Periodic Reporting for period 1 - SYMBIOLOSS (Mutualism abandonment in land plants and the origin of novel adaptations)
Reporting period: 2024-02-01 to 2026-01-31
Summary of the context and overall objectives of the project
The ability to perform symbiotic associations with arbuscular mycorrhizal (AM) fungi is an ancient and highly conserved trait in land plants that evolved half a billion years ago. Around 80% of species benefit from the improved access to soil nutrients provided by AM fungi, while delivering in exchange carbohydrates and lipids to the fungi. However, plants from several lineages are not able to perform AM symbiosis. These include aquatic, parasitic and carnivorous plants, as well as some plants with atypical root morphologies (e.g. proteoid roots) or that engage in derived forms of symbiosis (e.g. ectomycorrhiza, orchid mycorrhiza). Some vascular and non-vascular plant species that have lost the AM symbiosis, however, are not associated with any of such alternative nutrient acquisition strategies, including the Brassicaceae, Caryophyllaceae, Amaranthaceae, most mosses and other bryophyte lineages in liverworts and hornworts. It is not known if such nonmycorrhizal (NM) plants bear novel adaptations that compensate for the loss of symbiosis, nor to what extent similar adaptations might have evolved in unrelated NM lineages. In this project, I investigated this problem using comparative genomic analyses coupled with ecological and genetic approaches. The main objective was to identify genetic changes correlated with the loss of AM symbiosis, which could reveal unknown mechanisms of nutrient uptake and/or defence against pathogens that are unique to NM plants. Therefore, while addressing an evolutionary question, such an achievement could have important implications for the understanding of plant ecology and physiology, and be a valuable contribution to inform plant breeding efforts.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
A database with more than 400 genomes including arbuscular mycorrhizal (AM) and nonmycorrhizal (NM) species encompassing all major lineages of vascular and non-vascular plants was built for this project. Gene orthology relationships across species were established using a phylogenetic approach, and the identified groups of orthologous genes served as the basis for comparative genomic analyses. Plant symbiotic status is often ambiguous in the literature, and for this reason an approach to confirm NM species was developed based on knowledge of the genetic basis of plant symbioses. Previous studies have shown that NM species consistently lost six genes related to the infection process and establishment of AM symbiosis, and these are highly conserved in all land plant lineages. The presence/absence of such genes in the genome was used here to classify species as AM or NM. This approach confirmed the status of NM species described in the literature, and extended it to whole clades whenever possible, as in the case of the angiosperm clades Cyperaceae-Juncaceae and Caryophyllales. This genomic approach is expected to serve as a reference for screenings of NM species, bypassing microscopy-based studies that have been the gold standard in the field for decades, which are laborious and prone to false negative results.
To identify genetic changes specific to NM species, comparative genomic analyses were performed. Gene family losses and expansions were correlated with symbiotic status using a statistical approach. The results revealed several genes that were convergently lost in NM species across land plants, recapitulating previous studies. However, no case of convergent gene family expansion across NM lineages was observed. Likewise, when analysing the cellular and metabolic functions of gene families that were duplicated in NM lineages, no evidence of convergence at the functional level was observed. This suggested that the mechanisms of adaptation associated with the loss of AM symbiosis are not shared across all NM lineages but rather species, or lineage, specific. To explore this hypothesis, gene family contractions and expansions were identified and characterized independently in five major lineages containing multiple AM and NM species: the angiosperm orders Brassicales and Poales, and the three clades of bryophytes (liverworts, mosses, hornworts). Experimental validation of candidate genes associated with the loss of symbiosis was performed using the model liverwort species Marchantia paleacea (AM) and M. polymorpha (NM). Two candidate genes were selected for the experimental study. The first encodes a receptor-like kinase (RLK) that is present as single-copy in AM liverworts, and as duplicates in NM species (including four copies in M. polymorpha). Based on published and unpublished results from members of the host team, this putative receptor was postulated to be activated during mycorrhization in M. paleacea, while in M. polymorpha it was expected to be involved in immune responses. The second gene that was selected has a putative role in phosphate metabolism, and it is present in two or more copies in most land plant AM species, but absent in all NM species, except in mosses and some liverworts, including M. polymorpha, which kept one copy. A CRISPR/Cas9-based approach was used to generate knockout (KO) mutants for both gene families (single mutants for M. paleacea, and single and higher order mutants for M. polymorpha), and mutants were phenotyped for AM symbiosis and pathogen susceptibility to test the initial functional predictions.
The role of the candidate RLK in mycorrhization in M. paleacea was characterized using a well-established assay to quantify AM colonization upon infection by the AM fungus Rhizophagus irregularis. This work was performed on the KO mutants, as well as in plants transformed with a phosphomimetic version of the gene. We found evidence of delayed colonization in KO mutants, but no clear differences in overall colonization and arbuscule formation at later developmental stages when compared to wild type plants. Conversely, we found evidence of accelerated colonization in the phosphomimetic transformants, but no differences in overall colonization at later stages compared to the wild type. In addition, patterns of gene expression during mycorrhization were analysed using a promoter-reporter assay, which indicated that the gene is preferentially expressed around air pores on the thallus surface, but at similar levels in mycorrhized and nonmycorrhized plants. The working hypothesis is that the candidate RLK is not essential for AM symbiosis establishment, although it may play an accessory role during the progress of AM fungi infection. The observed phenotypes are currently being evaluated with additional experimental replicates.
