Analysis of alternative splicing events in the root tips and nodules of Pisum sativum L
- Authors: Zorin E.A.1, Kulaeva O.A.1, Afonin A.M.1, Zhukov V.A.1, Tikhonovich I.A.1,2
-
Affiliations:
- All-Russia Research Institute for Agricultural Microbiology
- Saint Petersburg State University
- Issue: Vol 17, No 1 (2019)
- Pages: 53-63
- Section: Genetic basis of ecosystems evolution
- URL: https://journals.rcsi.science/ecolgenet/article/view/10645
- DOI: https://doi.org/10.17816/ecogen17153-63
- ID: 10645
Cite item
Abstract
Background. Legumes establish symbioses with nitrogen-fixing bacteria from the Rhizobium group. In exchange for nutrients, bacteria provide fixed nitrogen needed to support plant growth. At the moment, information about the involvement of alternative splicing (AS) in the establishment and maintenance this symbiotic relationships is almost absent, but, as it is a powerful mechanism for the regulation of proteome diversity of the cell, it therefore may participate in cellular response to microsymbionts.
Materials and methods. Alternative splicing was analyzed using the assembly of “supertranscripts” and alignment of the reads from nodules and root tips to this reference. Target genes expression levels was estimated in tips of non-inoculated roots, and in nodules (2, 4, and 6 weeks post inoculation) with use of RT-qPCR.
Results.In this study, the analysis of AS events in the nodules and root tips of the pea was carried out. The presence of isoforms of four pea genes (PsSIP1, PsIGN, PsWRKY40, PsPR-10) was confirmed and their expression level was estimated.
Conclusion. Pea nodules were shown to be more enriched with AS events compared to root tips. Among the functional groups of genes that demonstrate AS events, one of the most enriched functional groups is the pathogens stress response. Intron retention probably leads to degradation of the transcript via NMD-system or to change of the protein function, that modulates the activity of genes in nodules.
Keywords
Full Text
##article.viewOnOriginalSite##About the authors
Evgeny A. Zorin
All-Russia Research Institute for Agricultural Microbiology
Author for correspondence.
Email: kjokkjok8@gmail.com
Technician, Laboratory of Genetics of Plant-Microbe Interactions
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Olga A. Kulaeva
All-Russia Research Institute for Agricultural Microbiology
Email: okulaeva@arriam.ru
PhD, Senior Scientist, Laboratory of Genetics of Plant-Microbe Interactions
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Alexey M. Afonin
All-Russia Research Institute for Agricultural Microbiology
Email: afoninalexeym@gmail.com
Researcher, Laboratory of Genetics of Plant-microbe Interactions
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Vladimir A. Zhukov
All-Russia Research Institute for Agricultural Microbiology
Email: vzhukov@arriam.ru
PhD, Head of the Lab, Laboratory of Genetics of Plant-Microbe Interactions
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608Igor A. Tikhonovich
All-Russia Research Institute for Agricultural Microbiology; Saint Petersburg State University
Email: arriam2008@yandex.ru
Sc.D., Professor PI, Academician of RAS; Dean of the Faculty, Faculty of Biology
Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608; 7/9, Universitetskaya embankment, Saint-Petersburg, 199034References
- Тихонович И.А., Андронов Е.Е., Борисов А.Ю., и др. Принцип дополнительности геномов в расширении адаптационного потенциала растений // Генетика. — 2015. — Т. 51. — № 9. — С. 973–990. [Tikhonovich IA, Andronov EE, Borisov AY. The principle of genome complementarity in the enhancement of plant adaptive capacities. Genetika. 2015;51(9): 973-990. (In Russ.)]. https://doi.org/10.7868/S001667581509012X.
- Shtark OY, Sulima AS, Zhernakov AI, et al. Arbuscular mycorrhiza development in pea (Pisum sativum L.) mutants impaired in five early nodulation genes including putative orthologs of NSP1 and NSP2. Symbiosis. 2016;68(1-3):129-144. https://doi.org/10.1007/s13199-016-0382-2.
