Overexpression of the Chromosome Partitioning Gene parA in Azorhizobium caulinodans ORS571 Alters the Bacteroid Morphotype in Sesbania rostrata Stem Nodules.

Hsiao-Lin Chien,Ming-Yen Tsai,Chi-Te Liu,Chiung-Hsiang Cheng,Wan-Zhen Huang

doi:10.3389/fmicb.2019.02422

Abstract

Azorhizobium caulinodans ORS571 is a diazotroph that forms N2-fixing nodules on the roots and stems of the tropical legume Sesbania rostrata. Deletion of the parA gene of this bacterium results in cell cycle defects, pleiomorphic cell shape, and formation of immature stem nodules on its host plant. In this study, we constructed a parA overexpression mutant (PnptII-parA) to complement a previous study and provide new insights into bacteroid formation. We found that overproduction of ParA did not affect growth, cell morphology, chromosome partitioning, or vegetative nitrogen fixation in the free-living state. Under symbiosis, however, distinctive features, such as a single swollen bacteroid in one symbiosome, relatively narrow symbiosome space, and polyploid cells were observed. The morphotype of the PnptII-parA bacteroid is reminiscent of terminal differentiation in some IRLC indeterminate nodules, but S. rostrata is not thought to produce the NCR peptides that induce terminal differentiation in rhizobia. In addition, the transcript patterns of many symbiosis-related genes elicited by PnptII-parA were different from those elicited by the wild type. Accordingly, we propose that the particular symbiosome formation in PnptII-parA stem-nodules is due to cell cycle disruption caused by excess ParA protein in the symbiotic cells during nodulation.

Highlights

Nodule formation in plants of the Fabaceae or Leguminosae family can be classified as determinate or indeterminate; the major difference between these types is the presence or absence of an active meristem in the fully developed organ (Hirsch, 1992; Sprent, 2007)
We constructed a ParA-overproducing A. caulinodans ORS571 strain (PnptII-parA) harboring a plasmid that constitutively expressed the parA gene under the control of the nptII promoter (Supplementary Figure S1)
When the bacteria were cultivated in minimal broth (L2-N) under microaerobic conditions, high constitutive expression of the ParA protein was detected in the PnptII-parA cells (Figure 1B)

Summary

Introduction

Nodule formation in plants of the Fabaceae or Leguminosae family can be classified as determinate or indeterminate; the major difference between these types is the presence or absence of an active meristem in the fully developed organ (Hirsch, 1992; Sprent, 2007). In other IRLC legume, rhizobia form highly elongated or branched cells (i.e., swollen bacteroids) in indeterminate nodules, such as those of Medicago, Pisum, Trifolium or Vicia The features of such bacteroids are cell enlargement, genome amplification (endoreduplication) and membrane permeabilization (Mergaert et al, 2006). Because these polyploid bacteroids lose their ability to resume growth, this type of cellular process is called terminal bacteroid differentiation (TBD); the resulting bacteroids are proposed to be more effective in nitrogen fixation than the reversibly differentiated, reproductive bacteroids (Oono and Denison, 2010). TBD is known to be triggered by plant factors that show similarities to defensin-like innate immunity factors, designated as nodulespecific cysteine-rich (NCR) peptides (Mergaert et al, 2006; Wang et al, 2010)

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