Gray Mold Fungus Botrytis Cinerea Research Articles

Plant triterpenoid saponins function as susceptibility factors to promote the pathogenicity of Botrytis cinerea

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen that causes diseases in hundreds of plant species, including high-value crops. Its polyxenous nature and pathogenic success are due to its ability to perceive host signals in its favor. In this study, we found that laticifer cells of Euphorbia lathyris are a source of susceptibility factors required by B. cinerea to cause disease. Consequently, poor-in-latex (pil) mutants, which lack laticifer cells, show full resistance to this pathogen, whereas lot-of-latex mutants, which produce more laticifer cells, are hypersusceptible. These S factors are triterpenoid saponins, which are widely distributed natural products of vast structural diversity. The downregulation of laticifer-specific oxydosqualene cyclase genes, which encode the first committed step enzymes for triterpene and, therefore, saponin biosynthesis, conferred disease resistance to B. cinerea. Likewise, the Medicago truncatula lha-1 mutant, compromised in triterpenoid saponin biosynthesis, showed enhanced resistance. Interestingly, the application of different purified triterpenoid saponins pharmacologically complemented the disease-resistant phenotype of pil and hla-1 mutants and enhanced disease susceptibility in different plant species. We found that triterpenoid saponins function as plant cues that signal transcriptional reprogramming in B. cinerea, leading to a change in its growth habit and infection strategy, culminating in the abundant formation of infection cushions, the multicellular appressoria apparatus dedicated to plant penetration and biomass destruction in B. cinerea. Taken together, these results provide an explanation for how plant triterpenoid saponins function as disease susceptibility factors to promote B. cinerea pathogenicity.

Genome Comparisons between Botrytis fabae and the Closely Related Gray Mold Fungus Botrytis cinerea Reveal Possible Explanations for Their Contrasting Host Ranges.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

Role of BcSfb3, the subunit of COPII vesicles, in fungal development and pathogenicity, ER-phagy and autophagy in the gray mold fungus Botrytis cinerea

Cytoplasmic coat protein complex II (COPII) plays a multifunctional role in the transport of newly synthesized proteins, autophagosome formation, and endoplasmic reticulum (ER)—ER-phagy. However, the molecular mechanisms of the COPII subunit in ER-phagy in plant pathogens remain unknown. Here, we identified the subunit of COPII vesicles (BcSfb3) and explored the importance of BcSfb3 in Botrytis cinerea. BcSfb3 deletion affected vegetative growth, conidiation, conidial morphology, and plasma membrane integrity. We confirmed that the increase in infectious hyphal growth was delayed in the ΔBcSfb3 mutant, reducing its pathogenicity in the host plant. Furthermore, the ΔBcSfb3 mutant was sensitive to ER stress, which caused massive ER expansion and induced the formation of ER whorls that were taken up into the vacuole. Further examination demonstrated that BcSfb3 deletion caused ER stress initiated by unfolded protein response, and which led to the promotion of ER-phagy and autophagy that participate in sclerotia formation. In conclusion, these results demonstrate that BcSfb3 plays an important role in fungal development, pathogenesis, ER-phagy and autophagy in B. cinerea.

The Gβ-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gβ-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gβ-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gβ-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Agroecological Management of the Grey Mould Fungus Botrytis cinerea by Plant Growth-Promoting Bacteria.

Botrytis cinerea is the causal agent of grey mould and one of the most important plant pathogens in the world because of the damage it causes to fruits and vegetables. Although the application of botrycides is one of the most common plant protection strategies used in the world, the application of plant-beneficial bacteria might replace botrycides facilitating agroecological production practices. Based on this, we reviewed the different stages of B. cinerea infection in plants and the biocontrol mechanisms exerted by plant-beneficial bacteria, including the well-known plant growth-promoting bacteria (PGPB). Some PGPB mechanisms to control grey mould disease include antibiosis, space occupation, nutrient uptake, ethylene modulation, and the induction of plant defence mechanisms. In addition, recent studies on the action of anti-Botrytis compounds produced by PGPB and how they damage the conidial and mycelial structures of the pathogen are reviewed. Likewise, the advantages of individual inoculations of PGPB versus those that require the joint action of antagonist agents (microbial consortia) are discussed. Finally, it should be emphasised that PGPB are an excellent option to prevent grey mould in different crops and their use should be expanded for environmentally friendly agricultural practices.

