The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogensCladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu \u3e61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation

Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand

Dothistromin is a virulence factor produced by the fungal pine needle pathogen Dothistroma septosporum. It is similar in structure to a precursor of aflatoxin and sterigmatocystin. Unlike most secondary metabolite genes in fungi, the genes for dothistromin biosynthesis are not clustered but spread over six loci on one chromosome. Another characteristic feature of dothistromin synthesis is that dothistromin is produced mainly during the early exponential growth phase in culture. These unusual features have been proposed to be adaptations for the biological role of dothistromin in the disease process. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of aflatoxin and sterigmatocystin in Aspergillus spp. and to address the question of whether genes in a fragmented cluster can be co-regulated. The availability of the D. septosporum genome facilitated identification of orthologs of the aflatoxin pathway regulatory genes aflR, aflJ and the global regulatory genes veA and laeA. These genes were functionally characterised by knockout and complementation assays and the effects of these mutations on the expression of dothistromin genes and the production of dothistromin were assessed. Inactivation of the DsAflR gene (?DsAflR) resulted in a 104 fold reduction in dothistromin production, but some dothistromin was still made. This contrasted with ?AflR mutants in Aspergillus species that produced no aflatoxin. Expression patterns in ?DsAflR mutants helped to predict the complete set of genes involved in dothistromin biosynthesis. AflJ was proposed to act as a transcriptional co-activator of AflR in Aspergillus spp. Disruption of DsAflJ resulted in a significant decrease in dothistromin production and dothistromin gene expression. Interestingly the expression of DsAflR was not affected by deleting DsAflJ, while conversely DsAflJ transcript levels increased significantly in a DsAflR mutant compared to the wild type. Heterologous complementation with A. parasiticus, A. nidulans and C. fulvum AflJ failed to revert the dothistromin level to wild type suggesting species-specific function of AflJ. VeA is an important regulator of secondary metabolism and development in fungi. Inactivation of the D. septosporum ortholog (DsVeA) resulted in reduced dothistromin production and showed the influence of DsVeA on the expression of other secondary metabolite backbone genes. Asexual sporulation was reduced but mutants were not compromised in pathogenicity. Overall, D. septosporum DsVeA showed functional conservation of the usual role in fungi. LaeA is a global regulator of secondary metabolism and morphogenetic development, first identified in Aspergillus nidulans. Unexpectedly, DsLaeA exhibited an unusual repressive function on the dothistromin pathway and DsLaeA mutants exhibited an extended period of dothistromin production compare to WT in vitro. The mutation of DsLaeA showed varied responses in expression of other secondary metabolite genes and had differences in sporulation and hydrophobicity compared to the wild type

Cloning and Expression of Fol E Coding for GTP Cyclohydrolase I from E. coli

Abstract: Folates are essential cofactors for one-carbon transfer reactions in most living organisms. Inadequate dietary levels of the vitamin folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer. Unlike plants and micro organisms, humans cannot synthesize folates de novo and must acquire themfrom the diet, primarily from plant foods so good strategy to improve folate intake worldwide would be to genetically engineer common food plants to make more folates. So the main goal of this work was cloning and expressing of the Fol E gene encoding GTP-Cyclohydrolase (which catalyses the conversion of GTP to dihydroneopterin triphosphate a rate-determining step in de novo pterin and folate biosynthesis in plants) from E. coli local isolate. E. coli culture was isolated from the stagnant water around the university campus. The PCR primers was designed to amplify FolE gene from E. coli, the single sharp amplicon of approximately Fol.E (670bp) gene was clone into pTZ57R/T cloning vector and confirmed by sequencing and was later sub-cloned into pET43A(+) prokaryotic expression vector and the expression of which was confirmed by PAGE analysis

Cloning and Expression of Endoglucanase genes from Trichoderma species in Saccharomyces cerevisiae

Abstract: The present study was conducted to isolate and clone the endoglucanase gene from Trichoderma harzianum and to study the expression of endoglucanase genes cloned from different species in Saccharomyces cerevisiae (INVSc1 strain). Using specific primers, gene encoding β-1, 6 endoglucanase (1.3 kb) from T. harzianum was cloned into pTZ57R/T vector. The clone was confirmed through PCR and restriction analysis. The clone was sequenced and analyzed for the homology at the nucleotide and at the protein level for domain analysis. Gene encoding β-1, 6 endoglucanase has shown 97% with reported sequence both at the nucleotide and protein level. Endoglucanase genes cloned from different species were expressed in Saccharomyces cerevisiae using pYES2/CT vector. The recombinant clones of T. reesei has shown three times more, T. harzianum and T. virens has shown twice the activity when compared to control INVSc1 having pYES2/CT vector. Further, the SDS-PAGE analysis of the recombinant clone β-1, 6 endoglucanase from T. harzianum has shown the presence of corresponding to β-1, 6 endoglucanase

Reduced Virulence of an Introduced Forest Pathogen over 50 Years

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys

Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains

Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.Supplementary Material: Fig. S1 Predicted duplications and deletions in chromosomes 1‐14 for 18 strains of D. septosporum.Fig. S2 Initial evidence for a reciprocal chromosome translocation in the NZE10 genome. (A) Assembled contigs from the SLV genome were aligned with NZE10 reference chromosomes (scaffolds). Two contigs (circled) mapped to both chromosomes 5 and 13 of the NZE10 reference genome. This was found in many of the other genome sequences. (B) Visualisation of reads from the ALP3 genome mapped onto a region of chromosome 13 show a gap, in which mate pairs are mapped to chromosome 5.Fig. S3 A reciprocal translocation involving chromosomes 5 and 13 in the NZE10 genome. (A) The reciprocal translation was centred on an identical sequence (GCGCGGT) found at positions 1459800‐1459806 in NZE10 chromosome 5 and 717926‐717932 in chromosome 13. Chromosomes 5 and 13 are shaded grey and pale blue respectively with ends coloured to distinguish the two arms in each case. Coloured sequences surrounding the breakpoint indicate which arm they are from. (B) In strains from regions other than Australasia, the two long sections of NZE10 chromosomes 5 and 13 are joined to make a 2.2 Mb chromosome and two short sections to make a 1.4 Mb chromosome. Sequences around the common 7 bp sequence are shown for strain ALP3 as an example. (C, D) Pairs of divergently transcribed genes straddle the breakpoints on NZE10 chromosomes 5 (C) and 13 (D). A GC content of about 70% was seen at the breakpoint regions (50 bp sliding window) as shown by the %GC (blue) profiles.Fig. S4 Alignment of pathway regulator AflR from 19 D. septosporum strains. Amino acid changes compared to strain NZE10 are highlighted in blue (these sites are also variant between AflR sequences of D. septosporum , Cladosporium fulvum , Aspergillus parasiticus and Aspergillus nidulans ; (Chettri et al ., 2013)) or in green (at sites conserved between those four species). The Zn2Cys6 zinc binuclear domain is highlighted in pink; the linker sequence thought to determine DNA binding specificity in grey; the acidic glutamine rich motif in yellow and C terminal arginine residues implicated in AflJ binding in red.Fig. S5 Secondary structure predictions for AflR from D. septosporum NZE10 and ALP3. Pairwise alignment predicted by HHpred. The arrow indicates the location of the N349K polymorphism in ALP3.Table S1 Transposable elements in the Dothistroma septosporum genomes.Table S2 Genes deleted in the 18 genomes compared to Dothistroma septosporum NZE10.Table S3 Genes deleted from chromosome 14 and their expression levels in NZE10.Table S4 Single Nucleotide Polymorphisms (SNPs) in dothistromin genes, grouped by dothistromin gene loci.Table S5 Deleted genes on Dothistroma septosporum chromosome 12.Table S6 Gene duplications predicted by CNV (copy number variant) analysis.Table S7 (a) Polymerase Chain Reaction (PCR) primers used for verification of 5:13 translocation (b) Primers used for copy number variant (CNV) verification (quantitative PCR [qPCR]).The Bio‐Protection Research Centre of New Zealand, Scion and Massey University.https://onlinelibrary.wiley.com/journal/13643703pm2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Global population genomics of the forest pathogen Dothistroma septosporum

Supplementary Material: Fig. S1 Predicted duplications and deletions in chromosomes 1‐14 for 18 strains of D. septosporum.Fig. S2 Initial evidence for a reciprocal chromosome translocation in the NZE10 genome. (A) Assembled contigs from the SLV genome were aligned with NZE10 reference chromosomes (scaffolds). Two contigs (circled) mapped to both chromosomes 5 and 13 of the NZE10 reference genome. This was found in many of the other genome sequences. (B) Visualisation of reads from the ALP3 genome mapped onto a region of chromosome 13 show a gap, in which mate pairs are mapped to chromosome 5.Fig. S3 A reciprocal translocation involving chromosomes 5 and 13 in the NZE10 genome. (A) The reciprocal translation was centred on an identical sequence (GCGCGGT) found at positions 1459800‐1459806 in NZE10 chromosome 5 and 717926‐717932 in chromosome 13. Chromosomes 5 and 13 are shaded grey and pale blue respectively with ends coloured to distinguish the two arms in each case. Coloured sequences surrounding the breakpoint indicate which arm they are from. (B) In strains from regions other than Australasia, the two long sections of NZE10 chromosomes 5 and 13 are joined to make a 2.2 Mb chromosome and two short sections to make a 1.4 Mb chromosome. Sequences around the common 7 bp sequence are shown for strain ALP3 as an example. (C, D) Pairs of divergently transcribed genes straddle the breakpoints on NZE10 chromosomes 5 (C) and 13 (D). A GC content of about 70% was seen at the breakpoint regions (50 bp sliding window) as shown by the %GC (blue) profiles.Fig. S4 Alignment of pathway regulator AflR from 19 D. septosporum strains. Amino acid changes compared to strain NZE10 are highlighted in blue (these sites are also variant between AflR sequences of D. septosporum , Cladosporium fulvum , Aspergillus parasiticus and Aspergillus nidulans ; (Chettri et al ., 2013)) or in green (at sites conserved between those four species). The Zn2Cys6 zinc binuclear domain is highlighted in pink; the linker sequence thought to determine DNA binding specificity in grey; the acidic glutamine rich motif in yellow and C terminal arginine residues implicated in AflJ binding in red.Fig. S5 Secondary structure predictions for AflR from D. septosporum NZE10 and ALP3. Pairwise alignment predicted by HHpred. The arrow indicates the location of the N349K polymorphism in ALP3.Table S1 Transposable elements in the Dothistroma septosporum genomes.Table S2 Genes deleted in the 18 genomes compared to Dothistroma septosporum NZE10.Table S3 Genes deleted from chromosome 14 and their expression levels in NZE10.Table S4 Single Nucleotide Polymorphisms (SNPs) in dothistromin genes, grouped by dothistromin gene loci.Table S5 Deleted genes on Dothistroma septosporum chromosome 12.Table S6 Gene duplications predicted by CNV (copy number variant) analysis.Table S7 (a) Polymerase Chain Reaction (PCR) primers used for verification of 5:13 translocation (b) Primers used for copy number variant (CNV) verification (quantitative PCR [qPCR]).Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.The Bio‐Protection Research Centre of New Zealand, Scion and Massey University.https://onlinelibrary.wiley.com/journal/13643703pm2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog