Discovery of a secreted Verticillium dahliae protease that cleaves cotton CRR1 and induces plant cell death

Authors: Naumann, Todd; Hao, Guixia; Dowd, Patrick; Johnson, Eric; NALDRETT, MICHAEL - University Of Nebraska; Price, Neil

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2022
Publication Date: 11/30/2022
Citation: Naumann, T.A., Hao, G., Dowd, P.F., Johnson, E.T., Naldrett, M.J., Price, N.P.J. 2022. Discovery of a secreted Verticillium dahliae protease that cleaves cotton CRR1 and induces plant cell death. Physiological and Molecular Plant Pathology. 123. Article 101941. https://doi.org/10.1016/j.pmpp.2022.101941.
DOI: https://doi.org/10.1016/j.pmpp.2022.101941

Interpretive Summary: Crop diseases caused by fungi reduce yield, costing farmers money. They can also contaminate harvested grains with toxins that harm people, pets, and livestock. Crop diseases are caused by pathogen proteins that manipulate the plant immune system. Discovering specific pathogen proteins for a crop disease and how they work can lead to improved plant health through plant breeding. In this work ARS researchers in Peoria, Illinois, discovered a fungal protein that causes disease by cutting a specific plant defense protein. This fungal protein is involved in Verticillium wilt of cotton, a devastating disease that causes economic losses for US farmers, especially in western Texas and the San Joaquin Valley of California. But the same protein appears to be involved in other plant diseases including Fusarium diseases of corn, wheat, and barley that produce toxins that are a major safety concern. This discovery will help crop breeders produce seed for farmers with improved disease resistance. This research will ultimately reduce economic losses for farmers and improve the safety and security of food and feeds.

Technical Abstract: 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.