea, known to localize at the plasma membrane (Tamaki et al., 1991). We infiltrated Pst CUCPB5500 containing AvrRPM1pro:AvrB-HA-2xsfGFP11 into the leaves of transgenic Arabidopsis plants expressing cytoplasmic CYTO-sfGFP1-10OPT. At 3 h postinfiltration (hpi), we observed reconstituted sfGFP signal as small foci at the plasma membrane of epidermal cells (Figure 5A). Counterstaining with propidium iodide (PI) showed the foci of sfGFP fluorescence at the periphery of the PI-stained cell wall (Figure 5B). To further confirm these results, we infiltrated Pst CUCPB5500 expressing AvrB-HA-2xsfGFP11 into one-half of the leaves of transgenic Arabidopsis plants expressing plasma membrane (PM)-targeted PM-sfGFP1-10OPT. At 3 hpi, we observed the reconstituted sfGFP signal at the plasma membrane (Figure 5C, right panel), while no signal was detectable at the other half of the leaves of the same plants where PstCUCPB5500 expressing AvrB without tag were infiltrated (Figure 5C, left panel).

Figure 5.

Figure 5.

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

(A) Pst CUCPB5500 containing AvrB-sfGFP11 was infiltrated into leaves of transgenic Arabidopsis expressing CYTO-sfGFP1-10OPT. A projection of Z slices of infected cells 3 hpi showed reconstituted sfGFP signals (left panel, arrows). Middle panel corresponds to magnified image corresponding to white boxed area in the left panel. Occasionally, small spot-like localization was observed along the plasma membrane (right panel, arrows). Strong green fluorescence at stomata 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 CUCPB5500 containing 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 plasma membrane targeted sfGFP1-10OPT (PM-sfGFP1-10OPT) with Pst CUCPB5500 containing AvrB-2xsfGFP11 consistently showed complemented sfGFP fluorescence at the plasma membrane at 3 hpi (right panel, arrows). The other half leaf of the same plants infected with Pst CUCPB5500 containing AvrB without any tag do not show detectable GFP fluorescence (left panel). Magenta, cell wall 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 fluorescence was observed as puncta structures frequently, 6 hpi with Pst CUCPB5500, suggesting trafficking of AvrB containing membrane structure. In (D) and (E), the fluorescence images were merged to differential interference contrast images to show plant cell shape. Bars = 20 μm. To generate results shown in this figure, 1 × 106 cfu/mL−1 bacteria for Arabidopsis and 1 × 107 cfu/mL−1 bacteria for N. benthamiana were used for infection.

To determine the localization in N. benthamiana plants, we first expressed CYTO-sfGFP1-10OPT by Agrobacterium-mediated transient expression for 2 d. Then, Pst CUCPB5500 containing AvrRPM1pro:AvrB-HA-2xsfGFP11 was infiltrated into the same leaves. At 3 hpi, we observed the reconstituted sfGFP11 signal at the plasma membrane in the epidermal and mesophyll cells (Figure 5D). Interestingly, at 6 hpi of Pst CUCPB5500 containing AvrRPM1pro:AvrB-HA-2xsfGFP11 into the leaves of N. benthamiana plants expressing CYTO-sfGFP1-10OPT, we observed vesicular localization of sfGFP signal compared with 3 hpi, which was primarily in the plasma membrane (cf. Figures 5D and 5E). These results suggest that AvrB effector may undergo trafficking in response to plant immune responses.

