TML1 and TML2 synergistically regulate nodulation and affect arbuscular mycorrhiza in Medicago truncatula

Abstract

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of nodulation (AON) and autoregulation of mycorrhizal symbiosis (AOM) both negatively regulate their respective processes and share multiple components-plants that make too many nodules usually have higher arbuscular mycorrhiza (AM) fungal root colonization. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus. Medicago truncatula has two sequence homologs: MtTML1 and MtTML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in MtTML1 or MtTML2 produced two to three times the nodules of wild-type plants, whereas plants containing mutations in both genes displayed a synergistic effect, forming 20× more nodules compared to wild-type plants. Examination of expression and heterozygote effects suggests that genetic compensation may play a role in the observed synergy. Plants with mutations in both TMLs only showed mild increases in AM fungal root colonization at later timepoints in our experiments, suggesting that these genes may also play a minor role in AM symbiosis regulation. The mutants created will be useful tools to dissect the mechanism of synergistic action of MtTML1 and MtTML2 in M. truncatula symbiosis with beneficial microbes.

Keywords: AOM; AON; Medicago truncatula; TML; mycorrhization; nodulation.

Figure 1

Phylogenetic analysis and structure of TML sequence homologs. Analysis by the maximum likelihood method using sequence homologs from public databases (Gene IDs in Supplementary Table 1 ) as described in Materials and Methods of in Medicago truncatula (Mt), Arabidopsis thaliana (At), Nicotiana tabacum (Nt), Pisum sativum (Ps), Trifolium pratense (Tp), Glycine max (Gm), Phaseolus vulgaris (Pv), Solanum lycopersicum (Sl), Populus trichocarpa (Pt), Zea mays (Zm), Oryza sativa (Os), and Sorghum bicolor (Sb). Percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1,000 replicates are shown next to the branches.

Figure 2

TML alleles used in this work. Diagrams are based on the translation of the R108 genotype sequence of Medtr7g029290 (MtTML1) and Medtr6g023805 (MtTML2). Protein features are shown by colored boxes: F-box in orange, kelch repeats in blue, and Nuclear Localization signal (NLS) in green. MiRNA2111 binding sites are in the coding sequence (CDS) indicated in Supplementary Figure 1 . (A) TML1 alleles. tml1-2 is a 334-bp deletion with the addition of an A that results in a V-to-K change at amino acid 11, and deletion of 111 amino acids, removing the F-box and NLS. tml1-4 is a 104-bp deletion in the 5′ UTR and beginning of the coding sequence that removes the start codon and the first 20 amino acids. (B) tml2-1 is a Tnt1 insertion 119 bp from the start of the coding region, resulting in addition of 20 new amino acids starting at position 47 before eventually terminating the protein at position 67. tml2-2 is an insertion of a C, creating a stop codon at amino acid position 60 in the F-box.

Figure 3

Phenotype of single and double mutant alleles in MtTML1 and MtTML2 genes. (A) Whole plants displaying root nodules at 10 dpi in an aeroponic system inoculated with Sinorhizobium meliloti strain RM41. Two representative plants per genotype L to R: R108 (wild type), tml1-2, tml1-4, tml2-1, tml2-2, tml1-1 tml2-2, tml1-2 tml2-2, and sunn-5. Scale bar = 2 cm. (B–H) Magnification of nodules from a representative plant for genotypes (B) wild-type R108, (C) tml1-2, (D) tml1-4, (E) tml2-1, (F) tml2-2, (G) tml1-1 tml2-2, and (H) tml1-2 tml2-2. A single root was folded in panel (H) to show wider area of nodulation in same picture; (B–H), scale bar = 2 mm.

Figure 4

Nodulation phenotypes of single and double mutant lines in MtTML1 and MtTML2 genes. (A) Nodule number and (B) nodules per cm root length at 10 dpi of wild type, single mutant single mutant (tml1 in black and tml2 in blue), sunn-5 plants from the same experiment in Figure 3 . N = 12 plants for R108 wild type and N = 15 plants for sunn-5 in all graphs. N = 7 plants for tml1-2, N = 10 for tml1-4, N = 11 for tml2-1, and N = 19 for tml2-2. Groups not connected by the same letter are statistically different. (C) Nodule number and (D) nodules per cm root length at 10 dpi of wild type, double mutant, and sunn-5 plants from the same experiment in Figure 3 . Groups not connected by the same letter are statistically different. N = 8 plants for tml1-1 tml 2-2 and N = 14 for the tml1-2 tml2-2 double mutant. Significant differences between means were tested by Tukey’s comparison for all pairs or non-parametric Steel–Dwass comparison (see Materials and Methods for detailed description). Dots indicate values for individual plants.

Figure 5

Spatiotemporal and differential expression of MtTML genes over 72 hours in plants responding to rhizobia. (A) Diagrams produced from ePlant resource (Schnabel et al., 2023) with gradient display adjusted to identical color representation to allow comparison of the expression levels as well as localization of TML1 and TML2 across time in wild-type roots responding to rhizobia up to 72 hpi. (B) Relative expression of TML1 in a tml2-2 mutant at 0 and 72 hours post-inoculation (hpi), normalized to wild type at 0 hpi. (C) Relative expression of TML2 in a tml1-4 mutant at 0 and 72 hours post-inoculation normalized to wild type at 0 hpi. Note Y-axis in B is five times the Y-axis in (A). **p < 0.05. Each graph is the results of three technical replicates of each of three biological replicates from 10 plants compared to the reference gene PIK (Kakar et al., 2008).

Figure 6

Molecular genotype analysis and photographs supporting heterozygous effect. (A) Testing 60% (84) of the total population of 141 (indicated in red dots in panel 1) broken down in panels by molecular genotype determined by PCR. Blue dots (panel 2) are homozygous wild type TML1, green dots (panel 3) are heterozygous tml1/TML1, and black dots (panel 4) are homozygous tml1/tml1. Lines indicate median nodule number for each group. (B) Box plot showing the distribution of nodule number in each genotype. Groups indicated by different letters are significantly different as tested by Tukey all pair test (p<0.001). (C) One representative plant per genotype L to R: R108 (wild type), tml1-2, tml1-2 tml2-2/TML2, tml1-2 tml2-2 and (D) R108 (wild type), tml2-2, tml1-2/TML1 tml2-2, and tml1-2 tml2-2. Scale bar = 2 cm.

Figure 7

Arbuscular mycorrhiza phenotype of tml1, tml2 double mutants. (A) Overall root length colonization in R108, tml1-1, tml2-2, and tml1-1, tml2-2 is similar at 4.5 weeks post-inoculation (wpi), whereas at 6 wpi, increased colonization levels were observed in the tml1-2, tml2-2 double mutants relative to R108 controls. Statistical differences were calculated separately for each timepoint (ANOVA followed by Tukey’s HSD; different letters denote significant differences in pairwise comparisons with p<0.05). (B) Representative image of Rhizophagus irregularis symbiotic structures in an R108 wild-type root (6wpi). (C) Representative image of a R. irregularis-colonized root of a tml1-1 tml 2-2 double mutant (6wpi). (D) Representative image of a R. irregularis-colonized root of a tml1-2 tml2-2 double mutant (6 wpi). (B–D) R. irregularis fungal structures are visualized with WGA-Alexafluor488. M. truncatula root is outlined with a dashed line. Scale bar: 250 μm.