The chitin deacetylase family of U. maydis

Chitin present in the cell wall of fungi is a potent MAMP end elicits strong plant defense responses. To downregulate these defenses or to protect hyphae from attack by plant chitinases, fungi have developed an arsenal of different strategies including the production of effectors which either bind chitin fragments in the apoplast or act as stealth molecules by binding to fungal hyphae. A less explored fungal strategy involves the transformation of chitin to chitosan by chitin deacetylases (CDAs). Chitosan is a poor substrate for chitinases and it does not activate chitin receptors if its fraction of acetylation is low enough. We have shown that U. maydis encodes six active CDA genes as well as one pseudogene. These genes are differentially expressed during colonization. One of the CDAs (Cda4) is predicted to be secreted and to reside in the apoplast and ﬁve are GPI-anchored CDAs of which Cda7 is the ﬁrst member of a new class of fungal CDAs. We have now provided a comprehensive functional study of the entire family. In budding cells of U. maydis we have detected a discrete pattern of chitosan staining which changes upon plant colonization when biotrophic hyphae become surrounded by a chitosan layer.

Chitin and chitosan staining of U. maydis hyphae during infection. U. maydis hyphae were analyzed four days after infection. To discriminate hyphae on the leaf surface from those that had penetrated, hyphae on the leaf surface were stained with calcofluor white, a stain unable to penetrate the epidemis and appear in blue. Chitin in hyphae on the leaf surface is detected in cyan. Intracellular hyphae are stained for chitin in green and for chitosan in red. Yellow indicates the presence of chitin and chitosan in intracellular hyphae. This shows that intracellular hyphae become surrounded by chitosan layer. — **Chitin and chitosan staining of *U. maydis* hyphae during infection.** *U. maydis* hyphae were analyzed four days after infection. To discriminate hyphae on the leaf surface from those that had penetrated, hyphae on the leaf surface were stained with calcofluor white, a stain unable to penetrate the epidemis and appear in blue. Chitin in hyphae on the leaf surface is detected in cyan. Intracellular hyphae are stained for chitin in green and for chitosan in red. Yellow indicates the presence of chitin and chitosan in intracellular hyphae. This shows that intracellular hyphae become surrounded by chitosan layer.

**Chitin and chitosan staining of *U. maydis* hyphae during infection.** *U. maydis* hyphae were analyzed four days after infection. To discriminate hyphae on the leaf surface from those that had penetrated, hyphae on the leaf surface were stained with calcofluor white, a stain unable to penetrate the epidemis and appear in blue. Chitin in hyphae on the leaf surface is detected in cyan. Intracellular hyphae are stained for chitin in green and for chitosan in red. Yellow indicates the presence of chitin and chitosan in intracellular hyphae. This shows that intracellular hyphae become surrounded by chitosan layer.

We puriﬁed all six active CDAs and showed their activity on different chitin substrates in collaboration with the group of Prof. Dr. Bruno Moerschbacher at Münster University. Single as well as multiple cda mutants were generated and revealed a virulence defect for mutants lacking cda7 which manifests itself already during the early infection stages. We implicated cda4 in production of the chitosan layer surrounding biotrophic hyphae and demonstrated that the loss of this layer does not reduce virulence. This surprising result indicates that the chitin layer surrounding biotrophic hyphae does not have a stealth function. However, certain combinations of mutants were signiﬁcantly reduced in virulence, suggesting functional redundancy. The loss in virulence was accompanied with reduced adherence, appressoria formation, penetration, and with activation of plant defenses. Attempts to inactivate all seven cda genes were unsuccessful, illustrating an essential role of chitosan for cell wall integrity in U. maydis.