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

AvrRps4 effector has been shown to localize to the cytoplasm, nucleus, and plastid in Agrobacterium-mediated transient expression system in N. benthamiana plants (Bhattacharjee et al., 2011; Heidrich et al., 2011; Li et al., 2014a) and in Arabidopsis transgenic plants overexpressing AvrRps4 under the control of an inducible promoter (Bhattacharjee et al., 2011; Heidrich et al., 2011; Li et al., 2014a). To determine the localization of AvrRps4 when delivered from bacteria, we generated full-length AvrRps4, AvrRps4 R112L noncleavable mutant (AvrRps4RL), AvrRps4N, and AvrRps4C fused to the HA-2xsfGFP11 tag under the control of AvrRpm1 promoter. Since AvrRps4C lacks T3SS signal peptide, we fused AvrRpm1 T3SS signal peptide at the N terminus. Upon infiltration of Pst CUCPB5500 with AvrRps4-HA-sfGFP11 into N. benthamiana leaves expressing CYTO-sfGFP1-10OPT, we occasionally observed a few cells showing reconstituted sfGFP signal in the cytoplasm 3 hpi (Supplemental Figure 8A). At 6 hpi, the cytosolic sfGFP signal was more frequently detected (Figure 6A, left panel). We also observed bright sfGFP signal in the nucleus at 6 hpi (Figure 6A, middle panel). Interestingly, we observed small vesicles that are localized in cytosol occasionally (Figure 6A, right panel, white arrows; Supplemental Figure 8).

Figure 6.

Figure 6.

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AvrRps4 Effector and Its Variants Tagged with sfGFP11 Delivered through P. syringae Are Detectable in Arabidopsis and N. benthamiana Plants.

(A) Reconstituted sfGFP signals were detected in the cytoplasm (left panel), nucleus (middle panel), and unidentified punctate structures (right panel), 6 hpi of Pst CUCPB5500 with AvrRps4-sfGFP11 in leaves of N. benthamiana transiently expressing sfGFP1-10OPT. Fluorescence images were merged to differential interference contrast images to show cell shape.

(B) Weak reconstituted sfGFP signal was observed in the cytoplasm of Arabidopsis 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 CUCPB5500 expressing AvrRps4-2xsfGFP11. The original confocal images were cropped and the fluorescence intensity was digitally enhanced for better visualization. The original images are shown in Supplemental Figure 10A.

(C) Strong reconstituted sfGFP signal was observed in the cytoplasm of Arabidopsis 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), 6 hpi with Pst CUCPB5500 expressing noncleavable AvrRps4RL-2xsfGFP11 mutant. Images were cropped and the fluorescence intensity was digitally enhanced to better 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, white arrow), and no signal in the plastids was detected in Arabidopsis transgenic plants expressing sfGFP1-10OPT in the plastids (right panel), 24 hpi with Pst CUCPB5500 expressing AvrRps4N-2xsfGFP11. Fluorescence images were merged to differential interference contrast images to show cell shape.

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

To further confirm these results, we infiltrated Pst CUCPB5500 with AvrRps4-HA-2xsfGFP11 into leaves of transgenic Arabidopsis Col-0 plants expressing sfGFP1-10OPT in the cytoplasm and in the nucleus. We observed reconstituted sfGFP signal 6 hpi in the cytoplasm (Figure 6B; left panel; Supplemental Figures 9A and 10A) and a weak signal in the nucleus (Figure 6B; right panel, white arrows; Supplemental Figure 10A). To confirm that the observed fluorescence was generated by reconstituted sfGFP by cytosolic sfGFP1-10OPT and secreted AvrRps4-HA-2xsfGFP11 in the infected cells, Arabidopsis Col-0 plants were infected with Pst CUCPB5500 expressing AvrRps4-HA-2xsfGFP11. We observed no fluorescence in these cells (Supplemental Figure 9B). Similarly, transgenic Arabidopsis Col-0 plants expressing sfGFP1-10OPT with Pst CUCPB5500 expressing AvrRps4 without any tag failed to show a fluorescent signal (Supplemental Figure 9C). Similar to AvrRps4-HA-2xsfGFP11, at 6 hpi, AvrRps4RL mutant was observed in the cytoplasm and in the nucleus (Figure 6C; Supplemental Figure 10B). We were unable to 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 reconstituted sfGFP signal at small foci in the cytoplasm (Figure 6D, left panel, white arrow). Previously, AvrRps4N has been shown to localize to chloroplasts when overexpressed under an inducible promoter in Arabidopsis transgenic plants (Li et al., 2014a). Therefore, we tested AvrRps4N-HA-2XsfGFP11 in plastid-targeted PT-sfGFP1-10OPT expressing transgenic Arabidopsis plants. However, we could not detect any sfGFP signals at 3, 6, and 24 hpi (Figure 6D, right panel for 24 hpi; data for 3 and 6 hpi are not shown). The AvrRps4C fused to the T3SS signal peptide of AvrRpm1 was successfully delivered into the plant cell and the reconstituted sfGFP signal was visible 6 hpi in the cytoplasm (Figure 6E, left panel). In addition, strong expression in the nucleus was detected (Figure 6E, right panel). Nuclear localization of reconstituted sfGFP was confirmed by PI staining (Supplemental Figures 9D and 9E). Together, our sfGFP11-T3SS delivery system combined with sfGFP1-10OPT targeted to subcellular compartments in the host cells successfully demonstrated temporal and spatial information of P. syringae effector delivery through the T3SS.