- Reddy AS, Marquez Y, Kalyna M, Barta A. Complexity of the alternative splicing landscape in plants. Plant Cell. 2013;25(10):3657-3683. https://doi.org/10.1105/tpc.113.117523.
- Gracz J. Alternative splicing in plant stress response. Biotechnologia. 2016;97(1):9-17. https://doi.org/10.5114/bta.2016.57719.
- Laloum T, Martin G, Duque P. Alternative Splicing Control of Abiotic Stress Responses. Trends Plant Sci. 2018;23(2):140-150. https://doi.org/10.1016/j.tplants.2017.09.019.
- Shang X, Cao Y, Ma L. Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants. Int J Mol Sci. 2017;18(2). https://doi.org/10.3390/ijms18020432.
- Zhiguo E, Wang L, Zhou J. Splicing and alternative splicing in rice and humans. BMB Rep. 2013;46(9):439-447. https://doi.org/10.5483/BMBRep.2013.46.9.161.
- Zenoni S, Ferrarini A, Giacomelli E, et al. Characterization of transcriptional complexity during berry development in Vitis vinifera using RNA-Seq. Plant Physiol. 2010;152(4):1787-1795. https://doi.org/10.1104/pp.109.149716.
- Wang C, Zhu H, Jin L, et al. Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus. Plant Mol Biol. 2013;82(1-2):97-111. https://doi.org/10.1007/s11103-013-0042-3.
- Walters B, Lum G, Sablok G, Min XJ. Genome-wide landscape of alternative splicing events in Brachypodium distachyon. DNA Res. 2013;20(2):163-171. https://doi.org/10.1093/dnares/dss041.
- Iniguez LP, Ramirez M, Barbazuk WB, Hernandez G. Identification and analysis of alternative splicing events in Phaseolus vulgaris and Glycine max. BMC Genomics. 2017;18(1):650. https://doi.org/10.1186/s12864-017-4054-2.
- Kelemen O, Convertini P, Zhang Z, et al. Function of alternative splicing. Gene. 2013;514(1):1-30. https://doi.org/10.1016/j.gene.2012.07.083.
- Wang Y, Liu J, Huang BO, et al. Mechanism of alternative splicing and its regulation. Biomed Rep. 2015;3(2):152-158. https://doi.org/10.3892/br.2014.407.
- Zhukov VA, Zhernakov AI, Kulaeva OA, et al. De Novo Assembly of the Pea (Pisum sativum L.) Nodule Transcriptome. Int J Genomics. 2015;2015:695947. https://doi.org/10.1155/2015/695947.
- Kumagai H, Hakoyama T, Umehara Y, et al. A Novel Ankyrin-Repeat Membrane Protein, IGN1, Is Required for Persistence of Nitrogen-Fixing Symbiosis in Root Nodules of Lotus japonicus. Plant Physiol. 2007;143(3):1293-1305. https://doi.org/10.1104/pp.106.095356.
- Alves-Carvalho S, Aubert G, Carrere S, et al. Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species. Plant J. 2015;84(1):1-19. https://doi.org/10.1111/tpj.12967.
- Jiao K, Li X, Guo W, et al. High-Throughput RNA-Seq Data Analysis of the Single Nucleotide Polymorphisms (SNPs) and Zygomorphic Flower Development in Pea (Pisum sativum L.). Int J Mol Sci. 2017;18(12). https://doi.org/10.3390/ijms18122710.
- Sudheesh S, Sawbridge TI, Cogan NO, et al. De novo assembly and characterisation of the field pea transcriptome using RNA-Seq. BMC Genomics. 2015;16:611. https://doi.org/10.1186/s12864-015-1815-7.
- Bioinformatics.babraham.ac.uk [Internet]. FASTQC [cited 2018 Nov 2]. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
- Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinforma tics. 2014;30(15):2114-2120. https://doi.org/10.1093/bioinformatics/btu170.
- jgi.doe.gov/data-and-tools [Internet]. bbtools; [cited 2018 Nov 2]. Available from: https://jgi.doe.gov/data-and-tools/bbtools/.
- Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21. https://doi.org/10.1093/bioinformatics/bts635.
