About twenty-five years after the sequencing of the first fungal genome and a dozen years after the first plant pathogenic fungi genomes were sequenced, unprecedented international efforts have led to an impressive collection of genomes available for the community of mycologists in international databases (Goffeau et al. 1996, Dean et al. 2005; Spatafora et al. 2017). For instance, to date, the Joint Genome Institute Mycocosm database has collected more than 2,100 fungal genomes over the fungal tree of life (https://mycocosm.jgi.doe.gov). Such resources are paving the way for comparative genomics, population genomics and phylogenomics to address a large panel of questions regarding the biology and the ecology of fungal species. Early on, population genomics applied to pathogenic fungi revealed a great diversity of genome content and organization and a wide variety of variants and rearrangements (Raffaele and Kamoun 2012, Hartmann 2022). Such plasticity raises questions about how to choose a representative genome to serve as an ideal reference to address pertinent biological questions.

Cryphonectria parasitica is a fungal pathogen that is infamous for the devastation of chestnut forests in North America after its accidental introduction more than a century ago (Anagnostakis 1987). Since then, it has been a quarantine species under surveillance in various parts of the world. As for other fungi causing diseases on forest trees, the study of adaptation to its host in the forest ecosystem and of its reproduction and dissemination modes is more complex than for crop-targeting pathogens. A first reference genome was published in 2020 for the chestnut blight fungus C. parasitica strain EP155 in the frame of an international project with the DOE JGI (Crouch et al. 2020). Another genome was then sequenced from the French isolate YVO003, which showed a few differences in the assembly suggesting possible rearrangements (Demené et al. 2019). Here the sequencing of a third isolate ESM015 from the native area of C. parasitica in Japan allows to draw broader comparative analysis and particularly to compare between native and introduced isolates (Demené et al. 2022).

Demené and collaborators report on a new genome sequence using up-to-date long-read sequencing technologies and they provide an improved genome assembly. Comparison with previously published C. parasitica genomes did not reveal dramatic changes in the overall chromosomal landscapes, but large rearrangements could be spotted. Despite these rearrangements, the genome content and organization – i.e. genes and repeats – remain stable, with a limited number of genes gains and losses. As in any fungal plant pathogen genome, the repertoire of candidate effectors predicted among secreted proteins was more particularly scrutinized. Such effector genes have previously been reported in other pathogens in repeat-enriched plastic genomic regions with accelerated evolutionary rates under the pressure of the host immune system (Raffaele and Kamoun 2012). Demené and collaborators established a list of priority candidate effectors in the C. parasitica gene catalog likely involved in the interaction with the host plant which will require more attention in future functional studies. Six major inter-chromosomal translocations were detected and are likely associated with double break strands repairs. The authors speculate on the possible effects that these translocations may have on gene organization and expression regulation leading to dramatic phenotypic changes in relation to introduction and invasion in new continents and the impact regarding sexual reproduction in this fungus (Demené et al. 2022).

I recommend this article not only because it is providing an improved assembly of a reference genome for C. parasitica, but also because it adds diversity in terms of genome references availability, with a third high-quality assembly. Such an effort in the tree pathology community for a pathogen under surveillance is of particular importance for future progress in post-genomic analysis, e.g. in further genomic population studies (Hartmann 2022). 

References

Anagnostakis SL (1987) Chestnut Blight: The Classical Problem of an Introduced Pathogen. Mycologia, 79, 23–37. https://doi.org/10.2307/3807741

Crouch JA, Dawe A, Aerts A, Barry K, Churchill ACL, Grimwood J, Hillman BI, Milgroom MG, Pangilinan J, Smith M, Salamov A, Schmutz J, Yadav JS, Grigoriev IV, Nuss DL (2020) Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen. Phytopathology®, 110, 1180–1188. https://doi.org/10.1094/PHYTO-12-19-0478-A

Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu J-R, Pan H, Read ND, Lee Y-H, Carbone I, Brown D, Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E, Rehmeyer C, Li W, Harding M, Kim S, Lebrun M-H, Bohnert H, Coughlan S, Butler J, Calvo S, Ma L-J, Nicol R, Purcell S, Nusbaum C, Galagan JE, Birren BW (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature, 434, 980–986. https://doi.org/10.1038/nature03449

Demené A., Laurent B., Cros-Arteil S., Boury C. and Dutech C. 2022. Chromosomal rearrangements with stable repertoires of genes and transposable elements in an invasive forest-pathogenic fungus. bioRxiv, 2021.03.09.434572, ver.6 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2021.03.09.434572

Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 Genes. Science, 274, 546–567. https://doi.org/10.1126/science.274.5287.546

Hartmann FE (2022) Using structural variants to understand the ecological and evolutionary dynamics of fungal plant pathogens. New Phytologist, 234, 43–49. https://doi.org/10.1111/nph.17907

Raffaele S, Kamoun S (2012) Genome evolution in filamentous plant pathogens: why bigger can be better. Nature Reviews Microbiology, 10, 417–430. https://doi.org/10.1038/nrmicro2790

Spatafora JW, Aime MC, Grigoriev IV, Martin F, Stajich JE, Blackwell M (2017) The Fungal Tree of Life: from Molecular Systematics to Genome-Scale Phylogenies. Microbiology Spectrum, 5, 5.5.03. https://doi.org/10.1128/microbiolspec.FUNK-0053-2016

Since the opening of the Rhine-Main-Danube-Channel, four goby species are known to have invaded the River Rhine. Of these, the most recent and numerous is the Caucasian Dwarf Goby, which has been found in the Rhine since 2019. This study presents a new high-quality genome for this species (Knipowitschia cf. caucasica) (Schoenle et al. 2024). Currently, chromosome-scale genome assemblies represent a key first step in invasion biology, allowing the reconstruction of a species’ invasion history and monitoring its progress, as well as identifying and characterizing candidate genes that control invasiveness (McCartney et al. 2019). 

The authors sequenced the nuclear and mitochondrial genomes of this species using state-of-the art methods including long-read sequencing techniques, scaffolded based on chromatin conformation data, and annotated using both direct transcriptomic and protein homology evidence. Data analyses follow currently established pipelines for genome assembly, scaffolding, annotation, and downstream bioinformatic analyses. The quality of the final genome was thoroughly assessed and conforms to what is expected from other genomes of fishes in the family Gobiidae. This study follows other recent endeavors that generated high-quality genomes to improve our understanding of invasion biology (e.g. Shao et al. 2020 and Kitsoulis et al. 2023). These studies are successfully contributing to increasing the genomic resources for the world’s most damaging invasive species, which were not available for even a third of the top 100 invasive species just five years ago (McCarthy et al. 2019). Beyond invasion biology, the Dwarf Goby genome is also an important resource for many other applications, including evolutionary genomic analyses and phylogeography of this species and closely related ones in their native ranges.

                
References          

Kitsoulis CV, Papadogiannis V, Kristoffersen JB, Kaitetzidou E, Sterioti A, Tsigenopoulos CS, Manousaki T (2023) Near-chromosome level genome assembly of devil firefish, Pterois miles. Peer Community Journal 3:e64. https://doi.org/10.24072/pcjournal.295  

McCartney MA, Mallez S, Gohl DM (2019) Genome projects in invasion biology. Conservation Genetics 20:1201–1222. https://doi.org/10.1007/s10592-019-01224-x

Schoenle A, Guiglielmoni N, Mainz T, Greve C, Hamadou AB, Heermann L, Borcherding J, Waldvogel A-M (2024) Chromosome level genome reference of the Caucasian dwarf goby Knipowitschia cf. caucasica, a new alien Gobiidae invading the River Rhine. bioRxiv, ver. 3 peer-reviewed and recommended by PCI Genomics. https://doi.org/10.1101/2024.04.22.590508

Shao F, Ludwig A, Mao Y, Liu N, Peng Z (2020). Chromosome-level genome assembly of the female western mosquitofish (Gambusia affinis). GigaScience 9:giaa092. https://doi.org/10.1093/gigascience/giaa092

Parasitoid wasps lay their eggs inside another arthropod, whose body is physically consumed by the parasitoid larvae. Phylogenetic inference suggests that Parasitoida are monophyletic, and that this clade underwent a strong radiation shortly after branching off from the Apocrita stem, some 236 million years ago (Peters et al. 2017). The increase in taxonomic diversity during evolutionary radiations is usually concurrent with an increase in genetic/genomic diversity, and is often associated with an increase in phenotypic diversity. Gene (or genome) duplication provides the evolutionary potential for such increase of genomic diversity by neo/subfunctionalisation of one of the gene paralogs, and is often proposed to be related to evolutionary radiations (Ohno 1970; Francino 2005).

