African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

BackgroundTea-oil tree (Camellia oleifera) is a unique edible-oil tree in China, and anthracnose occurs in wherever it is cultivated, causing great economic losses each year. We have previously identified the Ascomycete fungus Colletotrichum fructicola as the major pathogen of anthracnose in Ca. oleifera. The purpose of this study was to characterize the biological function of Snf1 protein, a key component of the AMPK (AMP-activated protein kinase) pathway, for the molecular pathogenic-mechanisms of C. fructicola.ResultsWe characterized CfSnf1 as the homolog of Saccharomyces cerevisiae Snf1. Targeted CfSNF1 gene deletion revealed that CfSnf1 is involved in the utilization of specific carbon sources, conidiation, and stress responses. We further found that the ΔCfSnf1 mutant was not pathogenic to Ca. oleifera, resulting from its defect in appressorium formation. In addition, we provided evidence showing crosstalk between the AMPK and the cAMP/PKA pathways for the first time in filamentous fungi.ConclusionThis study indicate that CfSnf1 is a critical factor in the development and pathogenicity of C. fructicola and, therefore, a potential fungicide target for anthracnose control.

The tea-oil tree Camellia oleifera is native to China and is cultivated in many parts of southern China. This plant has been grown for over 2,000 years, mainly for its high-quality cooking oil. Anthracnose is the main disease of tea-oil tree and results in a huge loss annually. Colletotrichum fructicola is a major pathogen causing anthracnose on tea-oil tree. In a previous study, we characterized that the bZIP transcription factor CfHac1 controlled the development and pathogenicity of C. fructicola. Here, we identified and characterized the function of CfVAM7 gene, which was significantly downregulated at the transcriptional level in the ΔCfhac1 strain under dithiothreitol stress. Targeted gene deletion revealed that CfVam7 is important in growth, pathogenicity, and responses to endoplasmic reticulum-related stresses. Further analysis revealed that CfVam7 is required for appressorium formation and homotypic vacuole fusion, which are important for fungal pathogen invasion. Cytological examinations revealed that CfVam7 is localized to vacuole membranes in the hyphal stage. The Phox homology (PX) and SNARE domains of CfVam7 were indispensable for normal cellular localization and biological function. Taken together, our results suggested that CfVam7-mediated vacuole membrane fusion promotes growth, stress response, and pathogenicity of C. fructicola.

Colletotrichum spp. is ranked in the top 10 plant fungal pathogens and serves as a model for the study of hemibiotrophic pathogens, but its molecular mechanisms of pathogenesis remain largely unknown. Among species of Colletotrichum , C. fructicola causes anthracnose disease on more than 50 plants, such as pears, apples, and the important, edible-oil plant Camellia oleifera .