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Mapping the genetic basis of symbiotic variation in legume-rhizobium interactions in Medicago truncatula.

ABSTRACT: Mutualisms are known to be genetically variable, where the genotypes differ in the fitness benefits they gain from the interaction. To date, little is known about the loci that underlie such genetic variation in fitness or whether the loci influencing fitness are partner specific, and depend on the genotype of the interaction partner. In the legume-rhizobium mutualism, one set of potential candidate genes that may influence the fitness benefits of the symbiosis are the plant genes involved in the initiation of the signaling pathway between the two partners. Here we performed quantitative trait loci (QTL) mapping in Medicago truncatula in two different rhizobium strain treatments to locate regions of the genome influencing plant traits, assess whether such regions are dependent on the genotype of the rhizobial mutualist (QTL × rhizobium strain), and evaluate the contribution of sequence variation at known symbiosis signaling genes. Two of the symbiotic signaling genes, NFP and DMI3, colocalized with two QTL affecting average fruit weight and leaf number, suggesting that natural variation in nodulation genes may potentially influence plant fitness. In both rhizobium strain treatments, there were QTL that influenced multiple traits, indicative of either tight linkage between loci or pleiotropy, including one QTL with opposing effects on growth and reproduction. There was no evidence for QTL × rhizobium strain or genotype × genotype interactions, suggesting either that such interactions are due to small-effect loci or that more genotype-genotype combinations need to be tested in future mapping studies.

SUBMITTER: Gorton AJ

PROVIDER: S-EPMC3484660 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Mapping the genetic basis of symbiotic variation in legume-rhizobium interactions in Medicago truncatula.

Gorton Amanda J AJ Heath Katy D KD Pilet-Nayel Marie-Laure ML Baranger Alain A Stinchcombe John R JR

G3 (Bethesda, Md.) 20121101 11

Mutualisms are known to be genetically variable, where the genotypes differ in the fitness benefits they gain from the interaction. To date, little is known about the loci that underlie such genetic variation in fitness or whether the loci influencing fitness are partner specific, and depend on the genotype of the interaction partner. In the legume-rhizobium mutualism, one set of potential candidate genes that may influence the fitness benefits of the symbiosis are the plant genes involved in th ...[more]

PMID: 23173081

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Project description:BACKGROUND: Medicago truncatula Gaertn. (barrel medic) is cultivated as a pasture legume for its high protein content and ability to improve soils through nitrogen fixation. Toxic concentrations of the micronutrient Boron (B) in agricultural soils hamper the production of cereal and leguminous crops. In cereals, the genetic analysis of B tolerance has led to the development of molecular selection tools to introgress and maintain the B tolerance trait in breeding lines. There is a comparable need for selection tools in legumes that grow on these toxic soils, often in rotation with cereals. RESULTS: Genetic variation for B tolerance in Medicago truncatula was utilised to generate two F2 populations from crosses between tolerant and intolerant parents. Phenotyping under B stress revealed a close correlation between B tolerance and biomass production and a segregation ratio explained by a single dominant locus. M. truncatula homologues of the Arabidopsis major intrinsic protein (MIP) gene AtNIP5;1 and the efflux-type transporter gene AtBOR1, both known for B transport, were identified and nearby molecular markers screened across F2 lines to verify linkage with the B-tolerant phenotype. Most (95%) of the phenotypic variation could be explained by the SSR markers h2_6e22a and h2_21b19a, which flank a cluster of five predicted MIP genes on chromosome 4. Three CAPS markers (MtBtol-1,-2,-3) were developed to dissect the region further. Expression analysis of the five predicted MIPs indicated that only MtNIP3 was expressed when leaf tissue and roots were assessed. MtNIP3 showed low and equal expression in the roots of tolerant and intolerant lines but a 4-fold higher expression level in the leaves of B-tolerant cultivars. The expression profile correlates closely with the B concentration measured in the leaves and roots of tolerant and intolerant plants. Whereas no significant difference in B concentration exists between roots of tolerant and intolerant plants, the B concentration in the leaves of tolerant plants is less than half that of intolerant plants, which further supports MtNIP3 as the best candidate for the tolerance trait-defining gene in Medicago truncatula. CONCLUSION: The close linkage of the MtNIP3 locus to B toxicity tolerance provides a source of molecular selection tools to pasture breeding programs. The economical importance of the locus warrants further investigation of the individual members of the MIP gene cluster in other pasture and in grain legumes.

Project description:BACKGROUND:We studied patterns of molecular adaptation in the wild Mediterranean legume Medicago truncatula. We focused on two phenotypic traits that are not functionally linked: flowering time and perception of symbiotic microbes. Phenology is an important fitness component, especially for annual plants, and many instances of molecular adaptation have been reported for genes involved in flowering pathways. While perception of symbiotic microbes is also integral to adaptation in many plant species, very few reports of molecular adaptation exist for symbiotic genes. Here we used data from 57 individuals and 53 gene fragments to quantify the overall strength of both positive and purifying selection in M. truncatula and asked if footprints of positive selection can be detected at key genes of rhizobia recognition pathways. RESULTS:We examined nucleotide variation among 57 accessions from natural populations in 53 gene fragments: 5 genes involved in nitrogen-fixing bacteria recognition, 11 genes involved in flowering, and 37 genes used as control loci. We detected 1757 polymorphic sites yielding an average nucleotide diversity (pi) of 0.003 per site. Non-synonymous variation is under sizable purifying selection with 90% of amino-acid changing mutations being strongly selected against. Accessions were structured in two groups consistent with geographical origins. Each of these two groups harboured an excess of rare alleles, relative to expectations of a constant-sized population, suggesting recent population expansion. Using coalescent simulations and an approximate Bayesian computation framework we detected several instances of genes departing from selective neutrality within each group and showed that the polymorphism of two nodulation and four flowering genes has probably been shaped by recent positive selection. CONCLUSION:We quantify the intensity of purifying selection in the M. truncatula genome and show that putative footprints of natural selection can be detected at different time scales in both flowering and symbiotic pathways.

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Project description:One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

Dataset Information

Mapping the genetic basis of symbiotic variation in legume-rhizobium interactions in Medicago truncatula.

Publications

Mapping the genetic basis of symbiotic variation in legume-rhizobium interactions in Medicago truncatula.

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