Characterization of three non-canonical N-glycosylation motifs indicates Nglyco-A reduces DNA N6-methyladenine and Nglyco-D alters G/F actin ratio in Phytophthora sojae.

ABSTRACT

Asparagine (Asn, N)-linked glycosylation is an abundant post-translational modification in which Asn, typically in Nglyco-X-S/T; X ≠ P motifs, are modified with N-glycans. It has essential regulatory roles in multicellular organisms. In this study, we systematically investigate the function of three N-glycosylation motifs (Nglyco-A, Nglyco-D and Nglyco-S) previously identified in Phytophthora sojae, through site-directed mutagenesis and functional assays. In P. sojae expressing glycosylation-dead variants pre-PsDMAP1N70A (Nglyco-A motif) or PsADFN64A (Nglyco-D motif), zoospore release or cyst germination is impaired. In particular, the pre-PsDMAP1N70A mutant reduces DNA methylation levels, and the PsADFN64A mutant disrupts the actin forms, which could explain the decrease in pathogenicity after N-glycosylation is destroyed. Similarly, P. sojae expressing PsNRXN132A (Nglyco-S motif) shows increased sensitivity to H2O2 and heat. Through autophagy or 26S proteasome pathway inhibition assays, we found that unglycosylated pre-PsDMAP1N70A and PsADFN64A are degraded via the 26S proteasome pathway, while the autophagy pathway is responsible for PsNRXN132A clearance. These findings demonstrate that glycosylation of these motifs regulates the stability and function of glycoproteins necessary for P. sojae growth, reproduction and pathogenicity, which expands the scope of known N-glycosylation regulatory functions in oomycetes.