Maria J. Manzanares-Dauleux

Association mapping of quantitative resistance for Leptosphaeria maculans in oilseed rape (Brassica napus L.)

Stem canker caused by the fungus Leptosphaeriamaculans is a major disease of Brassicanapus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.

Arginase Induction Represses Gall Development During Clubroot Infection in Arabidopsis

Arginase induction can play a defensive role through the reduction of arginine availability for phytophageous insects. Arginase activity is also induced during gall growth caused by Plasmodiophora brassicae infection in roots of Arabidopsis thaliana; however, its possible role in this context has been unclear. We report here that the mutation of the arginase-encoding gene ARGAH2 abrogates clubroot-induced arginase activity and results in enhanced gall size in infected roots, suggesting that arginase plays a defensive role. Induction of arginase activity in infected roots was impaired in the jar1 mutant, highlighting a link between the arginase response to clubroot and jasmonate signaling. Clubroot-induced accumulation of the principal amino acids in galls was not affected by the argah2 mutation. Because ARGAH2 was previously reported to control auxin response, we investigated the role of ARGAH2 in callus induction. ARGAH2 was found to be highly induced in auxin/cytokinin-triggered aseptic plant calli, and callus development was enhanced in argah2 in the absence of the pathogen. We hypothesized that arginase contributes to a negative control over clubroot symptoms, by reducing hormone-triggered cellular proliferation.

Partial resistance to clubroot in Arabidopsis is based on changes in the host primary metabolism and targeted cell division and expansion capacity

To date, studies of the molecular basis of disease resistance mainly focused on qualitative resistance. However, deciphering mechanisms underlying quantitative resistance could lead to insights into the relationship between qualitative and quantitative resistance and guide the utilization of these two types of resistance to produce durably resistant cultivars. A functional genomics approach, using the CATMA whole-genome microarray, was used to detect changes in gene expression associated with partial quantitative resistance in the Arabidopsis thaliana–Plasmodiophora brassicae pathosystem. The time course of transcript abundance during partial clubroot resistance response was monitored at the whole plant level, and direct comparisons between partial resistance and susceptibility responses were made using the same host genotype. An increasingly complex host response was revealed, as was the differential influence of P. brassicae infection on the transcription of Arabidopsis genes according to the isolate used. We observed, at the transcriptomic level, that metabolic diversion by the pathogen was reduced or delayed, classical plant defense responses were induced earlier and/or more strongly, and cell enlargement and proliferation were actively inhibited in the partial quantitative resistance response compared to the susceptible one.

Genetic and physiological analysis of the relationship between partial resistance to clubroot and tolerance to trehalose in Arabidopsis thaliana

In Arabidopsis thaliana the induction of plant trehalase during clubroot disease was proposed to act as a defense mechanism in the susceptible accession Col-0, which could thereby cope with the accumulation of pathogen-synthesized trehalose. In the present study, we assessed trehalose activity and tolerance to trehalose in the clubroot partially resistant accession Bur-0. We compared both accessions for several trehalose-related physiological traits during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny. Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of trehalase by validamycin treatments resulted in the enhancement of clubroot symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-induced toxicity symptoms than Col-0. A QTL analysis identified one locus involved in tolerance to trehalose overlapping the confidence interval of a QTL for resistance to Plasmodiophora brassicae. This colocalization was confirmed using heterogeneous inbred family (HIF) lines. Although not based on trehalose catabolism capacity, partial resistance to clubroot is to some extent related to the tolerance to trehalose accumulation in Bur-0. These findings support an original model where contrasting primary metabolism-related regulations could contribute to the partial resistance to a plant pathogen.

