Extracellular Vesicles of the Plant Pathogen Botrytis cinerea.

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.

Evidencing New Roles for the Glycosyl-Transferase Cps1 in the Phytopathogenic Fungus Botrytis cinerea.

The fungal cell wall occupies a central place in the interaction between fungi and their environment. This study focuses on the role of the putative polysaccharide synthase Cps1 in the physiology, development and virulence of the grey mold-causing agent Botrytis cinerea. Deletion of the Bccps1 gene does not affect the germination of the conidia (asexual spores) or the early mycelial development, but it perturbs hyphal expansion after 24 h, revealing a two-phase hyphal development that has not been reported so far. It causes a severe reduction of mycelial growth in a solid medium and modifies hyphal aggregation into pellets in liquid cultures. It strongly impairs plant penetration, plant colonization and the formation of sclerotia (survival structures). Loss of the BcCps1 protein associates with a decrease in glucans and glycoproteins in the fungus cell wall and the up-accumulation of 132 proteins in the mutant's exoproteome, among which are fungal cell wall enzymes. This is accompanied by an increased fragility of the mutant mycelium, an increased sensitivity to some environmental stresses and a reduced adhesion to plant surface. Taken together, the results support a significant role of Cps1 in the cell wall biology of B. cinerea.

Snf1 Kinase Differentially Regulates Botrytis cinerea Pathogenicity according to the Plant Host.

The Snf1 kinase of the glucose signaling pathway controls the response to nutritional and environmental stresses. In phytopathogenic fungi, Snf1 acts as a global activator of plant cell wall degrading enzymes that are major virulence factors for plant colonization. To characterize its role in the virulence of the necrotrophic fungus Botrytis cinerea, two independent deletion mutants of the Bcsnf1 gene were obtained and analyzed. Virulence of the Δsnf1 mutants was reduced by 59% on a host with acidic pH (apple fruit) and up to 89% on hosts with neutral pH (cucumber cotyledon and French bean leaf). In vitro, Δsnf1 mutants grew slower than the wild type strain at both pH 5 and 7, with a reduction of 20-80% in simple sugars, polysaccharides, and lipidic carbon sources, and these defects were amplified at pH 7. A two-fold reduction in secretion of xylanase activities was observed consequently to the Bcsnf1 gene deletion. Moreover, Δsnf1 mutants were altered in their ability to control ambient pH. Finally, Δsnf1 mutants were impaired in asexual sporulation and did not produce macroconidia. These results confirm the importance of BcSnf1 in pathogenicity, nutrition, and conidiation, and suggest a role in pH regulation for this global regulator in filamentous fungi.

Clathrin Is Important for Virulence Factors Delivery in the Necrotrophic Fungus Botrytis cinerea.

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.

The infection cushion of Botrytis cinerea: a fungal 'weapon' of plant-biomass destruction.

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.

A Similar Secretome Disturbance as a Hallmark of Non-pathogenic Botrytis cinerea ATMT-Mutants?

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.

A secreted metal-binding protein protects necrotrophic phytopathogens from reactive oxygen species.

Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.

The pH regulator PacC: a host-dependent virulence factor in Botrytis cinerea.

The phytopathogenic fungus Botrytis cinerea is able to infect a wide variety of plants and plant tissues with differing chemical compositions. During its interaction with the host, this pathogen modulates its ambient pH by secreting acids or ammonia. In this work, we examined the Pal/Pac pathway, the fungal ambient pH-responsive signalling circuit, and investigated the role of the PacC transcription factor. Characterization of the BcpacC deletion mutant revealed an alteration of both fungal growth and virulence depending on the pH of the culture medium or of the host tissue. The pathogenicity of the mutant was altered on plants exhibiting a neutral pH and not on plants with acidic tissues. The capacity of the mutant to acidify its environment and, more particularly, to produce oxalic acid was affected, as was production of reactive oxygen species. Finally, proteomic profiling of the mutant secretome revealed significant changes in plant cell wall polysaccharides proteins and lipid degradation and oxidoreduction, highlighting the importance of BcPacC in the necrotrophic lifestyle of B. cinerea.

Analysis of the Molecular Dialogue Between Gray Mold (Botrytis cinerea) and Grapevine (Vitis vinifera) Reveals a Clear Shift in Defense Mechanisms During Berry Ripening.

