Medicago truncatula quantitative resistance to a new strain of Verticillium alfalfae from Iran revealed by a genome-wide association study.

Verticillium wilt is a major threat to many crops, among them alfalfa (Medicago sativa). The model plant Medicago truncatula, a close relative of alfalfa was used to study the genetic control of resistance towards a new Verticillium alfalfae isolate. The accidental introduction of pathogen strains through global trade is a threat to crop production and such new strains might also be better adapted to global warming. Isolates of V. alfalfae were obtained from alfalfa fields in Iran and characterized. The Iranian isolate AF1 was used in a genome-wide association study (GWAS) involving 242 accessions from the Mediterranean region. Root inoculations were performed with conidia at 25°C and symptoms were scored regularly. Maximum Symptom Score and Area under Disease Progess Curve were computed as phenotypic traits to be used in GWAS and for comparison to a previous study with French isolate V31.2 at 20°C. This comparison showed high correlation with a shift to higher susceptibility, and similar geographical distribution of resistant and susceptible accessions to AF1 at 25°C, with resistant accessions mainly in the western part. GWAS revealed 30 significant SNPs linked to resistance towards isolate AF1. None of them were common to the previous study with isolate V31.2 at 20°C. To confirm these loci, the expression of nine underlying genes was studied. All genes were induced in roots following inoculation, in susceptible and resistant plants. However, in resistant plants induction was higher and lasted longer. Taken together, the use of a new pathogen strain and a shift in temperature revealed a completely different genetic control compared to a previous study that demonstrated the existence of two major QTLs. These results can be useful for Medicago breeding programs to obtain varieties better adapted to future conditions.

Temperature increase modifies susceptibility to Verticillium wilt in Medicago spp and may contribute to the emergence of more aggressive pathogenic strains.

Global warming is expected to have a direct impact on plant disease patterns in agro-eco-systems. However, few analyses report the effect of moderate temperature increase on disease severity due to soil-borne pathogens. For legumes, modifications of root plant-microbe interactions either mutualistic or pathogenic due to climate change may have dramatic effects. We investigated the effect of increasing temperature on the quantitative disease resistance to Verticillium spp., a major soil-borne fungal pathogen, in the model legume Medicago truncatula and the crop M. sativa. First, twelve pathogenic strains isolated from various geographical origin were characterized with regard to their in vitro growth and pathogenicity at 20°C, 25°C and 28°C. Most of them exhibited 25°C as the optimum temperature for in vitro parameters, and between 20°C and 25°C for pathogenicity. Second, a V. alfalfae strain was adapted to the higher temperature by experimental evolution, i.e. three rounds of UV mutagenesis and selection for pathogenicity at 28°C on a susceptible M. truncatula genotype. Inoculation of monospore isolates of these mutants on resistant and susceptible M. truncatula accessions revealed that at 28°C they were all more aggressive than the wild type strain, and that some had acquired the ability to cause disease on resistant genotype. Third, one mutant strain was selected for further studies of the effect of temperature increase on the response of M. truncatula and M. sativa (cultivated alfalfa). The response of seven contrasted M. truncatula genotypes and three alfalfa varieties to root inoculation was followed using disease severity and plant colonization, at 20°C, 25°C and 28°C. With increasing temperature, some lines switched from resistant (no symptoms, no fungus in the tissues) to tolerant (no symptoms but fungal growth into the tissues) phenotypes, or from partially resistant to susceptible. Further studies in greenhouse evidence the reduction in plant fitness due to disease in susceptible lines. We thus report that root pathogenic interactions are affected by anticipated global warming, with trends towards increased plant susceptibility and larger virulence for hot-adapted strains. New threats due to hot-adapted strains of soil-borne pathogens, with possibly wider host range and increased aggressiveness, might occur.

WhoGEM: an admixture-based prediction machine accurately predicts quantitative functional traits in plants.

The explosive growth of genomic data provides an opportunity to make increased use of sequence variations for phenotype prediction. We have developed a prediction machine for quantitative phenotypes (WhoGEM) that overcomes some of the bottlenecks limiting the current methods. We demonstrated its performance by predicting quantitative disease resistance and quantitative functional traits in the wild model plant species, Medicago truncatula, using geographical locations as covariates for admixture analysis. The method's prediction reliability equals or outperforms all existing algorithms for quantitative phenotype prediction. WhoGEM analysis produces evidence that variation in genome admixture proportions explains most of the phenotypic variation for quantitative phenotypes.

Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways.

The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short-term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics-identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up-regulation of SA-dependent PR proteins.