In their recent preprint, Dominique Colinet and coworkers have explored the genetic and functional diversity of a Rho GTPase activating protein (RhoGAP) multigene family in two very divergent wasp clades within Parasitoida, namely Leptopilina (Figitidae) and Venturia (Ichneumonidae) (Colinet et al. 2024). Some members of the RhoGAP family are present in the venom of the parasitoid wasp Leptopilina boulardi as well as in other Leptopilina species, and are probably involved in the parasitic lifestyle by binding and inactivating host’s Rho GTPases, thereby interfering with the host’s immune response (Colinet et al. 2007).

Venom protein composition is highly variable, even between very closely related species, and is subject to rapid evolutionary changes. Although gene duplication and subsequent neo/subfunctionalisation have been frequently proposed as the main mechanism underlying this evolutionary diversification, observations are often compatible with alternative explanations, such as horizontal gene transfer, gene co-option or multifunctionalisation (Martinson et al. 2017; Alvarado et al. 2020; Huang et al. 2021; Undheim and Jenner 2021). Furthermore, high mutation rates in venom protein-encoding genes hinder phylogenetic hypothesis testing, and venom proteomics can be needed to verify transcriptomic predictions (Smith and Undheim 2018; von Reumont et al. 2022).

Colinet and coworkers (2024) have applied a combined transcriptomic, proteomic and functional approach to i) identify potential transcripts of the RhoGAP family in Leptopilina species using experimental and bioinformatic approaches; ii) experimentally identify proteins of the RhoGAP family in the venom of three Leptopilina species; iii) identify transcripts and proteins of the RhoGAP family in the ovarian calyx of Venturia canescens; and iv) perform phylogenetic and selection analyses on the extant sequences of these RhoGAP family genes to propose an evolutionary scenario for their origin and diversification. The most striking results are first the large diversity of RhoGAP sequences retrieved in the transcriptomes and proteomes of Leptopilina and of V. canescens, and second the high number of branches and positions identified to have evolved under positive selection. All the retrieved hits share a RhoGAP domain, either alone or in tandem, preceded in the case of Leptopilina RhoGAPs by a signal peptide that may be responsible for protein vehiculation for venom secretion. Further, for some of the protein positions identified to have evolved under positive selection, the authors have experimentally verified the functional impact of the changes by reverse genetic engineering.

The authors propose an evolutionary scenario to interpret the phylogenetic relationships among extant RhoGAP diversity in the clades under study. They posit that two independent, incomplete duplication events from the respectively ancestral RacGAP gene, followed by subsequent, lineage- and paralog-specific duplication events, lie at the origin of the wealth of diversity of in the Leptopilina venom RhoGAPs and of V. canescens ovarian calyx RhoGAPs. Notwithstanding, the global relationships presented in the work are not systematically consistent with this interpretation, e.g. regarding the absence of monophyly for Leptopilina RhoGAPs and Leptopilina RacGAP, and the same holds true for the respective V. canescens sequences. It may very well be that the high evolutionary rate of these genes has eroded the phylogenetic signal and prevented proper reconstruction, as the large differences between codon-based and amino acid-based phylogenies and the low support suggest. Explicit hypothesis testing, together with additional data from other taxa, may shed light onto the evolution of this gene family.

The work by Colinet and coworkers communicates sound, novel transcriptomic, proteomic and functional data from complex gene targets, consolidated from an important amount of experimental and bioinformatic work, and related to evolutionarily intriguing and complex phenotypes. These results, and the evolutionary hypothesis proposed to account for them, will be instrumental for our understanding of the evolution and diversity of vesicle-associated RhoGAPs in divergent parasitoid wasps.

References

Alvarado, G., Holland, S., R., DePerez-Rasmussen, J., Jarvis, B., A., Telander, T., Wagner, N., Waring, A., L., Anast, A., Davis, B., Frank, A., et al. (2020). Bioinformatic analysis suggests potential mechanisms underlying parasitoid venom evolution and function. Genomics 112(2), 1096–1104. https://doi.org/10.1016/j.ygeno.2019.06.022

Colinet, D., Cavigliasso, F., Leobold, M., Pichon, A., Urbach, S., Cazes, D., Poullet, M., Belghazi, M., Volkoff, A-N., Drezen, J-M., Gatti, J-L., and Poirié, M. (2024). Convergent origin and accelerated evolution of vesicle-associated RhoGAP proteins in two unrelated parasitoid wasps. bioRxiv, ver. 3 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2023.06.05.543686

Colinet, D., Schmitz, A., Depoix, D., Crochard, D., and Poirié, M. (2007). Convergent Use of RhoGAP Toxins by eukaryotic parasites and bacterial pathogens. PLoS Pathogens 3(12), e203. https://doi.org/10.1371/journal.ppat.0030203

