Author Archives: diane

FIR analysis: genes encoding predicted secreted proteins occur in both gene sparse and gene dense regions of the H. pseudoalbidus genome

The contributors

Daniel Bunting (Nuffield student), Kentaro Yoshida, Dan MacLean and Diane Saunders at TSL.

The material

We used the potential Hymenoscyphus pseudoalbidus KW1 effector candidates identified in (http://oadb.tsl.ac.uk/?m=20130910).

Background information

In filamentous plant pathogens such as the late blight oomycete pathogen Phytophthora infestans, a repeat-driven expansion has created repeat and transposable element (TE) rich, gene-sparse regions that are distinct from the gene-dense conserved regions, known as a two-speed genome architecture. Determining the distance of a gene to its closest coding gene neighbours, (designated flanking intergenic regions, FIRs), can be used to determine whether a gene resides in a gene-dense or gene-sparse environment. Given that genes associated with pathogenicity tend to have long FIRs in pathogen genomes, genome architecture could be used to identify new candidate pathogenicity genes.

The analysis

To investigate whether a similar organisation occurs in the genome of H. pseudoalbidus we firstly identified candidate effector genes in the gene annotations  (http://oadb.tsl.ac.uk/?m=20130910). In order to determine whether genes encoding secreted proteins are in gene sparse or dense regions of the genome we modified the de novo gene calls using RNA-seq data to extend based on overlaps with transcripts, to create the file extended_genes.gff by aligning the RNAseq reads from KW1 against the KW1 assembly, using BWA. For each gene model in the TGAC gene predictions that was within 100nt of another gene we extracted reads on the same strand that fell within -1000nt of the start or 1000nt of the end. With these reads, starting with the start and end of the gene we followed read overlaps as far as possible, until reads no longer overlapped. The most distal read then counted as the new gene start/end.

The FIR distribution for genes in the H.pseudoalbidus genome can be seen below and is indicative of a single speed genome, with genes encoding secreted proteins dispersed both in gene-sparse and gene-dense regions of the genome.

 

Overlayed

Figure. The single speed H.pseudoalbidus genome. Distribution of H.pseudoalbidus genes according to the length of their 5′ and 3′ flanking intergenic regions (FIRs). Red circles, core genes; blue circles, genes encoding predicted secreted proteins.

Potential inhibition of a putative alternative oxidase identified in C. fraxinea reduces fungal growth in culture

The contributors

Mary Albury, Luke Young, Julia Shearman, Ben May and Tony Moore (University of Sussex); Kentaro Yoshida and Diane Saunders (The Sainsbury Laboratory)

The material

The Chalara fraxinea isolate KW1 was tested for growth in the presence/absence of two compounds; a traditional fungicide (compound A) or an inhibitor of the putative alternative oxidase protein identified in the C. fraxinea proteome (http://oadb.tsl.ac.uk/?p=526) (compound C).

The analysis

Agar plugs of C. fraxinea KW1 isolate (5 mm diameter) were used to inoculate glucose agar plates or potato dextrose broth, supplemented with 1, 1.5 or 2 uM of compounds A or C (Figure 1). DMSO was used as a negative control. The presence of compound A reduced C. fraxinea KW1 growth.

To further access the inhibition activity of the two compounds, agar plugs of C. fraxinea KW1 isolate (5 mm diameter) were used to inoculate glucose agar plates supplemented with 1.5 uM of both compounds A and C or DMSO. Agar plugs in potato dextrose broth were supplemented with 1.5 uM of compound A, C, both combined or DMSO (Figure 2). When both compounds were combined there was a significant reduction in C. fraxinea growth in culture.

Three biological and three technical replicates were undertaken for each experiment.

trials

Figure 1. Compound A reduced C. fraxinea KW1 isolate growth in culture. Agar plugs of C. fraxinea KW1 isolate (5 mm diameter) were used to inoculate glucose agar plates or potato dextrose broth, supplemented with 1, 1.5 or 2 uM of compounds A or C or DMSO. Pictures captured 12 days post-inoculation.

