Nicolas Rispail

Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes

SummaryBiotic and abiotic stresses cause significant yield losses in legumes and can significantly affect their productivity. Biotechnology tools such as marker-assisted breeding, tissue culture, in vitro mutagenesis and genetic transformation can contribute to solve or reduce some of these constraints. However, only limited success has been achieved so far. The emergence of “omic” technologies and the establishment of model legume plants such as Medicago truncatula and Lotus japonicus are promising strategies for understanding the molecular genetic basis of stress resistance, which is an important bottleneck for molecular breeding. Understanding the mechanisms that regulate the expression of stress-related genes is a fundamental issue in plant biology and will be necessary for the genetic improvement of legumes. In this review, we describe the current status of biotechnology approaches in relation to biotic and abiotic stresses in legumes and how these useful tools could be used to improve resistance to important constraints affecting legume crops.

Genetics of Symbiosis in Lotus japonicus: Recombinant Inbred Lines, Comparative Genetic Maps, and Map Position of 35 Symbiotic Loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

Breeding approaches for crenate broomrape (Orobanche crenata Forsk.) management in pea (Pisum sativum L.).

BACKGROUND Pea cultivation is strongly hampered in Mediterranean and Middle East farming systems by the occurrence of Orobanche crenata Forsk. Strategies of control have been developed, but only marginal successes have been achieved. Most control methods are either unfeasible, uneconomical, hard to achieve or result in incomplete protection. The integration of several control measures is the most desirable strategy. RESULTS [corrected] Recent developments in control are presented and re-evaluated in light of recent developments in crop breeding and molecular genetics. These developments are placed within a framework that is compatible with current agronomic practices. CONCLUSION The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid pea improvement for resistance against O. crenata and/or the herbicide.

Fusarium oxysporum Ste12 Controls Invasive Growth and Virulence Downstream of the Fmk1 MAPK Cascade

A conserved mitogen-activated protein kinase (MAPK) cascade homologous to the yeast Fus3/Kss1 mating/filamentation pathway regulates virulence in fungal plant pathogens. In the soilborne fungus Fusarium oxysporum, the MAPK Fmk1 is required for infection and development of vascular wilt disease on tomato plants. Knockout mutants lacking Fmk1 are deficient in multiple virulence-related functions, including root adhesion and penetration, invasive growth, secretion of pectinolytic enzymes, and vegetative hyphal fusion. The transcription factors mediating these different outputs downstream of the MAPK cascade are currently unknown. In this study, we have analyzed the role of ste12 which encodes an orthologue of the yeast homeodomain transcription factor Ste12p. F. oxysporum mutants lacking the ste12 gene were impaired in invasive growth on tomato and apple fruit tissue and in penetration of cellophane membranes. However, ste12 was not required for adhesion to tomato roots, secretion of pectinolytic enzymes, and vegetative hyphal fusion, suggesting that these Fmk1-dependent functions are mediated by other downstream MAPK targets. The Delta ste12 strains displayed dramatically reduced virulence on tomato plants, similar to the Delta fmk1 mutant. These results indicate that invasive growth is the major virulence function controlled by the Fmk1 MAPK cascade and depends critically on the transcription factor Ste12.

Achievements and Challenges in Legume Breeding for Pest and Disease Resistance

Yield stability of legume crops is constrained by a number of pest and diseases. Major diseases are rusts, powdery and downy mildews, ascochyta blights, botrytis gray molds, anthracnoses, damping-off, root rots, collar rots, vascular wilts and white molds. Parasitic weeds, viruses, bacteria, nematodes and damages caused by chewing and sap-sucking insects add to this long list of constraints for legume production. Their incidence and relative importance together with current understanding of their interactions with the host plants are presented. State of the art of current achievements and limitations for breeding for biotic stress resistance are listed and critically discussed. The recent development of large scale phenotyping, genome sequencing and analysis of gene, protein and metabolite expressions can be of great help to further decipher plant-pathogen interactions and identify key resistance components that may be introgressed into crop plants through breeding.

Genetic Diversity and Population Structure Among Oat Cultivars and Landraces

In this study, genetic diversity among 177 oat (Avena sativa L.) accessions including both white and red oat landraces and 36 commercial cultivars was studied for simple sequence repeat (SSR) loci. Thirty-one genomic and expressed sequence tags (EST)-derived primer pairs were selected according to high polymorphism from an initial 66 SSR batch. Markers revealed a high level of polymorphism, detecting a total of 454 alleles. The average gene diversity for the whole sample was 0.29. Genetic similarity, calculated using the Dice coefficient, was used for cluster analysis, and principal component analysis was also applied. In addition, population structure using a Bayesian clustering approach identified discrete subpopulation based on allele frequency and showed similar clustering of oat genotypes in four groups. Accessions could be classified into four main clusters that clearly separated the commercial cultivars, the red oat landraces and two clusters of white oat landraces. Cultivars showed less diversity than the landraces indicating a reduction of genetic diversity during breeding, whereas white oat landraces showed higher diversity than red ones. The average polymorphic information content of 0.80 for the SSR loci indicated the usefulness of many of the SSR for genotype identification. In particular, two markers, MAMA5 and AM04, with a total of 50 alleles and a high discrimination power (>0.90), were sufficient to discriminate among all commercial cultivars studied highlighting their potential use for variety identification.

Quantum dot and superparamagnetic nanoparticle interaction with pathogenic fungi: internalization and toxicity profile.

For several years now, nanoscaled materials have been implemented in biotechnological applications related to animal (in particular human) cells and related pathologies. However, the use of nanomaterials in plant biology is far less widespread, although their application in this field could lead to the future development of plant biotechnology applications. For any practical use, it is crucial to elucidate the relationship between the nanomaterials and the target cells. In this work we have evaluated the behavior of two types of nanomaterials, quantum dots and superparamagnetic nanoparticles, on Fusarium oxysporum, a fungal species that infects an enormous range of crops causing important economic losses and is also an opportunistic human pathogen. Our results indicated that both nanomaterials rapidly interacted with the fungal hypha labeling the presence of the pathogenic fungus, although they showed differential behavior with respect to internalization. Thus, whereas magnetic nanoparticles appeared to be on the cell surface, quantum dots were significantly taken up by the fungal hyphae showing their potential for the development of novel control approaches of F. oxysporum and related pathogenic fungi following appropriate functionalization. In addition, the fungal germination and growth, accumulation of ROS, indicative of cell stress, and fungal viability have been evaluated at different nanomaterial concentrations showing the low toxicity of both types of nanomaterials to the fungus. This work represents the first study on the behavior of quantum dots and superparamagnetic particles on fungal cells, and constitutes the first and essential step to address the feasibility of new nanotechnology-based systems for early detection and eventual control of pathogenic fungi.

Legume breeding for rust resistance: Lessons to learn from the model Medicago truncatula

Rusts are major biotic constraints of legumes worldwide. Breeding for rust resistance is regarded as the most cost efficient method for rust control. However, in contrast to common bean for which complete monogenic resistance exists and is efficiently used, most of the rust resistance reactions described so far in cool season food legumes are incomplete and of complex inheritance. Incomplete resistance has been described in faba bean, pea, chickpea and lentil and several of their associated QTLs have been mapped. However, the relatively large distance between the QTLs and their associated molecular markers hampers their efficient use for marker assisted selection. Their large genome size drastically hampers the development of genomic resource and limits the saturation of their genetic maps. The use of model plants such as the model legume Medicago truncatula may circumvent this drawback. The important genetic and genomic resources and tools available for this model legume can considerably speed up the discovery and validation of new genes and QTLs in resistance to legume pathogens. Here, the potential of M. truncatula as a model to study rust resistance in legumes, and to transfer rust resistance genes to cool season grain legumes is reviewed.

Genome-wide association study for crown rust (Puccinia coronata f. sp. avenae) and powdery mildew (Blumeria graminis f. sp. avenae) resistance in an oat (Avena sativa) collection of commercial varieties and landraces

Diseases caused by crown rust (Puccinia coronata f. sp. avenae) and powdery mildew (Blumeria graminis f. sp. avenae) are among the most important constraints for the oat crop. Breeding for resistance is one of the most effective, economical, and environmentally friendly means to control these diseases. The purpose of this work was to identify elite alleles for rust and powdery mildew resistance in oat by association mapping to aid selection of resistant plants. To this aim, 177 oat accessions including white and red oat cultivars and landraces were evaluated for disease resistance and further genotyped with 31 simple sequence repeat and 15,000 Diversity Arrays Technology (DArT) markers to reveal association with disease resistance traits. After data curation, 1712 polymorphic markers were considered for association analysis. Principal component analysis and a Bayesian clustering approach were applied to infer population structure. Five different general and mixed linear models accounting for population structure and/or kinship corrections and two different statistical tests were carried out to reduce false positive. Five markers, two of them highly significant in all models tested were associated with rust resistance. No strong association between any marker and powdery mildew resistance at the seedling stage was identified. However, one DArT sequence, oPt-5014, was strongly associated with powdery mildew resistance in adult plants. Overall, the markers showing the strongest association in this study provide ideal candidates for further studies and future inclusion in strategies of marker-assisted selection.

Identification of Sources of Quantitative Resistance to Fusarium oxysporum f. sp. medicaginis in Medicago truncatula

The resistance of 267 Medicago truncatula accessions was determined against the soilborne pathogen Fusarium oxysporum, one of the major constraints of forage and grain legumes worldwide. The initial screening of the collection revealed a wide range of disease response from completely resistant to highly susceptible to one strain of F. oxysporum f. sp. medicaginis. As a result, 26 accessions were identified as resistant, 9 as susceptible, and all other accessions as partially resistant. The phenotype of 12 resistant accessions was confirmed in two independent experiments on a subset of 23 accessions. Quantification of F. oxysporum f. sp. medicaginis within plant tissue indicated that the resistance level of the accessions is correlated with the amount of F. oxysporum f. sp. medicaginis within its shoot. Inoculation with a different F. oxysporum f. sp. medicaginis isolate indicated that the resistance phenotype was stable because accession response to both F. oxysporum f. sp. medicaginis strains followed similar trends. However, grouping accessions according to their geographic origin did not reveal foci of resistance, which supports the idea that resistance arose from independent events. The identification of 12 resistant accessions will be useful for further cellular and molecular studies to unravel the basis of resistance to F. oxysporum in this model species and to transfer resistance to legume crop.