DISCUSSION

Microbial pathogens deliver effector proteins into host cells to interfere with various cellular functions to cause diseases. The location and the timing of delivery of these effectors determine successful pathogenesis. Thus, a reliable method to monitor the spatiotemporal dynamics of effectors in host cells is necessary to understand the function of these effectors. Translocation of the Xanthomonas bacterial pathogen effector AvrBs2 through the T3SS has been validated using the adenylate cyclase (Cya) domain of the Bordetella pertussis cyclolysin fused to the AvrBs2 that allows measurement of cAMP production after translocation (Casper-Lindley et al., 2002). In animal cells, an effector fused to β-lactamase that cleaves a fluorescence resonance energy transfer (FRET)-based sensor was developed to monitor a change in FRET signal (Enninga and Rosenshine, 2009). However, these systems fail to provide spatial information of effectors in the infected cells. The self-assembling split sfGFPOPT system described in this study allows direct, real-time visualization of effectors delivered through the T3SS in plant cells. In addition, Arabidopsis transgenic lines with a series of organelle targeted sfGFP1-10OPT lines will be useful to detect effector localization in a variety of organelles and subcellular sites. Furthermore, T3SS-compatible broad-host-range Gateway vectors that we developed provide a convenient and efficient route for cloning multiple T3SS effectors.

Using the self-assembling split sfGFPOPT system, we demonstrate the delivery and visualization of P. syringae effector AvrB in the plasma membrane of the infected plant cells. Interestingly, the AvrB changes its localization from the plasma membrane to unknown vesicles at different time points after inoculation, suggesting a potential trafficking of AvrB. Further investigation will determine the precise molecular mode of AvrB trafficking and function during pathogenesis. Our imaging results using the split sfGFPOPT also revealed that both cleavable and noncleavable P. syringae AvrRps4 effector are localized to the cytoplasm and nucleus. Interestingly, AvrRps4 is also localized to unknown vesicles that were not previously observed using transient expression systems (Figure 6A). AvrRps4C has been detected in the microsomal fraction extracted from the Arabidopsis transgenic plants expressing AvrRps4C under an inducible promoter (Bhattacharjee et al., 2011; Heidrich et al., 2011), suggesting that our system might support the previous biochemical data. Further characterization of these vesicles might provide mechanism of AvrRps4 processing inside the plant cells. Previously, in Arabidopsis transgenic plants expressing AvrRps4N or full-length AvrRps4 under an inducible promoter has been shown to localize to plastids (Li et al., 2014a). However, both the full-length AvrRps4 and AvrRps4N delivered from bacteria failed to reconstitute sfGFP fluorescence in transgenic Arabidopsis plants expressing sfGFP1-10OPT targeted to the plastids.

Our study showed that localization to some organelles could be detected only when sfGFP1-10OPT targeted to the corresponding organelles. Reconstitution of sfGFP in the chloroplast, mitochondria, ER, and Golgi occurs only when both sfGFP11 and sfGFP1-10OPT are targeted to the same subcellular compartment. Several P. syringae effectors have been shown to target different organelles in plants cells to alter plant physiology and promote pathogenesis. The Pst DC3000 effector HopM1 has been biochemically shown to localize to the endomembrane compartment in transgenic Arabidopsis plants (Nomura et al., 2012). HopM1 interacts with Arabidopsis AtMIN7, which is involved in vesicular trafficking. Expression and interaction of Pst DC3000 effector HopD1 and Arabidopsis NTL9 protein in ER was shown by Agrobacterium-mediated transient expression in N. benthamiana (Block et al., 2014). Our various organelle-targeted sfGFP1-10OPT transgenic Arabidopsis lines should facilitate dynamic localization studies of effectors in real time.

Recently, the split sfGFP system was used to visualize effector translocation from fungal pathogen, U. maydis, without success (Tanaka et al., 2015). Lack of detection of the reconstituted GFP signal in this study could be due to a strong background signal at the infection site that masked the low complemented GFP signal (Tanaka et al., 2015). Since the original sfGFP1-10 (Cabantous et al., 2005) was used in the study, GFP complementation might have occurred less efficiently due to the aggregation of sfGFP1-10 (Cabantous et al., 2013). Here, we generated a plant expression system using sfGFP1-10OPT, which is optimized to overcome the protein aggregation issue (Cabantous et al., 2013). Our vector set with the AvrRpm1 effector signal peptide should facilitate localization 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 dynamics. The observed reconstituted sfGFP fluorescent signal is relatively weak. This could be due to the low level of expression of effectors when delivered directly from P. syringae compared with Agrobacterium-mediated transient overexpression. The signal could be improved using sfYFP1-10OPT since YFP is brighter than GFP (Heim and Tsien, 1996). In addition, fluorescence signal could also be increased by multimerizing sfGFP11 tag. In mammalian cells, seven repeats of sfGFP11 increased the signal significantly compared with single sfGFP11 tag (Kamiyama et al., 2016). We confirmed that transient expression of sfGFP11 tandem repeat yielded a brighter signal than those of single sfGFP11 tag (Figure 1B). However, effector fused to the tandem sfGFP11 tag delivered from bacteria to the plant cells still showed weak reconstituted sfGFP signal, presumably due to low expression of effectors. It will be interesting to test in the future if four or seven repeat sfGFP11s fused to effector can be efficiently delivered into plant cells to increase the fluorescence signal. In conclusion, the self-assembling split sfGFPOPT system described here will facilitate studies of direct visualization of pathogen effectors delivered by bacterial pathogens at subcellular level.

METHODS

Plant Materials and Growth Conditions

Nicotiana benthamiana plants were grown in a controlled growth room at ∼24°C, 65% humidity, in 16/8-h light/dark photoperiod with light intensity of ∼140 μE m−2 s−1 using fluorescent tubes. Six-week-old plants were used for all experiments. Arabidopsis thaliana plants were grown in a controlled environment growth chamber (Conviron) at 23°C, 70% humidity, in 10/14-h light/dark photoperiod with light intensity of ∼100 μE m−2 s−1 using fluorescent tubes. Four-week-old plants were used 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 and variants for expressing in subcellular compartments were constructed by inserting the UBQ10 promoter and corresponding fragments of sfGFP1-10OPT into the pCAMBIA1380 binary vector (GenBank accession number AF234301). For targeting to plasma membrane, plastid, mitochondria, peroxisome, ER, and Golgi, targeting signals were amplified based on Nelson et al. (2007). For nuclear-targeted sfGFP1-10OPT, the sfGFP1-10OPT was fused to nuclear localization signal at the N terminus (Kalderon et al., 1984). mCherry-sfGFP11 and variants for the transient gene expression in N. benthamiana leaves were generated by replacing sfGFP1-10OPT with mCherry-sfGFP11. Sequence information of sfYFP1-10OPT, sfCFP1-10OPT, sfCherry, and sfCherry11 were obtained from Kamiyama et al. (2016) and constructed as described above. Plasmids were sequenced to confirm the identity 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) were modified to introduce AvrRpm1 promoter (Upadhyaya et al., 2014) followed by the Gateway cassette with AvrRpm1 terminator. The series of sfGFP11 and linker were amplified using synthetic oligos and the corresponding fragments were introduced after the Gateway cassette in frame (Supplemental Figure 1B).

