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Open Access Highly Accessed Research article

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays

Jessica L Erickson1, Jörg Ziegler2, David Guevara34, Steffen Abel2, Ralf B Klösgen1, Jaideep Mathur4, Steven J Rothstein4 and Martin H Schattat14*

Author Affiliations

1 Abteilung Pflanzen Physiologie, Institut für Biologie-Pflanzenphysiologie, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, Halle/Saale 06120, Germany

2 Abteilung Molekulare Signalverarbeitung, Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, Halle/Saale 06120, Germany

3 Present Address: Pioneer Hi-Bred, 12111 Mississauga Rd, Georgetown, ON L7G 4S7, Canada

4 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2 W1, Canada

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BMC Plant Biology 2014, 14:127  doi:10.1186/1471-2229-14-127

Published: 9 May 2014

Abstract

Background

Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes.

Results

Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation.

Conclusion

Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as ‘normal’ as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy.

Keywords:
Agrobacterium tumefaciens; Nicotiana benthamiana; Transient assays; GV3101(pMP90); LBA4404; Plastid; Stromules; Bacteria-derived; Cytokinin; Trans-zeatin synthase