Ten Broad Spectrum Resistances to Downy Mildew Physically Mapped on the Sunflower Genome

Resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.) is conferred by major resistance genes, denoted Pl. Twenty-two Pl genes have been identified and genetically mapped so far. However, over the past 50 years, wide-scale presence of only a few of them in sunflower crops led to the appearance of new, more virulent pathotypes (races) so it is important for sunflower varieties to carry as wide a range of resistance genes as possible. We analyzed phenotypically 12 novel resistant sources discovered in breeding pools derived from two wild Helianthus species and in eight wild H. annuus ecotypes. All were effective against at least 16 downy mildew pathotypes. We mapped their resistance genes on the sunflower reference genome of 3,600 Mb, in intervals that varied from 75 Kb to 32 Mb using an AXIOM® genotyping array of 49,449 SNP. Ten probably new genes were identified according to resistance spectrum, map position, hypersensitive response to the transient expression of a P. halstedii RXLR effector, or the ecotype/species from which they originated. The resistance source HAS6 was found to carry the first downy mildew resistance gene mapped on chromosome 11, whereas the other resistances were positioned on chromosomes 1, 2, 4, and 13 carrying already published Pl genes that we also mapped physically on the same reference genome. The new genes were designated Pl23–Pl32 according to the current nomenclature. However, since sunflower downy mildew resistance genes have not yet been sequenced, rules for designation are discussed. This is the first large scale physical mapping of both 10 new and 10 already reported downy mildew resistance genes in sunflower

A tell tail sign : a conserved C-terminal tail-anchor domain targets a subset of pathogen effectors to the plant endoplasmic reticulum

The endoplasmic reticulum (ER) is the entry point to the secretory pathway and, as such, is critical for adaptive responses to biotic stress, when the demand for de novo synthesis of immunity-related proteins and signalling components increases significantly. Successful phytopathogens have evolved an arsenal of small effector proteins which collectively reconfigure multiple host components and signalling pathways to promote virulence; a small, but important, subset of which are targeted to the endomembrane system including the ER. We identified and validated a conserved C-terminal tail-anchor motif in a set of pathogen effectors known to localize to the ER from the oomycetes Hyaloperonospora arabidopsidis and Plasmopara halstedii (downy mildew of Arabidopsis and sunflower, respectively) and used this protein topology to develop a bioinformatic pipeline to identify putative ER-localized effectors within the effectorome of the related oomycete, Phytophthora infestans, the causal agent of potato late blight. Many of the identified P. infestans tail-anchor effectors converged on ER-localized NAC transcription factors, indicating that this family is a critical host target for multiple pathogens

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background: In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle. Methodology/Principal Findings: We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of 'wait anaphase', plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants. Conclusions/Significance: We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division

A simple method for high molecular-weight genomic DNA extraction suitable for long-read sequencing from spores of an obligate biotroph oomycete

International audienceLong-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read se-quencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes

Extraction, caractérisation et purification de bactériocines provenant du complexe d'espèces bactérien Ralstonia solanacearum : vers une stratégie innovante dans la lutte contre le flétrissement bactérien

International audienceExtraction, caractérisation et purification de bactériocines provenant du complexe d'espèces bactérien Ralstonia solanacearum : vers une stratégie innovante dans la lutte contre le flétrissement bactérie

Extraction, caractérisation et purification de bactériocines provenant du complexe d'espèces bactérien Ralstonia solanacearum : vers une stratégie innovante dans la lutte contre le flétrissement bactérien

International audienceExtraction, caractérisation et purification de bactériocines provenant du complexe d'espèces bactérien Ralstonia solanacearum : vers une stratégie innovante dans la lutte contre le flétrissement bactérie

RXLR and CRN Effectors from the Sunflower Downy Mildew Pathogen Plasmopara halstedii Induce Hypersensitive-Like Responses in Resistant Sunflower Lines

