Functional characterization of the first filamentous fungal tRNA ‐isopentenyltransferase and its role in the virulence of Claviceps purpurea

Summary In plants, cytokinins ( CK s) are synthesized de novo or by the degradation of modified tRNA s. Recently, the first fungal de novo pathway was identified within the plant pathogen Claviceps purpurea . As the deletion of the de novo pathway did not lead to a complete loss of CK s, this work focuses on the tRNA ‐modifying protein tRNA ‐isopentenyltransferase (Cpt RNA ‐ IPT ). The contribution of this enzyme to the CK pool of Claviceps and the role of CK s in the host–pathogen interaction are emphasized. The effects of the deletion of cpt RNA ‐ipt and the double deletion of cpt RNA ‐ipt and the key gene of de novo biosynthesis cpipt‐log on growth, CK biosynthesis and virulence were analyzed. In addition, the sites of action of Cpt RNA ‐ IPT were visualized using reporter gene fusions. In addition to CK ‐independent functions, Cpt RNA ‐ IPT was essential for the biosynthesis of cis ‐zeatin ( c Z ) and contributed to the formation of isopentenyladenine ( iP ) and trans ‐zeatin ( t Z ). Although Δcpt RNA ‐ipt was reduced in virulence, the ‘ CK ‐free’ double deletion mutant was nearly apathogenic. The results prove a redundancy of the CK biosynthesis pathway in C. purpurea for iP and tZ formation. Moreover, we show, for the first time, that CK s are required for the successful establishment of a host–fungus interaction.