Interdependence of plasma membrane nanoscale dynamics of a kinase and its cognate substrate underliesArabidopsisresponse to viral infection

Abstract

Plant viruses represent a risk to agricultural production and as only few treatments exist, it is urgent to identify resistance mechanisms and factors. In plant immunity, plasma membrane (PM)-localized proteins are playing an essential role in sensing the extracellular threat presented by bacteria, fungi or herbivores. Viruses being intracellular pathogens, the role of the plant PM in detection and resistance against viruses is often overlooked. We investigated the role of the partially PM-bound Calcium-dependent protein kinase 3 (CPK3) in viral infection and we discovered that it displayed a specific ability to hamper viral propagation over CPK isoforms that are involved in immune response to extracellular pathogens. More and more evidence support that the lateral organization of PM proteins and lipids underlies signal transduction in plants. We showed here that CPK3 diffusion in the PM is reduced upon activation as well as upon viral infection and that such immobilization depended on its substrate, Remorin (REM1.2), a scaffold protein. Furthermore, we discovered that the viral infection induced a CPK3-dependent increase of REM1.2 PM diffusion. Such interdependence was also observable regarding viral propagation. This study unveils a complex relationship between a kinase and its substrate that contrasts with the commonly described co-stabilisation upon activation while it proposes a PM-based mechanism involved in decreased sensitivity to viral infection in plants.

Photochromic reversion enables long-term tracking of single molecules in living plants

“…Seeds were stratified at 4°C for 2 days and germinated on half strength Murashige and Skoog (½ MS) medium supplemented with sucrose (10 g.L -1 ) and agar (8 g.L -1 ) 33 and grown in chambers under control conditions with 70 % relative humidity and cycles of 16 h of light (~150 µE m -2 sec -1 ) and 8 h of dark at 21 °C. The Arabidopsis transgenic lines Col-0/pUb::mEOS2-AHA2 34 , Col-0/pPIP2;1::PIP2;1-mEOS2, Col-0/pUb::Lti6a-mEOS2 3 and Col-0/pUb::mEOS2-ROP6 15 17,35 with pREM1.2::mEOS3.2-REM1.2 plasmid using floral dipping 36 . 3-weeks-old Nicotiana benthamiana leaves were transiently transformed using solution (OD 600 =0.1) of Agrobacterium tumefaciens strain GV3101 carrying corresponding constructs as previously described 37 .…”

“…The Arabidopsis transgenic lines Col-0/pUb::mEOS2-AHA2 34 , Col-0/pPIP2;1::PIP2;1-mEOS2, Col-0/pUb::Lti6a-mEOS2 3 and Col-0/pUb::mEOS2-ROP6 15 , were previously described. The rem1.2/rem1.3/rem1.4/ pREM1.2::mEOS3.2-REM1.2 line was generating by transforming the rem1.2/rem1.3/rem1.4 T-DNA mutant line 17,35 with pREM1.2::mEOS3.2-REM1.2 plasmid using floral dipping 36 . 3-weeks-old Nicotiana benthamiana leaves were transiently transformed using solution (OD 600 =0.1) of Agrobacterium tumefaciens strain GV3101 carrying corresponding constructs as previously described 37 .…”

OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization

“…However, it was shown previously that mEos2 tends to form oligomers and aggregates in animal cells, especially when fused to membrane proteins (Zhang et al, 2012). We therefore recommend to prefer the improved version mEos3.2 that is monomeric and also works in plants (Jolivet et al, 2023). As with mEos2, the native green form of mEos3.2 can be converted to a red form using light of ~400 nm wavelength, allowing to adjust the density of the visible fluorophores in the red imaging channel.…”

“…Therefore, alternative illumination methods such as highly inclined thin illumination (HiLo) (Tokunaga et al, 2008), also known as variable angle epifluorescence microscopy (VAEM) (Konopka and Bednarek, 2008), are widely used for plants. Moreover, due to the limited permeability of the cell wall, plant cell biologists cannot use organic dyes common in the animal or human field (Lelek et al, 2021) for live cell imaging but must rely on a limited selection of genetically encoded fluorophores fused to the gene of interest (Hosy et al, 2015;McKenna et al, 2019;Jolivet et al, 2023).…”

OneFlowTraX: A User-Friendly Software for Super-Resolution Analysis of Single-Molecule Dynamics and Nanoscale Organization

“…Therefore, alternative illumination methods such as highly inclined thin illumination (HiLo) (Tokunaga et al, 2008), also known as variable angle epifluorescence microscopy (VAEM) (Konopka and Bednarek, 2008), are widely used for plants. Moreover, due to the limited permeability of the cell wall, plant cell biologists cannot use organic dyes common in the animal or human field (Lelek et al, 2022) for live cell imaging but must rely on a limited selection of genetically encoded fluorophores fused to the gene of interest (Hosy et al, 2015; Jolivet et al, 2023; McKenna et al, 2019).…”