Whole Genome Bisulfite Sequencing of Botrytis cinerea
ABSTRACT: Analysis of the genome-wide DNA methylation pattern of Botrytis cinerea. Results provide new and important information that DNA methylation is critical for pathogenicity and development of Botrytis cinerea by regulating gene expression. Overall design: Bisulphite converted DNA from the 4 samples were sequenced by using the HighSeq4000.
Project description:Histone methylation is widely present in animals, plants and fungi, and the methylation modification of histone H3 has important biological functions. Methylation of Lys9 of histone H3 (H3K9) has been proven to regulate chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. In this work, we investigated the functions of a H3K9 methyltransferase gene BcDIM5 in Botrytis cinerea, which contains a PreSET domain, a SET domain and a PostSET domain. Characterization of BcDIM5 knockout transformants showed that the hyphal growth rate and production of conidiophores and sclerotia were significantly reduced, while complementary transformation of BcDIM5 could restore the phenotypes to the levels of wild type. Pathogenicity assays revealed that BcDIM5 was essential for full virulence of B. cinerea. BcDIM5 knockout transformants exhibited decreased virulence, down-regulated expression of some pathogenic genes and drastically decreased H3K9 trimethylation level. However, knockout transformants of other two genes heterochromatin protein 1 (HP1) BcHP1 and DNA methyltransferase (DIM2) BcDIM2 did not exhibit significant change in the growth phenotype and virulence compared with the wild type. Our results indicate that H3K9 methyltransferase BcDIM5 is required for H3K9 trimethylation to regulate the development and virulence of B. cinerea.
Project description:Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.
Project description:Botrytis cinerea, a fungal pathogen that causes gray mold, displays a high degree of phenotypic diversity. Light emitting diodes (LEDs) with specific light spectrum are increasingly used as lighting resource for plant greenhouse production. The chosen light spectrum can also have an effect on the pathogens in this production system. In this study, we investigated the phenological diversity in 15 B. cinerea isolates upon different light treatments. Daylight, darkness, and LED lights with different wavelengths (white, blue, red, blue+red) were chosen as treatments. The 15 Botrytis isolates differed in their mycelial growth rate, conidia production, and sclerotia formation. Light quality had a limited effect on growth rate. All isolates sporulated under daylight treatment, red light resulted in lower sporulation, while white, blue, and blue+red light inhibited sclerotia formation in all isolates, and sporulation in most, but not all isolates. Pathogenicity of the Botrytis isolates was studied on 2-week-old strawberry (Fragaria × ananassa 'Elsanta') leaves grown under white, blue, and red LED lights. The isolates differed in virulence on strawberry leaves, and this was positively correlated to oxalic acid production by B. cinerea in vitro. Red LED light improved leaf basal resistance to all the tested Botrytis isolates. Blue light pretreatment resulted in decreased leaf resistance to some isolates. Furthermore, we used image analysis to quantify the virulence of the different Botrytis isolates based on changes in photosynthetic performance of the strawberry leaves: chlorophyll fluorescence (Fv/Fm), chlorophyll index (ChlIdx) and anthocyanin content (modified anthocyanin reflection index, mAriIdx). Fv/Fm showed a strong negative correlation with disease severity and can be an indicator for the early detection of gray mold on strawberry leaves.
Project description:The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. The genome of B. cinerea has been fully sequenced while the importance of horizontal gene transfer (HGT) to extend the host range in plant pathogenic fungi has been recently appreciated. However, recent data confirm that the B. cinerea fungus shares conserved virulence factors with other fungal plant pathogens with narrow host range. Therefore, interkingdom HGT may contribute to the evolution of phytopathogenicity in B. cinerea. In this study, a stringent genome comparison pipeline was used to identify potential genes that have been obtained by B. cinerea but not by other fungi through interkingdom HGT. This search led to the identification of four genes: a UDP-glucosyltransferase (UGT), a lipoprotein and two alpha/beta hydrolase fold proteins. Phylogenetic analysis of the four genes suggests that B. cinerea acquired UGT from plants and the other 3 genes from bacteria. Based on the known gene functions and literature searching, a correlation between gene acquision and the evolution of pathogenicity in B. cinerea can be postulated.
