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An elevated environmental temperature impairs accumulation of the pattern recognition receptor FLS2

Bryony C.I.C. Jacobs, Kyle W. Bender, Emma Six, Cyril Zipfel, Marc R. Knight

https://doi.org/10.1101/2025.10.20.683271 (BioRxiv)

1. Summary of the findings

This study addresses a timely and biologically significant topic: the regulation of the immune response, more specifically of the FLS receptor, in response to a transient increase in temperature from 20 to 28 °C.treatment. The authors propose that the receptor levels are reduced upon heat treatment, correlating it to increased membrane fluidity. While the molecular phenotypic response is solid, the mechanistic function underlying this effect and to what extent it can be generalized to other PAMP receptors are not investigated in this manuscript.

2. Major issues

We are concerned about the ambient temperature treatment (24h at 28°C). The choice of these conditions is based on (Penfield, 2008) and (Liu et al., 2015). Yet we cannot exclude that this treatment might be perceived by the plant as a stress signal. To test this, you could for example do a time-course of a high temperature stress signal read-out to show that after 24h the stress-response is alleviated. In line with this it would be a valuable information to test for FLS2 protein levels not only after a 24h treatment but as well in plants grown longer than 24h or continuously at 28°C, to rule out that your results are an artifact of a stress-induced response.

3. Minor issues

a) We wonder whether the temperature treatment you chose (24h at 28°C) is representative of a real-life scenario, since in most climates there is a temperature fluctuation between day and night, while in your experiments the seedlings stay at 28°C for 24h.

b) We think it would be interested to know whether a similar effect is seen in fully grown plants or whether it is specific to in seedlings.

c) It is unclear whether the measurements (ROS, calcium spikes) are made at 28°C or all at room temperature. Please specify this important information in your method section. In case the measurement was not taken at 28°C we are concerned that the seedlings grown at high temperatures might have a stress-response to the change to a lower temperature.

d) We would recommend using a more direct read-out for immune activation by FLS2, eg MAPK-phosphorylation. Also for the qPCRs including direct marker genes of PTI signaling such as FRK1 and NHL10 would be informative instead of relying entirely on the downstream read-out of SA/EDS1 signaling used here.

e) Figures 3 and 4: a comparison is made between the bands of the western blot, yet no quantification values are reported. Even though the difference is striking in Fig.3, we would welcome quantification, especially for Fig.4.

f) In the qPCR experiments only PEX4 was used to normalise the expression levels. Do you have data showing that PEX4 expression is temperature independent? We would recommend showing the validation of your reference gene. Additionally, as PEX4 is responsible for the transport of receptors, we recommend using at least another gene for the normalisation of the qPCR data.

g) We would warmly welcome if you would publish your qPCR data as non-relative data, with the raw Ct values or normalized values (e.g., ΔCt) as supplementary data. This would enable a better interpretation of your data when compared to other datasets. Please also think of publishing your raw data as a dataset.

h) In figure 1 you show both the calcium response and the ROS results. Yet in figure 5 you show only the calcium response. As it is an equivalent figure to 1, but with the pMDC7FLS2 construct, we would have expected to see a ROS result as well.

i) For future investigations, we would suggest determining whether the biochemistry is still functional at 28°C, for example by purifying the kinase domain in vitro and testing its activity.

j) Minor comments:

1) Figure 1+5: Due to the overlap, it is not possible to see the height of the calcium spike of the different treatments. Maybe you could add the value or mark the height?

2) In the supplementary figure 1 “fec” seedlings are mentioned. Did you mean the fls2C mutant?

3) “Our work shows that similar effects can be seen in response to much more moderate temperature elevations, which are much closer to relevant field temperatures, suggesting this phenomenon is of significant contemporary relevance to agriculture“ The temperature you chose is probably relevant for agricultural fields in specific climates, but not for everywhere in the world. Please specify which climates you are referring to.

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.