Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.     

Genetic dissection of resistance to anthracnose and powdery mildew in Medicago truncatula

Medicago truncatula was used to characterize resistance to anthracnose and powdery mildew caused by Colletotrichum trifolii and Erysiphe pisi, respectively. Two isolates of E. pisi (Ep-p from pea and Ep-a from alfalfa) and two races of C. trifolii (races 1 and 2) were used in this study. The A17 genotype was resistant and displayed a hypersensitive response after inoculation with either pathogen, while lines F83005.5 and DZA315.16 were susceptible to anthracnose and powdery mildew, respectively. To identify the genetic determinants underlying resistance in A17, two F7 recombinant inbred line (RIL) populations, LR4 (A17 x DZA315.16) and LR5 (A17 x F83005.5), were phenotyped with E. pisi isolates and C. trifolii races, respectively. Genetic analyses showed that i) resistance to anthracnose is governed mainly by a single major locus to both races, named Ct1 and located on the upper part of chromosome 4; and ii) resistance to powdery mildew involves three distinct loci, Epp1 on chromosome 4 and Epa1 and Epa2 on chromosome 5. The use of a consensus genetic map for the two RIL populations revealed that Ct1 and Epp1, although located in the same genome region, were clearly distinct. In silico analysis in this region identified the presence of several clusters of nucleotide binding site leucine-rich repeat genes. Many of these genes have atypical resistance gene analog structures and display differential expression patterns in distinct stress-related cDNA libraries.     

The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines

In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.     

Effect of salinity on root-nodule conductance to the oxygen diffusion in the Medicago truncatula–Sinorhizobium meliloti symbiosis

In order to determine the effect of salinity on the nodule conductance, oxygen uptake by the nodulated roots was measured by registering the concentration of O₂ as a function of time in a tight incubator of known volume containing the nodulated roots of Medicago truncatula. Four lines, namely TN8.20 and TN6.18, originated from local populations, F83005.5 originated from Var (France) and Jemalong 6, a cultivar from Australia, were hydroponically grown in 250 ml glass bottles under semi-controlled conditions in a glasshouse, after germination and inoculation with the strain Sinorhizobium meliloti 2011. The saline treatment was applied gradually to reach 75 mM after 2 weeks. Results show that oxygen uptake increased significantly with salinity in TN6.18 and F83005.5, but not in Jemalong nor in TN8.20. Without salt, Jemalong showed a significantly higher O₂ uptake of 240 μmol O₂ per h per plant than the mean of 130 μmol O₂ per h per plant for other lines. Salinity increased significantly the nodule conductance in all genotypes. This salt effect was significantly higher for TN6.18 than for TN8.20, and for Jemalong than for F83005.5. Without salt there was less genotypic variation in nodule conductance in the range of 5–8 μm s^–1 for F83005.5 and TN8.20, respectively. Thus the sensitivity to salinity appears to be associated with an increase in nodule conductance that supports the increased respiration of N₂-fixing nodules under salinity.     

Involvement of Hydrogen Peroxide, Peroxidase and Superoxide Dismutase in Response of Medicago truncatula Lines Differing in Susceptibility to Phoma medicaginis Infection

This study was undertaken to assess the involvement of hydrogen peroxide (H₂O₂), peroxidase (POX; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1) in Medicago truncatula in relation with susceptibility to Phoma medicaginis infection. Several M. truncatula lines were studied in terms of their response to P. medicaginis infection. Fifteen days after inoculation (dai), differences in susceptibility were found. DZA45.5 was the least susceptible line and F83005.5 was the most susceptible line. Microscopic analysis of fungal development was performed in inoculated detached leaves of the DZA45.5 and F83005.5 lines. No significant difference was observed in events from conidia germination to penetration. Differences became apparent during the colonization process as the pathogen was able to sporulate rapidly increasing its concentration on the tissue of F83005.5 in comparison with DZA45.5. To characterize the susceptibility of the two lines, histochemical detection of H₂O₂ was made in detached leaves. H₂O₂ detection showed an early accumulation of this component in cells of DZA45.5 at 1 dai. However, H₂O₂ was detected in few, if any, cells in the tissues of the most susceptible line, F83005.5. The activity of POX and SOD were determined spectrophotometrically in leaves of intact inoculated plants of both lines. Phoma medicaginis inoculation of DZA45.5 and F83005.5 did not affect POX activity level in leaves when compared with control uninoculated plants. SOD activity showed a significant decrease in F83005.5 and DZA45.5 leaves at 4 dai and 9 dai, respectively, in comparison with control plants. In control plants POX activity was significantly higher in the least susceptible line DZA45.5 in comparison with F83005.5. Early and higher production of H₂O₂ and elevated basal POX activity in cells of the least susceptible line, DZA45.5 could explain its ability to be less favourable to the colonization and reproduction of P. medicaginis in comparison with the most susceptible line, F83005.5.     

