Medicago truncatula DNF2 is a PI‐PLC‐XD‐containing protein required for bacteroid persistence and prevention of nodule early senescence and defense‐like reactions

Abstract

Summary Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen‐fixing nodules. Nodule cells contain large numbers of bacteroids which are differentiated, nitrogen‐fixing forms of the symbiotic bacteria. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria. In order to better understand the molecular mechanism sustaining the phenomenon, we searched for new plant genes required for effective symbiosis. We used a combination of forward and reverse genetics approaches to identify a gene required for nitrogen fixation, and we used cell and molecular biology to characterize the mutant phenotype and to gain an insight into gene function. The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C‐like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Furthermore, mutant nodules senesce rapidly and exhibit defense‐like reactions. This atypical phenotype amongst Fix− mutants unravels dnf2 as a new actor of bacteroid persistence inside symbiotic plant cells.

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis

“…Phenotypic characterization suggests that dnf4 causes problems in the symbiosis at stages later than other dnf mutants, such as dnf1, dnf2, and dnf5, because a number of late symbiotic genes from bacteria (nodF, bacA and nifH) and plants (LB1, CAM1 and Nodulin 31) are still expressed in dnf4 (12). This classification is supported by our microscopic analyses demonstrating that differentiating bacteroids are present in dnf4 mutant nodules, in contrast to those of dnf1 and dnf2, in which the bacteroids barely differentiate (6,13). Furthermore, PI staining of differentiated bacteroids in the dnf4 mutant suggests that NCR211 functions to maintain the viability of differentiating bacteroids.…”

“…Furthermore, dnf4 bacteroids, including ones staining positive for PI, appeared elongated and differentiated (Fig. 2D), unlike other early-stage mutants (6,13), although the degree of bacteroid differentiation was unclear. Coupling PI staining with SYTO9, a commonly used viability dye, yielded a similar pattern.…”

The Symbiosis-Related ERN Transcription Factors Act in Concert to Coordinate Rhizobial Host Root Infection

“…proportion of ern2 nodules (Hossain et al, 2006;Kumagai et al, 2007;Pislariu et al, 2012;Bourcy et al, 2013;Berrabah et al, 2014). It has also been suggested that nodule senescence can be triggered by the host to avoid the energetic cost of maintaining ineffective nodules (Suganuma et al, 2003;Magori and Kawaguchi, 2009).…”

Insertional Mutagenesis Using Tnt1 Retrotransposon in Potato

“…Tnt1 was used to generate approximately 12,000 independent lines that represent over 300,000 insertions in M. truncatula (Tadege et al, 2008;Cheng et al, 2011). Many M. truncatula genes have been identified through forward and reverse genetics approaches using the Tnt1 retrotransposon insertion population (Pang et al, 2009;Zhao et al, 2010;Laurie et al, 2011;Tadege et al, 2011;Zhou et al, 2011;Cheng et al, 2012;Bourcy et al, 2013).…”