Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability

Authors: AMEEN, GAZALA - Washington State University; SOLANKI, SHYAM - Washington State University; SAGER-BITTARA, LAUREN - North Dakota State University; RICHARDS, JONATHAN - Louisiana State University Agcenter; Tamang, Prabin; Friesen, Timothy; BRUEGGEMAN, ROBERT - Washington State University

Submitted to: PLoS Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2021
Publication Date: 12/16/2021
Citation: Ameen, G., Solanki, S., Sager-Bittara, L., Richards, J., Tamang, P., Friesen, T.L., Brueggeman, R.S. 2021. Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability. PLoS Genetics. 17(12). Article e1009473. https://doi.org/10.1371/journal.pgen.1009473.
DOI: https://doi.org/10.1371/journal.pgen.1009473

Interpretive Summary: The ability of plants to resist pathogens and pests is essential to protecting plant health and development. However, the genes and genetic networks involved in governing resistance are not completely understood, especially in barley. Here we used a genetic strain of barley that was deficient in its ability to normally activate a biological mechanism known to be associated with disease and pest resistance in plants referred to as programmed cell death. Artificial inoculation of this genetic strain of barley with a fungal pathogen led to the formation of abnormally large disease lesions on the barley leaves indicating this barley strain was compromised in its ability to resist the pathogen. Genetic analysis indicated that the barley strain contained a mutated form of a gene known as P450, which affected the stability of the outer leaf layer, the first layer of defense against pests and pathogens. This work increases our understanding of plant defense and will be of interest to researchers working on plant signaling, plant defense, and plant breeding.

Technical Abstract: Disease lesion mimic mutants (DLMMs) are characterized by spontaneous development of necrotic spots with various phenotypes designated as necrotic ( nec ) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Also, barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3- .1 and variety Quest segregated as a single recessive gene post inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch . Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.