QTLs mapping of morphological traits related to salt tolerance in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To understand the complex inheritance of tolerance to salt stress in Medicago truncatula, quantitative trait loci (QTLs) analysis was performed using a set of recombinant inbred lines (RILs) derived from a cross between the tolerant line Jemalong A17 and susceptible line F83005.5. The RILs and parental lines were grown in individual pots filled with sterilized sand in a greenhouse under 0 and 50 mM NaCl. Plants were harvested after a period of 60 days. Fourteen quantitative traits related to aerial and root growths were measured. Broad-sense heritability of measured traits ranged from 0.21 to 0.83 and from 0.05 to 0.62 in control and in salt-stressed conditions, respectively. Established correlations between measured traits are dependent on treatment effect. We identified and mapped 10 QTLs in control conditions and 19 in salt stress. No major QTL was identified indicating that tolerance to salt stress is governed by several genes with low effects. The QTLs detected under control and under salt-stressed conditions almost did not share the same map locations suggesting that the loci that are not stable across treatments reflect adaptation to this constraint. The maximum of QTLs was observed on the chromosome 8. The usefulness of these QTLs, identified in greenhouse conditions, for marker-assisted selection should therefore be evaluated under field conditions, and validated in other genetic backgrounds.     

Identification of distinct quantitative trait loci associated with defence against the closely related aphids Acyrthosiphon pisum and A. kondoi in Medicago truncatula

Aphids are a major family of plant insect pests. Medicago truncatula and Acyrthosiphon pisum (pea aphid, PA) are model species with a suite of resources available to help dissect the mechanism underlying plant-aphid interactions. A previous study focused on monogenic and relatively strong resistance in M. truncatula to PA and other aphid species. In this study a moderate resistance to PA was characterized in detail in the M. truncatula line A17 and compared with the highly susceptible line A20 and the more resistant line Jester. The results show that PA resistance in A17 involves both antibiosis and tolerance, and that resistance is phloem based. Quantitative trait locus (QTL) analysis using a recombinant inbred line (RIL) population (n=114) from a cross between A17 and A20 revealed that one locus, which co-segregated with AIN (Acyrthosiphon-induced necrosis) on chromosome 3, is responsible for the reduction of aphid biomass (indicator of antibiosis) for both PA and bluegreen aphid (BGA, A. kondoi), albeit to a lesser degree for PA than BGA. Interestingly, two independent loci on chromosomes 5 and 3 were identified for the plant biomass reduction (indicator of plant tolerance) by PA and BGA, respectively, demonstrating that the plant's tolerance response to these two closely related aphid species is distinct. Together with previously identified major resistant (R) genes, the QTLs identified in this study are powerful tools to understand fully the spectrum of plant defence against sap-sucking insects and provide opportunities for breeders to generate effective and sustainable strategies for aphid control.     

Independent action and contrasting phenotypes of resistance genes against spotted alfalfa aphid and bluegreen aphid in Medicago truncatula

Host resistance to aphids is poorly understood. Medicago truncatula, a model legume and cultivated pasture species, was used to elucidate defense against two aphid species, Therioaphis trifolii f. maculata (spotted alfalfa aphid, SAA) and Acyrthosiphon kondoi (bluegreen aphid, BGA). Aphid performance and plant damage were compared between near-isogenic cultivars, Mogul and Borung, that differ in resistance to both aphids. Analyses of aphid resistance in Mogul x Borung F2 plants and their progeny revealed modes of action and chromosome locations of resistance genes. Separate genes were identified for SAA resistance (TTR) and BGA resistance (AKR); both mapped to chromosome 3 but were found to act independently to reduce survival and growth of their target aphid species. The TTR locus controls distinct, and contrasting, local and systemic plant responses between the near-isogenic cultivars. TTR-mediated plant responses imply interaction between a resistance factor(s) in vascular tissue and a bioactive component(s) of SAA saliva. Features of both resistance traits suggest homology to aphid resistance in other legumes; elucidation of their molecular mechanisms will likely apply to other aphid-plant interactions.     

Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures

We have cloned a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene from Medicago truncatula (MtSERK1) and examined its expression in culture using real time PCR. In the presence of the auxin 1-naphthaleneacetic acid (NAA) alone, root differentiation occurs from the proliferating calli in both the cultured highly embryogenic seed line (2HA) and a low to nonembryogenic seed line (M. truncatula cv Jemalong). Auxin stimulated MtSERK1 expression in both 2HA and M. truncatula cv Jemalong. Embryo induction in proliferating calli requires a cytokinin in M. truncatula and unlike root formation is substantively induced in 2HA, not M. truncatula cv Jemalong. On embryo induction medium containing NAA and the cytokinin 6-benzylaminopurine (BAP), expression of MtSERK1 is elevated within 2 d of initiation of culture in both M. truncatula cv Jemalong and 2HA. However, MtSERK1 expression is much higher when both NAA and BAP are in the medium. BAP potentiates the NAA induction because MtSERK1 expression is not up-regulated by BAP alone. The 2HA genotype is able to increase its embryo formation because of the way it responds to cytokinin, but not because of the cytokinin effect on MtSERK1. Although the studies with M. truncatula indicate that somatic embryogenesis is associated with high SERK expression, auxin alone does not induce somatic embryogenesis as in carrot (Daucus carota) and Arabidopsis. Auxin in M. truncatula induces roots, and there is a clear up-regulation of MtSERK1. Although our analyses suggest that MtSERK1 is orthologous to AtSERK1, which in Arabidopsis is involved in somatic embryogenesis, in legumes, MtSERK1 may have a broader role in morphogenesis in cultured tissue rather than being specific to somatic embryogenesis.     

