Transcriptome analysis of Stagonospora nodorum: gene models, effectors, metabolism and pantothenate dispensability

Abstract

The wheat pathogen Stagonospora nodorum, causal organism of the wheat disease Stagonospora nodorum blotch, has emerged as a model for the Dothideomycetes, a large fungal taxon that includes many important plant pathogens. The initial annotation of the genome assembly included 16,586 nuclear gene models. These gene models were used to design a microarray that has been interrogated with labelled transcripts from six cDNA samples: four from infected wheat plants at time points spanning early infection to sporulation, and two time points taken from growth in artificial media. Positive signals of expression were obtained for 12,281 genes. This represents strong corroborative evidence of the validity of these gene models. Significantly differential expression between the various time points was observed. When infected samples were compared with axenic cultures, 2882 genes were expressed at a higher level in planta and 3630 were expressed more highly in vitro. Similar numbers were differentially expressed between different developmental stages. The earliest time points in planta were particularly enriched in differentially expressed genes. A disproportionate number of the early expressed gene products were predicted to be secreted, but otherwise had no obvious sequence homology to functionally characterized genes. These genes are candidate necrotrophic effectors. We have focused attention on genes for carbohydrate metabolism and the specific biosynthetic pathways active during growth in planta. The analysis points to a very dynamic adjustment of metabolism during infection. Functional analysis of a gene in the coenzyme A biosynthetic pathway showed that the enzyme was dispensable for growth, indicating that a precursor is supplied by the plant.

Figure 1

Leaf infections and axenic cultures used for RNA extraction. (A–D) Infected leaf samples from 3, 5, 7 and 10 days post‐inoculation (dpi), corresponding to early infection, vegetative state, sporulation and late infection stages, respectively. (E, F) Samples in minimal medium for 4 and 16 days, corresponding to vegetative growth and sporulation, respectively.

Figure 2

Principal component analysis of the expression profile from 18 sample datasets using The Unscrambler® software (CAMO, Oslo, Norway). The samples used consisted of materials in triplicate from detached leaf assays infected with Stagonospora nodorum SN15 [3 dpi in planta (), 5 dpi in planta (◆), 7 dpi in planta (●) and 10 dpi in planta (▴)] and axenically grown SN15 [4 dpi in vitro () and 16 dpi in vitro ()]. Principal component 1 (PC1) accounted for 37% of the variation and principal component 2 (PC2) described 22% of the variation. PC1 mainly differentiates samples according to the growth medium, whereas PC2 mainly differentiates samples according to their developmental stage. dpi, days post‐inoculation.

Figure 5

The expression level of genes responsible for the assimilation of sulphate (A) and the conversion of sulphates (B). IP, in planta; IV, in vitro.

Figure 3

Growth of SN15 (1 and 2) and the Pbl1 deleted strain (3 and 4) on minimal medium with (1 and 3) and without (2 and 4) added pantothenate. Detached leaf assay (5, from left to right) of two Pbl1 strains and ectopic and wild‐type control.

Figure 4

The expression of CYP51 genes and other regulated genes involved in ergosterol biosynthesis. IP, in planta; IV, in vitro.

Figure 6

The expression of genes encoding phosphate transporters. IP, in planta; IV, in vitro.

Figure 7

The expression profile of genes encoding enzymes responsible for the catabolism of phenylalanine and tyrosine. IP, in planta; IV, in vitro.

Figure 8

The expression profile of genes encoding enzymes involved in the biosynthesis of melanin. DHN, 1,8‐dihydroxynaphthalene; IP, in planta; IV, in vitro.