Biological characterization of the melanin biosynthesis gene Bcscd1 in the plant pathogenic fungus Botrytis cinerea

The gray mold fungus Botrytis cinerea produces dark-colored conidia and sclerotia due to deposition of melanin on the cell wall of these structures. However, the role of melanin biosynthesis on development and function of conidia and sclerotia have not been well elucidated in this fungus. This study disrupted the melanin biosynthesis gene Bcscd1 (for scytalone dehydratase) in the wild type B05.10, and the resulting mutants were compared with B05.10 and complementary mutants (COM) for growth and development, virulence and response to biotic/abiotic stresses. Three disruption mutants were obtained, and they did not differ from B05.10 and COM in mycelial growth rate on potato dextrose agar, however, they formed brownish conidia and scleotia deficient in melanogenesis, whereas B05.10 and COM formed grayish conidia and black sclerotia with normal melanogenesis. The disruption mutants were as aggressive as B05.10 and COM in infection of tobacco leaves. TEM observation showed that the disruption mutant ΔScd1-85 formed numerous tiny grooves in the conidial cell wall, thereby causing uneven thickness in the cell wall. In contrast, B05.10 and COM rarely formed tiny grooves in their conidial cell wall with even thickness. Moreover, the sclerotial cortex cell wall of ΔScd1-85 lost rigidity and the cells became collapsed, whereas the sclerotial cortex cell wall of B05.10 and COM appeared rigid, and the cells appeared plump in shape. The disruption mutants were more sensitive than B05.10 and COM in response to chemical stresses (H2O2, NaCl, SDS, sorbitol) for conidial germination and sclerotial survival. The sclerotia of the disruption mutants were more susceptible than the sclerotia of B05.10 and COM to infection by the mycoparasite Trichoderma koningiopsis. These results confirmed previous studies about the effect of melanin production on pathogenicity of B. cinerea, and expanded our knowledge about the role of Bcscd1 in cell wall integrity and in response to biotic and abiotic stresses.

Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

‘Isabel’ grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 μg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

The autophagy protein BcAtg2 regulates growth, development and pathogenicity in the gray mold fungus Botrytis cinerea

Autophagy is an intracellular degradation process that facilitates material recycling to maintain cellular homeostasis in eukaryotes. Atg2 is a phospholipid transfer protein involved in cellular autophagy in Saccharomyces cerevisiae. To date, the role of Atg2 in growth, development and pathogenicity of the gray mold fungus Botrytis cinerea remains unknown. In this study, we identified and characterized an Atg2 ortholog, designated as BcAtg2, in B. cinerea. Deletion of BcATG2 resulted in a block of the autophagic process in B. cinerea. The ΔBcAtg2 mutant failed to produce sclerotia, and showed significant reduction in mycelial growth rate, formation of aerial mycelium and conidiation. In addition, the ΔBcAtg2 mutant lost the ability to form infection structures and cause symptom on host plants. All of these phenotypic changes in ΔBcAtg2 mutant were restored by targeted gene complementation. Moreover, BcAtg2 was demonstrated to physically interact with the phosphoinositide binding protein BcAtg18. Taken together, these results indicate that BcAtg2 plays an important role in vegetative growth, development and pathogenicity in B. cinerea.

Involvement of the Autophagy Protein Atg6 in Development and Virulence in the Gray Mold Fungus Botrytis cinerea.

Gray mold caused by Botrytis cinerea is a devastating disease that leads to huge economic losses worldwide. Autophagy is an evolutionarily conserved process that maintains intracellular homeostasis through self-eating. In this study, we identified and characterized the biological function of the autophagy-related protein Atg6 in B. cinerea. Targeted deletion of the BcATG6 gene showed block of autophagy and several phenotypic defects in aspects of mycelial growth, conidiation, sclerotial formation and virulence. All of the phenotypic defects were restored by targeted gene complementation. Taken together, these results suggest that BcAtg6 plays important roles in the regulation of various cellular processes in B. cinerea.

