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Bousset, L.

Publications and source records attributed to Bousset, L..

16 recordsLinked to original sources

Text guidance is powerful but prompt-sensitive for weakly-supervised leaf symptom segmentation

Accurate segmentation of plant disease symptoms is essential for crop monitoring and phenotyping, yet it typically requires costly pixel-level annotations. Weakly supervised semantic segmentation (WSSS) alleviates this burden using image-level labels, but its performance depends on the quality of spatial priors such as class activation maps (CAMs). We investigate whether text-guided segmentation with the Segment Anything Model 3 (SAM3) can serve as an alternative weak supervision signal. Three pseudo-mask generation strategies are compared: (i) CAMs refined with SAM or SAM3, (ii) zero-shot text-guided SAM3, and (iii) a hybrid approach combining weak spatial cues with text prompts. The resulting pseudo-masks are used to train a DeepLabV3 model. Text guidance alone matches or outperforms conventional WSSS, achieving up to 0.46 IoU without spatial supervision and 0.61 IoU on a public dataset, although performance is sensitive to text prompt formulation. The hybrid strategy improves robustness, reaching 0.50 IoU on the primary dataset and 0.58 IoU on the additional dataset while reducing prompt sensitivity. Overall, text guidance is a promising alternative to conventional weak supervision, while hybrid approaches provide a more robust solution for plant disease segmentation.

plant biology↗

Perilipin 4 repetitive region forms amyloid fibrils promoted by a genetic expansion found in myopathy patients

Lipid-binding amphipathic proteins play key roles in cellular organization, yet their conformational flexibility makes them prone to aggregation. Here we study the aggregation of perilipin 4 (PLIN4), a protein of the lipid droplet (LD)-binding perilipin family. PLIN4 contains an unusually long ([~]1000 amino acid, aa) amphipathic helix composed of tandem 33-aa repeats, which folds on the LD surface. A genetic expansion that increases the number of repeats is associated with skeletal muscle dysfunction in patients suffering from a late-onset vacuolar myopathy (MRUPAV). We show that the genetic expansion and the precise sequence of the 33 aa repeats do not significantly alter PLIN4 interaction with LDs in cells. By contrast, purified fragments of the PLIN4 repetitive region form amyloid fibrils as characterized by cryo-EM and atomic force microscopy. Repeat expansion strongly accelerates fibril formation, consistent with enhanced self-assembly driven by the accumulation of identical repeats. PLIN4 aggregation is attenuated by the presence of LDs, indicating that lipid binding modulates the balance between membrane association and self-assembly. Together, these results establish PLIN4 as a previously unrecognized amyloid-forming protein and suggest that repeat expansion enhances amyloidogenic self-assembly, providing a foundation for future studies of its role in degenerative muscle disease.

biochemistry↗

1.94 Angstrom structure of synthetic alpha-synuclein fibrils seeding MSA neuropathology

Summary ParagraphMultiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease of unknown cause, typically affecting individuals aged 50-60 and leading to death within a decade1-3. It is characterized by glial cytoplasmic inclusions (GCIs) composed of fibrillar alpha-synuclein (aSyn)4-8, whose formation shows parallels with prion propagation9,10. While fibrils extracted from MSA brains have been structurally characterized11, their ability to replicate in a "protein-only" manner has been questioned12, and their capacity to induce GCIs in vivo remains unexplored. By contrast, the synthetic fibril strain 1B13,14, assembled from recombinant human aSyn, self-replicates in vitro and induces GCIs in mice15 - suggesting direct relevance to MSA - but awaited scrutiny at an atomic scale. Here, we report high-resolution structural analyses of 1B fibrils and of fibrils extracted from diseased mice injected with 1B that developed GCIs (1BP). We show in vivo that conformational templating enables fibril strain replication, resulting in MSA-like inclusion pathology. Remarkably, the structures of 1B and 1BP are highly similar and mimic the fold of aSyn observed in one protofilament of fibrils isolated from MSA patients11. Moreover, reinjection of crude mouse brain homogenates containing 1BP into new mice reproduces the same MSA-like pathology induced by the parent synthetic seed 1B. Our findings identify 1B as a synthetic pathogen capable of self-replication in vivo and reveal structural features of 1B/1BP that may underlie MSA pathology, offering insights for therapeutic strategies.

neuroscience↗

Identifying fungal leaf spots on oilseed rape: could pictures help?

