Improved gene annotation of the fungal wheat pathogenZymoseptoria triticibased on combined Iso-Seq and RNA-Seq evidence

Abstract

Despite large omics datasets, the establishment of a reliable gene annotation is still challenging for eukaryotic genomes. Here, we used the reference genome of the major fungal wheat pathogen Zymoseptoria tritici (isolate IPO323) as a case study to develop methods to improve eukaryotic gene prediction. Four previous IPO323 annotations identified 10,933 to 13,260 gene models, but only one third of these coding sequences (CDS) have identical structures. To resolve these discrepancies and improve gene models, we generated full-length transcripts using long-read sequencing. This dataset was used together with other evidence (RNA-Seq transcripts and protein sequences) to generate novel ab initio gene models. The selection of the best structure among novel and existing gene models was performed according to transcript and protein evidence using InGenAnnot, a novel bioinformatics suite. Overall, 13,414 re-annotated gene models (RGMs) were predicted, including 671 new genes among which 53 encoded effector candidates. This process corrected many of the errors (15%) observed in previous gene models (coding sequence fusions, false introns, missing exons). While fungal genomes have poor annotations of untranslated regions (UTRs), our Iso-Seq long-read sequences outlined 5' and 3'UTRs for 73% of the RGMs. Alternative transcripts were identified for 13% of RGMs, mostly due to intron retention (75%), likely corresponding to unprocessed pre-mRNAs. A total of 353 genes displayed alternative transcripts with combinations of previously predicted or novel exons. Long non-coding transcripts (lncRNAs) and double-stranded RNAs from two fungal viruses were also identified. Most lncRNAs corresponded to antisense transcripts of genes (52%). lncRNAs that were up or down regulated during infection were enriched in antisense transcripts (70%), suggesting their involvement in the control of gene expression. Our results showed that combining different ab initio gene predictions and evidence-driven curation using InGenAnnot improved the quality of gene annotations of a compact eukaryotic genome. Our analysis also provided new insights into the transcriptional landscape of Z. tritici, helping develop an increasingly complex picture of its biology.

The expression landscape and pangenome of long non-coding RNA in the fungal wheat pathogen Zymoseptoria tritici

“…Comparison with the new annotation of the reference genome [52], in which similar numbers of sense and antisense lncRNAs were found, suggests that technical issues are the most likely cause of the lack of antisense lncRNA in our predictions. In some cases, reads originating from the loci in the new annotation are present but at low depth, and/or they do not cover the full length of the transcript [52]. As such, transcripts were not properly assembled and were never assessed by the prediction tool.…”

“…Despite a clear transition in the sRNA transcriptome under stressful conditions, no direct role in host colonization has been demonstrated for sRNAs, in contrast to several other fungal plant pathosystems [ 51 ]. Recently, an improved genome annotation based on long-read transcript sequencing was established [ 52 ] reporting the production of several lncRNAs during growth in vitro . The study further demonstrated that lncRNAs were differentially expressed between in vitro and in planta conditions, and that some lncRNAs showed interesting expression correlation patterns with nearby genes during infection [ 52 ].…”

Plasticity of the MFS1 promotor is not the only driver of Multidrug resistance in Zymoseptoria tritici

“…The fifth insert, however, does not exhibit any similarity with the type II inserts and was therefore referred to as the type VII insert. Further analysis using the reannotated genome of Z. tritici (Lapalu 2023) revealed that the conserved domains (totaling five) of type II inserts show similarities with retrotransposons from the RLX_Lard_Gridr family (Figure 1B).…”

“…Aligning the type VI insert sequence on the Z. tritici reference genome (Goodwin et al, 2011(Lapalu et al, 2023 shows 91.57% identity to 89 bp of the internal sequence of another DTX MITE of a total size of 402 bp localized on chromosome 12 (Fig. 2).…”

“…(annotated with Ingenannot Lapalu et al, 2023). For the Indel VIII the 678 bp deletion affects a portion of the 5'UTR region, removing 58 bp out of 166 bp.…”