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Functional analysis of mating type genes and transcriptome analysis during fruiting body development of botrytis cinerea
Rodenburg, Sander Y.A. ; Terhem, Razak B. ; Veloso, Javier ; Stassen, Joost H.M. ; Kan, Jan A.L. van - \ 2018
mBio 9 (2018)1. - ISSN 2161-2129
Ascospore - Epigenetic regulation - Plant disease - Sexual reproduction - Transcriptome
Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (com-prising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction. IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies (“apothecia”) of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea. The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.
A gapless genome sequence of the fungus Botrytis cinerea
Kan, Jan A.L. Van; Stassen, Joost H.M. ; Mosbach, Andreas ; Lee, Theo A.J. Van Der; Faino, Luigi ; Farmer, Andrew D. ; Papasotiriou, Dimitrios G. ; Zhou, Shiguo ; Seidl, Michael F. ; Cottam, Eleanor ; Edel, Dominique ; Hahn, Matthias ; Schwartz, David C. ; Dietrich, Robert A. ; Widdison, Stephanie ; Scalliet, Gabriel - \ 2017
Molecular Plant Pathology 18 (2017)1. - ISSN 1464-6722 - p. 75 - 89.
Genetic map - Grey mould - Optical map - SMRT sequencing
Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.
The Zn2Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea
Zhang, Lisha ; Lubbers, Ronnie ; Simon, Adeline ; Stassen, J.H.M. ; Vargas Ribera, Pablo ; Viaud, Muriel ; Kan, J.A.L. van - \ 2016
BioSpektrum (2016)Tagungsband. - ISSN 0947-0867 - p. 116 - 116.
A novel Zn2Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea
Zhang, Lisha ; Lubbers, Ronnie J.M. ; Simon, Adeline ; Stassen, Joost H.M. ; Vargas Ribera, Pablo R. ; Viaud, Muriel ; Kan, Jan A.L. van - \ 2016
Molecular Microbiology 100 (2016)2. - ISSN 0950-382X - p. 247 - 262.
Summary: D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa. Short Abstract: The transcriptional regulator that controls D-galacturonic acid (GalA) utilization in fungi was unknown. We identified in Botrytis cinerea a novel Zn2Cys6 transcription factor (TF), designated BcGaaR, which is required for the induction of GalA-utilization genes. The BcGaaR protein was predominantly localized in nuclei in cultures grown on GalA, but remained in the cytoplasm in cultures grown on glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.
|Transcriptome and functional analysis of apothecium development in Botrytis cinerea
Kan, J.A.L. van; Terhem, R.B. ; Rodenburg, A. ; Stassen, J.H.M. - \ 2015
In: Book of Abstracts 28th Fungal Genetics Conference. - - p. 261 - 261.
Botrytis cinerea is a plant pathogenic ascomycete producing sexual fruiting bodies named apothecia. We performed RNA-seq analysis of sclerotia, three stages of apothecium development and ascospores. Transcriptomes were compared between successive developmental stages in order to describe transcriptional changes occurring during developmental transitions. Strikingly, more than 5000 genes were differentially expressed between mature apothecia and ascospores. Ascospores expressed several genes encoding virulence factors, even prior to interaction with host plants. Secondly, cluster analysis identified six sets of genes with common transcriptional profiles over the developmental stages. Interestingly, ~80% of the genes that were specifically expressed in mature apothecia were of unknown function and exclusively had homologs within the order Helotiales, but no homologs in fungi producing other types of ascocarps. This suggest that the apothecium is a structure that evolved independently from other fruiting bodies (cleistothecia, perithecia or pseudothecia).
Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative D-galacturonic acid transporters
Zhang, L. ; Hua, C. ; Stassen, J.H.M. ; Chatterjee, S. ; Cornelissen, M. ; Kan, J.A.L. van - \ 2014
Fungal Genetics and Biology 72 (2014). - ISSN 1087-1845 - p. 182 - 191.
wall degrading enzymes - saccharomyces-cerevisiae - filamentous fungi - endopolygalacturonase genes - colletotrichum fungi - hexose transporters - confocal microscopy - rna-seq - acid - virulence
The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5~5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.
Specific In Planta Recognition of Two GKLR Proteins of the Downy Mildew Bremia lactucae Revealed in a Large Effector Screen in Lettuce
Stassen, J.H.M. ; Boer, E. den; Vergeer, P.W.J. ; Andel, A. ; Ellendorff, U. ; Pelgrom, K.T.B. ; Pel, M. ; Schut, J. ; Zonneveld, O. ; Jeuken, M.J.W. ; Ackerveken, G. van den - \ 2013
Molecular Plant-Microbe Interactions 26 (2013)11. - ISSN 0894-0282 - p. 1259 - 1270.
backcross inbred lines - genetic-linkage map - disease resistance - phytophthora-infestans - nonhost resistance - avirulence genes - wild lettuce - pathogen - saligna - potato
Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.
Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica
Jiang, R.H.Y. ; Bruijn, I. de; Haas, B.J. ; Belmonte, R. ; Löbach, L. ; Christie, J. ; Ackerveken, G. van den; Bottin, A. ; Bulone, V. ; Díaz-Moreno, S.M. ; Dumas, B. ; Fan, L. ; Gaulin, E. ; Govers, F. ; Grenville-Briggs, L.J. ; Horner, N.R. ; Levin, J.Z. ; Mammella, M. ; Meijer, H.J.G. ; Morris, P. ; Nusbaum, C. ; Oome, S. ; Phillips, A.J. ; Rooyen, D. van; Rzeszutek, E. ; Saraiva, M. ; Secombes, C.J. ; Seidl, M.F. ; Snel, B. ; Stassen, J.H.M. ; Sykes, S. ; Tripathy, S. ; Berg, H. ; Vega-Arreguin, J.C. ; Wawra, S. ; Young, S.K. ; Zeng, Q. ; Dieguez-Uribeondo, J. ; Russ, C. ; Tyler, B.M. ; West, P. van - \ 2013
Plos Genetics 9 (2013)6. - ISSN 1553-7404 - 20 p.
expressed sequence tags - anthocidaris-crassispina eggs - fully automated process - phytophthora-sojae - aphanomyces-euteiches - plant-pathogens - infestans - cells - evolution - reveals
Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.
|The regulation of D-galacturonic acid utilization in Botrytis cinerea
Zhang, L. ; Stassen, J.H.M. ; Chatterjee, S. ; Cornelissen, M. ; Kan, J.A.L. van - \ 2013
In: Book of Abstracts 27th Fungal Genetics Conference, Asilomar, Pacific Grove, California, USA, 12-17 March 2013. - - p. 212 - 212.
The plant cell wall is the first barrier to pathogen invasion. The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an important cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. D-galacturonic acid is the most abundant component of pectin and effective utilization of D-galacturonic acid is important for virulence of B. cinerea. The D-galacturonic acid catabolic pathway comprises three enzymatic steps, involving D-galacturonate reductase (encoded by Bcgar1 and Bcgar2), L-galactonate dehydratase (encoded by Bclgd1), and 2-keto-3-deoxy-L-galactonate aldolase (encoded by Bclga1). Therefore, an effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete pectin utilization by B. cinerea. In this study, RNA sequencing was performed to compare genome wide transcriptional profiles in B. cinerea grown in media containing glucose and pectate as sole carbon sources. We identified 31 genes that are significantly upregulated in pectate containing culture, including Bcgar2, Bclga1, and a putative monosaccharide transporter. In addition, conserved cis-regulatory elements were predicted in the promoters of genes involved in pectate decomposition and D-galacturonic acid utilization. Functional analysis was carried out of the bidirectional promoter of the Bcgar2-Bclga1 gene cluster to study which of the cis-regulatory elements is required for induction by D-galacturonic acid. Furthermore, potential regulatory protein(s) were isolated by DNA-protein pull down assays using one important cis-regulatory element.
|Functional analysis of genes in the mating type locus of Botrytis cinerea
Terhem, R.B. ; Stassen, J.H.M. ; Kan, J.A.L. van - \ 2013
In: Book of Abstracts 27th Fungal Genetics Conference, Asilomar, Pacific Grove, California, USA, 12-17 March 2013. - - p. 147 - 147.
Botrytis cinerea is a heterothallic ascomycete with two mating types, MAT1-1 and MAT1-2, each containing two genes. Besides the archetypal genes encoding the MAT1-1-1 (alpha-domain) protein and the MAT1-2-1 (HMG-box) protein, each idiomorph contains one additional gene, designated MAT1-1-5 and MAT1-2-4, respectively. Homologs of these genes are only found in closely related taxa, and their function is as yet unknown. Knockout mutants were generated in all four genes in the B. cinerea MAT locus, either in the MAT1-1 strain SAS56 or in the MAT1-2 strain SAS405. Mutants were crossed with a strain of the opposite mating type, either the wild type or a knockout mutant, in all possible combinations. Knockout mutants in the MAT1-1-1 gene and the MAT1-2-1 gene fail to show any sign of primordial outgrowth and are entirely sterile. This confirms the essential role of the alpha-domain protein and the HMG-box protein in the mating process. By contrast, mutants in the MAT1-1-5 gene and the MAT1-2-4 gene do produce stipes, but these fail to develop further into an apothecial disk. The MAT1-1-5 and MAT1-2-4 mutants show identical phenotypes, suggesting that these two genes jointly control the transition from stipe to disk development. RNA-seq data were obtained from a cross between two wild type strains and from a cross involving a MAT1-1-5 knockout mutant, from tissue at the stage of transition from stipe to disk. Differential gene expression analysis was performed to identify genes that are possibly involved in development of the apothecial disk.