Analysis of epitope distribution of arabinogalactan protein-extensins in pea (Pisum Sativum) nodules of wild-type and mutants impaired in infection thread growth

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Abstract

Background. During the colonization of root and nodule tissues of legumes by rhizobia, bacterial cells are immersed in a plant extracellular matrix which includes arabinogalactan protein-extensins (AGPE).

Materials and methods. Immunogold electron microscopy with monoclonal antibodies MAC204 and MAC236 was used to analyse the distribution and abundance of epitopes of AGPE in wild-type and symbiotically defective pea mutants.

Results. In the nodules of the wild-type line SGE, both AGPE epitopes were detected to the same extent in the matrix of infection threads and infection droplets. In the nodules of the mutant line SGEFix−-1 (sym40), the level of labelling by MAC204 was significantly higher than with SGE in both infection threads and infection droplets, but the level of labelling by MAC236 was only increased in the infection droplets. In the mutant line SGEFix−-2 (sym33-3), a relatively high level of both epitopes was observed among all analysed genotypes. The double mutant line RBT3 (sym33-3, sym40) showed an intermediate level of labelling for both epitopes in infection threads compared with the parental mutants. In SGEFix−-1, an abnormal distribution of both epitopes was observed in the intercellular space matrix. The MAC204 epitope was found in the cell walls of SGEFix−-1 and in the infection thread walls of SGEFix−-2, whereas in RBT3 this epitope was detected in both types of walls.

Conclusions. The sym33-3 and sym40 mutations have different effects on the accumulation of AGPE epitopes recognised by MAC204 and MAC236. This indicates that both the Sym33 and the Sym40 genes affect the composition of AGPE in the matrix of infection threads and infection droplets.

About the authors

Anna V. Tsyganova

All-Russia Research Institute for Agricultural Microbiology

Author for correspondence.
Email: isaakij@mail.ru
ORCID iD: 0000-0003-3505-4298
SPIN-code: 9149-5662
http://arriam.ru/departments/laboratoriya-molekulyarnoj-i-kletochnoj-biologii/

Candidate of Biological Sciences, Leading Scientist of the Laboratory of Molecular and Cellular Biology, Department of Biotechnology

Russian Federation, 196608, Saint Petersburg, Pushkin-8, Podbelsky chaussee 3

Nicholas J. Brewin

John Innes Centre

Email: nick.brewin@jic.ac.uk
ORCID iD: 0000-0001-6120-2464
https://www.jic.ac.uk/people/professor-nick-brewin/

Emeritus Fellow, Professor

United Kingdom, NR4 7UH, Norwich, Norwich Research Park

Viktor E. Tsyganov

All-Russia Research Institute for Agricultural Microbiology; Saint-Petersburg Scientific Center RAS

Email: tsyganov@arriam.spb.ru
ORCID iD: 0000-0003-3105-8689
SPIN-code: 6532-1332
http://arriam.ru/departments/laboratoriya-molekulyarnoj-i-kletochnoj-biologii/

Doctor of Biological Sciences, Head of the Laboratory of Molecular and Cellular Biology, Department of Biotechnology; Senior Scientist of Saint Petersburg Scientific Center RAS

Russian Federation, 196608, Saint Petersburg, Pushkin-8, Podbelsky chaussee 3; 199034, Saint Petersburg, Universitetskaya embankment 5

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2. Fig. 1. Immunogold labelling showing the distribution of arabinogalactan protein-extensin epitopes in thin sections from the nodules of pea wild-type SGE: a–c – МАС204; d–f – МАС236. IT – infection thread, ITW – infection thread wall, ID – infection droplet, B – bacterium, RB – releasing bacterium, Ba – bacteroid, * – newly synthesised matrix; arrows indicate gold particles. Bar: 500 nm

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3. Fig. 2. Distribution of arabinogalactan protein-extensin epitopes in the nodules of pea mutant line SGEFix–-1 (sym40): a, b – МАС204; c, d – МАС236. IT – infection thread, ID – infection droplet, B – bacterium, M – mitochondrion; arrows indicate gold particles. Bar: a, b = 1 µm, c, d = 500 nm

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4. Fig. 3. Distribution of arabinogalactan protein-extensin epitopes in cell walls in the nodules of pea mutant lines SGEFix–-1 (sym40) (a, b) and SGEFix–-2 (sym33-3) (c, d): a, c, d – МАС204, b – МАС236. V – vacuole, M – mitochondrion, A – amyloplast, CW – cell wall, ICS – intercellular space, IT – infection thread, ITW – infection thread wall, B – bacterium; arrows indicate gold particles. Bar: 500 nm

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5. Fig. 4. Distribution of arabinogalactan protein-extensin epitopes in the nodules of pea mutant line SGEFix–-2 (sym33-3): a, b – МАС204; c, d – МАС236. IT – infection thread, ITW – infection thread wall, ID – infection droplet, B – bacterium; arrows indicate gold particles. Bar: 500 nm.

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6. Fig. 5. Distribution of arabinogalactan protein-extensin epitopes in the nodules of pea double mutant line RBT3 (sym33-3, sym40): a–c – МАС204; d–f – МАС236. IT – infection thread, B – bacterium, ITW – infection thread wall, CW – cell wall, M – mitochondrion, ^ – transport vesicles with intercellular matrix; arrows indicate gold particles. Bar: 500 nm

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Copyright (c) 2019 Tsyganova A.V., Brewin N., Tsyganov V.E.

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