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

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-gamma 1 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. Author summaryAt the site of pathogen infection, plant defense mechanisms rely on controlled programmed cell death (PCD) to sequester biotrophic pathogens that require living cells to extract nutrients from the host. However, these defense mechanisms are hijacked by necrotrophic plant pathogens that purposefully induce PCD to feed on the dead cells, thus facilitating further disease development. Thus, understanding PCD responses is important for resistance to both classes of pathogens. We characterized five independent disease lesion mimic mutants of barley designated necrotic 3 (nec3) that show aberrant regulation of PCD responses upon pathogen challenge. A cytochrome P450 gene was identified as Nec3 encoding a Tryptamine 5-Hydroxylase that functions as a terminal serotonin biosynthetic enzyme in the Tryptophan pathway of plants. We posit that nec3 mutants have disrupted serotonin biosynthesis resulting in expanded PCD, necrotrophic pathogen susceptibility and cutin layer instability. The nec3 mutants show expanded PCD and disease susceptibility of pathogen-induced necrotic lesions, suggesting a role of serotonin to sequester PCD and suppress pathogen colonization. The identification of Nec3 will facilitate functional analysis to elucidate the role that serotonin plays in the elicitation or suppression of PCD immunity responses to diverse pathogens and the effects it has on cutin layer biosynthesis.