With the exponential development of algal aquaculture and blue biotechnology, there is a strong demand for simple, inexpensive, high-throughput, quantitative phenotyping assays to measure the biomass, growth and fertility of algae and other marine protists. Here, we validate nephelometry, a method that relies on measuring the scattering of light by particles in suspension, as a non-invasive tool to measure in real-time the biomass of aquatic micro-organisms, such as microalgae, filamentous algae, as well as non-photosynthetic protists. Nephelometry is equally applicable to optic density and chlorophyll fluorescence measurements for the quantification of some microalgae, but outperforms other spectroscopy methods to quantify the biomass of biofilm-forming and filamentous algae, highly pigmented species and non-photosynthetic eukaryotes. Thanks to its insensitivity to the sample's pigmentation, nephelometry is also the method of choice when chlorophyll content varies between samples or time points, for example due to abiotic stress or pathogen infection. As examples, we illustrate how nephelometry can be combined with fluorometry or image analysis to monitor the quantity and time-course of spore release in fertile kelps or the progression of symptoms in diseased algal cultures.

Background: Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection.Results: Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues.Conclusions: Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

During their life cycle, fungi face adverse environmental conditions associated with alterations in water status. Phytopathogenic fungi are faced with this type of stress during the infection process, especially when they colonize seeds. Although these organisms are particularly effective to adapt to these water potential decreases, these coping mechanisms are so far very poorly described, particularly in filamentous fungi. Alternaria brassicicola is a seed-borne fungal pathogen responsible for the black spot disease on Brassicaceae plants. Alteration of Brassicaceae seed quality is one of the most damaging effects of the black spot. Beyond contribution to pathogen dissemination, the presence of the fungus on the seeds compromises seedling germination and survival. To better understand the determinism of fungus transmission to seeds, we previously established a reliable Arabidopsis-based pathosystem allowing investigations of A. brassicicola transmission to seeds. In particular, we showed that two mutants strain ∆abhog and ∆abnik exhibiting higher susceptibility to osmotic and water stress were highly impaired in seed transmission ability. Transcriptomic analyzes, carried out under different experimental in vitro conditions inducing these types of stress (addition of sorbitol or Poly Ethylene Glycol (PEG) or by incubation under low relative humidity (1% RH)), allowed us to identify additional mechanisms potentially involved in the fungal adaptive responses. In particular, these analyzes revealed a pool of over-expressed genes encoding putative proteins which share physiochemical features typical of hydrophilin-like proteins. We initiated studies of some of these hydrophilins by generating respective Knock-Out mutants. Functional studies has been carried out to determine whether these mutants were impaired in their adaptive response to water stress and other types of stress (such as oxidative stress) and whether hydrophilins are involved in pathogenicity. Additional transcriptomic assays conducted on ∆abhog and ∆abnik strain growth under sorbitol exposure revealed that numerous hydrophilins are regulated by these two genes.

Glucosinolates are brassicaceous secondary metabolites that have long been considered as chemical shields against pathogen invasion. Isothiocyanates (ITCs), are glucosinolate-breakdown products that have negative effects on the growth of various fungal species. We explored the mechanism by which ITCs could cause fungal cell death using Alternaria brassicicola, a specialist Brassica pathogens, as model organism. Exposure of the fungus to ICTs led to a decreased oxygen consumption rate, intracellular accumulation of reactive oxygen species (ROS) and mitochondrial-membrane depolarization. We also found that two major regulators of the response to oxidative stress, i.e., the MAP kinase AbHog1 and the transcription factor AbAP1, were activated in the presence of ICTs. Once activated by ICT-derived ROS, AbAP1 was found to promote the expression of different oxidative-response genes. This response might play a significant role in the protection of the fungus against ICTs as mutants deficient in AbHog1 or AbAP1 were found to be hypersensitive to these metabolites. Moreover, the loss of these genes was accompanied by a significant decrease in aggressiveness on Brassica. We suggest that the robust protection response against ICT-derived oxidative stress might be a key adaptation mechanism for successful infection of host plants by Brassicaceae-specialist necrotrophs like A. brassicicola.

The fungal genus Alternaria contains many destructive plant pathogens, including Alternaria brassicicola, which causes black spot disease on a wide range of Brassicaceae plants and which is routinely used as a model necrotrophic pathogen in studies with Arabidopsis thaliana. During host infection, many fungal proteins that are critical for disease progression are processed in the endoplasmic reticulum (ER)/Golgi system and secreted in planta. The unfolded protein response (UPR) is an essential part of ER protein quality control that ensures efficient maturation of secreted and membrane-bound proteins in eukaryotes. This review highlights the importance of the UPR signaling pathway with respect to the ability of A. brassicicola to efficiently accomplish key steps of its pathogenic life cycle. Understanding the pathogenicity mechanisms that fungi uses during infection is crucial for the development of new antifungal therapies. Therefore the UPR pathway has emerged as a promising drug target for plant disease control.