Homoeologous duplicated regions are involved in quantitative resistance of Brassica napus to stem canker

BackgroundSeveral major crop species are current or ancient polyploids. To better describe the genetic factors controlling traits of agronomic interest (QTL), it is necessary to understand the structural and functional organisation of these QTL regions in relation to genome duplication. We investigated quantitative resistance to the fungal disease stem canker in Brassica napus, a highly duplicated amphidiploid species, to assess the proportion of resistance QTL located at duplicated positions.ResultsGenome-wide association analysis on a panel of 116 oilseed rape varieties genotyped with 3228 SNP indicated that 321 markers, corresponding to 64 genomic regions, are associated with resistance to stem canker. These genomic regions are relatively equally distributed on the A (53%) and C (47%) genomes of B. napus. Overall, 44% of these regions (28/64) are duplicated homoeologous regions. They are located in duplications of six (E, J, R, T, U and W) of the 24 ancestral blocks that constitute the B. napus genome. Overall, these six ancestral blocks have 34 duplicated copies in the B.napus genome. Almost all of the duplicated copies (82% of the 34 regions) harboured resistance associated markers for stem canker resistance, which suggests structural and functional conservation of genetic factors involved in this trait in B. napus.ConclusionsOur study provides information on the involvement of duplicated loci in the control of stem canker resistance in B. napus. Further investigation of the similarity/divergence in sequence and gene content of these duplicated regions will provide insight into the conservation and allelic diversity of the underlying genes.

Both the Jasmonic Acid and the Salicylic Acid Pathways Contribute to Resistance to the Biotrophic Clubroot Agent Plasmodiophora brassicae in Arabidopsis

The role of salicylic acid (SA) and jasmonic acid (JA) signaling in resistance to root pathogens has been poorly documented. We assessed the contribution of SA and JA to basal and partial resistance of Arabidopsis to the biotrophic clubroot agent Plasmodiophora brassicae. SA and JA levels as well as the expression of the SA-responsive genes PR2 and PR5 and the JA-responsive genes ARGAH2 and THI2.1 were monitored in infected roots of the accessions Col-0 (susceptible) and Bur-0 (partially resistant). SA signaling was activated in Bur-0 but not in Col-0. The JA pathway was weakly activated in Bur-0 but was strongly induced in Col-0. The contribution of both pathways to clubroot resistance was then assessed using exogenous phytohormone application and mutants affected in SA or JA signaling. Exogenous SA treatment decreased clubroot symptoms in the two Arabidopsis accessions, whereas JA treatment reduced clubroot symptoms only in Col-0. The cpr5-2 mutant, in which SA responses are constitutively induced, was more resistant to clubroot than the corresponding wild type, and the JA signaling-deficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(δ)-acetylornithine biosynthesis in infected Col-0 roots. The 35S::NATA1 and nata1 lines displayed reduced or enhanced clubroot symptoms, respectively, thus suggesting that in Col-0 this pathway was involved in the JA-mediated basal clubroot resistance. Overall, our data support the idea that, depending on the Arabidopsis accession, both SA and JA signaling can play a role in partial inhibition of clubroot development in compatible interactions with P. brassicae.

The evolution of the Gp-Rbp-1 gene in Globodera pallida includes multiple selective replacements.

SUMMARY The Globodera pallida SPRYSEC Gp‐Rbp‐1 gene encodes a secreted protein which induces effector‐triggered immunity (ETI) mediated by the Solanum tuberosum disease resistance gene Gpa2. Nonetheless, it is not known how the Andes orogeny, the richness in Solanum species found along the Cordillera or the introduction of the nematode into Europe have affected the diversity of Gp‐Rbp‐1 and its recognition by Gpa2. We generated a dataset of 157 highly polymorphic Gp‐Rbp‐1 sequences and identified three Gp‐Rbp‐1 evolutionary pathways: the ‘Northern Peru’, ‘Peru clade I/European’ and ‘Chilean’ paths. These may have been shaped by passive dispersion of the nematode and by climatic variations that have influenced the nature and diversity of wild host species. We also confirmed that, by an analysis of the selection pressures acting on Gp‐Rbp‐1, this gene has evolved under positive/diversifying selection, but differently among the three evolutionary pathways described. Using this extended sequence dataset, we were able to detect eight sites under positive selection. Six sites appear to be of particular interest because of their predicted localization to the extended loops of the B30.2 domain and/or support by several computational methods. The P/S 187 position was previously identified for its effect on the interaction with GPA2. The functional importance of the other five amino acid polymorphisms observed was investigated using Agrobacterium transient transformation assays. None of these new residues, however, appears to be directly involved in Gpa2‐mediated plant defence mechanisms. Thus, the P/S polymorphism observed at position 187 remains the sole variation sufficient to explain the recognition of Gp‐Rbp‐1 by Gpa2.

