The APSES transcription factor LmStuA is required for sporulation, pathogenic development and effector gene expression in Leptosphaeria maculans

Leptosphaeria maculans causes stem canker of oilseed rape (Brassica napus). The APSES transcription factor StuA is a key developmental regulator of fungi, involved in morphogenesis, conidia production and also more recently described as required for secondary metabolite production and for effector gene expression in phytopathogenic fungi. We investigated the involvement of the orthologue of StuA in L.maculans, LmStuA, in morphogenesis, pathogenicity and effector gene expression. LmStuA is induced during mycelial growth and at 14 days after infection, corresponding to the development of pycnidia on oilseed rape leaves, consistent with the function of StuA described so far. We set up the functional characterization of LmStuA using an RNA interference approach. Silenced LmStuA transformants showed typical phenotypic defects of StuA mutants with altered growth in axenic culture and impaired conidia production and perithecia formation. Silencing of LmStuA abolished the pathogenicity of L.maculans on oilseed rape leaves and also resulted in a drastic decrease in expression of at least three effector genes during inplanta infection, suggesting either that LmStuA regulates, directly or indirectly, the expression of several effector genes in L.maculans or that the infection stage in which effectors are expressed is not reached when LmStuA expression is silenced

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus

Leptosphaeria maculans is a hemibiotophic ascomycete which causes stem canker of oilseed rape. That phytopathogenic fungus interacts with its host (Brassica napus) according to the gene-for-gene concept. The most economically and environment friendly method of control of stem canker is the genetic control by using host resistance. Single gene resistance is extremely efficient, but races of the pathogen virulent towards a resistance gene can appear in a few years and necessitates continuously new breeding programs. Moreover, specific resistances are rare in oilseed rape, and a lot of efforts are made to find other resistance genes in other Brassica species. To date, 11 interactions were genetically characterized between L. maculans avirulence genes and corresponding resistance genes in Brassica, and 5 of those avirulence genes were cloned. Recently, the avirulence gene AvrLm10 which is recognized by the resistance gene Rlm10 of the black mustard (Brassica nigra) has been cloned. AvrLm10 corresponds in fact to two avirulence genes AvrLm10_1 and AvrLm10_2 which are located in the same AT-rich genomic region. They encore for small secreted proteins (SSP), are co-regulated and over-expressed 7 days post-infection. Each of them is necessary but not sufficient to induce resistance towards Rlm10. Silencing of one of those genes is sufficient to abolish recognition by Rlm10. Silencing by RNA interference of AvrLm10-1 induces an increase of lesion size on oilseed rape leaves while silencing of AvrLm10-2 has no major effect on aggressiveness of the fungus. That interaction of two avirulence genes against one resistance gene is therefore different from the classical gene-for-gene concept. It suggests that AvrLm10_1 and AvrLm10_2 could directly interact and / or that they could target the same plant protein. A Y2H screen suggested a direct interaction between AvrLm10-1 and AvrLm10-2. This interaction was confirmed with Bimolecular Fluorescence Complementation (BiFC) experiments. Coimmunoprecipitation experiments are also in progress to confirm this interaction

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus. 7th Effectome meetin

Identification et analyse fonctionnelle des effecteurs tardifs impliqués dans la colonisation systémique du colza par Leptosphaeria maculans

National audienc

Leptosphaeria maculans effectors involved in the oilseed rape systemic colonization.

The stem canker disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood. By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome. Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem. The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates. Reference Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape. Mol. Plant Pathol

Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia

High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (similar to 11), low-temperature (< 40 degrees C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Therrnoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ

Microbial communities associated with alkaline hydrothermal systems of Prony Bay, New Caledonia

