Thermotolerance and post-fire growth in Rhizina undulata is associated with the expansion of heat stress-related protein families

Abstract

Background: Rhizina undulata is an important tree pathogen, infecting a wide variety of conifer species, including those in the genus Pinus. The fungus relies on heat shock-mediated activation of its ascospores, which require high temperatures to initiate germination. Consequently, disease due to R. undulata often occurs after fire events in both natural and managed forests. The genetic mechanisms contributing to the pyrophilous nature of R. undulata have not been investigated. We sought to identify key genes that may be responsible for thermotolerance in the fungus. A comparative genomics approach was used, by sequencing the genome of R. undulata for the first time and comparing its predicted proteome to those from other Pezizomycetes, both with and without known associations with fire.

Results: Three protein families were shown to be expanded in R. undulata; heat shock protein 20 (HSP20), glutathione-S transferases (GST), and aromatic compound dioxygenases (ACD). While HSP20 was uniquely over-represented in R. undulata, the expansions of the GST and ACD families were also identified in other fire-associated species.

Conclusion: HSP20s are known to protect cells against heat stress, GSTs are involved in the detoxification of reactive oxygen species, and ACDs play a role in the metabolism of recalcitrant compounds present in post-fire environments. The expansion of these families thus suggests that they may play an important role in protecting and stimulating R. undulata ascospores during and after fire-induced heat shock, activating the fungus, and enabling it to colonise the root systems of conifers.

Keywords: Rhizina undulata; Aromatic compound dioxygenases; Glutathione S-transferase; Heat shock proteins; Thermotolerance.

Fig. 1

Ecological niches of fungi included in this study, with particular reference to their known associations with fire. Pezizomycetes not known to be associated with fire or other heat shock events include a variety of truffles as well as a coprophilous fungus. The remaining species are known to have some association with fire and include morels, cup fungi, and others. Those for which data relating to the time of colonization exist are categorized as either “early colonizers” or “late colonizers”, based on the classification system of [57]. Those for which no such data exists are categorized as “colonizers”. Published literature suggests that R. undulata is the only species dependent on a heat shock, such as fire, for the completion of its life cycle

Fig. 2

Genome comparisons across the Pezizomycetes. A An annotated phylogeny illustrating differences in genome statistics. The phylogeny was generated using 783 shared BUSCO proteins and represents the known relationships between these species. The full phylogeny is included in Figure S2 with additional information and node support data. Species known to associate with fire are in orange text and the R. undulata isolate sequenced for this study is in bold, red text (See Fig. 1 and Table S1 for details on fire associations). The Pezizomycete genomes ranged from ~ 27 to ~ 171 Mb and encoded between ~ 7 800 and ~ 24 000 genes. All of the genomes were considered complete (94.3 to 99.1% with respect to the fungal BUSCO dataset) and none were contaminated with bacterial sequences (5.6 to 11.3% with respect to the bacterial BUSCO dataset). B Plots comparing the genome size, GC content, and number of protein coding genes in the genomes of the 22 species. The R. undulata genome (represented by the red dots) is within the expected length, gene count and GC content statistics of the other Pezizomycetes (represented by the blue dots)

Fig. 3

The presence of HSP-associated PFAM domains across the Pezizomycetes. A An annotated phylogeny illustrating the numbers of HSP20 (PF00011) and GST N-Terminal (PF02798) proteins in each of the genomes. The full phylogeny is included in Figure S2 with additional information and node support data. The phylogeny was generated using 783 shared BUSCO proteins and represents the known relationships between these species. Species known to associate with fire are presented in orange text and the R. undulata isolate sequenced for this study is in bold, red text. The R. undulata genome harbours many more proteins in the HSP20 and GST families than the remaining Pezizomycetes. B A heatmap illustrating the number of HSP-associated PFAM domains encoded in each genome. R. undulata is highlighted in a black box. Note that each column is normalized individually, and the absolute values can be found in Table S5. C Phylogenies of the orthologues from two orthogroups harbouring protein families that are expanded in R. undulata. OG0000190 represents the HSP20 proteins and is comprised of 18 proteins with HSP20 PFAM domains and an additional protein identified by OrthoFinder. OG0000279 represents GST proteins and is comprised of nine proteins with GST-N PFAM domains and two additional proteins identified by OrthoFinder. The R. undulata orthologues are represented by red circles and red text, while those from the other 21 species are represented by blue circles and blue text. Note that each orthologue ID begins with four letters, made up of the first letter of the genus and the first three letters of the species epithet (e.g., Rhizina undulata = RUND)

Fig. 4

Comparison in orthologue count between fire associated and non-fire associated species with respect to the expanded heat shock-associated protein families in R. undulata. In all three families, R. undulata is an outlier (red dots in dotted black circles), harbouring more orthologue copies than the other 21 species. In the case of the HSP20 protein family, there is not a significant difference in copy number between fire associated (red circles and boxes) and non-fire associated (blue circles and boxes) species. In contrast, the fire-associated fungi harbour more copies of the GST and ACD proteins than the non-fire associated species. This pattern holds true when R. undulata is removed from the analysis (Table S7). ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001

Fig. 5

The presence of orthologues from OG0000266 across the Pezizomycetes. The genes in this orthogroup encode aromatic compound dioxygenase (ACD) enzymes. A An annotated phylogeny illustrating the number of ACD proteins in each of the genomes. The full phylogeny is included in Figure S2 with additional information and node support data. The phylogeny was generated using 783 shared BUSCO proteins and represents the known relationships between these species. Species known to have fire associations are presented in orange text and the R. undulata isolate sequenced for this study is in bold, red text. The R. undulata genome harbours many more proteins in the ACD family than the remaining Pezizomycetes. B A phylogeny of the orthologues from OG0000266, a protein family that is expanded in R. undulata. The R. undulata orthologues are represented by red circles and red text, while those from the other 21 species are represented by blue circles and blue text. Note that each orthologue ID begins with four letters, made up of the first letter of the genus and the first three letters of the species epithet (e.g., Rhizina undulata = RUND)

Fig. 6

The molecular mechanisms hypothesized to play a role in the thermotolerance and post-fire activation of R. undulata. Ascospores of R. undulata are airborne and remain dormant in the soil in areas where the fungus has previously sporulated. When a heat-inducing event, such as a fire, occurs in conifer plantations or forests, the spores are activated and germinate, after which they infect the roots of the trees, causing disease. In the presence of fire, HSP20 proteins likely protect the ascospores from heat-induced stress by binding to heat-denatured proteins, thereby preventing aggregation and further damage. GST enzymes detoxify the ROS molecules that are produced in response to the heat stress. In the post-fire environment, when the spores have germinated, nutrients may be provided through the ACD-facilitated metabolism of PyOm produced by the fire. Again, the GST enzymes would detoxify ROS molecules produced through this metabolism. The germinated ascospores subsequently give rise to mycelium that colonizes roots of the associated conifers in the post-fire environment, resulting in disease and producing apothecial fruiting bodies (ascocarps). These fruiting bodies produce large numbers of forcibly ejected ascospores, which are dispersed to new environments. The expansion of the HSP20 protein family is unique to R. undulata, whereas the expansions of the GST and ACD protein families, while particularly notable in R. undulata, are also found in the other heat-associated Pezizomycetes. Together, the expansion of these protein families in R. undulata likely enables the pathogen to be activated by fire events and to thrive in post-fire environments