Cadmium-induced changes in antioxidative systems and differentiation in roots of contrasted Medicago truncatula lines.

Defense pathways and stress responses induced under Cd stress were illustrated in roots of hydroponically grown Medicago truncatula seedlings. Actually, the ascorbate-glutathione and antioxidative system, secondary metabolism events including peroxidases, phenolic compounds, and lignification launching, and developmental modifications were described. Cd (100 µM) initially increased reactive oxygen species, enhanced antioxidative (total SOD, CAT, and PRX) and ascorbate-glutathione-related metabolism enzymes (APX and MDAR), except in A17 and TN1.11. In agreement with peroxidase enhancement, physiological measurement and in situ observation illustrated soluble phenolic compound accumulation under Cd treatment. However, lignification was restricted to recently created protoxylem elements established in the root tip area, usually constituting the elongation zone. Cell death was increased. In the absence of necrotic reactions, developmental changes including lignin deposition, increase in cellulose and pectin contents, intercellular meatus, and condensed and deformed hairs were noticed in Cd-treated roots.

Quantitative Resistance to Verticillium Wilt in Medicago truncatula Involves Eradication of the Fungus from Roots and Is Associated with Transcriptional Responses Related to Innate Immunity.

Resistance mechanisms to Verticillium wilt are well-studied in tomato, cotton, and Arabidopsis, but much less in legume plants. Because legume plants establish nitrogen-fixing symbioses in their roots, resistance to root-attacking pathogens merits particular attention. The interaction between the soil-borne pathogen Verticillium alfalfae and the model legume Medicago truncatula was investigated using a resistant (A17) and a susceptible (F83005.5) line. As shown by histological analyses, colonization by the pathogen was initiated similarly in both lines. Later on, the resistant line A17 eliminated the fungus, whereas the susceptible F83005.5 became heavily colonized. Resistance in line A17 does not involve homologs of the well-characterized tomato Ve1 and V. dahliae Ave1 genes. A transcriptomic study of early root responses during initial colonization (i.e., until 24 h post-inoculation) similarly was performed. Compared to the susceptible line, line A17 displayed already a significantly higher basal expression of defense-related genes prior to inoculation, and responded to infection with up-regulation of only a small number of genes. Although fungal colonization was still low at this stage, the susceptible line F83005.5 exhibited a disorganized response involving a large number of genes from different functional classes. The involvement of distinct phytohormone signaling pathways in resistance as suggested by gene expression patterns was supported by experiments with plant hormone pretreatment before fungal inoculation. Gene co-expression network analysis highlighted five main modules in the resistant line, whereas no structured gene expression was found in the susceptible line. One module was particularly associated to the inoculation response in A17. It contains the majority of differentially expressed genes, genes associated with PAMP perception and hormone signaling, and transcription factors. An in silico analysis showed that a high number of these genes also respond to other soil-borne pathogens in M. truncatula, suggesting a core of transcriptional response to root pathogens. Taken together, the results suggest that resistance in M. truncatula line A17 might be due to innate immunity combining preformed defense and PAMP-triggered defense mechanisms, and putative involvement of abscisic acid.

Naturally occurring diversity helps to reveal genes of adaptive importance in legumes.

Environmental changes challenge plants and drive adaptation to new conditions, suggesting that natural biodiversity may be a source of adaptive alleles acting through phenotypic plasticity and/or micro-evolution. Crosses between accessions differing for a given trait have been the most common way to disentangle genetic and environmental components. Interestingly, such man-made crosses may combine alleles that never meet in nature. Another way to discover adaptive alleles, inspired by evolution, is to survey large ecotype collections and to use association genetics to identify loci of interest. Both of these two genetic approaches are based on the use of biodiversity and may eventually help us in identifying the genes that plants use to respond to challenges such as short-term stresses or those due to global climate change. In legumes, two wild species, Medicago truncatula and Lotus japonicus, plus the cultivated soybean (Glycine max) have been adopted as models for genomic studies. In this review, we will discuss the resources, limitations and future plans for a systematic use of biodiversity resources in model legumes to pinpoint genes of adaptive importance in legumes, and their application in breeding.

Effect of cadmium pollution on mobilization of embryo reserves in seedlings of six contrasted Medicago truncatula lines.

