DOI: 10.5281/zenodo.14450693
title: Confocal microscopy data associated with "Aphid effectors suppress plant immunity via recruiting defence proteins to processing bodies"

description: Confocal microscopy data associated with “Aphid effectors suppress plant immunity via recruiting defence proteins to processing bodies” (Liu et al., 2024). 

In Liu et al. (2024), we reported that the cathepsin B (CathB) effectors of the peach-potato aphid, Myzus persicae, suppress plant immunity via recruiting plant proteins with key roles in regulating immune response to processing bodies. Here we provide the raw data for all confocal microscopy analyses conducted in this paper. Please refer to Liu et al. (2024) for a full description of the data and the methods.

 

Subcellular localization

Colocalization analysis using confocal microscopy

Colocalization analysis was conducted by co-expression of different fluorescence protein-tagged proteins in N. benthamiana epidermal cells or A. thaliana protoplasts followed by observation with a Leica TCS SP8X upright confocal laser scanning microscope. Please refer to Liu et al. (2024) for full methods.

 

Figure 2. CathB6 forms mobile puncta and colocalizes with processing body markers VCS and DCP5 in N. benthamiana leaf cells.

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Fig.2A: Confocal images of CathB6-GFP-induced puncta.

Fig.2B: Time-lapse confocal images of a CathB6-GFP puncta fusion.

Fig.2C: Confocal images of CathB6-GFP colocalization with VCS-mCherry and intensity profiles below the images indicating overlap.

Fig.2D: Confocal images of CathB6-GFP colocalization with DCP5-mCherry and intensity profiles below the images indicating overlap.

 

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Fig.2_E-F: Analysis of VCS-mCherry puncta size distributions in the presence of GFP or CathB6-GFP.

 

Figure 3. CathB6 interacts with EDS1 and relocates EDS1, PAD4 and ADR1 to p-bodies. 

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Fig.3C: Confocal images illustrating EDS1-mCherry localized in puncta with CathB6-GFP.

Fig.3D: Confocal images illustrating colocalization of EDS1-GFP and VCS-mCherry with HA-CathB6.

Fig.3E: Confocal images illustrating colocalization of EDS1-GFP and PAD4-mCherry in puncta with HA-CathB6.

Fig.3F: Confocal images illustrating colocalization of EDS1-GFP and ADR1-L1-mCherry in puncta with Flag-PAD4 and HA-CathB6.

 

Figure 5. Acd28.9 counteracts CathB6 recruitment of EDS1 to p-bodies and contributes to plant resistance against aphids. 

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Fig.5B: Confocal images illustrating Acd28.9-mCherry co-expression depletes CathB6-GFP puncta in N. benthamiana.

Fig.5C: Confocal images illustrating EDS1-GFP forms puncta with HA-CathB6, and does not form puncta with Acd28.9-mCherry and HA-CathB6.

 

Figure S5. M. persicae CathB6 locates to mobile puncta within plant cells.

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Fig.S5A: Confocal images of GFP and CathB6-GFP in N. benthamiana cells with mCherry serving as a reference marker.

Fig.S5B (top): Confocal image of CathB6-GFP puncta.

 

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Fig.S5B (Bottom): Classification of CathB6-GFP puncta into three size categories: Large (L), Medium (M), and Small (S) based on area. Puncta frequency was determined as the ratio of cells with CathB6-GFP puncta to the total number of cells observed in the field. Data were compiled from 61 observation fields across three independent experiments, with 22–40 cells per field.

 

Fig. S7. M. persicae CathB6 and CathB3, and to a lesser extent CathB9, form cytoplasmic puncta in N. benthamiana epidermal cells and A. thaliana protoplasts. 

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Fig.S7A: CathB6 forms puncta in N. benthamiana cells when fused to GFP (CathB6-GFP) or RFP (CathB6-RFP), with free RFP or GFP as internal references.

Fig.S7B: CathB3-GFP forms puncta of varying sizes in N. benthamiana cells, whereas CathB9-GFP generates significantly fewer puncta.

Fig.S7C: CathB6-GFP forms puncta of varying sizes in A. thaliana protoplasts.

 

Fig. S8. CathB6-GFP colocalizes with p-body markers VCS, DCP5 and DCP1 in plant cells.

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Fig.S8A (left): CathB6-GFP colocalizes with VCS-mCherry in N. benthamiana cells.

Fig.S8B (left): CathB6-GFP colocalizes with DCP5-mCherry in N. benthamiana cells.

Fig.S8C (left): CathB6-GFP colocalizes with DCP1-RFP in N. benthamiana cells.

Fig.S8D: Confocal images of YFP-DCP1 and VCS-mCherry in N. benthamiana cells, showing YFP-DCP1 localizing within VCS-mCherry puncta.

 

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Fig.S8A (Right): Quantification of colocalization from three independent experiments across all observation fields. Top: ratios of cells expressing CathB6-GFP and VCS-mCherry. Middle: proportions of cells with large, medium, and small CathB6-GFP puncta colocalized with VCS-mCherry. Bottom: Proportions of VCS-mCherry puncta colocalized with CathB6-GFP in cells with CathB6-GFP.

 

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Fig.S8B (Right): Quantification of colocalization from three independent experiments across all observation fields. Top: ratios of cells expressing CathB6-GFP and DCP5-mCherry. Middle: proportions of cells with large, medium, and small CathB6-GFP puncta colocalized with DCP5-mCherry. Bottom: Proportions of DCP5-mCherry puncta colocalized with CathB6-GFP in cells with CathB6-GFP.

 

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Fig.S8C (Right): Quantification of colocalization from three independent experiments across all observation fields. Top: ratios of cells expressing CathB6-GFP and DCP1-RFP. Middle: proportions of cells with large, medium, and small CathB6-GFP puncta colocalized with DCP1-RFP. Bottom: Proportions of DCP1-RFP puncta colocalized with CathB6-GFP in cells with CathB6-GFP.

 

Figure S9. CathB6-RFP colocalizing with p-body markers YFP-VCS and YFP-DCP1 in N. benthamianaleaf cells and A. thaliana protoplasts.

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Fig.S9A: CathB6-RFP colocalizing with p-body markers YFP-VCS and YFP-DCP1 in N. benthamiana leaf cells.

Fig. S9B: CathB6-RFP colocalizing with p-body markers YFP-VCS and YFP-DCP1 in A. thaliana protoplasts.

 

Figure S10. CathB6-GFP puncta partially colocalize with the stress granule marker RBP47b-mCherry, while no obvious colocalizations were observed with RFP-ATG8 or NPR1-mCherry in N. benthamiana epidermal cells.

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Fig.S10A: CathB6-GFP puncta partially colocalize with the stress granule marker RBP47b-mCherry.

Fig.S10B: CathB6-GFP puncta don’t colocalize with RFP-ATG8.

Fig.S10C: CathB6-GFP puncta don’t colocalize with NPR1-mCherry.

 

Figure S14. CathB6-RFP relocates EDS1-GFP to puncta in N. benthamiana epidermal cells and A. thaliana protoplasts.

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Fig.S14A: CathB6-RFP relocates EDS1-GFP to puncta in N. benthamiana epidermal cells

Fig.S14B: CathB6-RFP relocates EDS1-GFP to puncta in A. thaliana protoplasts.

 

Fig. S15. EDS1 alone localizes to cytoplasmic puncta/p-bodies, while the EDS1-SAG101 complex remains localized to nuclei, in the presence of M. persicae CathB6 in N. benthamiana leaves.

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Fig.S15A: EDS1 alone localizes to cytoplasmic puncta/p-bodies in the presence of M. persicae CathB6 in N. benthamiana leaves.

Fig.S15B: EDS1-SAG101 complex remains localized to nuclei in the presence of M. persicae CathB6 in N. benthamiana leaves.

 

Time-lapse recording using confocal microscopy

Time-lapse analysis was performed by transient expression of GFP-tagged CathB in N. benthamianaepidermal cells followed by observation with a Leica Stellaris 8 FALCON upright confocal microscope. Please refer to Liu et al. (2024) for full methods.

 

Movie S1. M. persicae CathB6 locates to mobile puncta within cells of N. benthamiana leaves. 

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MovieS1: Time-lapse recording of CathB6-GFP in N. benthamiana leaves.

 

Fluorescence Recovery After Photobleaching (FRAP)

CathB6-GFP was transiently expressed in N. benthamiana epidermal cells. CathB6-GFP punctum was photobleached with laser, and the recovery was recorded post-bleaching with a Leica Stellaris 8 FALCON upright confocal microscope. Please refer to Liu et al. (2024) for full methods.

 

Fig. S5. M. persicae CathB6 locates to mobile puncta within plant cells.

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Fig.S5C: Fluorescence recovery after photobleaching (FRAP) analysis of CathB6-GFP puncta. Three representative FRAP images show fluorescence recovery over time, with Time 0 marking the photobleaching pulse.

 

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Fig.S5D: Time-course plot of fluorescence recovery for CathB6-GFP puncta after photobleaching. Recovery was quantified as the percentage of fluorescence intensity post-bleaching relative to pre-bleaching intensity. Data are analyzed from 25 independent experiments.

 

FLIM-FRET analysis

Constructs for genes of interest were co-expressed in N. benthamiana epidermal cells. Leaf sections were examined using a Leica Stellaris 8 FALCON scanning confocal microscope. FLIM experiments were conducted in TCSPC (Time-Correlated Single Photon Counting) mode. FLIM data were recorded using Leica LAS X software. FRET efficiency was calculated as 1 – (τₓ / τ₀). Please refer to Liu et al. (2024) for full methods.

 

Figure 5. Acd28.9 counteracts CathB6 recruitment of EDS1 to p-bodies and contributes to plant resistance against aphids. 

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Fig.5D: FLIM-FRET analysis showing reduced fluorescence lifetime of CathB6-GFP with EDS1 and Acd28.9, but not mCherry and VCS, with CathB6-GFP fluorescence and FLIM images.

 

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Fig.5E: Lifetime measurements of CathB6-GFP of FLIM in Fig.5D.

Fig.5F: Statistical lifetime and FRET efficiency of all FLIM.

 

Acknowledgements

We are grateful to JIC Bioimaging Facility for all confocal microscopy included in this submission.

This work was funded by UK Research and Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) grants to SAH (BB/V008544/1 and BB/R009481/1) with help from The Gatsby Charitable Foundation, The Sainsbury Laboratory, Norwich, UK. Additional Support is provided by the BBSRC Institute Strategy Programmes (BBS/E/J/000PR9797 and BBS/E/JI/230001B) awarded to the JIC. The JIC is grant-aided by the John Innes Foundation.

 

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Reference

Liu, Q., Neefjes, A.C.M., Kobylinska, R., Mugford, S.T., Marzo, M., Canham, J., Schuster, M., van der Hoorn, R.A.L., Chen, Y., and Hogenhout, S.A. (2024). Aphid effectors suppress plant immunity via recruiting defence proteins to processing bodies. bioRxiv, 2024.11.20.624400.