A wheat tandem kinase and NLR pair confers resistance to multiple fungal pathogens

  1. Ping Lu
  2. Gaohua Zhang
  3. Jing Li
  4. Zhen Gong
  5. Gaojie Wang
  6. Lingli Dong
  7. Huaizhi Zhang
  8. Guanghao Guo
  9. Min Su
  10. Yueming Wang
  11. Keyu Zhu
  12. Qiuhong Wu
  13. Yongxing Chen
  14. Miaomiao Li
  15. Baoge Huang
  16. Beibei Li
  17. Wenling Li
  18. Lei Dong
  19. Yikun Hou
  20. Xuejia Cui
  21. Hongkui Fu
  22. Dan Qiu
  23. Chengguo Yuan
  24. Hongjie Li
  25. Jianmin Zhou
  26. Guan-Zhu Han
  27. Yuhang Chen
  28. Zhiyong Liu

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Abstract

Recently discovered tandem kinase proteins (TKPs) are pivotal to the innate immune systems of cereal plants, yet how they initiate plant immune responses remains unclear. This report identifies the wheat protein WTN1, a non-canonical NLR receptor featuring tandem NB-ARC domains, as crucial for WTK3- mediated disease resistance. Both WTK3 and its allelic variant Rwt4, known for conferring resistance to wheat powdery mildew and blast respectively, are capable of recognizing the blast effector PWT4, and activate WTN1 to form calcium-permeable channels, akin to ZAR1 and Sr35. This study unveils a unique plant defense mechanism wherein TKPs and associated NLRs operate as “sensor-executor” pairs against fungal pathogens. Additionally, evolutionary analyses reveal a co-evolutionary trajectory of the TKP-NLR module, highlighting their synergistic role in triggering plant immunity.

One Sentence Summary

An ancient synergistic TKP-NLR pair triggers innate immunity for multiple disease resistance in wheat.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14395253.

    General Remarks

    In wheat and barley, tandem kinase proteins (TKPs) have emerged as a crucial class of resistance determinants. Previously, Lu et al. cloned the powdery mildew fungal resistance TKP, known as WHEAT TANDEM KINASE 3 (WTK3), and others published a related TKP, RWT4, which confers resistance to blast fungus. This paper demonstrates that the WTK3 TKP relies on an atypical NLR protein, WTN1, to function. The authors suggest that this represents a decoy sensor/NLR immune system in cereal grasses.

    The paper starts off promisingly with the cloning of WTN1 via EMS mutagenesis, confirmed independently through CRISPR mutants. The genetics in Figure 1 looks solid. However, the paper then moves into a series of experiments that lack proper controls, leading to serious doubts about the overall quality of the work and the validity of the conclusions. The experiments fall short of the standards expected for a high-quality paper. 

    Below is a list of critical comments and observations:

    • Figure 2A-B: These figures require western blot checks to ensure that the proteins are properly expressed, intact, and not subject to processing. Additionally, stability tests are needed to confirm that the stability of these proteins isn't compromised in different combinations.

    • Figure 2C: This experiment lacks a proper negative control, which is seriously concerning. It's a FLAG immunoprecipitation (IP) of an HA-tagged protein, but there's no HA-tagged negative control protein. As a result, after IP-FLAG, there should be no HA-tagged proteins that suddenly appear. So where is the negative control? Conducting and submitting such an experiment without proper controls is unacceptable.

    • Figure 2D: There are bands the size of Flag-WTK3 in lanes that shouldn't contain any Flag-tagged proteins of this size, indicating issues with the experiment design or execution.

    • Figure 3D: This figure suffers from the same problem as Figure 2C. It features an IP without a negative control for PWT4-HA, raising questions about the experiment's reliability.

    • Figure 3F: This limited experiment could benefit from additional controls and treatments. It would be helpful to know how RWT4 and WTN1 behave when tested individually. Is RWT4 even tagged? How do the effectors perform in these assays? The lack of adequate controls makes this an incomplete experiment. The much-emphasized point about WTN1 oligomerization relies on these two poorly controlled strips, which is problematic.

    • Figure 3F non-denaturing gels: This approach could be used to validate effector binding to TKP in the presence or absence of the NLR, which would be crucial for supporting the proposed model.

    • Many of the comments above also apply to Figure 4, which has 12 panels (A-M), many of which are incomplete and lack essential controls. Without these controls, it's hard to draw reliable conclusions from these experiments.

    • Panels E-H in Figure 4 feel out of place as this figure is mostly focused on calcium channels. Panel 4H is also essentially 3E. The placement of the two in two separate figures is confusing.

    • Figure S2: Similar to the biochemistry experiments structural modeling figures also miss proper controls and details such as confidence scores.

    • Figure S6: For HR assays no western blots showing proper accumulation of proteins is shown. S6A also lacks a positive control.

    • Figures S8, S9, S10: Despite the evolutionary model in Figure S9, the authors do not show any information on the Oryzoideae and Poaceae lineages. In Figure S9, the presence-absence plot and number of pairs in each species are limited to Pooideae. Drawing conclusions based on phylogenetic trees lacking the other lineages is problematic.

    • None of the reported phylogenetic trees are annotated with support values to examine the robustness of the tree topology. Authors only gave support values for the subclades of interest.

    • Figure S11: Authors don't mention why they chose NBD1 as a representative to draw the framework.

    • Overall, the phylogenetic analysis would've benefited significantly from a more in-depth dataset showing the overall placement of the TKD kinases and NLR clades relative to other kinases and NLRs rather than only extracting the close orthologs using the mentioned methods. Given the absence of sequence data and control reference sequences to assess the robustness of the analysis, the claims on the evolutionary origin of the kinases and the two NBD domains are rather inconclusive.

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2024.08.30.610185v1 on bioRxiv