Lirong Jin; Guanglin Li; Dazhao Yu; Wei Huang; Chao Cheng; Shengjie Liao; Qijia Wu; Yi Zhang

“ Herein, our study developed specific algorithms to accurately reveal the alternative splicing events in the fungal pathogen V. dahliae. Four different transcriptome data were generated by deep sequencing, being used to better annotate the 5′ and 3′ UTRs of the protein coding genes and for identification of the previously unannotated introns in all genic regions. Alternative splicing by intron retention and those associated with novel/alternative splice junctions has been separately classified. ”

“ In contrast to the predominant exon-skipping events in animals, plant and fungi AS events were reported to be predominantly involved in intron retention, while the other AS forms are rare [ [7] – [9] ] .
AS controls almost all aspects of biological processes in mammalian cells and is involved in many human diseases [ [10] – [11] ] , it is logical to expect it plays a role in biological functions of fungi. PscRXLR1 was the first example of a non-effector transformed to a functional effector protein by alternative splicing in Pseudoperonospora cubensis [ [12] ] . ”

“ Two different methods were applied to generate cDNA libraries for Illumina sequencing platform, showing the expression of over 95% of the annotated genes under the in vitro vegetative growth condition. Using computational algorithms developed in this study, we showed that about 50% of the intron-containing genes of V. dahliae were potentially under alternative splicing regulation. In addition to the large amount of intron retention AS events expected, we revealed over a thousand of AS events covering most non-intron-retention AS types. ”

“ During the validation process, we also found some other combinatory alternative splicing events, including the combination of Cassette Exon with A5SS and with IR (Additional file 1: Figure S6B and C) .
Alternative splicing does not appreciably contribute to the fungal mRNA levels The alternative spliced mRNAs containing premature termination codons (PTC) within an appropriate distance to the last exon-exon junction tend to be linked to the nonsense mediated decay (NMD) machinery in mammals [ [21] ] . ”

“ Combined with the functional clustering results of the AS genes, our results strongly suggest that this plant fungal pathogen has acquired sophisticated AS mechanisms to maximize its protein encoding potential and to control essential biological functions such as mycelium development, sporulation, signal response etc., and thus very likely AS contributes to the pathogenicity after the fungus encounters its hosts. ”

“ The cDNA was prepared from the polyadenylated RNA and subjected to the high-throughput sequencing using Illumina GAIIx platform. We obtained a total of 1.37 and 1.43 millions of cDNA reads from the two strains, respectively, which unambiguously mapped onto the sequenced regions of the annotated genes (10,535) [ [17] ] . Both strains showed a major distribution of the reads in the protein-coding regions (~70%) , and the remaining reads matched the non-coding regions including 5′ UTR, 3′ UTR and intron (Additional file 1: Figure S1A and Additional file 2: Table S1A) . ”

“ Alternative splicing events in Verticillium transcriptome In this study, the known/model splice junctions (SJs) was defined by joining splice sites at the exon-intron boundaries of all annotated introns in the recently published genome [ [17] ] , generating a total of 19,150 such model/known SJs. All other SJs involving only one or none of the known splice site were considered as novel junction. SJs in the transcriptome of V. dahliae supported by cDNA reads were identified using TopHat tool [ [31] ] . Junction reads were required to have at least 8-nt mapped on each of the adjacent exons. ”

“ Base coverage of each mRNA by the mapped cDNA reads was first calculate, and the number of genes was then plotted against the coverage from 0 (no coverage) to 1 (100% coverage) . d The expression level of all expressed genes represented by RPKM. RPKM stands for mapped Reads Per Kilo base of mRNA per Million reads. e Distribution of the mapped reads across the first 1000 kb of the supercontig 1 of the V. dahliae genome Interestingly, the second dataset showed a drastic increase in the sequence depth in the CDS region, and a sharp decrease in the 5′ UTR region. ”

“ Abstract Background Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied.
Results This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction ”

“ The statistics of their length distribution (left) ; RT-PCR validation of one representative of these introns (right) . b Exonic introns involving extended UTRs. c Intronic intron. Labels are similar to those in Fig. 2 When we investigated the cDNA reads layout on the genome, we found that there were also cases of exonic intron with most of the intron residing in the known exon, and a small fraction extending into the 5′ UTR region (Fig. 3b) . This type of splicing resulted in 5′ UTR-containing mRNA isoforms having truncated 5′ coding sequences. ”

“ Competing interests I have read the journal’s policy and the authors of this manuscript have the following competing interests: CC, SL and YZ are employed by a company, ABLife Inc, which is a biological scientific research company. YZ is the chief scientist of the company. ”

“ Schroth, GP; et al. (2008) , Alternative isoform regulation in human tissue transcriptomes, vol. 456, pp. 470–6, 10.1038/nature0750918978772, pmid:18978772, DOI 10.1038/nature07509 ↑ Filichkin, SA; Priest, HD; Givan, SA; Shen, R; Bryant, DW; Fox, SE; Wong, WK & Mockler, TC (2010) , Genome-wide mapping of alternative splicing in Arabidopsis thaliana, vol. ”

“ Alternative 3′ splice site and exon skipping;A5SS: Alternative 5′ splice site;A5SS&ES: Alternative 5′ splice site and exon skipping;aJAS: Alternative splice Junction associated AS events;AS: Alternative splicing;CE: Cassette exon;DEGs: Differentially expressed genes;ES: Exon skipping;FDR: False discovery rate;iRAS: Intron Rentention associated AS event;MXE: mutually exclusive exons;RPKM: Reads per kilo base per million mapped reads;RT-PCR: Real-time Polymerase Chain Reaction;SJs: Splice junctions;V. dahlia: Verticillium dahliaeAcknowledgementsThe authors are grateful to Prof. ”

“ PCR Primers used in this study. (DOCX 32 kb) File:12864 2017 3507 MOESM3 ESM.xlsxIntronR-1st dataset. (XLSX 125 kb) File:12864 2017 3507 MOESM4 ESM.xlsxIntronR-2nd dataset. (XLSX 203 kb) File:12864 2017 3507 MOESM5 ESM.xlsxPotential IR-induced PTCs for non-sense mediated decay. (XLSX 36 kb) File:12864 2017 3507 MOESM6 ESM.xlsxExonic Intron-1st & 2nd datasets. (XLSX 43 kb) File:12864 2017 3507 MOESM7 ESM.zipGFF annotation. V. dahliae genome annotation_additional UTRs and Exonic intron_for AS analysis. (ZIP 1332 kb) File:12864 2017 3507 MOESM8 ESM.xlsxaJAS & Intronic intron-1st dataset. ”