A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

Catalina I Pislariu,Vagner A Benedito,Andry Andriankaja,Rajasekhara Reddy Duvvuru Muni,Rujin Chen,Million Tadege,Pascal Ratet,Shulan Zhang,Jiangqi Wen,Xiaofei Cheng,Mingyi Wang,Jeremy D Murray,Viviane Cosson,Samuel Mondy,Ivone Torres Jerez,Kirankumar S Mysore,Mark E Taylor,Michael K Udvardi

doi:10.1104/pp.112.197061

Abstract

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

Highlights

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation
Plant cells differentiate during nodule development and provide rhizobia with a carbon source(s) and other nutrients for metabolism, assimilate ammonium produced by the bacteria into organic compounds, and export these nitrogen compounds to the rest of the plant (Udvardi and Day, 1997; Prell and Poole, 2006; White et al, 2007)
Segregating progeny of R0 and R1 seeds from 230 of these lines were subjected to rescreening under symbiotic conditions to confirm their phenotypes

Summary

Introduction

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Symbiotic Tnt1-Insertion Mutants of Medicago truncatula rhizobia colonize cells of developing nodules via infection threads, which begin in hair cells of the root epidermis and, deposit the bacteria in cortical cells via endocytosis This process results in a unique “organelle” called the symbiosome that consists of one or more bacteria surrounded by a plant membrane (Udvardi and Day, 1997; Brewin, 2004). Developed DNA-insertion mutant populations provide a rapid way of linking mutant phenotypes to defective genes and are poised to revolutionize the discovery of genes required for SNF, via both forward and reverse genetics (Tadege et al, 2005; Young and Udvardi, 2009; Urbanski et al, 2012)

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