Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea

Abstract

In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite. IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Subcellular localization of fungal specialized metabolites

“…Investigation into the biosynthesis of melanin in Botrytis cinerea led to the observation that scytalone is secreted extracellularly and accumulation of this metabolite inhibits the growth of B. cinerea [ 46 ]. Similar to melanin biosynthesis in A. fumigatus , melanin biosynthesis in B. cinerea requires six biosynthetic enzymes: the PKS BcPKS12 (or BcPKS13 with the hydrolase BcYGH1); the reductases BcBRN1 and BcBRN2; and the dehydratase BcSCD1 (Scheme 3 C) [ 47 ].…”

“…The subcellular location of the different melanin biosynthetic enzymes was determined via fusion with GFP in combination with organelle stains. The two PKS, BcPKS12 and BcPKS13, and the hydrolase BcYGH1 were demonstrated to be localized in peroxisomes, the two reductases BcBRN1 and BcBRN2 were detected within endosomes initially, and BcSCD1 is localized at the cell wall (Scheme 3 C) [ 46 ]. The specific locations of BcBRN1 and BcBRN2 is further described in " Transient recruitment of enzymes and trafficking " section.…”

“…Melanin biosynthesis in B. cinerea is also proposed to involve enzyme trafficking since the subcellular localization of BcBRN1/2 is observed in the cytoplasm, endosomes, and the cell wall [ 46 ]. This correlates well with the observed toxicity of scytalone and the requirement of BcBRN1/2 in its synthesis and subsequent conversion (Scheme 3 C).…”

“…The trafficking of redox enzymes to the cell wall / outside of the cell has been reported in the biosynthesis of melanin [ 19 , 46 ], patulin [ 53 ], and fumiquinazoline [ 112 ], perhaps indicating a frequent self-resistance mechanism. Finally, the containment of specialized metabolites has been also reported within specific tissues e.g.…”

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence

“…In M. oryzae and Colletotrichum gloeosporioides , melanin is a prerequisite for the formation of functional appressoria that accumulate turgor pressure necessary to penetrate plant cell walls [ 22 – 24 ]. Melanized sclerotia of Sclerotinia sclerotiorum and B. cinerea are essential for vegetative growth, long-term survival, and pathogenicity [ 25 – 27 ]. In Hortaea werneckii , melanin is important to adapt to osmotic stress, maintenance of cell wall integrity, and normal cell division [ 28 ].…”