Long-read RNA sequencing reveals extensive alternative splicing repertoires in fungal plant pathogens.

Shaw, P. L.*, Wingfield, B. D., Slippers, B., Duong, T. A.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa

Alternative splicing (AS) is a dynamic process that generates multiple mRNA isoforms from a single gene, thereby expanding the transcriptome and the eventual proteome. In fungi, AS is associated with many biological processes, including development, environmental adaptation, and pathogenicity. The Ceratocystis fimbriata s.l. is comprised of several closely related economically important plant pathogens that are both morphologically and genetically similar. Despite their close relatedness, these pathogens have a diverse range of hosts and display host specificity. Economically important hosts affected by these pathogens include acacia, eucalypt, mango, pomegranate and sweet potato. Given their close relatedness but displaying diversity in host interactions, species of the C. fimbriata complex are great models to investigate the genetic mechanisms underline host adaptation in fungal plant pathogens. The objective of this study was to identify, characterize and compare alternative splicing events between two closely related species of the C. fimbriata complex, namely C. eucalypticola and C. fimbriata. The genomes of both species were sequenced and assembled into nine contigs, with respective genome sizes of 32.37 and 31.73 Mb. Employing both long-read and short-read RNA sequencing, we sequenced the transcriptome of both species under in-vitro and in-planta conditions. We used this transcriptomic data to generate high-quality gene annotations. We discovered an extensive range of alternatively spliced genes in both C. fimbriata and C. eucalypticola, with 35.49% and 42.39% of multi-exonic genes undergoing AS in each species, respectively. When comparing AS genes between the two species, more than 43% underwent alternative splicing in C. eucalypticola exclusively, while in C. fimbriata, this figure exceeded 29%. Intron retention was the dominant form of AS observed and constituted 84.18% and 85.26% of AS events in C. eucalypticola and C. fimbriata respectively. This was followed by alternative 3 prime splicing (9.27% and 8.62%), alternative 5 prime splicing (5.38% and 5.32%) and exon skipping (1.17% and 0.8%). Substantial variation regarding differential gene expression and differential transcript usage in AS genes were observed between in-vitro and in-planta conditions in the two species, including those encoding for effectors and CAZymes. These findings suggest that while approximately 90% of genes are shared between the two species, their regulation and usage differ significantly, likely contributing to the differences in host specificity but this needs further investigation.

Keywords: adaptation, alternative splicing, Ceratocystis, host specificity, pathogenicity