Francino, M.P. (2005). An adaptive radiation model for the origin of new gene functions. Nature Genetics 37, 573–577. https://doi.org/10.1038/ng1579

Huang, J., Chen, J., Fang, G., Pang, L., Zhou, S., Zhou, Y., Pan, Z., Zhang, Q., Sheng, Y., Lu, Y., et al. (2021). Two novel venom proteins underlie divergent parasitic strategies between a generalist and a specialist parasite. Nature Communications 12, 234. https://doi.org/10.1038/s41467-020-20332-8

Martinson, E., O., Mrinalini, Kelkar, Y. D., Chang, C-H., and Werren, J., H. 2017. The evolution of venom by co-option of single-copy genes. Current Biololgy 27(13), 2007-2013.e8. https://doi.org/10.1016/j.cub.2017.05.032

Ohno, S. (1970). Evolution by gene duplication. New-York: Springer-Verlag.

Peters, R., S., Krogmann, L., Mayer, C., Donath, A., Gunkel, S., Meusemann, K., Kozlov, A., Podsiadlowski, L., Petersen, M., Lanfear, R., et al. (2017). Evolutionary history of the Hymenoptera. Current Biology 27(7), 1013–1018. https://doi.org/10.1016/j.cub.2017.01.027

von Reumont, B., M., Anderluh, G., Antunes, A., Ayvazyan, N., Beis, D., Caliskan, F., Crnković, A., Damm, M., Dutertre, S., Ellgaard, L., et al. (2022). Modern venomics—Current insights, novel methods, and future perspectives in biological and applied animal venom research. GigaScience 11, giac048. https://doi.org/10.1093/gigascience/giac048

Smith, J., J., and Undheim, E., A., B. (2018). True lies: using proteomics to assess the accuracy of transcriptome-based venomics in centipedes uncovers false positives and reveals startling intraspecific variation in Scolopendra subspinipes. Toxins 10(3), 96. https://doi.org/10.3390/toxins10030096

Undheim, E., A., B., and Jenner, R., A. (2021). Phylogenetic analyses suggest centipede venom arsenals were repeatedly stocked by horizontal gene transfer. Nature Communications 12, 818. https://doi.org/10.1038/s41467-021-21093-8

Third-generation sequencing has revolutionised de novo genome assembly. Thanks to this technology, genome reference sequences have evolved from fragmented drafts to gapless, telomere-to-telomere genome assemblies. Long reads produced by Oxford Nanopore and PacBio technologies can span structural variants and resolve complex repetitive regions such as centromeres, unlocking previously inaccessible genomic information. Nowadays, many research groups can afford to sequence the genome of their working model using long reads. Nevertheless, genome assembly poses a significant computational challenge. Read length, quality, coverage and genomic features such as repeat content can affect assembly contiguity, accuracy, and completeness in almost unpredictable ways. Consequently, there is no best universal software or protocol for this task. Producing a high-quality assembly requires chaining several tools into pipelines and performing extensive comparisons between the assemblies obtained by different tool combinations to decide which one is the best. This task can be extremely challenging, as the number of tools available rises very rapidly, and thorough benchmarks cannot be updated and published at such a fast pace. 

In their paper, Orjuela and collaborators present CulebrONT [1], a universal pipeline that greatly contributes to overcoming these challenges and facilitates long-read genome assembly for all taxonomic groups. CulebrONT incorporates six commonly used assemblers and allows to perform assembly, circularization (if needed), polishing, and evaluation in a simple framework. One important aspect of CulebrONT is its modularity, which allows the activation or deactivation of specific tools, giving great flexibility to the user. Nevertheless, possibly the best feature of CulebrONT is the opportunity to benchmark the selected tool combinations based on the excellent report generated by the pipeline. This HTML report aggregates the output of several tools for quality evaluation of the assemblies (e.g. BUSCO [2] or QUAST [3]) generated by the different assemblers, in addition to the running time and configuration parameters. Such information is of great help to identify the best-suited pipeline, as exemplified by the authors using four datasets of different taxonomic origins. Finally, CulebrONT can handle multiple samples in parallel, which makes it a good solution for laboratories looking for multiple assemblies on a large scale. 