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.

The homeobox BcHOX8 gene in Botrytis cinerea regulates vegetative growth and morphology.

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

Proteomic analysis of proteins secreted by Botrytis cinerea in response to heavy metal toxicity.

Although essential in many cellular processes, metals become toxic when they are present in excess and constitute a global environmental hazard. To overcome this stress, fungi have evolved several mechanisms at both intracellular and extracellular levels. In particular, fungi are well known for their ability to secrete a large panel of proteins. However, their role in the adaptation of fungi to metal toxicity has not yet been investigated. To address this question, here, the fungus Botrytis cinerea was challenged to copper, zinc, nickel or cadmium stress and secreted proteins were collected and separated by 2D-PAGE. One hundred and sixteen spots whose volume varied under at least one tested condition were observed on 2D gels. Densitometric analyses revealed that the secretome signature in response to cadmium was significantly different from those obtained with the other metals. Fifty-five of these 116 spots were associated with unique proteins and functional classification revealed that the production of oxidoreductases and cell-wall degrading enzymes was modified in response to metals. Promoter analysis disclosed that PacC/Rim101 sites were statistically over-represented in the upstream sequences of the 31 genes corresponding to the varying unique spots suggesting a possible link between pH regulation and metal response in B. cinerea.

pH modulation differs during sunflower cotyledon colonization by the two closely related necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum.

During pathogenesis on sunflower cotyledons, Botrytis cinerea and Sclerotinia sclerotiorum show a striking resemblance in symptom development. Based on pH change profiles, the colonization process of both fungi can be divided into two stages. The first stage is associated with a pH decrease, resulting from an accumulation of citric and succinic acids. The second stage is correlated with a pH increase, resulting from an accumulation of ammonia. In this article, we also report that oxalic acid is produced at the late stage of the colonization process and that ammonia accumulation is concomitant with a decrease in free amino acids in decaying tissues. Sclerotinia sclerotiorum produces eight-fold more oxalic acid and two-fold less ammonia than B. cinerea. Consequently, during sunflower cotyledon colonization by B. cinerea, pH dynamics differ significantly from those of S. sclerotiorum. In vitro assays support the in planta results and show that decreases in pH are linked to glucose consumption. At different stages of the colonization process, expression profiles of genes encoding secreted proteases were investigated. This analysis highlights that the expression levels of the B. cinerea protease genes are higher than those of S. sclerotiorum. This work suggests that the overt similarities of S. sclerotiorum and B. cinerea symptom development have probably masked our recognition of the dynamic and potentially different metabolic pathways active during host colonization by these two necrotrophic fungi.

Novel insights into mannitol metabolism in the fungal plant pathogen Botrytis cinerea.

In order to redefine the mannitol pathway in the necrotrophic plant pathogen Botrytis cinerea, we used a targeted deletion strategy of genes encoding two proteins of mannitol metabolism, BcMTDH (B. cinerea mannitol dehydrogenase) and BcMPD (B. cinerea mannitol-1-phosphate dehydrogenase). Mobilization of mannitol and quantification of Bcmpd and Bcmtdh gene transcripts during development and osmotic stress confirmed a role for mannitol as a temporary and disposable carbon storage compound. In order to study metabolic fluxes, we followed conversion of labelled hexoses in wild-type and DeltaBcmpd and DeltaBcmtdh mutant strains by in vivo NMR spectroscopy. Our results revealed that glucose and fructose were metabolized via the BcMPD and BcMTDH pathways respectively. The existence of a novel mannitol phosphorylation pathway was also suggested by the NMR investigations. This last finding definitively challenged the existence of the originally postulated mannitol cycle in favour of two simultaneously expressed pathways. Finally, physiological and biochemical studies conducted on double deletion mutants (DeltaBcmpdDeltaBcmtdh) showed that mannitol was still produced despite a complete alteration of both mannitol biosynthesis pathways. This strongly suggests that one or several additional undescribed pathways could participate in mannitol metabolism in B. cinerea.

Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic fungus Botrytis cinerea: from plant hexoses to mannitol.

