Although fungi lack adenosine deaminase acting on RNA (ADAR) enzymes, adenosine to inosine (A-to-I) RNA editing was reported recently in during sexual reproduction. In this study, we profiled the A-to-I editing landscape and characterized its functional and adaptive properties in the model filamentous fungus A total of 40,677 A-to-I editing sites were identified, and approximately half of them displayed stage-specific editing or editing levels at different sexual stages. RNA-sequencing analysis with the Δ-1 and Δ-1 mutants confirmed A-to-I editing occurred before ascus development but became more prevalent during ascosporogenesis. Besides fungal-specific sequence and secondary structure preference, 63.5% of A-to-I editing sites were in the coding regions and 81.3% of them resulted in nonsynonymous recoding, resulting in a significant increase in the proteome complexity. Many genes involved in RNA silencing, DNA methylation, and histone modifications had extensive recoding, including -1,-3, -1, and-2. Fifty pseudogenes harbor premature stop codons that require A-to-I editing to encode full-length proteins. Unlike in humans, nonsynonymous editing events in are generally beneficial and favored by positive selection. Almost half of the nonsynonymous editing sites in are conserved and edited in Furthermore, hundreds of them are conserved in and had higher editing levels. Two unknown genes with editing sites conserved between and were experimentally shown to be important for ascosporogenesis. This study comprehensively analyzed A-to-I editing in and showed that RNA editing is stage-specific and generally adaptive, and may be functionally related to repeat induced point mutation and meiotic silencing by unpaired DNA.

Summary Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological impact in filamentous fungi, however, is largely unknown. Here we combine PacBio Isoform‐Sequencing and strand‐specific RNA‐sequencing of multiple tissues and mutant characterization to reveal the landscape and regulation of AS and APA in Fusarium graminearum. We generated a transcript annotation comprising 51 617 isoforms from 17 189 genes. In total, 4997 and 11 133 genes are alternatively spliced and polyadenylated, respectively. Majority of the AS events alter coding sequences. Unexpectedly, the AS transcripts containing premature‐termination codons are not sensitive to nonsense‐mediated messenger RNA decay. Unlike in yeasts and animals, distal APA sites have strong signals, but proximal APA isoforms are highly expressed in F. graminearum. The 3′‐end processing factors FgRNA15, FgHRP1, and FgFIP1 play roles in promoting proximal APA site usage and intron splicing. A genome‐wide increase in intron inclusion and distal APA site usage and downregulation of the spliceosomal and 3′‐end processing factors were observed in older and quiescent tissues, indicating intron inclusion and 3′‐untranslated region lengthening as novel mechanisms in regulating aging and dormancy in fungi. This study provides new insights into the complexity and regulation of AS and APA in filamentous fungi.

Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in animals, and it occurs in fungi specifically during sexual reproduction. However, it is debatable whether A-to-I RNA editing is adaptive. Deciphering the functional importance of individual editing sites is essential for the mechanistic understanding of the adaptive advantages of RNA editing. Here, by performing gene deletion for 17 genes with conserved missense editing (CME) sites and engineering underedited (ue) and overedited (oe) mutants for 10 CME sites using site-specific mutagenesis at the native locus in Fusarium graminearum , we demonstrated that two CME sites in CME5 and CME11 genes are functionally important for sexual reproduction. Although the overedited mutant was normal in sexual reproduction, the underedited mutant of CME5 had severe defects in ascus and ascospore formation like the deletion mutant, suggesting that the CME site of CME5 is co-opted for sexual development. The preediting residue of Cme5 is evolutionarily conserved across diverse classes of Ascomycota, while the postediting one is rarely hardwired into the genome, implying that editing at this site leads to higher fitness than a genomic A-to-G mutation. More importantly, mutants expressing only the underedited or the overedited allele of CME11 are defective in ascosporogenesis, while those expressing both alleles displayed normal phenotypes, indicating that concurrently expressing edited and unedited versions of Cme11 is more advantageous than either. Our study provides convincing experimental evidence for the long-suspected adaptive advantages of RNA editing in fungi and likely in animals.

Alternative splicing (AS) and alternative polyadenylation (APA) of pre-mRNAs contribute greatly to transcriptome complexity and gene expression regulation in higher eukaryotes. Their biological impact in filamentous fungi, however, has been poorly studied. Here we combine PacBio Isoform Sequencing and strand-specific RNA-Seq of multiple tissues together with mutant characterization to reveal the landscape, complexity and regulation of AS and APA in the filamentous plant pathogenic fungus Fusarium graminearum. We updated the reference genome and generated a comprehensive annotation comprising 51,617 transcript isoforms from 17,189 genes. Majority of the transcripts represent novel isoforms, including 2,998 undiscovered protein-coding genes. In total, 42.7% of multi-exonic genes and 64.8% of genes have AS and APA isoforms, respectively, suggesting AS and APA increase previously unrecognized transcriptome complexity in fungi. Nonsense-mediated mRNA decay factor FgUPF1 may not degrade AS transcripts with premature-stop codons but regulate ribosome biogenesis. Distal polyadenylation sites have a strong signal but proximal polyadenylation isoforms are high expressed. The core 3’-end processing factors FgRNA15, FgHRP1, and FgFIP1 play important roles in promoting proximal polyadenylation site usage and also intron splicing. Genome-wide increase in the abundance of transcripts with retained introns and long 3’-UTRs and downregulation of the spliceosomal and 3’-end processing factors are found in older tissues and quiescent conidia, indicating that intron retention and 3’-UTR lengthening may be a transcriptional signature of aging and dormancy in fungi. Overall, our study generates a comprehensive full-length transcript annotation for F. graminearum and provides new insights into the complexity and regulation of transcriptome in filamentous fungi.