PREreview of The Receptor Kinase MEE39/ATHE Mediates Cell Wall Integrity Surveillance During Root Vascular Pathogen Infection

Summary

This manuscript by Montesinos et al. demonstrates that the Arabidopsis thaliana malectin-like leucine-rich repeat receptor kinase (LRR-RK) ATHENA (ATHE)/MEE39 confers resistance against the root pathogens Fusarium oxysporum (Fo) and Ralstonia solanacearum. Interestingly, ATHE was isolated via an organelle immunoprecipitation (IP) assay using the multivesicular body/prevacuolar compartment (MVB/PVC) marker ARA7. This approach was driven by the hypothesis that novel plasma membrane-localized pattern-recognition receptors could be identified by tracing clathrin-mediated endocytosis (CME) following pathogen infection.

The authors show that ATHE localization and abundance are dynamically altered upon Fo infection. Subsequent investigation into the ATHE signaling mechanism revealed an interaction with the LRR-RK MIK2. Furthermore, the authors provide genetic evidence indicating that ATHE is involved in the response to cell wall (CW) stress and Fo-RALF. Overall, this study provides novel mechanistic insights into how plants monitor cell wall integrity (CWI) and respond to pathogen-induced cell wall alterations, addressing a fundamental yet relatively unexplored aspect of plant immunity.

General Comments

The authors present substantial data characterizing the novel function of the ATHE receptor kinase. The experimental design yields several notable strengths:

・The ATHE knockout in Arabidopsis and the corresponding gene knock-in in tomato exhibit clear and contrasting phenotypes, with enhanced susceptibility and resistance to Fusarium oxysporum (Fo), respectively.

・The data demonstrating that CME is essential for the pathogen response are highly convincing. Furthermore, ATHE protein dynamics are beautifully captured using a combination of microscopy and biochemical assays.

Overall, the role of ATHE in conferring resistance to root pathogens is well supported. However, a gap remains in the mechanistic framework:

・The mechanisms by which ATHE (potentially in complex with MIK2) senses cell wall integrity (CWI), and how this process relates to RALF perception and broader root pathogen resistance, are not yet fully integrated. For example, it remains unclear whether FoRALF enhances or stabilizes the interaction between ATHE and MIK2.

Further investigation into the precise role of ATHE in CWI sensing will be important for developing a cohesive understanding of how CWI signaling interfaces with plant immune responses.

Specific Comments

・In the Introduction, the logical connection between CWI and CME is not entirely clear. Clarifying why CME is a necessary focus for uncovering novel mechanisms of CWI monitoring would strengthen the rationale.

・The phenotypes shown in Fig. S1C–F are striking; incorporating these panels into the main figures would improve visibility and impact.

・In Fig. 1F, since the data quantify BFA bodies per cell, the full single cell should be shown in the images. In addition, cell size should be comparable between the two treatment groups.

・In Fig. 2A, it would be useful to clarify whether MIK2 is included in the detected protein profile.

・In Fig. 3E, because chemiluminescence and staining have different sensitivities, normalization using an endogenous control detected by chemi-lumi (e.g., actin) would allow more accurate quantification.

・In Fig. 3G, if the treatments are independent, the statistical analysis may need revision. A repeated-measures t-test is appropriate only for paired samples.

・In Fig. 4A and C, please clarify whether the Input and IP samples were blotted on the same membrane. Inclusion of appropriate negative controls would also strengthen the data.

・In Fig. 4F–G, the image in F does not appear representative of the distribution shown in G; notably, the double knockout root appears shorter than mik2-1. This point is important for genetic interpretation and should be clarified.

・In Fig. 5G (main text), the statement that “athe-1 roots exhibited a reduced induction trend at 1 dpt” may not be sufficiently supported without evaluation of effect size.

・In Fig. 6C, please specify the protein used for normalization. At minimum, molecular weight information should be indicated on the Ponceau-stained membrane.

・In Fig. 7B, “survival rate” would be a more standard term than “non-dead plants.”

・In the Discussion, it would be valuable to clarify whether ATHE function is more closely associated with CWI responses or FoRALF signaling, or whether these pathways are integrated. A data-driven discussion that weighs these possibilities would improve clarity for readers.

・More broadly, is it possible that ATHE functions as a pattern-triggered immunity (PTI)-related receptor kinase? Incorporating foundational assays (e.g., ROS burst, MAPK activation) would help clarify ATHE function during pathogen infection.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.