Interdependence of a kinase and its cognate substrate plasma membrane nanoscale dynamics underlies Arabidopsis response to viral infection
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eLife assessment
This important study describes how the composition and stabilization of nanodomains in the plasma membrane are an integral part of plant defence against viruses, with a focus on the calcium-dependent kinase CPK3 and its apparent interaction with a plasma-membrane nano domain scaffold protein from the remorin family. While the evidence for a specific role of CPK3 in limiting viral spread is convincing, the claims regarding the CPK3-remorin interaction would be strengthened by additional experimental support. The work, which will be of interest to plant cell biologists and plant virology, opens new avenues for understanding the role of plasma membrane nanodomains in limiting viral spread.
This article has been Reviewed by the following groups
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- Evaluated articles (eLife)
- Plant Biology (eLife)
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.
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eLife assessment
This important study describes how the composition and stabilization of nanodomains in the plasma membrane are an integral part of plant defence against viruses, with a focus on the calcium-dependent kinase CPK3 and its apparent interaction with a plasma-membrane nano domain scaffold protein from the remorin family. While the evidence for a specific role of CPK3 in limiting viral spread is convincing, the claims regarding the CPK3-remorin interaction would be strengthened by additional experimental support. The work, which will be of interest to plant cell biologists and plant virology, opens new avenues for understanding the role of plasma membrane nanodomains in limiting viral spread.
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Reviewer #1 (Public Review):
Summary:
How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting …
Reviewer #1 (Public Review):
Summary:
How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting viral spread. The authors then go on to show that CPK3 diffusion in the membrane is reduced after viral infection and study the interaction between CPK3 and the remorins, which are a group of scaffold proteins important in nano domain organisation. The authors conclude that there is an interdependence between CPK3 and remorins to control their dynamics during viral infection in plants.
Strengths:
The dissection of which CPK was involved in the viral propagation was masterful and very conclusive. Identifying CPK3 through knockout time course monitoring of viral movement was very convincing. The inclusion of overexpression, constitutively active and point mutation non functioning lines further added to that.
Weaknesses:
My main concerns with the work are twofold.
Firstly, the imaging described and shown is not sufficient to support the claims made. The PM localisation and its non-PM localised form look similar and with no PM stain or marker construct used to support this. The sptPALM data conclusions are nice and fit the narrative. However, no raw data or movie is shown, only representative tracks. Therefore the data quality cannot be verified and in addition, the reporting of number of single particle events visualised per experiment is absent, only number of cells imaged is reported. Therefore it is impossible for the reader to appreciate the number of single molecule behaviours obtained and hence the quality of the data.
Secondly, remorins are involved in a lot of nano domain controlled processes at the PM. The authors have not conclusively demonstrated that during viral infection the remorin effects seen are solely due to its interaction with CPK3. The sptPALM imaging of REM1.2 in a cpk3 knockout line goes part way to solve this but more evidence would strengthen it in my opinion. How do we not know that during viral infection the entire PM protein dynamics and organisation are altered? Or that CPK3 and REM are at very distant ends of a signalling cascade. Negative control experiments are required here utilising other PM localised proteins which have no role during viral infection. In addition, if the interaction is specific, the transiently expressed CPK3-CA construct (shown to from nano domains) should be expressed with REM1.2-mEOS to show the alterations in single particle behaviour occur due to specific activations of CPK3 and REM1.2 in the absence of PIAMV viral infection and it is not an artefact of whole PM changes in dynamics during viral infection.
In addition, displaying more information throughout the manuscript (such as raw particle tracking movies and numbers of tracks measured) on the already generated data would strengthen the manuscript further.
Overall, I think this work has the potential to be a very strong manuscript but additional reporting of methods and data are required and additional lines of evidence supporting interaction claims would significantly strengthen the work and make it exceptional.
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Reviewer #2 (Public Review):
Summary:
The paper provides evidence that CPK3 plays a role in plant virus infection, and reports that viral infection is accompanied by changes in the dynamics of CPK3 and REM1.2, the phosphorylation substrate of CPK3, in the plasma membrane. In addition, the dynamics of the two proteins in the PM are shown to be interdependent.
Strengths:
The paper contains novel, important information.
Weaknesses:
The interpretation of some experimental data is not justified, and the proposed model is not fully based on the available data.
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Reviewer #3 (Public Review):
Summary:
This study examined the role that the activation and plasma membrane localisation of a calcium dependent protein kinase (CPK3) plays in plant defence against viruses.
The authors clearly demonstrate that the ability to hamper the cell-to-cell spread of the virus P1AMV is not common to other CPKs which have roles in defence against different types of pathogens, but appears to be specific to CPK3 in Arabidopsis. Further they show that lateral diffusion of CPK3 in the plasma membrane is reduced upon P1AMV infection, with CPK3 likely present in nano-domains. This stabilisation however, depends on one of its phosphorylation substrates a Remorin scaffold protein REM1-2. However, when REM1-2 lateral diffusion was tracked, it showed an increase in movement in response to P1AMV infection. These contrary …
Reviewer #3 (Public Review):
Summary:
This study examined the role that the activation and plasma membrane localisation of a calcium dependent protein kinase (CPK3) plays in plant defence against viruses.
The authors clearly demonstrate that the ability to hamper the cell-to-cell spread of the virus P1AMV is not common to other CPKs which have roles in defence against different types of pathogens, but appears to be specific to CPK3 in Arabidopsis. Further they show that lateral diffusion of CPK3 in the plasma membrane is reduced upon P1AMV infection, with CPK3 likely present in nano-domains. This stabilisation however, depends on one of its phosphorylation substrates a Remorin scaffold protein REM1-2. However, when REM1-2 lateral diffusion was tracked, it showed an increase in movement in response to P1AMV infection. These contrary responses to P1AMV infection were further demonstrated to be interdependent, which led the authors to propose a model in which activated CPK3 is stabilised in nano-domains in part by its interaction with REM1.2, which it binds and phosphorylates, allowing REM1-2 to diffuse more dynamically within the membrane.
The likely impact of this work is that it will lead to closer examination of the formation of nano-domains in the plasma membrane and dissection of their role in immunity to viruses, as well as further investigation into the specific mechanisms by which CPK3 and REM1-2 inhibit the cell-to-cell spread of viruses.
Strengths:
The paper provided compelling evidence about the roles of CPK3 and REM1-2 through a combination of logical reverse genetics experiments and advanced microscopy techniques, particularly in single particle tracking.
Weaknesses:
There is a lack of evidence for the downstream pathways, specifically whether the role that CPK3 has in cytoskeletal organisation may play a role in the plant's defence against viral propagation. Also, there is limited discussion about the localisation of the nano-domains and whether there is any overlap with plasmodesmata, which as plant viruses utilise PD to move from cell to cell seems an obvious avenue to investigate.
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