Characterization of resistance to multiple aphid species (Hemiptera: Aphididae) in Medicago truncatula

Aphids are phloem-feeding insects that damage many important crops throughout the world yet, compared to plant-pathogen interactions, little is known about the mechanisms by which plants become resistant to aphids. Medicago truncatula (barrel medic) is widely considered as the pre-eminent model legume for genetic and biological research and in Australia is an important pasture species. Six cultivars of M. truncatula with varying levels of resistance to two pests of pasture and forage legumes, the bluegreen aphid Acyrthosiphon kondoi Shinji and the spotted alfalfa aphid Therioaphis trifolii f. maculata. (Buckton) are investigated. Two resistance phenotypes against T. trifolii f. maculata are described, one of which is particularly effective, killing most aphids within 24 h of infestation. Each resistance phenotype provided a similar but somewhat less effective degree of resistance to the closely-related spotted clover aphid Therioaphis trifolii (Monell). In the case of A. kondoi only one resistance phenotype was observed, which did not vary among different genetic backgrounds. None of the observed resistance against A. kondoi or T. trifolii f. maculata significantly affected the performance of green peach aphid Myzus persicae (Sulzer) or cowpea aphid Aphis craccivora Koch. The existence of multiple aphid resistance mechanisms in similar genetic backgrounds of this model plant provides a unique opportunity to characterize the fundamental basis of plant defence to these serious agricultural pests.     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.     

Characterization of pea aphid resistance in Medicago truncatula

To achieve a thorough understanding of plant-aphid interactions, it is necessary to investigate in detail both the plant and insect side of the interaction. The pea aphid (PA; Acyrthosiphon pisum) has been selected by an international consortium as the model species for genetics and genomics studies, and the model legume Medicago truncatula is a host of this aphid. In this study, we identified resistance to PA in a M. truncatula line, 'Jester', with well-characterized resistance to a closely related aphid, the bluegreen aphid (BGA; Acyrthosiphon kondoi). The biology of resistance to the two aphid species shared similarity, with resistance in both cases occurring at the level of the phloem, requiring an intact plant and involving a combination of antixenosis, antibiosis, and plant tolerance. In addition, PA resistance cosegregated in 'Jester' with a single dominant gene for BGA resistance. These results raised the possibility that both resistances may be mediated by the same mechanism. This was not supported by the results of gene induction studies, and resistance induced by BGA had no effect on PA feeding. Moreover, different genetic backgrounds containing a BGA resistance gene from the same resistance donor differ in resistance to PA. These results suggest that distinct mechanisms are involved in resistance to these two aphid species. Resistance to PA and BGA in the same genetic background in M. truncatula makes this plant an attractive model for the study of both plant and aphid components of resistant and susceptible plant-aphid interactions.     

Aphid resistance in Medicago truncatula involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by NBS-LRR resistance gene analogs

Aphids and related insects feed from a single cell type in plants: the phloem sieve element. Genetic resistance to Acyrthosiphon kondoi Shinji (bluegreen aphid or blue alfalfa aphid) has been identified in Medicago truncatula Gaert. (barrel medic) and backcrossed into susceptible cultivars. The status of M. truncatula as a model legume allows an in-depth study of defense against this aphid at physiological, biochemical, and molecular levels. In this study, two closely related resistant and susceptible genotypes were used to characterize the aphid-resistance phenotype. Resistance conditions antixenosis since migratory aphids were deterred from settling on resistant plants within 6 h of release, preferring to settle on susceptible plants. Analysis of feeding behavior revealed the trait affects A. kondoi at the level of the phloem sieve element. Aphid reproduction on excised shoots demonstrated that resistance requires an intact plant. Antibiosis against A. kondoi is enhanced by prior infestation, indicating induction of this phloem-specific defense. Resistance segregates as a single dominant gene, AKR (Acyrthosiphon kondoi resistance), in two mapping populations, which have been used to map the locus to a region flanked by resistance gene analogs predicted to encode the CC-NBS-LRR subfamily of resistance proteins. This work provides the basis for future molecular analysis of defense against phloem parasitism in a plant model system.