Alejandra Martinez-D’Alto, Xia Yan, Tyler C. Detomasi,  Richard I. Sayler, William C. Thomas, Nicholas J. Talbot, and Michael A. Marletta.

PNAS; February 15, 2023; 120 (8) e2215426120

https://doi.org/10.1073/pnas.2215426120

Significance

Blast disease is a worldwide concern affecting crops like rice and wheat. During plant penetration, the causative fungus Magnaporthe oryzae secretes a polysaccharide monooxygenase (MoPMO9A). Here, we show that MoPMO9A is active on (1→3, 1→4)-β-glucans present in the cell wall of cereal-type plants, hydroxylating the C4 position of the glycosidic bond and using either oxygen or hydrogen peroxide as a cosubstrate. This PMO has a second domain of unknown function that is highly conserved within a subset of PMOs and is essential for PMO activity. Moreover, MoPMO9A deletion results in reduced pathogenicity in rice. Taken together, this work provides insight into the biochemistry of MoPMO9A and the domain architecture of PMOs, supporting its role in polysaccharide degradation during plant infection.

Abstract

Blast 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 (MoPMO9A) is increased. MoPMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the MoPMO9A family AA9 showed that 220 of the 223 sequences in the MoPMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two MoPMO9A 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), MoPMO9A is not active on cellulose but showed activity on cereal-derived mixed (1→3, 1→4)-β-D-glucans (MBG). Moreover, the DUF is required for activity. MoPMO9A 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. MoPMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for MoPMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of MoPMO9A results in reduced pathogenicity.

See https://www.pnas.org/doi/10.1073/pnas.2215426120

Figure 2: Representation of pSUMO-MoPMO9A constructs (T1, T2, and FL). The N-terminal tag is removed by Ulp1 to generate MoPMO9A with an N-terminal histidine. The amino acid length of each construct is indicated. Purification details are in SI Appendix, Fig. S1. MoPMO9A predicted domains are shown in gray. 1: PMO catalytic domain (AAs 1 to 210); 2: DUF (AAs 243 to 322); 3: chitin-binding domain (AAs 484 to 537).