Promotion of tomato growth by the volatiles produced by the hypovirulent strain QT5-19 of the plant gray mold fungus Botrytis cinerea

Our previous study identified a hypovirulent strain QT5-19 of Botrytis cinerea, the causal agent of the plant gray mold disease, and found that QT5-19 can produce volatile organic compounds (VOCs) with high antifungal activity and high control efficacy against B. cinerea. However, impact of the QT5-19 VOCs on plant growth remains unknown. This study was conducted to investigate the impact of the QT5-19 VOCs on tomato growth, and to elucidate the mechanisms for the plant growth-promoting (PGP) activity of the QT5-19 VOCs. Results showed that compared to the control treatment, the QT5-19 VOCs significantly (P < 0.05) promoted tomato growth, and the PGP activity of the QT5-19 VOCs acted in dose- and time-dependent manners. Results also showed that the values of photosynthetic assimilation, stomatal conductance and transpiration, water use efficiency and chlorophyll content in the treatments of the QT5-19 VOCs were significantly (P < 0.05) higher than the corresponding values in the control treatment. The QT5-19 VOCs up-regulated expression of the genes for expansins (EXP2, EXP9 and EXP18), IAA (SlIAA1, SlIAA3 and SlIAA9), cytokinins (SlCKX1) and gibberellins in leaves and/or roots, whereas down-regulated expression of the gene ACO1 for ethylene in both organs. Moreover, enhanced accumulation of auxins and decreased accumulation of ethylene were observed in tomato roots in the treatment of the QT5-19 VOCs, compared to the control treatment. These results suggest that the QT5-19 VOCs probably promote tomato growth through improving photosynthesis and biosynthesis of expansins and IAA, and reducing ethylene biosynthesis. This study suggests that QT5-19 is a versatile biocontrol control agent.

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea.

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

Inhibition of Botrytis cinerea and control of gray mold on table grapes by calcium propionate

Abstract Objectives The gray mold fungus Botrytis cinerea (B. cinerea) infects a wide range of crops before and after harvest, causing huge losses worldwide. Inhibition mechanisms of B. cinerea in vitro and in plants by calcium propionate (CP), generally recognized as a safe substance, are described in this study. Materials and methods Wild-type and transgenic mutant strains of B. cinerea were used in the study to evaluate the effects of CP on fungal growth and development in vitro. Plant materials including tomato leaves and table grapes were tested for controlling efficiency of CP against gray mold deterioration in vivo. Results Mycelial growth of B. cinerea was inhibited by CP in a dose-dependent manner with occasional disruption of hyphal tips, causing cellular collapse and efflux of cell contents. Staining with fluorescein diacetate and propidium iodide indicated that CP decreased fungal cell viability. Inhibition efficiency of CP against B. cinerea was enhanced by reducing pH. In contrast, the vel1 mutant, which exhibited deficiency in acid production, was more resistant to CP, suggesting that inhibition of B. cinerea by CP is enhanced by the acidification ability of the fungus itself. Additionally, CP inhibited infection cushion development by germlings of B. cinerea. Infection assays with tomato leaves and table grapes showed that CP inhibited decay development in both host tissues. Moreover, application of CP on grapes 3 days prior to harvest could contribute to management of deterioration caused by spontaneous fungal diseases during storage. Conclusion CP can suppress hyphal growth, inhibit infection cushion development, and reduce the virulence of B. cinerea. CP is thus promising for practical management of gray mold in fruit crops and merits further evaluation.

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.

Botcinic acid biosynthesis in Botrytis cinerea relies on a subtelomeric gene cluster surrounded by relics of transposons and is regulated by the Zn2Cys6 transcription factor BcBoa13.

Botcinic acid is a phytotoxic polyketide involved in the virulence of the gray mold fungus Botrytis cinerea. Here, we aimed to investigate the specific regulation of the cluster of Bcboa genes that is responsible for its biosynthesis. Our analysis showed that this cluster is located in a subtelomeric genomic region containing alternating G + C/A + T-balanced regions, and A + T-rich regions made from transposable elements that underwent RIP (Repeat-Induced Pointmutation). Genetic analyses demonstrated that BcBoa13, a putative Zn2Cys6 transcription factor, is a nuclear protein with a major positive regulatory role on the expression of other Bcboa1-to-Bcboa12 genes, and botcinic acid production. In conclusion, the structure and the regulation of the botcinic acid gene cluster show similar features with the cluster responsible for the biosynthesis of the other known phytotoxin produced by B. cinerea, i.e., the sesquiterpene botrydial. Both clusters contain a gene encoding a pathway-specific Zn2Cys6 positive regulator, and both are surrounded by relics of transposons which raise some questions about the role of these repeated elements in the evolution and regulation of the secondary metabolism gene clusters in Botrytis.

The Sensor Proteins BcSho1 and BcSln1 Are Involved in, Though Not Essential to, Vegetative Differentiation, Pathogenicity and Osmotic Stress Tolerance in Botrytis cinerea.

