Updated project metadata. For potato crops, host resistance is currently the most effective and sustainable tool to manage potato root and tuber diseases caused by the plasmodiophorid, Spongospora subterranea. Arguably, zoospore root attachment is the most critical phase of the pathogen infection, however, the mechanisms underlying zoospore root attachment remains unknown. This study investigated the potential role of root cell wall surface polysaccharides and proteins in zoospore root attachment in resistant and susceptible potato cultivars. We first compared the effects of enzymatic removal of root cell wall proteins, N-linked glycans or polysaccharides on S. subterranea attachment to root tissue of resistant and susceptible potato cultivars. Subsequently, mass spectrometry analysis of peptides released by trypsin shaving (TS) of root segments identified 1235 proteins, of which 262 were differentially abundant between the resistant and susceptible cultivars. In particular, proteins associated with glutathione metabolism and lignin biosynthesis were more abundant in the resistant cultivar. Comparison with whole-root proteomic analysis of the same resistant and susceptible cultivars led to identification of 226 proteins unique to the TS dataset, of which 188 were significantly different between cultivars. Among these, the pathogen defence-related cell wall protein stem 28 kDa glycoprotein and two major latex proteins were significantly less abundant in the resistant cultivar compared to the susceptible cultivar. A further major latex protein was detected at reduced levels in the resistant cultivar in both TS and whole-root proteomic datasets. In contrast, in the TS-specific dataset, three glutathione S-transferase proteins were more abundant in the resistant cultivar, while the protein glucan endo-1,3-beta-glucosidase was significantly increased in both the TS and whole-root datasets. These results imply a particular role of major latex proteins and glucan endo-1,3-beta-glucosidase in the regulation of host susceptibility to S. subterranea.