Genome-wide DNA methylation and transcriptomic profiles in the lifestyle strategies and asexual development of the forest fungal pathogen Heterobasidion parviporum

Figures & data

Figure 1. Inoculation and harvesting of samples. (a) Illustration of field inoculation. (b) Inoculated holes filled with sawdust pre-colonized by H. parviporum. (c) Area surrounding the inoculated holes after harvesting of sawdust. The arrow indicated the necrotrophic growth of H. parviporum (d) Stem after harvesting of the area surrounding the inoculated holes. The arrow indicated the necrosis caused by fungal infection.

Table 1. BS-seq, RNA-seq reads and mapping to the H. parviporum reference.

Sample	Clean trimmed BS-seq reads	Uniquely mapped BS-seq reads	Average depth (X)	Coverage (%)	C sites covered by reads (%)	Uniquely mapped RNA-seq read pairs
MYCEL1	14,870,260	9,654,870	23.40	88.33	86.80	20,310,912
MYCEL2	17,410,086	11,607,152	28.36	88.48	87.16	20,831,526
MYCEL3	16,709,532	9,758,454	24.07	88.26	86.68	18,176,796
NECT1	224,293,660	4,079,650	9.35	88.64	82.32	10,450,332
NECT2	110,926,664	3,106,566	7.52	88.81	80.70	12,242,118
NCET3	54,145,292	2,974,288	7.35	88.49	79.16	14,923,771
SAP1	36,020,680	6,413,302	15.54	88.11	85.55	27,594,109
SAP2	43,535,074	6,447,114	15.42	88.21	85.87	31,173,620
SAP3	36,003,232	7,018,926	17.28	88.21	86.11	34,372,560
SPORE1	15,205,834	10,393,944	25.43	87.35	87.24	36,022,809
SPORE2	18,230,646	13,091,254	32.20	86.82	87.49	39,921,109
SPORE3	15,107,956	10,257,480	25.13	86.90	87.14	31,634,395

Figure 2. H. parviporum DNA methylation pattern. (a) Relative proportions of mC sites in the three nucleotide contexts. R1, R2 and R3 indicated the three biological replicates of each condition. (b) Methylation density in CpG, CHG and CHH contexts. (c) Average methylation level in different functional genomic regions including TEs, 1.5 kb upstream of genes, gene, 1.0 kb downstream of genes, exons and introns. (d) Zoomed in average methylation level in gene and gene upstream till 1.5 kb and downstream till 1.0 kb. Vertical dash line represented the transcribed region of genes. Biological replicates were shown in the same color.

Figure 3. Distribution of DNA methylation levels in CpG, CHG and CHH contexts of all samples. Vertical dash lines represented the average methylation level in each biological replicate. The arrows denoted the average methylation level among the three biological replicates together with its standard error.

Figure 4. Genome-scale map of DNA methylation density in 10-kb windows of three biological replicates of MYCEL. Each circle from the outside to the inside depicts: (a) H. parviporum 287 scaffolds from the largest to the smallest size arranged in clockwise; (b) gene density; (c) transposable elements density; (d–f) methylation density in CpG, CHG and CHH context, respectively.

Figure 5. Selection of condition-specific genes and in planta-expressed genes. (a) Venn diagram showing the number of significantly up-regulated (unique and common) genes during mycelial growth in living trees (NECT) relative to mycelial growth in liquid media (MYCEL), sawdust (SAP) and conidiospores (SPORE). (b) Venn diagram showing the number of significantly up-regulated (unique and common) genes in SAP relative to MYCEL, NECT and SPORE. (c) Venn diagram showing the number of significantly up-regulated (unique and common) genes in SPORE relative to MYCEL, NECT and SAP. The numbers in bold red indicated the number of condition-specific genes. (d) Hierarchical clustering of condition-specific and in planta-expressed genes using their TPM values.

Table 2. Significantly enriched GO terms and KEGG pathways of selected gene sets.