Fungicide_exp1

 

Figure 2. Combining compounds A and C significantly reduced C. fraxinea KW1 growth in culture. Agar plugs of C. fraxinea KW1 isolate (5 mm diameter) were used to inoculate glucose agar plates or potato dextrose broth. The plates were supplemented with 1.5 uM of compounds A and C or DMSO. The liquid cultures were supplemented with 1.5 uM of compound A, C, both combined or DMSO. Pictures captured 15 days post inoculation.

Repeated mRNA-Seq analysis of Tree 35

The contributors

Martin Trick (JIC), Andrea Harper (CNAP, University of York), Leah Clissold (TGAC) and Ian Bancroft (CNAP, University of York)

The material

Young leaf material was harvested from a clone of Tree 35 in Denmark in 2013.

The analysis

mRNA was extracted and a paired-end (but this time not strand-specific) Illumina RNA-Seq library constructed. About 125 million read pairs were obtained from a single HiSeq 2500 lane – the raw data are available from The Sainsbury Laboratory’s FTP server, with details in the github repository here. Trinity was again used to assemble transcripts from the complete set of reads, this time generating 242,115 assemblies, and then RSEM transcript abundance analysis was carried out to select 130,978 principal isoforms which constitute our new reference sequence. 96% of the transcripts were located to scaffolds in the Tree 35 genome assembly developed by TGAC. Candidate open reading frames were extracted and the predicted peptides were queried against the UniProt protein database with BLASTP producing a functional annotation.

We have now sequenced the leaf transcriptomes of 186 trees that have been sampled from across Denmark and phenotyped for disease symptoms by our colleague Erik Dahl Kjaer’s group. SNPs and expression levels with respect to the Tree 35 reference have been calculated and we are about to start on the association work.

The top 100 ranked C. fraxinea candidate effector tribes

The contributors

Daniel Bunting (Nuffield student), Kentaro Yoshida and Diane Saunders at TSL.

The material

We used the potential C. fraxinea KW1 effector candidates identified in (http://oadb.tsl.ac.uk/?m=20130910).

The analysis

To select proteins with high likelihood as candidate effectors we focused on tribes that ranked highly in our scoring system. We used hierarchical clustering of the top 100 ranked C. fraxinea effector candidate tribes to group them further based on shared features.

 

Chalara_circos

Figure 1. The top 100 ranked protein tribes containing putative effectors. A. Combined score used to rank tribes based on their content of effector features. B. Score for number of members classified as secreted. C. Score for number of members identified as expressed during infection. D. Score for number of members with similarity to known fungal AVRs. E. Score for number of members with effector motifs or nuclear localisation signals (NLS). F. Score for number of members classified as repeat containing. G. Score for number of members classified as small and cysteine rich. H. Score for number of members encoded by genes with at least one flanking intergenic region > 10Kb. I. Score for number of members not annotated by PFAM domain searches. Red star indicates tribe that contains potential Nep1-like protein (http://oadb.tsl.ac.uk/?p=235).

Mining for putative effectors in the Chalara fraxinea KW1 genome

The contributors

Daniel Bunting (Nuffield student), Kentaro Yoshida and Diane Saunders at TSL.

The material

We used the C. fraxinea KW1 predicted proteome (data/ash_dieback/chalara_fraxinea/Kenninghall_wood_KW1/annotations/Gene_predictions/TGAC_Chalara_fraxinea_ass_s1v1_ann_v1.1) as a basis to mine for candidate effectors.

The analysis

  1. First, the predicted proteome of C. fraxinea KW1 was searched for potential secreted proteins using SignalP2 with parameters described in [1]. Transmembrane domain containing proteins and proteins with mitochondrial signal peptides were removed using TMHMM [2] and TargetP [3], respectively.
  2. We then clustered all proteins using TribeMCL [4], following the methods described in [5]. We identified clusters of proteins (known as tribes) that contained at least one secreted protein. These 593 tribes were then used for all further analysis.
  3. Next, we annotated the protein tribes for known effector features as described in [6].
  4. Finally, we assigned an e-value to each feature within a tribe using the method described in [7] in order to rank tribes based on their likelihood of containing effector proteins.