In this study, the physiological functions of fungal mannitol metabolism in the pathogenicity and protection against environmental stresses were investigated in the necrotrophic fungus Alternaria brassicicola. Mannitol metabolism was examined during infection of Brassica oleracea leaves by sequential HPLC quantification of the major soluble carbohydrates and expression analysis of genes encoding two proteins of mannitol metabolism, i.e., a mannitol dehydrogenase (AbMdh), and a mannitol-1-phosphate dehydrogenase (AbMpd). Knockout mutants deficient for AbMdh or AbMpd and a double mutant lacking both enzyme activities were constructed. Their capacity to cope with various oxidative and drought stresses and their pathogenic behavior were evaluated. Metabolic and gene expression profiling indicated an increase in mannitol production during plant infection. Depending on the mutants, distinct pathogenic processes, such as leaf and silique colonization, sporulation, survival on seeds, were impaired by comparison to the wild-type. This pathogenic alteration could be partly explained by the differential susceptibilities of mutants to oxidative and drought stresses. These results highlight the importance of mannitol metabolism with respect to the ability of A. brassicicola to efficiently accomplish key steps of its pathogenic life cycle.

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.

During infection, pathogens secrete small secreted proteins (SSPs), called effectors, that promote disease. Plant receptors encoded by resistance R genes might recognize such effectors (also called avirulence factors AVRs), resulting in plant immunity. Pathogens evade recognition thanks to the emergence of virulent alleles present in populations. It has been demonstrated that avirulent effectors are crucial for the pathogen infection cycle and that their loss-of-function may induce a substantial fitness cost. This kind of effector is expected to be under purifying selective pressure. Here, we aim at identifying the effector repertoire of Venturia inaequalis, the agent of apple scab, assessing its evolutionary dynamics and studying the role of candidate effectors in virulence. We sequenced de novo 90 strains, collected on apple and on their wild relatives and differing in their host range or virulence to study allelic polymorphism at 880 putative effector loci. The top-20 hits for highly conserved sequences were selected as candidates for further functional analyses. In planta gene expression showed a significant induction of these conserved SSP at the early stage of plant infection. Their functions were investigated using targeted deletion mutants. Remarkably, loss of two conserved SSPs resulted in reduced aggressiveness without any alteration in growth in vitro. GFP-tagged protein and heterologous expression were used to assess their sub-cellular localization in infected apple leaves. Involvement of theses SSP in the modulation of host defence was also investigated using an apple full-transcript microarray. Highly conserved effectors will be used to screen for novel R genes in Malus genotypes characterized for their high resistance to scab. This combined knowledge should enable us to understand strategies used by the pathogen to overcome defences in apple and consequently to build more durable resistance towards apple scab.

L'induction par un hote d'un stress oxydatif chez un parasite represente l'une des strategies de defense chimique les plus repandues. Chez les Brassicacees, famille de plantes comprenant des especes a fort interet agronomique comme le chou ou le colza, ou d'interet scientifique comme Arabidopsis thaliana, le stress oxydatif peut etre genere soit par des formes actives d'oxygene liberees au niveau de la zone d'agression lors du burst oxydatif soit par des molecules issues du metabolisme secondaire de la plante telles que les isothiocyanates (ITC). Bien que fortement expose a un tel stress lors de l'infection, Alternaria brassicicola, champignon pathogene necrotrophe infeode aux Brassicacees, est capable d'accomplir son cycle parasitaire et donc de s'y adapter. Cette these met en evidence que la tolerance du champignon aux stress oxydatif est partiellement dependante du metabolisme du mannitol qui participe a la resistance des conidies lors du burst oxydatif in planta ou a la protection contre les dommages intracellulaires des FAO generees par les ITC lors des phases precoces de l'interaction. Les travaux realises montrent egalement que la charge fongitoxique des derives des glucosinolates peut etre inhibee par leur conjugaison au glutathion via des Glutathion-Stransferases (GST) specifiques. L'alteration du metabolisme du mannitol ou l'inactivation de ces GST diminue significativement le pouvoir pathogene d'A. brassicicola et la regulation de ces voies metaboliques est donc a considerer dans l'optique de developper de nouvelles strategies de protection des cultures.

By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi-tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling.