Curled Flag Leaf 2, Encoding a Cytochrome P450 Protein, Regulated by the Transcription Factor Roc5, Influences Flag Leaf Development in Rice

Moderate curling generally causes upright leaf blades, which favors the establishment of ideal plant architecture and increases the photosynthetic efficiency of the population, both of which are desirable traits for super hybrid rice (Oryza sativa L.). In this study, we identified a novel curled-leaf mutant, curled flag leaf 2 (cfl2), which shows specific curling at the base of the flag leaf owing to abnormal epidermal development, caused by enlarged bulliform cells and increased number of papillae with the disordered distribution. Map-based cloning reveals that CFL2 encodes a cytochrome P450 protein and corresponds to the previously reported OsCYP96B4. CFL2 was expressed in all analyzed tissues with differential abundance and was downregulated in the clf1 mutant [a mutant harbors a mutation in the homeodomain leucine zipper IV (HD-ZIP IV) transcription factor Roc5]. Yeast one-hybrid and transient expression assays confirm that Roc5 could directly bind to the cis-element L1 box in the promoter of CFL2 before activating CFL2 expression. RNA sequencing reveals that genes associated with cellulose biosynthesis and cell wall-related processes were significantly upregulated in the cfl2 mutant. The components of cell wall, such as lignin, cellulose, and some kinds of monosaccharide, were altered dramatically in the cfl2 mutant when compared with wild-type “Jinhui10” (WT). Taken together, CFL2, as a target gene of Roc5, plays an important role in the regulation of flag leaf shape by influencing epidermis and cell wall development.

TheAspergillus fumigatusDamage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

ABSTRACTErgosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogenAspergillus fumigatushas developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochromeb5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapAstrains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme.In vivodata demonstrate that inactivated DapA combined with activated DapB yields anA. fumigatusmutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found inSaccharomyces cerevisiaeandSchizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli.IMPORTANCEKnowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochromeb5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.