Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

Abstract

Summary Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

Fungal Melanin Biosynthesis Pathway as Source for Fungal Toxins

“…Other prominent examples for PQs are the phytotoxic cercosporin or elsinochrome, which are produced by Cercospora subsp. and Elsinoë ( 47 ). The gene clusters required for those two toxins have been characterized to some extent ( 48 ).…”

“…There was some evidence that the melanin biosynthesis pathway may be involved in elsinochrome biosynthesis ( 49 , 50 ). However, later it was shown that the PQ and the DHN melanin synthesis routes are separate pathways in Colletotrichum beticola and in Elsinoe fawcettii ( 47 ). Hence, the biosynthesis of PQs is still controversial and not solved, despite the great importance of many of such substances as virulence facilitators in plant-pathogenic fungi as mycotoxins or novel anticancer drugs.…”

Sensibilidad y resistencia a fungicidas de <i>Cercospora kikuchii</i>, agente causal del tizón de la hoja y mancha púrpura de la semilla de la soja

“…pers. ) Durante la exploración de los genes de patogenicidad y virulencia conocidos en Cercospora, en la presente tesis se detectaron 4 genes previamente descriptos en C. beticola, involucrados en la biosíntesis de melanina-DHN (Ebert et al, 2019). Si bien el gen central, que codifica para la melanina-DHN PKS (una policétido sintasa), se encuentra conservado entre las especies exploradas, algunos genes como CB0940_09636 (tetrahidroxinaftaleno reductasa) y CB0940_09639 (prefoldina) presentan cierta variabilidad entre especies de Cercospora que quizás podrían contribuir en diferencias de patogenicidad.…”

“…Se cree que este cluster es más amplio, incluyendo genes adicionales (Schumacher, 2016;Ebert et al, 2019) (Martins et al, 2004).…”

“…La cercosporina es un pigmento púrpura-rojizo (tiene un color rojo sangre llamativo) que fue aislado por primera vez al ser extraído a partir de micelios secos de C. kikuchii en la década de 1950 (Kuyama y Tamura, 1957); y su estructura y propiedades químicas se caracterizaron entre principios de los años 1960 y 1970 (Kuyama, 1962;Lousberg et al, 1971;Yamazaki y Ogawa, 1972;Yamazaki et al, 1975). Esta molécula fue químicamente identificada como una quinona de perileno (perilenquinona) de origen policétido, es decir, producto del metabolismo secundario, de peso molecular (masa molar relativa) equivalente a 534 (Figura 5) (Lousberg et al, 1971;Yamazaki y Ogawa, 1972;Ebert et al, 2019). La cercosporina es estrictamente dependiente de la luz con respecto tanto a su producción como a su modo de acción.…”