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Characterization of a unique polysaccharide monooxygenase from the plant pathogen Magnaporthe oryzaeBlast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase ( Mo PMO9A) is increased. Mo PMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the Mo PMO9A family AA9 showed that 220 of the 223 sequences in the Mo PMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two Mo PMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), Mo PMO9A is not active on cellulose but showed activity on cereal-derived m ixed (1→3, 1→4)- β -D- g lucans (MBG). Moreover, the DUF is required for activity. Mo PMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. Mo PMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for Mo PMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of Mo PMO9A results in reduced pathogenicity.more » « less
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Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling) , encodes two linked genes, cwr-1 and cwr-2 . Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein–protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain.more » « less