In filamentous fungi, the secretory pathway plays a key role in polarized growth by delivering vesicles carrying lipids and enzymes required for cell wall synthesis and nutrition. In pathogenic species, it also supports the delivery of virulence factors. In metazoa and yeast, secretory vesicle formation occurs at the Golgi apparatus and endosomes through a complex and still partially characterized machinery involving clathrin and adaptor proteins such as AP-1. In filamentous fungi, the molecular mechanisms controlling these processes remain poorly understood. Using a mutant underexpressing the β-subunit of the heterotetrameric adaptor AP-1, we investigated the role of this adaptor in the phytopathogenic fungus Botrytis cinerea. Reduced expression of AP-1β resulted in pleiotropic developmental defects, altered cell wall integrity, and loss of polarized trafficking of a class III chitin synthase. Moreover, the AP-1β mutant is affected in the secretion of specific classes of hydrolytic enzymes and displays a severe defect in pathogenicity. Together, these results highlight the importance of AP-1-dependent trafficking pathways in the biology and virulence of B. cinerea.

In filamentous fungi, the secretory pathway plays a key role in polarized growth through the delivery of vesicles carrying enzymes and components necessary to cell wall synthesis and fungal nutrition. In pathogenic species, it moreover supports the delivery of virulence factors. In metazoa and yeast, the formation of several secretory vesicles occurs at the Golgi apparatus and endosomes through a complex and partially characterized machinery involving clathrin and adaptor proteins such as AP-1. Data on the molecular mechanisms controlling this process are still scarce in filamentous fungi. Using a mutant under-expressing the beta-subunit of the heterotetrameric AP-1 clathrin adaptor complex, we investigated the importance of intracellular vesicle trafficking in the phytopathogenic fungus Botrytis cinerea. The mutant exhibits pleiotropic developmental defects, an abnormality in cell wall integrity associated with a mislocalization of the plasma-membrane chitine synthase BcCHSIIIa. Moreover, the AP-1 mutant is affected in the secretion of hydrolytic enzymes and displays a severe defect in pathogenicity. This study confirms the importance of the AP1/clathrin machinery in the secretory pathway of B. cinerea. ### Competing Interest Statement The authors have declared no competing interest.

LysM effectors are suppressors of chitin-triggered plant immunity in biotrophic and hemibiotrophic fungi. In necrotrophic fungi, LysM effectors might induce a mechanism to suppress host immunity during the short asymptomatic phase they establish before these fungi activate plant defenses and induce host cell death leading to necrosis. Here, we characterize a secreted LysM protein from a major necrotrophic fungus, Botrytis cinerea, called BcLysM1. Transcriptional induction of BcLysM1 gene was observed in multicellular appressoria, called infection cushions, in unicellular appressoria and in the early phase of infection on bean leaves. We confirmed that BcLysM1 protein binds chitin in the fungus cell wall and protects hyphae against degradation by external chitinases. This effector is also able to suppress the chitin-induced ROS burst in Arabidopsis thaliana, suggesting sequestration of chitooligosaccharides in apoplast during infection. Moreover, contribution of BcLysM1 in infection initiation and in adhesion to bean leaf surfaces were demonstrated. Our data show for the first time that a LysM effector can play a dual role in mycelial adhesion and suppression of chitin-triggered host immunity, both of which occur during the early asymptomatic phase of infection by necrotrophic fungi.

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

### Competing Interest StatementThe authors have declared no competing interest.

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall-degrading enzymes and plant cell death-inducing proteins. Parallel upregulation of sugar transport and sugar catabolism-encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.

Fungi are the most prevalent plant pathogens, causing annually important damages. To infect and colonize their hosts, they secrete effectors including hydrolytic enzymes able to kill and macerate plant tissues. These secreted proteins are transported from the Endoplasmic Reticulum and the Golgi apparatus to the extracellular space through intracellular vesicles. In pathogenic fungi, intracellular vesicles were described but their biogenesis and their role in virulence remain unclear. In this study, we report the essential role of clathrin heavy chain (CHC) in the pathogenicity of Botrytis cinerea, the agent of gray mold disease. To investigate the importance of this protein involved in coat vesicles formation in eukaryotic cells, a T-DNA insertional mutant reduced in the expression of the CHC-encoding gene, and a mutant expressing a dominant-negative form of CHC were studied. Both mutants were strongly affected in pathogenicity. Characterization of the mutants revealed altered infection cushions and an important defect in protein secretion. This study demonstrates the essential role of clathrin in the infectious process of a plant pathogenic fungus and more particularly its role in virulence factors delivery.

AbstractPlant nitrogen (N) fertilization is known to affect disease; however, the underlying mechanisms remain mostly unknown. We investigated the impact of N supply on the Arabidopsis thaliana–Botrytis cinerea interaction. A. thaliana plants grown in low nitrate were more tolerant to all wild‐type B. cinerea strains tested. We determined leaf nitrate concentrations and showed that they had a limited impact on B. cinerea growth in vitro. For the first time, we performed a dual RNA‐Seq of infected leaves of plants grown with different nitrate concentrations. Transcriptome analysis showed that plant and fungal transcriptomes were marginally affected by plant nitrate supply. Indeed, only a limited set of plant (182) and fungal (22) genes displayed expression profiles altered by nitrate supply. The expression of selected genes was confirmed by quantitative reverse transcription PCR at 6 hr postinfection (hpi) and analysed at a later time point (24 hpi). We selected three of the 22 B. cinerea genes identified for further analysis. B. cinerea mutants affected in these genes were less aggressive than the wild‐type strain. We also showed that plants grown in ammonium were more tolerant to B. cinerea. Furthermore, expression of the selected B. cinerea genes in planta was altered when plants were grown with ammonium instead of nitrate, demonstrating an impact of the nature of N supplied to plants on the interaction. Identification of B. cinerea genes expressed differentially in planta according to plant N supply unveils two novel virulence functions required for full virulence in A. thaliana: a secondary metabolite (SM) and an acidic protease (AP).

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted proteins in the infection process. In the twelve non-pathogenic mutants, the differentiation of infection cushions was also impaired, suggesting a link between the penetration structures and the secretion of proteins involved in the virulence of the pathogen.