From sowing in late summer until harvest in following summer, oilseed rape can be infected by several fungi, which foliar symptoms (leaf spots) coexist on the crop. Training an expert at their identification is quick for the typical symptoms with characteristic appearance. However, in many cases the size, colour and morphology are similar and for the atypical symptoms, there is a risk of confusion or in-decidability. Also, scouting the fields for expert training is not possible at all seasons and all diseases might not be seen in all years and all places. The aim of our study was to produce large sets of pictures annotated by several experts, from which tables illustrating the diversity of symptom appearance were chosen. These tables will enable assistance to diagnostic and expert training.

ecology↗

C9orf72-derived poly-GA DPRs undergo endocytic uptake in iNPC-derived astrocytes and spread to motor neurons

Dipeptide repeat proteins (DPRs) are aggregation-prone polypeptides encoded by the pathogenic G4C2 repeat expansion in the C9orf72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic &[beta]-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and - independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to- motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly- GA cellular uptake and cell-to-cell propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

neuroscience↗

Quantitative study of alpha-synuclein prion-like spreading in fully oriented reconstructed neural networks reveals non-synaptic dissemination of seeding aggregates

The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinsons disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.

neuroscience↗

Cryo-EM structure of alpha-synuclein fibrils amplified by PMCA from PD and MSA patient brains

Synucleinopathies are neurodegenerative diseases related to the aggregation of the protein alpha-synuclein (aSyn). Among these diseases, Parkinsons disease (PD) and multiple system atrophy (MSA) are most prevalent. aSyn can readily form different fibrillar polymorphs, if exposed to an air-water interface or by templating with pre-existing fibrils. We here report the structures of three fibrillar polymorphs that were obtained after seeding monomeric aSyn with PD and MSA patients brain homogenates using protein misfolding cyclic amplification (PMCA). Seeding with a control brain homogenate did not produce fibrils, and seeding with other in vitro generated fibrillar polymorphs as a control faithfully produced polymorphs of a different type. The here determined fibril structures from PD and MSA brain tissue represent new folds, which partly resemble that of previously reported in vitro generated fibrils from Y39 phosphorylated aSyn protein. The relevance of these fibrils for synucleinopathies in humans remains to be further investigated. Impact StatementNeurodegenerative diseases such as Parkinsons disease (PD) and Multiple System Atrophy (MSA) are suspected to be causatively related to the prion-like propagation of aggregates of the protein alpha-synuclein (aSyn). The fibril structures reported here were obtained after seeding from diseased human brain homogenate and differ from all previously published aSyn fibril arrangements. In case these fibrils would turn out to be the long sought causative agents of these diseases, their structures might lead to the development of therapeutic strategies to modify these diseases and to a better understanding of the mechanistic processes that lead to neurodegeneration and spreading of the diseases.

neuroscience↗

Besides stem canker severity, oilseed rape host genotype matters for the production of Leptosphaeria maculans fruiting bodies

For fungal cyclic epidemics on annual crops, the pathogen carry-over is an important step in designing disease control strategies. However, it remains particularly difficult to estimate and predict. Plant resistance affects the pathogen development within the epidemics but we lack data on the inter-annual transmission of inoculum. We addressed this question by considering Leptosphaeria maculans on 15 oilseed rape genotypes in field during 4 growing seasons. Stem canker severity of host genotypes was visually scored at harvest while the number of fruiting bodies produced on incubated stubble was quantified using an automated image analysis framework. Our results confirm that higher severity at harvest leads to higher fruiting body production and is significantly affected by host genotype and Nitrogen supply. Most interestingly, we show that the production of fruiting bodies is significantly and substantially affected by host genotype, independently of severity at harvest. Tracking individual stems through incubation, we confirm for the first time that the oilseed rape genotype has a direct effect, not only through disease severity. While the genericity of this finding should be investigated on other fungi, this major effect of genotype on inoculum carry-over should be taken into account in models of varietal deployment strategies.

microbiology↗

Influence of the culture media on the aspect and growth of oilseed rape fungal pathogens

While culturing fungal pathogens on artificial media, the phenotype, i.e. colony aspect, growth kinetics and in some case the pigment production can be used as discrimination criteria. Our aim was to enable the comparison between the growth kinetics of 8 fungal species, cultured simultaneously on 3 different media, and imaged under standardised conditions. Using a camera stand, taking pictures was quick and easy, producing homogeneous pictures, facilitating their comparison. The colony growth kinetics varied widely across the 8 species. The mycelium aspect and pigment production depended on the media and changed over time. Having standardised the imaging setup and grown species simultaneously allows proposing reference sets of pictures.

