Chitin, a linear polysaccharide composed of (134)-linked 2-acetamido-2-deoxy-␤-D-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (134)-linked GlcNAc and 2-amino-2-deoxy-␤-D-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (134)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (134)-linked GlcN were not active as elicitors, whereas purified oligomers of (134)-linked GlcNAc with a degree of polymerization > 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Summary• Conversion of surface-exposed chitin to chitosan in cell walls of in vitro -and in vivo -differentiated infection structures of two rust fungi, the wheat stem rust fungus Puccinia graminis f. sp. tritici and the broad bean rust fungus Uromyces fabae , and of the causal agent of maize anthracnose, Colletotrichum graminicola , were studied.• Epi-fluorescence microscopy with the fluorescence-labeled lectin wheat germ agglutinin (WGA) revealed that surfaces of infection structures formed on the plant cuticle expose chitin, whereas surfaces of structures formed after invading the host do not.• To identify chitin modification by de-N -acetylation, we raised polyclonal antibodies specifically recognizing de-N -acetylated chitosan. These antibodies labeled only those infection structures that differentiate inside the plant, indicating that chitosan is exposed on cell wall surfaces post penetration.• Surface modification of the fungal cell walls by chitin de-N -acetylation is discussed as a fungal strategy to protect cell walls of pathogenic hyphae from enzymatic hydrolysis by host chitinases, and to avoid generation of an auto-catalytic defense response system in the invaded host tissue.

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Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli. The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications. IMPORTANCEWe successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycete Puccinia graminis f. sp. tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

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