Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae

Authors: CADDELL, DANIEL - UNIVERSITY OF CALIFORNIA, DAVIS; WEI, TONY - UNIVERSITY OF CALIFORNIA, DAVIS; SHARMA, SWETA - UNIVERSITY OF CALIFORNIA, DAVIS; OH, MAN-HO - UNIVERSITY OF ILLINOIS; WEI, TONG - UNIVERSITY OF CALIFORNIA, DAVIS; PARK, CHANG-JIN - UNIVERSITY OF CALIFORNIA, DAVIS; CANLAS, PATRICK - UNIVERSITY OF CALIFORNIA, DAVIS; Huber, Steven; RONALD, PAMELA - UNIVERSITY OF CALIFORNIA, DAVIS

Submitted to: PeerJ
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2018
Publication Date: 12/11/2018
Citation: Caddell, D.F., Wei, T., Sharma, S., Oh, M., Wei, T., Park, C., Canlas, P., Huber, S.C., Ronald, P.C. 2018. Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae. PeerJ. 6:e6074. https://doi.org/10.7717/peerj.6074.
DOI: https://doi.org/10.7717/peerj.6074

Interpretive Summary: Receptor kinases are often involved in the recognition of bacterial pathogens and initiate a signal transduction cascade that induces defense responses and resistance. Recognition of the pathogen triggers phosphorylation and activation of the protein kinase domain of the receptor kinase, and it is important to identify which residues are phosphorylated and their role in immune signaling. The rice receptor kinase, XA21, is required for resistance to Xanthomonas oryzae, which causes a serious blight disease. Previous work with XA21 established that the protein kinase domain of the receptor was active and could autophosphorylate on serine and threonine residues. Here we demonstrate that XA21 can also autophosphorylate on tyrosine residues, and three specific residues (Tyr-698, Tyr-907 and Tyr-909) are likely phosphosites. Preventing phosphorylation at the four potential tyrosine phosphosites (by substitution with phenylalanine) did not affect susceptibility to transgenic rice to the Xanthomonas pathogen. Therefore, either tyrosine phosphorylation of XA21 does not occur in vivo, or it is not playing a role in regulation of XA21 activity.

Technical Abstract: Tyrosine residues often play an important role in protein function, sometimes as a result of phosphorylation, which has emerged as an important regulator of the activity of plasma membrane localized immune receptors. However, a role for tyrosine phosphorylation in regulating the rice XA21 immune receptor has not been investigated. Here we report that the cytoplasmic domain of XA21 autophosphorylates on Tyr residues during expression in E. coli. Directed mutagenesis of the ten tyrosine residues in the cytoplasmic domain caused reduced autophosphorylation of Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain (JK), identifying these residues as potential phosphosites. Rice plants expressing XA21-GFP fusion proteins and fusion proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP) to prevent phosphorylation at the site, were resistant to Xanthomonas oryzae pv. oryzae (Xoo). However, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP), were susceptible. Ectopically expressed XA21JK, XA21JKY698F, XA21JKY907F, and XA21JKY909F were kinase active and autophosphorylated in vitro, while XA21JKY768F and the four XA21JKYD variants were not. In addition to kinase activity, the direct interaction between XA21 and OsSERK2 is critical for its biological function. The four XA21JKYF variants interacted with OsSERK2 in yeast, while XA21JKYD variants did not. Taken together, these results suggest that although XA21 has tyrosine kinase activity, either phosphotyrosine does not occur in vivo or it is not playing a role in activation of XA21-mediated immunity.