Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

• Published in: PLoS pathogens Vol. 17; no. 10; p. e1010001
• Main Authors: Feng, Hui, Wan, Chuanxu, Zhang, Zhichao, Chen, Han, Li, Zhipeng, Jiang, Haibin, Yin, Maozhu, Dong, Suomeng, Dou, Daolong, Wang, Yuanchao, Zheng, Xiaobo, Ye, Wenwu
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 14.10.2021; Public Library of Science (PLoS)
• Subjects: 3' Untranslated regions; Amino acids; Binding proteins; Binding sites; Biological activity; Biology and Life Sciences; Complementation; CRISPR; Flavodoxin; Fungi; Gene expression; Gene regulation; Genetic aspects; Genetic engineering; Genome editing; Genomes; Genomics; Kinases; Life cycles; Messenger RNA; Microbiological research; Mutants; Mutation; Oogonia; Oospores; Pathogens; Phenotypes; Physiological aspects; Plant species; Plasmids; Polyethylene glycol; Post-transcription; Protein-protein interactions; Proteins; Pythium ultimum; Reproduction; Reproduction (biology); Reproduction, Asexual; Research and Analysis Methods; Ribonucleic acid; RNA; RNA-binding protein; Sexual reproduction; Structure; Water molds; China
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1010001

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P . ultimum . We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium . PuM90 -knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP , which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90 -knockout mutants were observed with overexpression of PuFLP , mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP . The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P . ultimum .