Transcriptome analysis of programmed cell death associated with allorecognition in chestnut blight fungus Cryphonectria parasitica

Abstract

i The "dialectics" of this situation is that the evolutionary narratives certainly are oversimplified "myths" that have the unfortunate (and, in modern studies, unintended) teleological flavor (as in "selected for" or, worse, "selected for the purpose of"), yet the language of these narratives seems best suited to describing evolution and formulating falsifiable hypotheses that propel further research. At present, we hardly can give up these stories […] precisely because they are necessary means for the advancement of research, even though they tend to leave a scientist […] with feelings of uneasiness and dissatisfaction. It seems important not to forget that evolutionary narratives effectively are semantic devices that are constructed to structure and simplify our thinking about evolution and to facilitate the generation of hypotheses. These narratives should be prudently distrusted and by no account should be construed as "accurate representations of reality" (whatever that might mean […]). AbstractIn this thesis, I analysed transcriptional profiles of the chestnut blight fungus, Cryphonectria parasitica, during vegetative incompatibility (allorecognition) reactions due to differences at the vic3 locus. Out of a total 13944 identified expressed transcripts, including 2334 novel ones, only 1411 were differentially expressed during vic3 incompatibility reaction. Functional enrichment analysis showed increased expression of genes involved in detoxification and stress response (e.g.s Cytochrome p450, Glutathione S-transferase), and toxin biosynthesis. Surprisingly, even though the test strains were both the same mating type (MAT-2), genes involved in sexual reproduction (mf2-1, mf2-2and mat-2) showed the most dramatic increase in expression during allorecognition response. Further qPCR analysis showed that activation of mating pheromone genes occurs during incompatible reactions involving five of the six known vic incompatibility loci. The only exception was vic4, which elicits a weak incompatibility and showed almost no change in pheromone gene expression. Analysis suggests that mf2-1, mf2-2 and mat-2 expression is triggered by activation of asexual sporulation. Genes encoded at the vic3 locus, vic3a and vic3b, were upregulated in barraging samples along with seven HET-domain genes located at other regions of the genome. Among the seven HET genes activated, one is located at the vic1 locus and previously implicated in vic1incompatibility. Activation of these same HET domain genes also occurred in other vic incompatible pairings. For example, upregulation of dev3a and vic1a genes occurred during incompatibility reactions associated with each of the six known vic loci. This suggests that some HET genes serve as universal allorecognition factors. Furthermore, this data indicates that each incompatibility locus uses a set of several HET genes to iii activate Programmed Cell Death (PCD). In addition, I analysed the effects on barrage formation of p29, a protein-coding region from Cryphonectria hypovirus 1 (CHV1).Ex...

Secondary Metabolite Biosynthesis during Vegetative Incompatibility in Cryphonectria parasitica, Causal Agent of the American Chestnut Blight

“…Explained variance in PC2 separates monocultures from incompatible ones beginning on day 4, increasing approximately linearly over time. This data supports the transcriptomic analysis of VI in Cp, which suggested secondary metabolite-related chemical profiles should begin to change in VI interactions on or after day 3 from co-inoculation, the optimal day at which transcripts were harvested (Belov, 2018).…”

“…This method may more closely approximate VI phenotypes in nature, because VI and barrage formation ultimately involve the simultaneous interaction of hyphae detecting non-self without cell fusion, of incompatible cells anastomosing and undergoing PCD, and of global, colony-level changes in metabolic states and phenotypes. Methodological differences notwithstanding, a recent transcriptomic analysis of VI between co-inoculated strains of C. parasitica which are polymorphic at the vic3 locus supports many of the proposed patterns in heterokaryon incompatibility-associated differential expression discussed above (Belov, 2018). The vic3 locus encodes two genes: one with unknown function, and another with homology to Lifeguard1, whose product is a transmembrane protein shown to inhibit Fas-mediated apoptotic signals and is conserved among many species of animal (Somia et al, 1999)(Table 1).…”

“…pheromones in that there are two copies: mf2-1 and mf2-2, which are not thought to encode different finished products due to identical amino acid sequences in the processed, 'final' pheromone form (Zhang et al, 1998). Both mf2-1 and mf2-2 were significantly differentially expressed during VI between mat-2 strains incompatible at vic1, vic2, vic3, vic6 and vic7 loci (LFC = 13.31 and 9.55 for mf2-1 and mf2-2 respectively during vic3 VI, p-values < 0.05) (Belov, 2018). Additionally, an upregulated predicted farnesyltransferase (Cp266015, LFC = 2.09, pvalue < 0.05) is relevant to pheromone post-translational processing during vic3 incompatibility.…”

“…Both strains are of the same mating type (mat-2) and were sourced from the collection of Dr. Myron Smith (Carleton University). The transcriptomes of both strains in monoculture, as well as in a co-cultured mix, have been described in a recent study (Belov, 2018).…”

“…The genome of strain EP155 was scanned for predicted fungal BGCs using the freely available online program "antiSMASH" in January 2019 (Blin et al, 2019). Genes found to be differentially expressed in vic3 VI transcriptomes (Belov, 2018) were then compared with the predicted BGCs where possible or examined manually using the JGI online genome browser.…”