References

1. Orjuela J, Comte A, Ravel S, Charriat F, Vi T, Sabot F, Cunnac S (2022) CulebrONT: a streamlined long reads multi-assembler pipeline for prokaryotic and eukaryotic genomes. bioRxiv, 2021.07.19.452922, ver. 5 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2021.07.19.452922

2. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM (2015) BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics, 31, 3210–3212. https://doi.org/10.1093/bioinformatics/btv351

3. Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: quality assessment tool for genome assemblies. Bioinformatics, 29, 1072–1075. https://doi.org/10.1093/bioinformatics/btt086

 Comparative genomics is a general approach for understanding how genomes differ, which can be considered from many angles. For instance, this approach can delineate how gene content varies across organisms, which can lead to novel hypotheses regarding what those organisms do. It also enables investigations into the sequence-level divergence of orthologous DNA, which can provide insight into how evolutionary forces differentially shape genome content and structure across lineages. 
 
Such comparisons are often restricted to protein-coding genes, as these are sensible units for assessing putative function and for identifying homologous matches in divergent genomes. Although information is lost by focusing only on the protein-coding portion of genomes, this simplifies analyses and has led to crucial findings in recent years. Perhaps most dramatically, analyses based on hundreds of orthologous proteins across microbial eukaryotes are fundamentally changing our understanding of the eukaryotic tree of life (Burki et al. 2020).
 
These and other topics are highlighted in a new pre-print from Dr. Daniel Richter and colleagues, which describes EukProt (Richter et al. 2022): a database containing protein sets from 993 eukaryotic species. The authors provide a BLAST portal for matching custom sequences against this database (https://evocellbio.com/eukprot/) and the entire database is available for download (https://doi.org/10.6084/m9.figshare.12417881.v3). They also provide a subset of their overall dataset, ‘The Comparative Set’, which contains only high-quality proteomes and is meant to maximize phylogenetic diversity.
 
There are two major advantages of EukProt:
 
   1. It will enable researchers to quickly compare proteomes and perform phylogenomic analyses, without needing the skills or the time commitment to aggregate and process these data. The authors make it clear that acquiring the raw protein sets was non-trivial, as they were distributed across a wide variety of online repositories (some of which are no longer accessible!).
 
    2. Analyses based on this database will be more reproducible and easily compared across studies than those based on custom-made databases for individual studies. This is because the EukProt authors followed FAIR principles (Wilkinson et al. 2016) when building their database, which is a set of guidelines for enhancing data reusability. So, for instance, each proteome has a unique identifier in EukProt, and all species are annotated in a unified taxonomic framework, which will aid in standardizing comparisons across studies.
 
The authors make it clear that there is still work to be done. For example, there is an uneven representation of proteomes across different eukaryotic lineages, which can only be addressed by further characterization of poorly studied lineages. In addition, the authors note that it would ultimately be best for the EukProt database to be integrated into an existing large-scale repository, like NCBI, which would help ensure that important eukaryotic diversity was not ignored. Nonetheless, EukProt represents an excellent example of how reproducible bioinformatics resources should be designed and should prove to be an extremely useful resource for the field.
 
References

Burki F, Roger AJ, Brown MW, Simpson AGB (2020) The New Tree of Eukaryotes. Trends in Ecology & Evolution, 35, 43–55. https://doi.org/10.1016/j.tree.2019.08.008

Richter DJ, Berney C, Strassert JFH, Poh Y-P, Herman EK, Muñoz-Gómez SA, Wideman JG, Burki F, Vargas C de (2022) EukProt: A database of genome-scale predicted proteins across the diversity of eukaryotes. bioRxiv, 2020.06.30.180687, ver. 5 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2020.06.30.180687

Wilkinson MD, Dumontier M, Aalbersberg IjJ, Appleton G, Axton M, Baak A, Blomberg N, Boiten J-W, da Silva Santos LB, Bourne PE, Bouwman J, Brookes AJ, Clark T, Crosas M, Dillo I, Dumon O, Edmunds S, Evelo CT, Finkers R, Gonzalez-Beltran A, Gray AJG, Groth P, Goble C, Grethe JS, Heringa J, ’t Hoen PAC, Hooft R, Kuhn T, Kok R, Kok J, Lusher SJ, Martone ME, Mons A, Packer AL, Persson B, Rocca-Serra P, Roos M, van Schaik R, Sansone S-A, Schultes E, Sengstag T, Slater T, Strawn G, Swertz MA, Thompson M, van der Lei J, van Mulligen E, Velterop J, Waagmeester A, Wittenburg P, Wolstencroft K, Zhao J, Mons B (2016) The FAIR Guiding Principles for scientific data management and stewardship. Scientific Data, 3, 160018. https://doi.org/10.1038/sdata.2016.18