Disease lesion mimic mutants (DLMMs) are characterized by the 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. 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 F
2 progeny of a cross between
nec3-γ1 and variety Quest segregated as a single recessive susceptibility 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 the 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.
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