microbiology↗

A Leptosphaeria maculans set of isolates characterised on all available differentials and used as control to identify virulence frequencies in a current French population

The characterization of virulence frequencies has to be regularly updated to identify which genes are currently efficient and use this information to advise gene deployment by choosing varieties depending on the current composition of local pathogen population. In L. maculans on Brassica napus, because different genes were characterized by different teams, because new interactions are continuously identified and seed of differentials are difficult to obtain, we today still lack isolates characterized on all current resistance genes. On the one hand, we assembled a set of 12 isolates characterized on 13 of the 17 described resistance genes, having clearly compatible and clearly incompatible isolates for each interaction. This set can be used to characterize the L. maculans - B. napus interaction at cotyledon stage. Expanding the set of isolates with clearly virulent ones allowed us to detect inconsistent behaviour or intermediate (avirulent) phenotypes. On the other hand, we used this set of isolates as controls to identify virulence frequencies in a current French L. maculans population sampled in 2018 at Le Rheu. We provide the current status for 13 avirulence frequencies, including LepR1, LepR2 and LepR3 available in near isogenic lines of spring canola but not yet documented in France. Avirulence frequencies on the genes Rlm1, Rlm2, Rlm3, Rlm4, Rlm7, Rlm9 and LepR3 were low, indicating the lack of efficacy of these genes against the current population. In the opposite, all or most of isolates were avirulent for the genes Rlm5, Rlm6, Rlm10, Rlm11, LepR1 and LepR2. An optimistic point of view could conclude that there are ample resources for oilseed rape breeding. However, as compared to previous studies, so far all the resistance genes used on significant acreage without additional management practices have lost efficacy and only avirulences corresponding to resistance genes not deployed in France retain efficacy. While the call to wisely manage the available host resistance genes is not recent, it is still relevant. Adding, management practices to the deployment of resistance genes in order to reduce inoculum carry-over from one growing season to the next and to lower population sizes is key to maintain their efficacy over time.

pathology↗

Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species

The accumulation of amyloid Tau aggregates is implicated in Alzheimers disease and other Tauopathies. Molecular chaperones are known for their function in maintaining protein homeostasis by preventing the formation or promoting the disaggregation of amorphous and amyloid protein aggregates. Here we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro. This function is mediated by the core chaperone Hsc70, assisted by specific co-chaperones, in particular class B J-domain proteins and an Hsp110-type NEF. Recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell culture were processed by the Hsp70 disaggregation machinery, demonstrating the ability of this machinery to recognize a broad range of Tau aggregates. Chaperone treatment released monomeric, and small oligomeric Tau species, which induced the aggregation of self-propagating Tau species in a Tau cell culture model. We infer from these results that the activity of the Hsp70 disaggregation machinery is a double-sided sword as it attempts to eliminate Tau amyloids but with the price of generating new seeds. The Hsp70 disaggregase therefore has a crucial function in the Tau propagation cycle, rendering it a potential drug target in Tauopathies.

molecular biology↗

Two new polymorphic structures of alpha-synuclein solved by cryo-electron microscopy

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinsons disease (PD). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 [A] and 3.4 [A] resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability. Impact statementTwo new polymorphic structures of recombinant human alpha-synuclein fibrils show striking differences to previous structures, while familial PD mutation sites remain crucial for protofilament interaction and fibril stability.

neuroscience↗

LRRK2 modifies α-syn pathology and spread in mouse models and human neurons

Progressive aggregation of the protein alpha-synuclein (-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinsons disease (PD). Accruing evidence suggests that -syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of -syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors have been identified in studies of both familial and sporadic forms of PD. However, how these genes affect -syn aggregation and pathological spread, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of -syn aggregation, using an -syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated -syn aggregation in mouse primary neurons. Conversely, -syn aggregation increased in primary neurons from transgenic mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, we investigated -syn spreading in a LRRK2 G2019S transgenic mouse model and observed acceleration of -syn aggregation and degeneration of dopaminergic neurons in the SNpc. Furthermore, LRRK2 G2019S transgenic mice displayed more pronounced degeneration-associated neuroinflammation and exacerbated behavioral deficits. To validate our findings in a human context, we established a human -syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs). Recombinant human -syn PFFs triggered aggregation of endogenous -syn in a time-dependent manner in human iNs. In isogenic lines of human iNs, the G2019S mutation enhanced -syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and -syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, not just in the cases caused by LRRK2 mutations.

neuroscience↗

Automated image processing to support the analysis of between-year transmission of Leptosphaeria maculans in field conditions

Understanding the transmission of inoculum between periods where the host plants are present is central for predicting the development of plant diseases and optimising mitigation strategies. However, the production at the end of the growing period, the survival during the intercrop period, and the emergence or emission of inoculum after sowing or planting can be highly variable, difficult to assess and generally inferred indirectly from symptoms data. As a result, there is a lack of large data sets which is a major brake for the study of these epidemiological processes. Here we focus on Leptosphaeria maculans that causes the black leg of oilseed rape. After having infected leaves, at early stages of the plant, and migrating into the stem, it causes a basal stem canker before harvest. It then survives on stubble left in the field from which ascospores are emitted at the beginning of the next growing period. In this study we first developed an image processing framework to estimate the density of fruiting bodies produced on stubble. Then, we used this framework to analyse automatically a large number of stems collected in oilseed rape fields among a cultivated area. Having performed a quality assessment of the processing chain we used the output data to investigate how the potential level of inoculum may change with the source field, the considered year and the stem canker severity at harvest. Besides the insights gain into the blackleg of oilseed rape, this work shows how image-based phenotyping may support epidemiological studies by increasing substantially the precision of high throughput disease data.

epidemiology↗

Fungal growth is affected by and affects pH and redox potential (Eh) of the growth medium

Fungal plant pathogens live in the specific environment of plants. Understanding of the interaction between pathogens and their host plants might open new ways to control plant diseases. Yet, the specificities of the plant environment and its effects on fungal growth are not yet fully explored. Both pH and Eh play a key role during the interaction between the fungus and its host plants, but often studied independently or at different scales. To decipher the interrelation between plant growth and their soil environment, recent theoretical and methodological advances have been made through the joint characterization of the pH and Eh. This opens the prospect to develop similar methods for fungi. The aim of our study was to investigate whether the methods developed for soil could be transposed to fungi. We first worked on artificial media, assessing the impact of fungal growth on the media in cultures. The growth of all 16 species tested significantly altered either Eh, pH or both in agar media. Measuring Eh reveals that even the species not modifying pH can have an impact on the surrounding environment. Reciprocally, altered media were used to characterize sensitivity of fungal growth to both pH and Eh parameters. The response of the six fungi tested to the modified media was quantitative with a decrease in colony diameter. In addition, colony aspect was repeatedly and thoroughly modified. As a first step towards the same studies in conditions matching the natural environment of fungal pathogens, we tested how the measurement can be performed with fungi growing on oilseed rape plants. In infected plant stems, pH and Eh were significantly altered, in opposite directions for L. maculans and S. sclerotiorum. The observed alcalinisation or acidification correlates with canker length. Our series of experiments indicate that the procedure published for Eh and pH in soil can be extended for measurement in agar media and in infected plants. Further, the joint characterization of both parameters opens the way to a more precise understanding of the impact of fungi on their environment, and conversely, of the environment on fungal growth. The availability of methods for measurement opens the prospect to study combinations of stresses, either on agar media or in plants, and get an understanding of the involvement of pH and Eh modifications in these interactions.

microbiology↗

Careful deployment of oilseed rape crops with Rlm6 resistance gene against L. maculans is recommended to prevent the loss of efficacy of this resistance gene in French condiment mustard.

Breeding varieties for increased disease resistance is a major means to control epidemics. However, the deployment of resistance genes through space and time drives the genetic composition of the pathogen population, with predictable changes in pathotype frequencies. In France, Leptosphaeria maculans causes disease on Brassica napus oilseed rape crops but not on B. juncea condiment mustard. Prior to the deployment of winter B. napus varieties with Rlm6 resistance gene introduced from B. juncea, the aim of our study was to investigate if this deployment could impact disease control in condiment mustard. We assessed the presence of resistance genes against phoma stem canker in a set of current French B. juncea varieties and breeding lines. Rlm6 was detected in all the 12 condiment mustard varieties. Rlm5 was also detected in 8 varieties. No additional resistance genes were detected with the set of isolates used. Because frequency of isolates virulent on Rlm6 is very low, these results indicate that Rlm6 gene is a major component of disease control in the French B. juncea mustards tested. Using Rlm6 in oilseed rape varieties will very likely induce an increase in frequency of Rlm6 virulent isolates. This raises the acute concern of a wise deployment of oilseed rape around the condiment mustard growing area. Scientific knowledge on adaptation dynamics, spatial segregation of crops and cooperation between actors is currently available in order to mitigate the risk and advert negative consequences of the introduction of Rlm6 resistance gene in oilseed rape varieties.

epidemiology↗