All primers used for making plasmids are listed in Supplemental Table 2. The sequences of various plasmids have been deposited into the Addgene (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, and Survival 2 T1R (Thermo Fisher Scientific). Pseudomonas syringae was grown 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). Agrobacteria and E. coli were grown in Luria-Bertani (LB) agar plates or LB liquid media with shaking at 28°C or 37°C, respectively. Antibiotics were used at the final concentrations of 100 µg/mL rifampicin, 25 µg/mL kanamycin, and 50 µg/mL gentamycin. Effector fused to sfGFP11 tags was transformed into P. syringae CUCPB5500 using a standard electroporation.

Generation of Arabidopsis Transgenic Lines

Transgenic plants generated in this study are listed in Supplemental Table 1. Briefly, constructs were transformed into Arabidopsis Col-0 plants by Agrobacterium-mediated transformation (Clough and Bent, 1998). Stable transgenic plants were screened on 0.5× strength Murashige and Skoog medium containing 1% sucrose (pH 5.8) and solidified with 0.22% phytagel (Sigma-Aldrich) with hygromycin at a final concentration of 25 µg/mL. All transgenic lines seeds have been deposited to the ABRC and seed stock numbers are in Supplemental Table 1.

N. benthamiana Transient Expression Assay

Agrobacterium-mediated transient expression in N. benthamiana leaves was performed as described by Shamloul et al. (2014) with modifications. Briefly, agrobacteria containing corresponding constructs were infiltrated into the leaves of 4-week-old plants using a needleless syringe. For validation of the organelle-targeted self-assembling split sfXFP, agrobacteria containing the given sfXFP1-10OPT and the other strain containing the corresponding mCherry fused to sfGFP11 were grown in LB liquid media overnight. Two strains were then mixed at equal concentration to final OD600 to 0.5 and infiltrated into N. benthamiana leaves. Fluorescence signals in cells were imaged by confocal microscopy (described below). Experiments were repeated three times with three biological repeats for each experiment.

Arabidopsis Expression Assay

GFP complementation using transgenic Arabidopsis plants was performed as described by Li et al. (2009). Briefly, T2 transgenic plants expressing cytoplasmic, plasma membrane, nucleus, ER, and organelle targeted sfGFP1-10OPT were germinated on 0.5× Murashige and Skoog-0.22% phytagel (Caisson Labs) plate containing 1% sucrose and pH adjusted to 5.8. Three days prior to cocultivation, agrobacteria carrying the cytoplasmic, plasma membrane, nucleus, ER, and organelle targeted mCherry-sfGFP11 were streaked on agar plate containing LB medium with appropriate antibiotics. One day before cocultivation, a colony was inoculated in liquid LB media overnight. Bacteria cells were collected by centrifugation at 6000g for 5 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 seedlings were cocultivated with agrobacteria cells in a plant growth chamber in the dark. Fluorescence signals were observed 40 h after cocultivation. Experiments were repeated three times independently.

P. syringae Infection to Visualize Effectors in Plant Cells

P. syringae cells containing various constructs were grown on King’s B agar plates at 28°C for 2 d. A loopful of bacterial cells were inoculated in MG liquid media overnight and cells were resuspended in 10 mM MgCl2 and OD600 adjusted to 0.02 (1 × 107 cfu/mL) for N. benthamiana and to 0.002 (1 × 106 cfu/mL) for Arabidopsis. To observe effectors in N. benthamiana leaves, agrobacteria containing sfGFP1-10OPT were infiltrated into two 4-week-old N. benthamiana leaves 2 d prior to P. syringae infection. Three plants were included for each construct. For Arabidopsis, P. syringae was infiltrated into two 4-week-old short-day (14 h light/10 h dark) grown leaves and four plants were included for each construct. At specific time points, two 2-cm2 leaf discs from the single plant were imaged under confocal microscope. For cell wall staining, 20 μM PI was infiltrated into the leaf disc prior to observation. For nucleus staining, leaf discs were first submerged into 0.1% paraformaldehyde for 5 min followed by washing with water. Then, 10 μM PI was infiltrated into the leaf disc 5 to 10 min before microscopy. Experiments were repeated three times.