High-osmolarity glycerol (HOG) signaling pathway belongs to mitogen-activated protein kinase (MAPK) cascades that regulate responses of organism to diverse extracellular stimuli. The membrane spanning proteins Sho1 and Sln1 serve as biosensors of HOG pathway in Saccharomyces cerevisiae. In this study, we investigated the biological functions of BcSHO1 and BcSLN1 in the gray mold fungus Botrytis cinerea. Target gene deletion demonstrated that both BcSHO1 and BcSLN1 are important for mycelial growth, conidiation and sclerotial formation. The BcSHO1 and BcSLN1 double deletion mutant ΔBcSln1-Sho1 produced much more, but smaller sclerotia than ΔBcSho1 and the wild-type (WT) strain, while ΔBcSln1 failed to develop sclerotia on all tested media, instead, formed a large number of conidia. Infection tests revealed that the virulence of ΔBcSln1-Sho1 decreased significantly, however, ΔBcSho1 or ΔBcSln1 showed no difference with the WT strain. In addition, ΔBcSln1-Sho1 exhibited resistance to osmotic stress by negatively regulating the phosphorylation of BcSak1 (yeast Hog1). All the phenotypic defects of mutants were recovered by target gene complementation. These results suggest that BcSHO1 and BcSLN1 share some functional redundancy in the regulation of fungal development, pathogenesis and osmotic stress response in B. cinerea.

Involvement of the cysteine protease BcAtg4 in development and virulence of Botrytis cinerea.

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.

The Two Cryptochrome/Photolyase Family Proteins Fulfill Distinct Roles in DNA Photorepair and Regulation of Conidiation in the Gray Mold Fungus Botrytis cinerea.

The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system.IMPORTANCEBotrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation).

A new transformant selection system for the gray mold fungus Botrytis cinerea based on the expression of fenhexamid-insensitive ERG27 variants

The gray mold fungus Botrytis cinerea features a wide host range and causes severe economic losses, making it an important object for molecular research. Thus far, genetic modification of the fungus mainly is relied on two selection systems (nourseothricin and hygromycin), while other selection systems hold significant disadvantages. To broaden the spectrum of available molecular tools, a new selection system based on the cheap and widely used fungicide fenhexamid (hydroxyanilide group) was established. Fenhexamid specifically targets the 3-ketoreductase ERG27 from the ergosterol biosynthesis pathway. We generated a set of expression vectors suitable for deletion or expression of genes of interest (GOIs) in B. cinerea based on fenhexamid-insensitive ERG27 variants. Expression of BcERG27F412I and Fusarium fujikuroi ERG27 in the sensitive B. cinerea strain B05.10 causes resistance towards fenhexamid (fenR) and allows for the selection of transformants and their genetic purification. A modified split-marker approach facilitates the site-specific integration and expression of GOIs at the bcerg27 locus. No undesired secondary phenotypes regarding virulence, stress responses, the formation of reproductive structures or conidial germination were observed in strains expressing fenhexamid-insensitive ERG27 variants. Thus, the fenR system represents a third reliable selection system for genetic modifications of fenhexamid-sensitive B. cinerea strains.

The septin protein Sep4 facilitates host infection by plant fungal pathogens via mediating initiation of infection structure formation.

Many phytopathogenic fungi use infection structures (IFSs, i.e., appressoria and infection cushions) to penetrate host cuticles. However, the conserved mechanisms that mediate initiation of IFS formation in divergent pathogens upon sensing the presence of host plants remain obscure. Here, we demonstrate that a conserved septin gene SEP4 plays crucial roles in this process. Disruption of SEP4 in the plant grey mould fungus Botrytis cinerea completely blocked IFS formation and abolished the virulence of ΔBcsep4 mutants on unwounded hosts. During IFS formation, mutants lacking SEP4 could produce reactive oxygen species (ROS) normally. Inhibition of ROS production in strains harbouring the SEP4 gene resulted in disordered assembly of Sep4 and the subsequent failure to form infection cushions, suggesting that proper Sep4 assembly regulated by ROS is required for initiation of IFS formation and infection. Moreover, loss of SEP4 severely impaired mutant conidiation, melanin and chitin accumulation in hyphal tips and lesion expansion on wounded hosts, but significantly promoted germ tube elongation and sclerotium production. SEP4-mediated fungal pathogenic development, including IFS formation, was validated in the hemibiotroph Magnaporthe oryzae. Our findings indicate that Sep4 plays pleiotropic roles in B. cinerea development and specifically facilities host infection by mediating initiation of IFS formation in divergent plant fungal pathogens in response to ROS signaling.

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.