A spreadsheet that contains the above analysis is available at (data/ash_dieback/chalara_fraxinea/Kenninghall_wood_KW1/annotations/Effector_mining).

flow_diagram

Figure 1. Pipeline used to mine for potential effector proteins in C. fraxinea KW1 isolate. Programs are indicated in red.

References
1. Torto TA, Li S, Styer A, Huitema E, Testa A, Gow NAR, Van West P, Kamoun S: EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. Genome Res 2003, 13(7):1675–1685.
2. Krogh A, Larsson BÈ, Von Heijne G, Sonnhammer ELL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001, 305(3):567–580.
3. Emanuelsson O, Brunak S, von Heijne G, Nielsen H: Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2007, 2(4):953–971.
4. Enright AJ, Van Dongen S, Ouzounis CA: An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res 2002, 30(7):1575–1584.
5. Haas BJ, Kamoun S, Zody MC, Jiang RHY, Handsaker RE, Cano LM, Grabherr M, Kodira CD, Raffaele S, Torto-Alalibo T, et al: Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature 2009, 461(7262):393–398.
6. Cantu D, Seqovia V, MacLean D, Bayles R, Chen X, Kamoun S, Dubcovsky J, Saunders DGO, Uauy C: Genome analyses of the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. tritici reveal polymorphic and haustorial expressed secreted proteins as candidate effectors. BMC Genomics 2013, 14:270.
7. Saunders DGO, Win J, Cano LM, Szabo LJ, Kamoun S, Raffaele S: Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi. PLoS One 2012, 7(1):e29847.

mRNAseq analysis of primordial and mature fruiting bodies of H. pseudoalbidus

The contributors

Anne Edwards (JIC), Diane Saunders (TSL), Kentaro Yoshida (TSL) and Allan Downie (JIC).

The material

Dead ash raciness collected from Ashwellthorpe Lower Wood on 10th February 2013 were incubated on moist filter paper in a controlled environment at 18oC, under 16 hr of 62.5% light and black UV. Following 10 weeks of incubation primordial fruiting bodies were observed but did not develop into mature fruiting bodies.

Mature fruiting bodies (below) found on dead ash racines in Ashwellthorpe Wood on June 2nd were collected and incubated as above. They were harvested after one week.

The analysis

RNA was extracted from a single primordial or mature fruiting body and subjected to mRNA-seq analysis. The reads were assembled into transcripts, which are available in the github repository (data/ash_dieback/h_pseudoalbidus/Primordial_fruiting_body/assemblies/RNAseq/PFB1_Trinity.fasta and data/ash_dieback/h_pseudoalbidus/Mature_fruiting_body/assemblies/RNAseq/MFB1_Trinity.fasta, respectively).

_DSC1638_lwr

Both mating types of Hymenoscyphus pseudoalbidus (Chalara fraxinea) are present in Ashwellthorpe lower wood

The contributors

Kentaro Yoshida and Diane Saunders at TSL.

The material

We collected 24 samples of potentially infected Ash trees from Ashwellthorpe lower wood.

The analysis

We used the PCR method of Gross et al (2012) to identify the H. pseudoalbidus MAT 1-1 (MAT1-1-3) and MAT 1-2 (MAT-1-2-1) mating types from these samples. The size of the PCR products indicated that five samples were of the MAT1-1 mating type and three were of the MAT1-2 mating type.

Conclusion

This confirms that both mating types are present within Ashwellthorpe lower wood.

gel_pictures

Figure:

PCR amplification of H. pseudoalbidous mating loci. AT indicates samples taken from Ashwellthorpe lower wood, KW1 is an isolate from Kenninghall wood that was previously confirmed as displaying the MAT1-1 (MAT1-1-3) mating type. ITS (internal transcribed spacer) analysis was used to confirm infection by C. fraxinea.