Plasmopara halstedii is an obligate biotrophic oomycete causing downy mildew disease on sunflower, Helianthus annuus, an economically important oil crop. Severe symptoms of the disease (e.g., plant dwarfism, leaf bleaching, sporulation and production of infertile flower) strongly impair seed yield. PI resistance genes conferring resistance to specific P. halstedii pathotypes were located on sunflower genetic map but yet not cloned. They are present in cultivated lines to protect them against downy mildew disease. Among the 16 different P halstedii pathotypes recorded in France, pathotype 710 is frequently found, and therefore continuously controlled in sunflower by different PI genes. High throughput sequencing of cDNA from P. halstedii led us to identify potential effectors with the characteristic RXLR or CRN motifs described in other oomycetes. Expression of six P halstedii putative effectors, five RXLR and one CRN, was analyzed by qRT-PCR in pathogen spores and in the pathogen infecting sunflower leaves and selected for functional analyses. We developed a new method for transient expression in sunflower plant leaves and showed for the first time subcellular localization of P halstedii effectors fused to a fluorescent protein in sunflower leaf cells. Overexpression of the CRN and of 3 RXLR effectors induced hypersensitive-like cell death reactions in some sunflower near-isogenic lines resistant to pathotype 710 and not in susceptible corresponding lines, suggesting they could be involved in PI loci-mediated resistances

Prévalence contrastée des souches de l'agent causal du flétrissement bactérien (le complexe d'espèces Ralstonia solanacearum) dans les îles du sud-ouest de l'océan Indien

International audiencePrévalence contrastée des souches de l'agent causal du flétrissement bactérien (le complexe d'espèces Ralstonia solanacearum) dans les îles du sud-ouest de l'océan Indie

Prévalence contrastée des souches de l'agent causal du flétrissement bactérien (le complexe d'espèces Ralstonia solanacearum) dans les îles du sud-ouest de l'océan Indien

International audiencePrévalence contrastée des souches de l'agent causal du flétrissement bactérien (le complexe d'espèces Ralstonia solanacearum) dans les îles du sud-ouest de l'océan Indie

Bacteriocin Production Correlates with Epidemiological Prevalence of Phylotype I Sequevar 18 Ralstonia pseudosolanacearum in Madagascar

International audienceBacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a major threat to vegetable crops in Madagascar. For more effective disease management, surveys were carried out in the main vegetable production areas of the country, leading to the collection of 401 new RSSC isolates. Phylogenetic assignment of the isolates revealed a high prevalence of phylotype I sequevar 18. This result contrasts sharply with the epidemiological pattern of RSSC in neighboring islands, including Reunion Island, Comoros, Mayotte, Mauritius, Rodrigues, and the Seychelles, where phylotype I sequevar 31 is widespread. Molecular typing characterization of the Malagasy isolates allowed the identification of 96 haplotypes. Some are found in various plots located in different provinces, which suggests that they were probably disseminated via infected plant material. To find out a potential explanation for the observed epidemiological pattern, we examined the capacity of the Malagasy strains to produce bacteriocin. Interestingly, the highly prevalent genetic lineages I-18 produce bacteriocins that are active against all the genetic lineages present in the country. This work sheds light on the potential impact of bacteriocins in the epidemiology of Malagasy RSSC.IMPORTANCE Knowledge of the epidemiology of a plant pathogen is essential to develop effective control strategies. This study focuses on the epidemiological pattern of Ralstonia pseudosolanacearum phylotype I populations responsible for bacterial wilt in Madagascar. We identified, with the newly collected isolates in three provinces, four genetic lineages probably propagated via infected plant material in Madagascar. We revealed that the epidemiological situation in Madagascar contrasts with that of neighboring Indian Ocean islands. Interestingly, our study on the bacteriocin-producing capacity of Malagasy isolates revealed a correlation between the inhibitory activity of the producing strains and the observed epidemiology. These results suggested that the epidemiology of plant pathogens may be impacted by bacteriocin production