Camalexin contributes to the partial resistance of Arabidopsis thaliana to the biotrophic soilborne protist Plasmodiophora brassicae

Camalexin has been reported to play defensive functions against several pathogens in Arabidopsis. In this study, we investigated the possible role of camalexin accumulation in two Arabidopsis genotypes with different levels of basal resistance to the compatible eH strain of the clubroot agent Plasmodiophora brassicae. Camalexin biosynthesis was induced in infected roots of both Col-0 (susceptible) and Bur-0 (partially resistant) accessions during the secondary phase of infection. However, the level of accumulation was four-to-seven times higher in Bur-0 than Col-0. This was associated with the enhanced transcription of a set of camalexin biosynthetic P450 genes in Bur-0: CYP71A13, CYP71A12, and CYP79B2. This induction correlated with slower P. brassicae growth in Bur-0 compared to Col-0, thus suggesting a relationship between the levels of camalexin biosynthesis and the different levels of resistance. Clubroot-triggered biosynthesis of camalexin may also participate in basal defense in Col-0, as gall symptoms and pathogen development were enhanced in the pad3 mutant (Col-0 genetic background), which is defective in camalexin biosynthesis. Clubroot and camalexin responses were then studied in Heterogeneous Inbred Families (HIF) lines derived from a cross between Bur-0 and Col-0. The Bur/Col allelic substitution in the region of the previously identified clubroot resistance QTL PbAt5.2 (Chromosome 5) was associated with both the enhanced clubroot-triggered induction of camalexin biosynthesis and the reduced P. brassicae development. Altogether, our results suggest that high levels of clubroot-triggered camalexin biosynthesis play a role in the quantitative control of partial resistance of Arabidopsis to clubroot.

Connected populations for detecting quantitative resistance factors to phoma stem canker in oilseed rape (Brassica napus L.)

Improvement of effectiveness and durability of disease resistance in crops most often relies on the use of quantitative resistance, with the hypothesis that a wide range of quantitative resistance factors (QTL) makes the overcoming of the resistance by the pathogen more difficult. For an optimum use of these QTL in effective and durable strategies of resistance deployment, there is a need to precisely know their localization but also their stability/specificity and their allelic effects in various genetic backgrounds. Stem canker caused by the fungus Leptosphaeria maculans is one of the most important diseases in oilseed rape. In this Brassica napus- L. maculans pathosystem, QTL were previously identified by linkage analysis using populations derived from biparental crosses that were analyzed separately. In this study, we explored new quantitative resistance factors using a multi-cross connected design derived from four resistant lines crossed with a single susceptible line. Independent and connected mapping analyses revealed to be complementary to get an overview of QTL organization. We validated different QTL across different years and genetic backgrounds and identified novel QTL which had not yet been mapped. Population-common and population-specific QTL were identified. Knowledge of QTL organization and effects should help in the rational choice of relevant factors in breeding resistant genotypes to be integrated with other control means such as cultural practices and rotations for durable management of the disease.

Comparative genomic analysis of duplicated homoeologous regions involved in the resistance of Brassica napus to stem canker.

All crop species are current or ancient polyploids. Following whole genome duplication, structural and functional modifications result in differential gene content or regulation in the duplicated regions, which can play a fundamental role in the diversification of genes underlying complex traits. We have investigated this issue in Brassica napus, a species with a highly duplicated genome, with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker, a disease caused by the fungal pathogen Leptosphaeria maculans. Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E, J, R, U, and W were involved in resistance to stem canker. The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions, as overall in the whole B. napus genome. The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B. napus progenitors. About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block. Genes retained in several copies were mainly involved in response to stress, signaling, or transcription regulation. Genes with resistance-associated markers were mainly retained in more than two copies. These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes. Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions. Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype.