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Abstract: The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol. [Copyright &y& Elsevier]