Microscopy

Images were generated in a Zeiss 710 laser scanning confocal system using an Axio observer Z1 inverted microscope with 40×/1.2 NA C-Apochromat water immersion objective or 60×/0.8 NA C-Apochromat oil immersion objective (Carl Zeiss). About 2 to 15% of 488-nm argon laser, 514-nm argon laser, 405-nm diode laser, and 561-nm HeNe1 laser were used 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 to magenta. 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 appropriate antibiotics for 2 d. A colony was resuspended in KB liquid media with shaking at 28°C overnight (Sohn et al., 2009). Bacteria were collected by centrifugation and resuspended in 10 mM MgCl2 to a concentration of 1 × 105 cfu/mL cells. Cells were infiltrated into leaves of 4-week-old CYTO-sfGFP1-10OPT transgenic Arabidopsis plants. Bacterial growth was determined at 0 and 4 d postinfiltration. Two 2-cm2 leaf discs were collected from one leaf and grounded with a mini beadbeater (Biospec). Colonies were counted from a serial dilution of plant extracts after growing on KB agar plates at 28°C for 2 d. Four plants were used for one experiment with three repeat experiments. Statistical analysis and graph generation were performed using Prism7 (GraphPad). Numbers of dead cells were compared by one-way ANOVA (Supplemental Table 3A) followed by Tukey’s multiple comparisons using a statistic module in Prism7.

Cell Death Assay

For cell death assay in Arabidopsis, seedling flood inoculation assay was performed (Ishiga et al., 2011). CYTO-sfGFP1-10OPT Arabidopsis transgenic plants were grown in short-day conditions for 2 weeks, and seedlings were inoculated in 0.025% Silwet L-77 solution containing 1 × 107 cfu/mL of Pst CUCPB5500 expressing corresponding effectors. At 24 hpi, leaves were stained by heating for 10 min in lactophenol trypan blue solution followed by boiling for 1 min and cooling for 30 min (Koch and Slusarenko, 1990). Leaves were destained in chloral hydrate solution and mounted in 10% glycerol for imaging. Trypan blue-stained dead cells were photographed using a stereomicroscope (3.4× digital zoom) and counted using a cell counter plug-in in ImageJ (NIH). Eight leaves from four plants were used for each condition and experiments were repeated twice with similar results. Statistical analysis and graph generation were performed using Prism7 (GraphPad). Numbers of dead cells were compared by one-way ANOVA (Supplemental Table 3B) followed by Dunnett’s multiple comparisons using a statistic module in Prism7.

Accession Numbers

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

Supplemental Data

Acknowledgments

We thank Andreas Nebenfuehr for the organelle targeting sequence containing plasmids that were used as templates for PCR, Bo Huang for sfCFP1-10OPT, sfYFP1-10OPT, sfCherry1-10, and sfCherry11 plasmids, and Shisong Ma for AvrB entry vector. We thank Barry Chan for generating ER-sfYFP1-10OPT, sfCFP1-10OPT, GUS-sfCherry11 plasmids used in this study and Susan Wu for screening Arabidopsis sfGFP1-10OPT transgenic plants. We also thank Alan Collmer for various Pst DC3000 effector deletion strains. This work was supported by National Institutes of Health GM097587 and 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, and Future Planning (NRF-2015R1A2A1A01002327 to D.C.).

AUTHOR CONTRIBUTIONS

E.P. and S.P.D.-K. conceived research plan. E.P., H.-Y.L., J.W., and S.P.D.-K. designed experiments. E.P., H.-Y.L., J.W., and S.P.D.-K. constructed plasmids. E.P. and H.-Y.L. generated microscopy images for transient expression and generated transgenic plants. E.P. and J.W. performed other experiments and analyzed images. E.P., H.-Y.L., J.W., D.C., and S.P.D.-K. wrote the manuscript.

Glossary

T3SS

type III secretion system

T3E

type III effector

ER

endoplasmic reticulum

cfu

colony-forming units

PTI

pattern-triggered plant immunity

hpi

hours postinfiltration

PI

propidium iodide

PM

plasma membrane

KB

King’s B

LB

Luria-Bertani

Footnotes

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