Analysis of the Phialocephala subalpina Transcriptome during Colonization of Its Host Plant Picea abies

Abstract

Background: Phialocephala subalpina belongs to the Phialocephala fortinii s.l.-Acepphala applanata species complex (PAC) forming one of the major groups belonging to the dark septate endophytes (DSE). Depending on the strain, PAC was shown to form neutral to pathogenic associations with its host plant Picea abies. To understand PACs lifestyle we investigated the effect of presence/absence of Picea abies on the transcriptome of strain 6_70_1.

Materials and methods: PAC strain 6_70_1 was grown in liquid Pachlewski media either induced by its host plant Picea abies or without host plant as a control. Mycelia were harvested in a time course (1, 2, 3, 4, 7, 11, 18 days) with and without induction by the host plant and the fungal transcriptome revealed by Illumina sequencing. Differential gene expression analysis over the time course comparing control and treatment at each time point using the 'edgeR glm approach' and a gene enrichment analysis using GO categories were performed.

Results: The three main functional groups within differentially expressed genes were 'metabolism', 'transport' and 'cell rescue, defense and virulence'. Additionally, genes especially involved in iron metabolism could be detected by gene set enrichment analysis.

Conclusion: In conclusion, we found PAC strain 6_70_1 to be metabolically very active during colonization of its host plant Picea abies. A major shift in functional groups over the time course of this experiment could not be observed but GO categories which were found to be enriched showed different emphasis depending in the day post induction.

Fig 1. Validation of Illumina data using real-time RT PCR.

Sqrt-transformed, normalized Illumina counts were plotted against negative ∆C_T values for correlation. Colors represent different target genes (open rectangle = PAC_1440, closed circle = PAC_18739 and open circle = PAC_7783) which were validated. Housekeeping gene PAC_19651 served as reference gene.

Fig 2. MDS plot showing sample relations.

Sample relations are plotted using a multidimensional scaling plot (MDS) generated with edgeR showing the variability between replicates and different treatments in log2-fold-change distance. The axes represent gene expression levels between the different experimental factors. Samples from days 1, 7, 11 and 18 show best separation between treatments and best accordance of replicates. “c” = control, “t” = treatment and d1 –d18 = days after induction with the host plant.

Fig 3. Differentially expressed genes.

Number of DE genes belonging to the main functional categories according to FunCat annotations over time. Most genes fall into more than one functional category and therefore, contribute to several categories. LogFC cutoff was between -2 and 2 therefore DE genes within this range are displayed only.

Fig 4. Top 20 differentially expressed genes.

Heat map depicting the Top 20 genes including their main functional categories. Gene expression in blue shows down-regulated and in orange up-regulated genes. Genes were clustered by their expression pattern. The transcription level is depicted in logFC values. Genes displayed in blue are downregulated, therefore expressed in favor of the control and genes depicted in orange are expressed in favor of the treatment. Count corresponds to the number of reads covering the gene model at each timepoint.