TKPs form an important protein family capable of conferring resistance to diverse fungal pathogens. Here, we provide the first assessment of tandem kinases’ distribution across the plant kingdom. Our results demonstrate that the TKP protein family is widespread across the plant kingdom, with an average of 25.7 TKPs identified in each of the 104 studied plant genomes. Recently, two tandem KD-PKDs (TKP7 and TKP8) were found in sugarcane as the candidate genes for Bru1 brown rust resistance34. Wheat blast resistance protein RWT4, allelic to powdery mildew resistance protein WTK3, can also trigger immunity in rice protoplasts by perceiving the effector AvrPWT4, indicating TKPs can be transferred between plant genera22. Therefore, the current TKP Atlas sheds light on plant immunity, probably far beyond the Triticeae tribe.
The RLK Pelle kinase family is the plants' most extensive group of receptor-like kinases35. Within the TKP sequences discovered in this work, more than 95% of the KDs belonged to the RLK Pelle family. Our data revealed an almost equal number of TKPs with KD-KD or KD-PKD architectures. However, predicting unfunctional kinases based only on their sequence is still imperfect and some classified PKDs may possess kinase activity. The kinase activity of RLKs has been well studied, such as the RLK LecRK-IX.2 from Arabidopsis36. However, the TKP’s KDs kinase activity was only experimentally proved in three cases, RPG1, WTK7-TM and RWT420,22. For example, the two kinases of WTK7-TM were predicted as PKD based on their sequences, but when tested experimentally they were both shown to be functional20. This suggests that different TKPs exhibit diverse kinase activities as should be expected for a protein family that evolved by convergent molecular evolution and provides similar solutions under selection by pathogen stress. Further studies are required to determine the activity and function of the predicted KD and PKD domains.
Most of the experimentally confirmed functional TKPs (e.g. RPG1 and WTK1-WTK6) are cytoplasmic proteins, except for WTK7-TM which possesses a transmembrane domain shown to be essential for protein function20. Furthermore, a quarter of the TKPs in our data had both a transmembrane domain and a signal peptide indicating membrane association31. These accumulated data suggest that at least a portion of TKPs are associated with cell membranes and may detect pathogens in this subcellular location. Taken together, these data provide an important TKP atlas for the plant community and highlight many novel facets of TKP evolution and function.
Notably, all ten experimentally identified TKPs contained at least one domain from the RLK Pelle family11–21(Supplementary Table S1). Our study suggests that a significant proportion of TKPs with two RLK Pelle Lec domains are the result of a single ancient fusion event between distant members of the family. This fusion is indicative of an ancient event that has persisted across multiple species (monocots and dicots). However, the remainder of TKPs with two RLK Pelle L-Lec domains arise from numerous independent fusion events among closely related members (Fig. 6). This latter pattern is also typical of the DLSV family. RLKs are a large gene family and can be tandemly clustered, which could facilitate independent fusion events33. The occurrence of these independent events may demonstrate convergent molecular evolution, if indeed most of the events are related to plant biotic mechanisms. Different plant species have independently evolved similar receptor characteristics to adapt to various plant pathogens, as demonstrated for the RLP30 immune receptor, which plays a role in immunity against pathogens from two microbial kingdoms36.
In previous work, we proposed a model for the molecular function of TKPs with a combination of active and inactive domains38. In the context of immune perception, the pseudokinase domain is hypothesized to serve as a decoy to interact with pathogen components working together with the kinase for signal transduction38. However, a recent study Sung et al. (2024) found that the RWT4 can specifically bind the AvrPWT4 effector in both the kinase and pseudokinase regions, leading to the transcription of defense genes and inducing cell death. Moreover, the authors show tandem kinases may serve as a novel class of immune receptors capable of directly interacting with pathogens, independent of NLRs or PRRs22.
Another possible hypothesis for the activation of tandem kinases recognition of the effector occurs via integrated non-kinase domains. Like in NLR-ID receptors, the integration is the decoy domain, which mimics pathogen effector targets, and allows for direct interception of effector pathogen proteins10. Recently, two TKPs (WTK6-vWA and WTK6b-vWA) were demonstrated to include an integrated domain19,21. Mutations in these vWA domains resulted in a loss of resistance, which may suggest that the two IDs located at the C-terminus of these proteins are possibly involved in effector recognition19. More than half (56%) of the discovered TKPs contained at least one integrated non-kinase domain, with the most common being betta lectin, LRR VIII, and Stress-antifungal. Beta lectin (Legume lectin domain; PF00139) is a carbohydrate-binding domain often found in proteins involved in plant defense reactions, such as chitinases, glucanases, and thaumatins24–26. The functions of the LRR VIII domain are poorly understood, but LRR domains are associated with plant immune responses, often found in immune receptors like LRR-RLKs10. Another significant ID identified in NLR proteins is the HMA domain. Multiple NLRs with HMA IDs experimentally verified that the HMA domain directly interacts with the effector39. We identified TKPs that are fused with HMA, including the characterized TKP RPG1. Based on this evidence, it is plausible to propose that integrated domains present in TKPs could also participate in pathogen perception by acting as decoys of pathogen virulence targets.
We identified 637 TKPs with transmembrane domain, signal peptide, and integrated domains (such as stress antifungal or DUF26, LysM, beta lectin and etc), which also were found in another family of plant immune receptors — RLKs40. These TKPs probably have evolved from RLKs, however, RLKs have one kinase domain, whereas tandem kinases have two or more. This might indicate an evolutionary separation of functions after gene duplication or fusion events, leading to the birth of a new type of receptors in plants28.
In conclusion, our study provides new insights into the diversity of tandem kinase proteins across the plant kingdom. Our analysis revealed a high degree of variability in the number of these proteins across different species. We also found that many TKPs contain integrated non-kinase domains, which could affect their functional properties. Our findings highlight the importance of studying tandem kinase proteins' functional properties in plants and their potential contribution to plant resistance to pathogens. The animal Janus tandem kinases (JAKs) participate in complex regulatory networks governing cell differentiation, tissue regeneration, and innate immune responses41,42. For example, JAK3 differentially regulates Toll-like receptors (TLR)-mediated inflammatory cytokine production in innate immune cells. It is possible that plants’ TKPs may be involved in similar processes, offering another level of functional diversity.