The main steps for carbon acquisition and conversion by Botrytis cinerea during pathogenesis of sunflower cotyledon were investigated here. A sequential view of soluble carbon metabolites detected by NMR spectroscopy during infection is presented. Disappearance of plant hexoses and their conversion to fungal metabolites were investigated by expression analysis of an extended gene family of hexose transporters (Bchxts) and of the mannitol pathway, using quantitative PCR. In order to analyse the main fungal metabolic routes used by B. cinerea in real time, we performed, for the first time, in vivo NMR analyses during plant infection. During infection, B. cinerea converts plant hexoses into mannitol. Expression analysis of the sugar porter gene family suggested predominance for transcription induced upon low glucose conditions and regulated according to the developmental phase. Allocation of plant hexoses by the pathogen revealed a conversion to mannitol, trehalose and glycogen for glucose and a preponderant transformation of fructose to mannitol by a more efficient metabolic pathway. Uptake of plant hexoses by B. cinerea is based on a multigenic flexible hexose uptake system. Their conversion into mannitol, enabled by two simultaneously expressed pathways, generates a dynamic intracellular carbon pool.

pH controls both transcription and post-translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis cinerea.

During pathogenesis, the ascomycete Botrytis cinerea secretes a range of cell-wall-degrading enzymes such as polygalacturonases, glucanases and proteases. We report the identification of a new member of the G1 family of proteases, BcACP1, which is secreted by B. cinerea during infection. The production of BcACP1 correlates with the acidification of the plant tissue, and transcriptional analysis of the Bcacp1 gene showed that it is only expressed under acidic growth conditions. Using a transcriptional reporter system, we showed that pH regulation of Bcacp1 is not mediated by the canonical PacC transcription factor binding site. Like other G1 proteases, BcACP1 is produced as a pro-enzyme. Trapping of the zymogen form allowed investigation of its maturation process. Evidence is presented for an autocatalytic proteolysis of the enzyme that is triggered by acidic pH. Environmental pH therefore controls Bcacp1 production at both the transcriptional and post-translational level.

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by Sclerotinia sclerotiorum.

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on (13)C- and (31)P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon-nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags.

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessor's sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5' or 3' flanking regions.

Ambient pH controls the expression of endopolygalacturonase genes in the necrotrophic fungus Sclerotinia sclerotiorum.

In the necrotrophic fungus Sclerotinia sclerotiorum, secretion of polygalacturonases (PGs) and decrease of the environmental pH via oxalic acid production are considered as the main pathogenicity determinants. In order to evaluate the relationship between these two aspects of the infection process, we analyzed the expression of the endoPG-encoding genes pg1-3. Transcription of pg1-3 was not carbon regulated but was strictly controlled by pH and highly favored in a narrow range of acidic pH. During plant infection, a pH gradient was established in relation to oxalic acid secretion. Transcripts of pg1-3 were localized to the zone of colonization of healthy tissues while transcripts of genes encoding other lytic enzymes were restricted to the more acidic zones of the infected tissues. Our results show that progressive acidification of the ambient medium by the fungus is a major strategy for the sequential expression of pathogenicity factors.

Characterization of PG2, an early endoPG produced by Sclerotinia sclerotiorum, expressed in yeast.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated. We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons. The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified. Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5. Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer. This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action. These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum.

When grown in the presence of sunflower cell walls, Sclerotinia sclerotiorum, an ubiquitous necrotrophic fungus, secretes several acid proteases including a non-aspartyl protease. The gene acp1, encoding an acid protease, has been cloned and sequenced. The intronless ORF encodes a preproprotein of 252 aa and a mature protein of 200 residues. In vitro expression of acp1 is subject to several transcriptional regulatory mechanisms. Expression induced by plant cell-wall proteins is controlled by both carbon and nitrogen catabolite repression. Glucose on its own represses acp1 expression while ammonium repression requires the simultaneous presence of a carbon source. Ambient pH higher than pH 5 overrides induction resulting in full repression of acp1. These transcriptional regulatory mechanisms and the presence of several motifs in the promoter of acp1 that may encode binding sites for the regulators CREA, AREA and PacC suggest the involvement of these regulators in the control of acp1 expression. acp1 is expressed in planta during sunflower cotyledon infection. Expression is low at the beginning of infection but increases suddenly at the stage of necrosis spreading. Comparison of in vitro and in planta acp1 expression suggests that glucose and nitrogen starvation together with acidification can be considered as key factors controlling Scl. sclerotiorum gene expression during pathogenesis.