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 F2 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.

Identification of A Novel CYP4V Gene In The Polychaete Perinereis Aibuhitensis: Transcriptional Comparison With CYP4B Exposed To PAHs

Polychaete worms can biotransform polycyclic aromatic hydrocarbons (PAHs) in environments, and the cytochrome P450 (CYP) enzyme plays an important role in this process. Herein, a novel cytochrome P450 gene was identified and characterized from the polychaete worm Perinereis aibuhitensis. The full-length cDNA, which is named CYP4V82, is 1709 bp encoding a protein of 509 amino acids and has high similarity to CYP4V. The expression levels of CYP4V82 and CYP4BB4 (a CYP gene identified from P. aibuhitensis in a previous study, Chen et al., 2012) exposure to various concentrations of benzo[α]pyrene (B[α]P) (0.5, 2, 4, and 8 μg/L) and same mass concentrations of fluoranthene (Flu, 3.2 μg/L), phenanthrene (Phe, 2.9 μg/L), B[α]P (4.0 μg/L) were detected to identify the function of the CYP4 family in P. aibuhitensis. Compared with CYP4BB4, CYP4V82 mRNA was minimally expressed on day 7 but highly sensitive on day 14. Notably, the expression level of CYP4V82 and CYP4BB4 was relatively different in response to PAHs with different benzene rings of the same concentration. The expression of CYP4V82 in the B(a)P group was the highest, while that of CYP4BB4 in the Phe group was relatively higher than the two other groups. These findings suggest that PAHs are associated with the induction of CYP4V82 and CYP4BB4 expressions, which may have different efficiencies in the detoxification of PAHs.

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

ABSTRACTDisease 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-γ1 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.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 nutrient from the host. However, these defense mechanisms are hijacked by necrotrophic plant pathogens that purposefully induce PCD mechanism to feed from the dead cells 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. The nec3 mutants have disrupted serotonin biosynthesis resulting in expansive PCD, necrotrophic pathogen susceptibility and cutin layer instability. The nec3 mutants lacking serotonin deposition in pathogen induced necrotic lesions show expansive PCD and disease susceptibility suggesting a role of serotonin to sequester PCD and suppress pathogen colonization. The identification of Nec3 will facilitate functional analysis to elucidate the role serotonin plays in the elicitation or suppression of PCD immunity responses to diverse pathogens and effects it has on cutin layer biosynthesis.