Significance of Gene Expression Regulation for Environmental and Host Adaptation in Plant Pathogenic Fungi

Abstract

Pathogenic fungi impose a great threat to the global food production. They are globally distributed and display a huge amount of phenotypic diversity. They demonstrate high adaptability to diverse environments and to fluctuating conditions within their hosts. However, not much is known about the mechanisms underlying these processes of adaptation. Deciphering the genetic basis of phenotypic diversity may help us to get a deeper insight into the mechanisms that regulate complex adaptive traits. Furthermore, these findings may help in improving strategies for an effective and sustainable management of pathogens. In my PhD study, I aimed to understand the genetic basis of adaptation to variable environments using Zymoseptoria tritici. Z. tritici causes septoria leaf blotch of wheat and is considered to be the most devastating fungal wheat pathogen in Europe. In the first chapter of my thesis, I studied the role of melanin in adaptation of Z. tritici. I, demonstrated that melanin is essential for protection against harmful chemical fungicides such as bixafen. Remarkably, I observed high phenotypic variability in melanin accumulation levels in different Zymoseptoria tritici strains, most probably reflecting its role in adaptation to variable environments. Using genetic mapping, I was able to demonstrate that variability in melanization levels between two Swiss strains of Z. tritici was mediated by differential expression of the transcription factor Zmr1 (Zymoseptoria melanin regulation 1). I identified nucleotide mutations in the promoter region and an insertion of transposable elements (TEs) upstream of the promoter region as two mechanisms responsible for this observed differential expression of Zmr1. My findings clearly demonstrated the significance of gene expression regulation, mediated by the insertion of TEs and SNPs, in optimizing the growth and adaptation of the fungus under variable environments. Furthermore, these adaptive changes are responsible for the phenotypic diversity that we observe in Z. tritici strains from all over the world. In the second part of my thesis, I analyzed the expression of several secreted peptidases of Z. tritici at distinct stages during the infection process of wheat plants. Using computational genetic methods, I also analyzed nucleotide data sets of the peptidase genes for evolutionary signatures of genetic adaptation. By combining both approaches, I aimed at obtaining a more detailed picture of the relevance and the role of each enzyme during the infection process. I was able to show that the peptidases belonging to MEROPS families A1 and G1 were significantly up regulated during the asymptomatic phase of infection and displayed signatures of accelerated evolution, suggesting their key role in suppression of plant defenses and host specialization. In the third part of my thesis, I evaluated the role of a cellulase-encoding gene (JGI Prot. ID: 76589) using molecular biology techniques. This cellulase was only expressed during the final necrotrophic phase of Z. tritici infection and the nucleotide sequences of the gene showed signatures of diversifying selection. This pattern is consistent with the hypothesis of evasion from host recognition during the first phases of infection and subsequent induction of host-cell death by the secretion of an array of different cellulase isoforms by the fungus. To test this hypothesis, I constitutively expressed the cellulase-encoding gene in Z. tritici during all infection stages by inserting a constitutive promoter (originally from the Glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans) upstream of the start codon of the cellulase gene. I was able to show that the progress of infection in this mutant was delayed compared to the non-modified Z. tritici isolates. This suggests that the cellulase acts as an elicitor molecule that is detected by the plant and triggers the defense machinery that will hinder the growth of the pathogen. In summary, my PhD work demonstrated the significance of fine-tuning of gene expression regulation of various genes in plant pathogenic fungi to facilitate adaptation to different environmental conditions and host colonization. Remarkably, I was able to show that gene expression fine-tuning resulted in large amounts of phenotypic diversity in adaptive traits.