International audienceA shallow submarine hydrothermal field comparable to that of Lost City (http://www.lostcity.washington.edu/) was discovered in Prony Bay in the south of New Caledonia (SW Pacific). Both Prony and Lost City are ultramafic hydrothermal systems driven by serpentinization reactions of water with mantle rocks resulting in warm, anoxic, highly alkaline fluids (up to pH 11) rich in calcium, dissolved hydrogen and methane but depleted in carbon oxides and metals. Thus they sharply contrast with the very hot, acidic metal rich fluids typical of sulfidic vents (“black smokers”) associated with magmatic heat. Like in Lost City, the Prony Hydrothermal Field (PHF) is characterized by large carbonate chimneys (up to 30 m in height) venting clear fluids, which temperature however do not exceed 40°C (while it can reach up to 90°C in LCHF). The main difference between the two sites is the geological setting: oceanic (800 m depth) on ultrabasic massif off the MAR for LCHF and a costal environment (0 to 50 m depth) on perodotite nappe in a tectonic active area for PHF. Here, we report the first investigation on the microbial communities inhabiting the carbonate chimneys in PHF, focusing on the inner parts, most alkaline and warmest. For spatio-temporal analyses, samples were collected (2005, 2010 and 2011), from different sections of chimneys (top to bottom, outer to inner), located in seven sites (1 to 5 km distant) at varied depths (0 to 47 m) in the Prony bay. The structure and diversity of microbial communities and functional groups were compared by fingerprinting (SSCP/DGGE) and sequences analyses of clone libraries of both 16S rRNA and functional genes for methanogenesis (mcrA), methanotrophy (pmoA), and sulfate-reduction (dsrAB). We found that overall microbial patterns varied little inside a given chimney but changed in time and dramatically differs from one site to another, likely due to variation in hydrothermal activity and local geological settings. In each site and chimney sample, the specific richness of bacteria was an order higher than of archaea, in agreement with previous studies on LCHF. Archaea were dominated by few phylotypes of Methanosarcinales, related to the ANME group 2 (responsible for anaerobic methane oxydation) Lost City Methanosarcinales (LCMS). Bacteria were mostly affiliated to Firmicutes, α-, β-, and δ-Proteobacteria. Uncultivated lineages(candidate divisions OP1 and OP7), Chloroflexi and deep branching phylotypes were also identified. Interestingly, many bacterial phylotypes were related to alkaliphiles (Desulfonatronum, Alkaliphilus) or, surprisingly, to thermophiles (Thermotagales, Thermus) of volcanic hydrothermal systems. The main metabolic groups were methylotrophs, sulphate-reducers, sulphide oxidizers for bacteria and methanogens or methanotrophs (ANME-related) for archaea indicating that microbial cycling of sulphur and methane must be the dominant biogeochemical processes in PHF as previously observed in LCHF. The overall functions occurring in this original microbial ecosystem resemble those reported for LCHF, meanwhile the microbial actors fulfilling these roles corresponds to unique phylotypes, endemic to PHF. These data provide an important initial microbiological description of a novel example of submarine alkaline hydrothermal ecosystem for meaningful comparison to the well-studied LCHF

Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period

Active carbonate chimneys from the shallow marine serpentinizing Prony Hydrothermal Field were sampled 3 times over a 6 years period at site ST09. Archaeal and bacterial communities composition was investigated using PCR-based methods (clone libraries, Denaturating Gel Gradient Electrophoresis, quantitative PCR) targeting 16S rRNA genes, methyl coenzyme M reductase A and dissimilatory sulfite reductase subunit B genes. Methanosarcinales (Euryarchaeota) and Thaumarchaea were the main archaeal members. The Methanosarcinales, also observed by epifluorescent microscopy and FISH, consisted of two phylotypes that were previously solely detected in two other serpentinitzing ecosystems (The Cedars and Lost City Hydrothermal Field). Surprisingly, members of the hyperthermophilic order Thermococcales were also found which may indicate the presence of a hot subsurface biosphere. The bacterial community mainly consisted of Firmicutes, Chloroflexi, Alpha-, Gamma-, Beta-, and Delta-proteobacteria and of the candidate division NPL-UPA2. Members of these taxa were consistently found each year and may therefore represent a stable core of the indigenous bacterial community of the PHF chimneys. Firmicutes isolates representing new bacterial taxa were obtained by cultivation under anaerobic conditions. Our study revealed diverse microbial communities in PHF STO9 related to methane and sulfur compounds that share common populations with other terrestrial or submarine serpentinizing ecosystems