IDs	Annotation	Type^a	p-value	Expected	Significant	Annotated	Tested genes
GO:0044281	Small molecule metabolic process	BP	4.7E-05	76.94	92	597	SPORE-specific
GO:0019748	Secondary metabolic process	BP	0.0014	1.93	7	15	SPORE-specific
GO:0006457	Protein folding	BP	0.0093	5.80	12	45	SPORE-specific
GO:0008135	Translation factor activity, RNA binding	MF	0.0038	4.54	11	39	SPORE-specific
Kegg:hir00190	Oxidative phosphorylation	-	1.3E-04	-	24	80	SPORE-specific
Kegg:hir03040	Spliceosome	-	9.0E-04	-	23	88	SPORE-specific
Kegg:hir03020	RNA polymerase	-	0.0018	-	12	29	SPORE-specific
Kegg:hir00240	Pyrimidine metabolism	-	0.0046	-	18	70	SPORE-specific
Kegg:hir00230	Purine metabolism	-	0.0063	-	20	87	SPORE-specific
GO:0003700	DNA binding transcription factor activity	MF	3.0E-04	1.33	7	130	SAP-specific
GO:0005975	Carbohydrate metabolic process	BP	7.4E-06	1.27	8	249	NECT-specific
GO:0016798	Hydrolase activity, acting on glycosyl bonds	MF	1.0E-05	0.81	7	138	NECT-specific
GO:0005975	Carbohydrate metabolic process	BP	1.6E-12	22.24	57	249	In planta-expressed
GO:0055085	Transmembrane transport	BP	0.0081	16.88	27	189	In planta-expressed
GO:0016798	Hydrolase activity, acting on glycosyl bonds	MF	5.1E-06	12.72	30	138	In planta-expressed
GO:0022891	Substrate-specific transmembrane transporter activity	MF	0.0010	16.78	30	182	In planta-expressed
GO:0043167	Ion binding	MF	0.0079	128.49	151	1394	In planta-expressed
GO:0006259	DNA metabolic process	BP	9.2E-09	1.82	10	153	Amenable genes

^aThe GO categories, BP: biological process; MF: molecular function.

Figure 6. Expression levels and methylation status of different classes of TEs. (a) Occurrences of predicted TEs in H. parviporum 96026 genome. (b) Barplot showing the number of expressed TEs in the studied four conditions. ‘TOTAL’ represented the total number of TEs in each subclass. The biological replicates of each condition were indicated by using the same bar color. (c) Boxplot of TEs expression level represented by Log2 TPM values in the studied conditions. (d) Barplot showing the number of methylated TEs. ‘TOTAL’ represented the total number of TEs in each subclass. The biological replicates of each condition were indicated by using the same bar color.

Figure 7. Association of expression level represented by Log (TPM+1) with CpG methylation levels of (a,b) transcribed region of genes and (c,d) 1.5 kb upstream of genes in the four conditions. The gene expression levels were divided into five groups. The 1st group represented the non-expressed genes, and 2nd to 5th groups included the lowest 25% to the highest 25% of expressed genes in the four conditions. Genes of all biological replicates were plotted. (e) The distribution of DNA methylation level of transcribed region of 1st group of genes (non-expressed genes). (f) The distribution of DNA methylation level of 1.5 kb upstream of 1^st group of genes (non-expressed genes).

Figure 8. Examples of methylation patterns in relation to expression level under different conditions. (a) Methylation pattern I: One GMC oxidoreductase (evm.scaffold20.13) was heavily methylated in all conditions with lower methylation level and higher transcript abundances in NECT and SAP. (b) Methylation pattern II: One protein from cytochrome P450 superfamily (evm.scaffold2.271) had slightly higher methylation levels and higher transcript abundance in NECT and SAP. (c) Methylation pattern III: RNA polymerases III RPC1 subunit encoding gene (evm.scaffold29.111) had higher methylation levels and lower transcript abundance in NECT and SAP.

Figure 9. Summary of representative genes or biological processes in the generated gene sets of our study. All analyzed genes sets were summarized in square blocks.