DOI: 10.5281/zenodo.13752242
title: Confocal microscopy data associated with "The conserved aphid saliva chemosensory protein effector Mp10 targets plant AMSH deubiquitinases at cellular membranes to suppress pattern-triggered immunity"

description: Confocal microscopy data as described in "The conserved aphid saliva chemosensory protein effector Mp10 targets plant AMSH deubiquitinases at cellular membranes to suppress pattern-triggered immunity".

 

Data relate to Figure 2 (“Mp10 interacts with AMSH deubiquitinases in yeast and plants”) involving FLIM-FRET imaging data to determine the interaction between eGFP-tagged Myzus persicae Mp10 and mCherry-tagged Nicotiana benthamiana AMSH proteins in plant cells; and Figure 6 (“Mp10 affects the abundance and localisation of cell-surface receptor-like kinases) involving confocal microscopy showing the effect of Mp10-expression on the localisation of the GFP-tagged FLS2 receptor-kinase protein, and it’s colocalization with RFP-tagged markers of the plasma membrane and the tonoplast in plant cells.

Constructs encoding fluorescent protein fusions were transformed into Agrobacterium tumefaciens GV3101, and mixed Agrobacterium cultures were infiltrated into mature leaves of N. benthamiana plants to co-express the desired combinations of proteins. All image data was gathered from lower epidermal leaf cells of infiltrated leaves 2-3 days post infiltration.

 

FLIM-FRET assays.

eGFP-tagged Mp10, or eGFP-alone, was co-expressed with mCherry-tagged AMSH proteins, or mCherry fused to aquaeorin in N. benthamiana via agroinfiltration as described above. Lower epidermal cells of leaf sections were imaged 2-3 days after infiltration using a Leica Stellaris 8. Images were captured detecting fluorescence from eGFP (WLL laser, ex.488 nm, em 509-534 nm.) mCherry (lWLL aser, ex. 587 nm em 603-625 nm.) and chlorophyll (WLL laser, ex 587 nm. em 687-712 nm.). Regions of cells showing expression of both eGFP- and mCherry- tagged proteins but lacking chlorophyll were selected for FLIM analysis to avoid bleed through of chlorophyll fluorescence into the eGFP chanel. Fluorescence lifetime data of EGFP were collected from these regions in FLIM mode (WLL laser ex. 488nm, em 525-530 nm.), data were collected at 128x128 resolution until 1000 photons per pixel were collected for the most intense regions of the image. Instrument response function was captured using erythrosine on each day of data collection. FLIM data were analysed using Leica LASX FLIM FCS software. Fluorescence lifetime decay curves of free eGFP control samples were modelled as a 2-component exponential function, and all samples from each experimental set were modelled against the fluorescence lifetime from the corresponding control samples to derive values for fluorescent lifetime and %FRET efficiency for each image collected. %FRET efficiency was mapped to the images and phasor plots were generated for regions with the highest and lowest FRET efficiency, showing that the FRET signal was associated with a clockwise shift on the phasor plot consistent with bona-fide FRET. Full experimental metadata for each image set are included within the .lif files.

 

FLS2-GFP localisation experiments.

Confocal microscopy analysis was performed on a Leica TCS SP8X confocal DM6 microscope with a 63x water-immersion objective, using Leica Application Suite X (LAS X) software (3.5.7.23225). eGFP and chlorophyll signals were excited by a 488 nm Argon laser with emission, respectively, at 495–545 nm and 690-710 nm. RFP signal was excited by a 590 nm white light laser (WLL) with emission at 605–650 nm. Full experimental metadata for each image set are included within the .lif files.

 

Leica Image Files (.lif) that contain multiple images including metadata associated with image acquisition and processing.

 

FLIM030823.lif

FLIM130724.lif

FLIM140623.lif

FLIM160623.lif

FLIM240523.lif

FLIM250523.lif

Files include FLIM-FRET data as shown in Figure 2 parts D-L.

FLIM-data-files.xlsx includes a description of the individual image filenames, and the combinations of fluorophore imaged in each.

 

Fig6cSlFLS2-gfp_Mp10-RFP.lif

Fig6c-SlFLS2-GFP_EV-RFP.lif

Correspond to Figure 6 C showing co-expression of RFP-tagged Mp10 (or free RFP control) co-expressed with GFP-tagged FLS2

 

20230828_SlFLS2-GFP_Flag-Mp10_Flag-alone_Remorin-RFP-3-3-1x.lif

20230828_SlFLS2-GFP_Flag-Mp10_Flag-alone_Remorin-RFP-3-3-3x.lif

20230828_SlFLS2-GFP_Flag-Mp10_Flag-alone_Remorin-RFP-4-2-3x.lif

0230828_SlFLS2-GFP_Flag-Mp10_Flag-alone_Remorin-RFP4-2-1x.lif

Correspond to Figure 6 D showing co-expression of FLAG-tagged Mp10 (or free FLAG control) co-expressed with GFP-tagged FLS2 together with RFP-tagged plasma membrane marker Remorin

 

20230911_SlFLS2-GFP_Flag-Mp10_Flag-alone_StSUC4-RFP-14-5-1x.lif

20230911_SlFLS2-GFP_Flag-Mp10_Flag-alone_StSUC4-RFP-14-5-3x.lif

20230911_SlFLS2-GFP_Flag-Mp10_Flag-alone_StSUC4-RFP-16-1-1x.lif

20230911_SlFLS2-GFP_Flag-Mp10_Flag-alone_StSUC4-RFP-16-1a-3x.lif

20230911_SlFLS2-GFP_Flag-Mp10_Flag-alone_StSUC4-RFP-16-1b-1x.lif

Correspond to Figure 6 E showing co-expression of FLAG-tagged Mp10 (or free FLAG control) co-expressed with GFP-tagged FLS2 together with RFP-tagged tonoplast marker SUC4.

 

We are grateful to the John Innes Centre (JIC) Bioimaging Platform for training and technical support

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/N009169/1), Additional Support was provided by the BBSRC Institute Strategy Programmes (BBS/E/J/000PR9797 and BBS/E/JI/230001B) awarded to the John Innes Centre (JIC). The JIC is grant-aided by the John Innes Foundation.