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

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

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Free PMC article

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

Eunsook Park et al. Plant Cell. .
Free PMC article

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.

Figures

Figure 1.
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 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.
Figure 2.
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)-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.
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.
Figure 4.
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 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.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
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.

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