Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Knockout of SlYTH2, encoding a YTH domain-containing protein, caused plant dwarfing, delayed fruit internal ripening, and increased seed abortion rate in tomato.

The m6A is one of the most abundant and widespread modifications in eukaryotic mRNAs, which regulates gene expression at the post-transcriptional level and plays key roles in many physiological processes. The YT521-B homologous (YTH) domain-containing proteins act as m6A readers to regulate m6A-RNA metabolism processes and mediate the various functions of m6A modification. Previously, we reported tomato contains 9 YTH genes, among which SlYTH2 is relatively highly expressed. This study reports the physiological functions of SlYTH2 in tomato. SlYTH2 protein is distributed in the nucleus and cytoplasm, and its three-dimensional structure is highly similar to human HsYTHDF1. SlYTH2 knockout through Crispr/Cas9 gene editing technology leaded to pleiotropic phenotypes, including dwarfing plant, delayed fruit internal ripening process, and increased seed abortion rate. The deletion of SlYTH2 gene increased the accumulation of endogenous ABA, decreased the content of endogenous GA3, and enhanced the sensitivity of seed germination to exogenous ABA and seedling growth to exogenous GA3. RNA-Seq data showed that the expression levels of multiple hormone-related genes were altered in SlYTH2 knockout line. These facts suggested SlYTH2 plays its physiological roles related to ABA, gibberellin and other hormones in tomato.

Tomato SlYTH1 encoding a putative RNA m6A reader affects plant growth and fruit shape.

N6-methyladenosine (m6A), the most abundant and common modification on eukaryotic mRNA, plays crucial roles in multiple biological processes through controlling endogenous gene activity in organisms. The m6A reader specifically recognizes the m6A mark to mediate the regulation of m6A on mRNA, and determines the fate of its target mRNA. In plants, the currently confirmed m6A readers are YTH (YT521B homology) domain-containing proteins. We previously reported that tomato contains 9 YTH genes, of which SlYTH1 has the strongest expression. The present study reports the functional characterization of SlYTH1 in tomato. SlYTH1 mutants generated via CRISPR/Cas9 technology exhibited pleiotropic phenotypes, including low seed germination rate, shortened seedling root, retarded plant growth and development during vegetative development, and elongated and longitudinally flattened fruit with reduced the locule number. SlYTH1 knockout reduced GA3 content and downregulated the expression of related genes in gibberellin biosynthesis pathway. Moreover, exogenous GA3 application could partially restore the phenotypic defects caused by SlYTH1 mutations. SlYTH1 knockout could alleviate the inhibition of seedling root elongation by exogenous GA3 application at relatively low concentration. These facts indicated SlYTH1 is involved in regulating gibberellin biosynthesis and plays important roles in multiple physiological processes in tomato.