- Fu L, Niu B, Zhu Z, et al. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012;28(23):3150-3152. https://doi.org/ 0.1093/bioinformatics/bts565.
- Davidson NM, Oshlack A. Corset: enabling differential gene expression analysis for de novo assembled transcriptomes. Genome Biol. 2014;15(7):410. https://doi.org/10.1186/s13059-014-0410-6.
- Hawkins AD, Oshlack A, Davidson NM. SuperTranscript: a data driven reference for analysis and visualisation of transcriptomes. BioRxiv. 2016:077750. https://doi.org/10.1101/077750.
- Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14(2):178-192. https://doi.org/10.1093/bib/bbs017.
- Supek F, Bosnjak M, Skunca N, Smuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One. 2011;6(7):e21800. https://doi.org/10.1371/journal.pone.0021800.
- Huerta-Cepas J, Forslund K, Coelho LP, et al. Fast Genome-Wide Functional Annotation through Ortho logy Assignment by eggNOG-Mapper. Mol Biol Evol. 2017;34(8):2115-2122. https://doi.org/10.1093/molbev/msx148.
- Stanke M, Morgenstern B. AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 2005;33(Web Server issue): W465-467. https://doi.org/10.1093/nar/gki458.
- Gasteiger E, Gattiker A, Hoogland C, et al. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31(13):3784-3788. https://doi.org/10.1093/nar/gkg563.
- UniProt C. The universal protein resource (UniProt). Nucleic Acids Res. 2008;36(Database issue): D190-195. https://doi.org/10.1093/nar/gkm895.
- Finn RD, Bateman A, Clements J, et al. Pfam: The protein families database. Nucleic Acids Res. 2014;42(D1):D222-D230. https://doi.org/10.1093/nar/gkt1223.
- spss-tutorials.com [Internet]. ANOVA – Simple Introduction [cited 2018 Nov 2]. Available from: https://www.spss-tutorials.com/anova-what-is-it/.
- Kosterin OE, Rozov SM. Mapping of the new mutation blb and the problem of integrity of linkage group I. Pisum Genet. 1993;25:27-31.
- Serova TA, Tsyganova AV, Tsyganov VE. Early nodule senescence is activated in symbiotic mutants of pea (Pisum sativum L.) forming ineffective nodules blocked at different nodule developmental sta ges. Protoplasma. 2018;255(5):1443-1459. https://doi.org/ 10.1007/s00709-018-1246-9.
- Chakraborty J, Ghosh P, Sen S, Das S. Epigenetic and transcriptional control of chickpea WRKY40 promoter activity under Fusarium stress and its heterologous expression in Arabidopsis leads to enhanced resistance against bacterial pathogen. Plant Sci. 2018;276:250-267. https://doi.org/10.1016/j.plantsci.2018.07.014.
- Birkenbihl RP, Kracher B, Ross A, et al. Principles and characteristics of the Arabidopsis WRKY regulatory network during early MAMP-triggered immunity. Plant J. 2018;96(3):487-502. https://doi.org/ 10.1111/tpj.14043.
- Xu X, Chen C, Fan B, Chen Z. Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell. 2006;18(5):1310-1326. https://doi.org/10.1105/tpc.105.037523.
- Rayson S, Arciga-Reyes L, Wootton L, et al. A role for nonsense-mediated mRNA decay in plants: pathogen responses are induced in Arabidopsis thaliana NMD mutants. PLoS One. 2012;7(2):e31917. https://doi.org/10.1371/journal.pone.0031917.
- Ruszkowski M, Szpotkowski K, Sikorski M, Jaskolski M. The landscape of cytokinin binding by a plant nodulin. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 12):2365-2380. https://doi.org/10.1107/S0907444913021975.
- Liu JJ, Ekramoddoullah AKM. The family 10 of plant pathogenesis-related proteins: Their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol. 2006;68(1-3):3-13. https://doi.org/10.1016/j.pmpp.2006.06.004.
- mtgea.noble.org/v3/ [Internet]. The Medicago truncatula Gene Expression Atlas (MtGEA) Project [ci ted 2018 Nov 3]. Available from: https://mtgea.noble.org/v3/.