Discovery of a Secreted Verticillium Dahliae Protease That Cleaves Cotton CRR1 and Induces Plant Cell Death

Domain of unknown function 26 (DUF26) is a non-catalytic protein domain found only in land plant proteins. These proteins are often associated with defense. Cotton (Gossypium hirsutum L) CRR1 is a secreted protein consisting of two DUF26 domains connected by a linker. In this study we report that CRR1 is cleaved by an alkaline subtilase secreted by Verticillium dahliae, a hemi-biotrophic fungal pathogen. Recombinant CRR1 was converted from a 30 kDa glycoprotein into products of ∼15 kDa (CRR1-P) when incubated with secreted protein extracts from V. dahliae cultures. Using this activity as a guide, the protease was purified, and its tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. It was identified as alkaline subtilase G2X826_VERDV, which we named Vd-DUMP for V. dahliae DUF26 modifying protein. This identification was confirmed by producing active recombinant Vd-DUMP in the yeast Pichia pastoris. Biochemical analysis indicated that Vd-DUMP cleaves CRR1 between the DUF26 domains. Infiltration of Vd-DUMP into Arabidopsis (Arabidopsis thaliana) leaves or cotton cotyledons induced cell death, a response that was absent when Vd-DUMP was chemically inactivated prior to infiltration. This study identifies plant DUF26 proteins, associated with defense, as substrates for alkaline subtilases secreted by fungi that are known to function as elicitors and effectors.