Analysis of alternative splicing events in the root tips and nodules of Pisum sativum L

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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.

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, 196608

Olga 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, 196608

Alexey 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, 196608

Vladimir 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, 196608

Igor 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, 199034

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Supplementary files

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2. Fig. 1. Scheme of assembly of “supertranscripts” with the subsequent analysis of alternative splicing events

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3. Fig. 2. Layout of primers for quantitative assessment of isoforms. The upper figure is an isoform diagram with an intron (broad blocks are exons, narrow is an intron). The bottom figure is an intron-free isoform diagram (black blocks are exons, gray is the border of exons)

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4. Fig. 3. Distribution of different types of splice sites in the pea nodule transcriptome

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5. Fig. 4. Enrichment of various functional groups with alternative splicing events. Frequency is the percentage of total transcripts showing alternative splicing falling into this functional group

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6. Fig. 5. Comparison of expression of the pea gene isoforms PsPR-10, PsWRKY40, PsIGN, PsSIP1. In fig. a the lines are connected to the comparison group, demonstrating statistically significant differences (roots relative to nodules in terms of 2, 4, 6 weeks), in fig. d significant differences are shown for isoforms in roots relative to nodules at 2, 4, 6 weeks and separately isoforms with intron and without in nodules for 2 weeks (* p < 0,05, ** p < 0,0001)

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Copyright (c) 2019 Zorin E.A., Kulaeva O.A., Afonin A.M., Zhukov V.A., Tikhonovich I.A.

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This work is licensed under a Creative Commons Attribution 4.0 International License.
 


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