Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

Abstract

Background Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical Abstract

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony

“…Since the proliferation and differentiation of parasites during these stages are strictly governed by specific sets of genes, comprehending their biology through the perspective of gene regulation is essential. Taking advantage of genome and transcriptome sequencing [ 12 – 14 ], the dawn of deciphering the genetic regulation of development in eimerian parasites appears on the horizon. RNA sequencing (RNA-Seq) technology is widely used to assess expression patterns of genes in different developmental stages [ 15 , 16 ], as well as in exploring the differentially expressed genes (DEGs) among samples with different phenotypes such as drug-sensitive or drug-resistant [ 17 – 19 ].…”

Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix

“…Eimeria necatrix is a highly pathogenic coccidian that can cause high mortality in susceptible birds, particularly in chickens over 8 weeks old reared on litter floors (Mesa-Pineda et al, 2021). In our previous studies on the comparative transcriptome analysis of different developmental stages of E. necatrix (Su et al, 2017;Su et al, 2018;Gao et al, 2021a), we detected 37 transcripts contained AP2 domains, of which 2, 5, 7 and 12 transcripts were upregulated in SZ, MZ-2, MZ-3 and GAM, respectively. The expression of AP2 family TFs throughout the life cycle suggests that members of this family are crucial regulators of gene expression at all stages of E. necatrix development.…”