Project description:BcHpt is a core element of the high-osmolarity glycerol (HOG) transduction pathway in Botrytis cinerea. In contrast to other elements of the pathway, which have been characterized and proven to play important roles in vegetative differentiation, fungicide resistance, the multistress response, and virulence in B. cinerea, BcHpt (Histidine-containing phosphotransfer) is essential but uncharacterized in B. cinerea. Our previous study reported the first lysine acetylation site (Lys161) in BcHpt. In this study, the functions of this lysine acetylation site in BcHpt were characterized using site-directed mutagenesis. To mimic Lys161 acetylation, we generated the mutant strain ?BcHPt + BcHptK161Q-GFP, which exhibited a slower growth rate; lower pathogenicity; higher sensitivity to multiple stresses, including osmotic and oxidative stresses, dicarboximides, and demethylation inhibitors (DMIs); and lower BcSak1 phosphorylation levels than wild-type B. cinerea. Constitutive acetylation of BcHpt Ly161 apparently inhibits hyphal growth, the multistress response, and sensitivity to fungicides in B. cinerea. Moreover, the lysine acetylation site affected phosphorylation of the MAPK BcSak1.
Project description:A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea.
Project description:Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.
Project description:Lysine crotonylation (Kcr), a recently discovered post-translational modification, plays a key role in the regulation of diverse cellular processes. Botrytis cinerea is a destructive necrotrophic fungal pathogen distributed worldwide with broad ranging hosts. However, the functions of Kcr are unknown in B. cinerea or any other plant fungal pathogens. Here, we comprehensively evaluated the crotonylation proteome of B. cinerea and identified 3967 Kcr sites in 1041 proteins, which contained 9 types of modification motifs. Our results show that although the crotonylation was largely conserved, different organisms contained distinct crotonylated proteins with unique functions. Bioinformatics analysis demonstrated that the majority of crotonylated proteins were distributed in cytoplasm (35%), mitochondria (26%), and nucleus (22%). The identified proteins were found to be involved in various metabolic and cellular processes, such as cytoplasmic translation and structural constituent of ribosome. Particularly, 26 crotonylated proteins participated in the pathogenicity of B. cinerea, suggesting a significant role for Kcr in this process. Protein interaction network analysis demonstrated that many protein interactions are regulated by crotonylation. Furthermore, our results show that different nutritional conditions had a significant influence on the Kcr levels of B. cinerea. These data represent the first report of the crotonylome of B. cinerea and provide a good foundation for further explorations of the role of Kcr in plant fungal pathogens.
Project description:Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A -1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.
Project description:MADS-box transcription factors are highly conserved in eukaryotic species and involved in a variety of biological processes. Little is known, however, regarding the function of MADS-box genes in Botrytis cinerea, a fungal pathogen with a wide host range. Here, the functional role of the B. cinerea MADS-box gene, Bcmads1, was characterized in relation to the development, pathogenicity and production of sclerotia. The latter are formed upon incubation in darkness and serve as survival structures during winter and as the female parent in sexual reproduction. Bcmads1 is indispensable for sclerotia production. RT-qPCR analysis suggested that Bcmads1 modulated sclerotia formation by regulating the expression of light-responsive genes. Bcmads1 is required for the full virulence potential of B. cinerea on apple fruit. A comparative proteomic analysis identified 63 proteins, representing 55 individual genes that are potential targets of Bcmads1. Among them, Bcsec14 and Bcsec31 are associated with vesicle transport. Deletion of Bcsec14 and Bcsec31 resulted in a reduction in the virulence and protein secretion of B. cinerea. These results suggest that Bcmads1 may influence sclerotia formation by modulating light responsive gene expression and regulate pathogenicity by its effect on the protein secretion process.