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Fungal cell wall mannans are complex carbohydrate polysaccharides with different structures in yeasts and molds. In contrast to yeasts, their biosynthetic pathway has been poorly investigated in filamentous fungi. In Aspergillus fumigatus, the major mannan structure is a galactomannan that is cross-linked to the β-1,3-glucan-chitin cell wall core. This polymer is composed of a linear mannan with a repeating unit composed of four α1,6-linked and α1,2-linked mannoses with side chains of galactofuran. Despite its use as a biomarker to diagnose invasive aspergillosis, its biosynthesis and biological function were unknown. Here, we have investigated the function of three members of the Ktr (also named Kre2/Mnt1) family (Ktr1, Ktr4, and Ktr7) in A. fumigatus and show that two of them are required for the biosynthesis of galactomannan. In particular, we describe a newly discovered form of α-1,2-mannosyltransferase activity encoded by the KTR4 gene. Biochemical analyses showed that deletion of the KTR4 gene or the KTR7 gene leads to the absence of cell wall galactomannan. In comparison to parental strains, the Δktr4 and Δktr7 mutants showed a severe growth phenotype with defects in polarized growth and in conidiation, marked alteration of the conidial viability, and reduced virulence in a mouse model of invasive aspergillosis. In yeast, the KTR proteins are involved in protein 0- and N-glycosylation. This study provided another confirmation that orthologous genes can code for proteins that have very different biological functions in yeasts and filamentous fungi. Moreover, in A. fumigatus, cell wall mannans are as important structurally as β-glucans and chitin.IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It allows fungal pathogens to survive environmental challenge posed by nutrient stress and host defenses, and it also is central to polarized growth. The cell wall is mainly composed of polysaccharides organized in a three-dimensional network. Aspergillus fumigatus produces a cell wall galactomannan whose biosynthetic pathway and biological functions remain poorly defined. Here, we described two new mannosyltransferases essential to the synthesis of the cell wall galactomannan. Their absence leads to a growth defect with misregulation of polarization and altered conidiation, with conidia which are bigger and more permeable than the conidia of the parental strain. This study showed that in spite of its low concentration in the cell wall, this polysaccharide is absolutely required for cell wall stability, for apical growth, and for the full virulence of A. fumigatus. ; This research was funded by l'Agence Nationale pour la Recherche (AfuInf ANR-16-CE92-0039), la Fondation pour la Recherche Médicale (DEQ20150331722 LATGE Equipe FRM 2015), the French Government's Investissement d'Avenir program, and Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant ANR-10-LABX-62-IBEID). ; Sí