Six Medicago truncatula genotypes differing in cadmium susceptibility were used to test the effect of this heavy metal on mineral, carbohydrate and amino acid supply in growing radicles. Cadmium treatment caused alteration of macronutrient (Ca and K), microelement (Fe, Zn and Cu), carbohydrate (total soluble sugars (TSS), glucose, fructose and sucrose) and free amino acid (FAAS) accumulations. These mobilization changes differed in the tested genotypes. Carbohydrates were determining to susceptible lines' growth in control condition; free amino acids enabled tolerant lines to counteract cadmium intrusion. Transcriptional changes in response to cadmium treatment were analyzed on MtMST, a gene encoding a monosaccharide transport protein. A significant down-regulation was observed in the most susceptible line TN1.11. Glucose was over-consumed in tolerant lines. Thus, glucose metabolism integrity seems essential to maintain growth under cadmium exposure. Analysis of germination medium showed solute losses at the expense of suitable mobilization to the growing embryonic axis and highlights cadmium-triggered membrane alterations. FAAS and TSS leakages were reduced in tolerant lines while monosaccharide losses were accentuated in susceptible lines. This research work gave an overview of cadmium deleterious effects on biomass mobilization and membrane integrity. Carbon metabolism is shown to be primordial to enhance early embryonic growth and nitrogen metabolism is revealed to be crucial to establish seedling growth under cadmium stress.

Oxidative injury and antioxidant genes regulation in cadmium-exposed radicles of six contrasted Medicago truncatula genotypes.

Oxidative disorders were triggered in the presence of Cd toxicity in early seedling growth of six Medicago truncatula genotypes. Interactions between root growth inhibition, cadmium uptake, as well as the occurrence of oxidative injury suggest differential responses of the genotypes, with susceptible or tolerant accessions. ROS enhancement was observed in situ and H2O2 content was measured, that did not seem related to tolerance or susceptibility. Oxidative burst impact on cell membrane integrity was analyzed in agreement with MDA content and glucose exudation, which suggest an active role of this burst in susceptible lines. Transcriptional changes in response to cadmium treatment were analyzed on target genes involved in (1) ROS-scavenging enzymes (superoxide dismutase (SOD; EC1.15.1.1) and peroxidase (PRX; EC 1.11.1.7)), (2) reduced glutathione (γ-Glu-Cys-Gly, GSH) metabolism (glutathione-S-transferase (GST; EC: 2.5.1.18) and glutathione reductase (GR; EC 1.8.1.7)), and (3) metal-chelating metabolism (PCS). The susceptible line shows no response or non-timely gene expression patterns. This research work gave an overview of the deleterious effects and oxidative injury of cadmium stress in Medicago truncatula. Oxidative defense efficiency and gene upregulation should explain relative tolerance in tested genotypes.

Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt.

Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state.

Little data exist on the mechanism and stability of transformation in Phytophthora parasitica, a major oomycete parasite of plants. Here, we studied the stability of drug-resistant protoplast transformants by analyzing single-zoospore derivatives. We show that the transgenic sequences are not stably integrated into the chromosomes, resulting in the loss of drug resistance in single-zoospore derivatives. However, in strains where the P. parasitica gene encoding the CBEL elicitor was silenced by transformation with sense or antisense constructs, silencing is not reversed when the transgenic sequences are lost. This suggests that instability of P. parasitica transformants is not an obstacle for loss-of-function studies in this organism.

Cellulose binding domains of a Phytophthora cell wall protein are novel pathogen-associated molecular patterns.

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.

Antisense expression of a NBS-LRR sequence in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.): evidence for a dual role in plant development and fungal resistance.

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.

The CBEL glycoprotein of Phytophthora parasitica var-nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates.

The cell wall of the oomycete plant pathogen Phytophthora parasitica var. nicotianae contains a protein called CBEL that shows cellulose-binding (CB), elicitor (E) of defense in plants and lectin-like (L) activities. The biological role of this molecule in Phytophthora was investigated by generating transgenic strains suppressed in CBEL expression. Phenotypic characterization of these strains showed that they were severely impaired in adhesion to a cellophane membrane, differentiation of lobed structures in contact with cellophane, and formation of branched aggregating hyphae on cellophane and on flax cellulose fibres. Infection assays revealed that the strains suppressed in CBEL expression were not greatly affected in pathogenicity and formed branched aggregating hyphae in contact with the roots of the host plant, thereby indicating that CBEL is involved in the perception of cellulose rather than in the morphogenesis of hyphal aggregates. Interestingly, the absence of CBEL was correlated with abnormal formation of papillae-like cell wall thickenings in vitro, suggesting that CBEL is involved in cell wall deposition in Phytophthora. Reverse genetics in oomycetes has long been hampered by their diploid nature and difficulties in transformation and regeneration. The gene inactivation approach reported in this work provides the first direct evidence for intrinsic functions of an elicitor and cell wall protein in oomycetes.