ARBUSCULAR MYCORRHIZA-INDUCED KINASES AMK8 and AMK24 associate with the receptor-like kinase KINASE3 to regulate arbuscular mycorrhizal symbiosis in Lotus japonicus

A signaling pathway to accommodate arbuscular mycorrhizal fungi consists of receptor-like kinase-associated components at the periarbuscular membrane and is evolutionarily conserved in legumes and cereals. Arbuscular mycorrhizal (AM) symbiosis is a widespread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3, AMK8, or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171, the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation.

Automated summary

A signaling pathway involving receptor-like kinases and receptor-like cytoplasmic kinases plays a crucial role in arbuscular mycorrhizal (AM) symbiosis. This pathway is conserved in legumes and cereals. The expression of certain kinases is upregulated by AM transcription factors, and several of these kinases are specifically required for AM symbiosis. The CBX1 transcription factor directly regulates the expression of KIN3, a key kinase involved in AM symbiosis, and loss of function mutations in KIN3, AMK8, or AMK24 result in reduced mycorrhizal colonization.