A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation

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.

Figure 1.

Schematic representation of Tnt1 tagging in M. truncatula and overview of the mutant collection. Tobacco retrotransposon Tnt1 insertion mutagenesis in Medicago was developed from a parental line (Tnk88-7-7) carrying approximately five Tnt1 copies. During tissue culture, the Tnt1 retroelement transposes by a copy-and-paste mechanism involving an mRNA intermediate. This is reverse transcribed to complementary DNA that is stably inserted mostly in coding regions within the genome. Gene disruption by the 5.3-kb Tnt1 retroelement generally results in loss of function. Approximately 30% of the 9,300 lines screened so far at the Samuel Roberts Noble Foundation displayed visible phenotypes. Over 30,000 FSTs have been recovered by inverse (I)-PCR, touchdown (TD)-PCR, thermal asymmetric interlaced (TAIL)-PCR, as well as Solexa and 454 sequencing and were deposited, along with photographs of the mutants, into the mutant database (http://bioinfo4.noble.org/mutant/). PCR reverse screening, using gene-specific primers in combination with Tnt1 primers, was used to identify Tnt1 insertions in genes of interest.

Figure 2.

Examples of nodulation phenotypes in the Tnt1-insertion symbiotic mutant collection. Representative nodules and root segments are shown from wild-type R108 (A); Nod+ Fix−: NF0063, NF1883, NF4608, NF0414, NF0416, NF0313, NF0235, and NF0151 (B–I); Nod+ Fix+/−: NF0322, NF0124, NF0306, and NF0130 (J–M); Nod+/− Fix−: NF0342 and NF2811 (N and O); Nod++ Fix+/−: NF0984 (P); and Nod−: NF0197 and NF0117 (Q and R) phenotypes. Bars = 1 mm (A–P) and 1 cm (Q and R).

Figure 3.

Tnt1 insertions in known symbiotic genes. A, Gene coverage of the mutant collection was assessed by identifying Tnt1 insertions in known symbiotic genes using nested Tnt1 forward (T-F1 and T-F2) and reverse (T-R1 and T-R2) and gene-specific forward (GS-F1 and GS-F2) and reverse (GS-R1 and GS-R2) primers. Gene-specific primers were designed to amplify the entire gene plus 300 to 400 bp of the 5′ and 3′ untranslated regions. B, Schematic representations of Tnt1 insertion sites in known genes as determined by PCR reverse screening (black arrowheads) and by thermal asymmetric interlaced-PCR FST sequencing (white arrowheads) in lines with confirmed symbiotic phenotypes. Exons are depicted as black boxes, and introns and untranslated regions are represented by lines. Exact insertion sites are given in Supplemental Table S1.

Figure 4.

Symbiotic phenotypes of the wild type (R108) and potentially new Nod− Tnt1-insertion mutants. Plants were inoculated with the S. meliloti Sm2011 strain containing the chimeric hemA::LacZ gene and grown for 21 d post inoculation on Turface:vermiculite (2:1). Samples were harvested at different time points after inoculation, histochemically stained for β-galactosidase activity, and visualized as whole mounts. Rhizobia stain blue. A and B, Wild-type R108 infection in nodule primordium (A) and mature nodule (B). Successful infection was visualized at 5 d post inoculation, with branching infection threads (arrow) into the nodule primordium (NP), and the mature nodule was visualized at 15 d post inoculation. C to E, Symbiotic phenotype in the mutant isolated from NF0438. C, Root segment with numerous infections and nodule primordia that do not develop into nodules after 21 d post inoculation. D, Closeup imaging reveals highly branched and interrupted infection threads (arrows) alternating with sac-like structures (arrowheads) inside roots hairs. E, Infection thread maintenance is disrupted (arrow) and rhizobia are prematurely released in epidermal cells (arrowheads). F, Rhizobial microcolonies are seen on the root surface of the mutant isolated from NF2629 in the absence of root hair infection and cortical cell division. G, Extensive and uncontrolled cell division is seen in the nodulation window on roots of the mutant isolated from NF0359, while root hairs undergo deformation but no curling (arrows). H, Blue stain for β-galactosidase activity in the mutants of NF5794 delineates cell shapes, suggestive of intercellular infection. I, The only sites of β-galactosidase activity in the mutant isolated from NF2853 are where some lateral roots emerge. J to L, Symbiotic phenotypes of Nod− mutants isolated from NF1859 (J), NF0673 (K), and NF2811 (L) display infections arrested at the root epidermis, cortical cell division, and nodule primordia development but no nodule morphogenesis at 21 d post inoculation. M, Nod− phenotype in NF5654 with arrested infections, extensive and irregular cortical cell division (arrows), and proliferation of the vascular tissue (seen also in the inset), which gives the appearance of nodule bumps. Accumulation of brown material, suggestive of defense-like responses, is seen in most mutants (C, G, and I–M, arrowheads). Bars = 100 µm.

Figure 5.

Examples of AM phenotypes in Tnt1-insertion symbiotic mutants. Tnt1-insertion mutants defective in nodulation (Nod−) were inoculated with a mixture of soil and G. intraradices- and G. mossae-colonized root pieces. To visualize fungal structures (Arb, arbuscles; h, hyphae; v, vesicles), roots were cleared with 10% KOH at 95°C and subsequently stained with trypan blue. The wild-type Myc+ phenotype is shown in R108 (A). Myc+ phenotypes were also observed in NF0825 (C), which carries a Tnt1 insertion in NIN, and in NF0848 (D), which has a Tnt1 insertion in NSP1. Defective mycorrhization (Myc−) is seen in mutants NF0028 (B) and NF0814 (E), which have Tnt1 inserted in DMI3 and NSP2, respectively. An example of a Myc− mutant with no Tnt1 insertion linked to a known symbiotic gene is shown in F (NF0549). Bars = 100 µm.

Figure 6.

Potentially new Fix− symbiotic mutants isolated from the Tnt1-insertion mutant population. Nodules of wild-type R108 (A) and Fix− mutants isolated from lines NF0063 (B), NF5039 (C), NF2496 (D), NF0342 (E), NF4608 (F), NF0440 (G), NF0134 (H), NF4928 (I), and NF1320 (J) were harvested from plants grown in Turface and Metro-Mix layers at 21 d post inoculation with Sm2011-hemA::LacZ. After 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining and clearing with 70% ethanol, nodules were sliced into 50-µm-thick sections and photographed. Rhizobia stain blue. Bars = 100 µm.