The putative role of the candidate RLK in perceiving fungi signals and triggering symbiotic and/or immune responses was tested by probing cellular calcium influx upon treatment of Marchantia plants with chitooligosaccharides (here CO7), which are products of fungi cell wall degradation. This was possible once M. paleacea and M. polymorpha CRISPR mutants were generated on plants expressing a cytosolic calcium reporter gene (aequorin), which allows monitoring calcium influx using luminescence methods. No differences were observed in calcium influx upon CO7 treatment when comparing mutants (single in M. paleacea, and single, triple and quadruple mutants in M. polymorpha) and wild type in both species, which ruled out a role in the perception of this molecule. The mutant lines generated as part of this project will be screened with additional elicitors as part of the work of a current PhD student in the host team.
Finally, a role in defence of the candidate RLK was tested using a well-established pathogen assay in Marchantia plants with the pathogenic fungi Colletotrichum nymphaeae. Plants of M. polymorpha including single, triple and quadruple mutants were grown in vitro and inoculated with fungi spores or mock-inoculated, and the growth rate and tissue necrosis was quantified. No significant differences in growth rate and disease progression were observed between any of the mutant lines and wild type plants. These results therefore do not support the role of the candidate RLK in immunity-related responses, although the effect of alternative pathogens should be evaluated.
The second candidate gene under experimental investigation, which has a putative role in phosphate metabolism, was discovered at a later stage of the project, and for this reason no phenotypic data was obtained so far. Nonetheless, CRISPR mutants of M. paleacea were generated, and the role of this gene in mycorrhization is currently being evaluated. The strong pattern of presence/absence of this gene in AM and NM species, with retention in a few NM bryophyte lineages points to a case of neofunctionalization in NM species of a gene required for AM symbiosis.
To identify genetic changes specific to NM species, comparative genomic analyses were performed. Gene family losses and expansions were correlated with symbiotic status using a statistical approach. The results revealed several genes that were convergently lost in NM species across land plants, recapitulating previous studies. However, no case of convergent gene family expansion across NM lineages was observed. Likewise, when analysing the cellular and metabolic functions of gene families that were duplicated in NM lineages, no evidence of convergence at the functional level was observed. This suggested that the mechanisms of adaptation associated with the loss of AM symbiosis are not shared across all NM lineages but rather species, or lineage, specific. To explore this hypothesis, gene family contractions and expansions were identified and characterized independently in five major lineages containing multiple AM and NM species: the angiosperm orders Brassicales and Poales, and the three clades of bryophytes (liverworts, mosses, hornworts). Experimental validation of candidate genes associated with the loss of symbiosis was performed using the model liverwort species Marchantia paleacea (AM) and M. polymorpha (NM). Two candidate genes were selected for the experimental study. The first encodes a receptor-like kinase (RLK) that is present as single-copy in AM liverworts, and as duplicates in NM species (including four copies in M. polymorpha). Based on published and unpublished results from members of the host team, this putative receptor was postulated to be activated during mycorrhization in M. paleacea, while in M. polymorpha it was expected to be involved in immune responses. The second gene that was selected has a putative role in phosphate metabolism, and it is present in two or more copies in most land plant AM species, but absent in all NM species, except in mosses and some liverworts, including M. polymorpha, which kept one copy. A CRISPR/Cas9-based approach was used to generate knockout (KO) mutants for both gene families (single mutants for M. paleacea, and single and higher order mutants for M. polymorpha), and mutants were phenotyped for AM symbiosis and pathogen susceptibility to test the initial functional predictions.
The role of the candidate RLK in mycorrhization in M. paleacea was characterized using a well-established assay to quantify AM colonization upon infection by the AM fungus Rhizophagus irregularis. This work was performed on the KO mutants, as well as in plants transformed with a phosphomimetic version of the gene. We found evidence of delayed colonization in KO mutants, but no clear differences in overall colonization and arbuscule formation at later developmental stages when compared to wild type plants. Conversely, we found evidence of accelerated colonization in the phosphomimetic transformants, but no differences in overall colonization at later stages compared to the wild type. In addition, patterns of gene expression during mycorrhization were analysed using a promoter-reporter assay, which indicated that the gene is preferentially expressed around air pores on the thallus surface, but at similar levels in mycorrhized and nonmycorrhized plants. The working hypothesis is that the candidate RLK is not essential for AM symbiosis establishment, although it may play an accessory role during the progress of AM fungi infection. The observed phenotypes are currently being evaluated with additional experimental replicates.
The putative role of the candidate RLK in perceiving fungi signals and triggering symbiotic and/or immune responses was tested by probing cellular calcium influx upon treatment of Marchantia plants with chitooligosaccharides (here CO7), which are products of fungi cell wall degradation. This was possible once M. paleacea and M. polymorpha CRISPR mutants were generated on plants expressing a cytosolic calcium reporter gene (aequorin), which allows monitoring calcium influx using luminescence methods. No differences were observed in calcium influx upon CO7 treatment when comparing mutants (single in M. paleacea, and single, triple and quadruple mutants in M. polymorpha) and wild type in both species, which ruled out a role in the perception of this molecule. The mutant lines generated as part of this project will be screened with additional elicitors as part of the work of a current PhD student in the host team.
Finally, a role in defence of the candidate RLK was tested using a well-established pathogen assay in Marchantia plants with the pathogenic fungi Colletotrichum nymphaeae. Plants of M. polymorpha including single, triple and quadruple mutants were grown in vitro and inoculated with fungi spores or mock-inoculated, and the growth rate and tissue necrosis was quantified. No significant differences in growth rate and disease progression were observed between any of the mutant lines and wild type plants. These results therefore do not support the role of the candidate RLK in immunity-related responses, although the effect of alternative pathogens should be evaluated.
The second candidate gene under experimental investigation, which has a putative role in phosphate metabolism, was discovered at a later stage of the project, and for this reason no phenotypic data was obtained so far. Nonetheless, CRISPR mutants of M. paleacea were generated, and the role of this gene in mycorrhization is currently being evaluated. The strong pattern of presence/absence of this gene in AM and NM species, with retention in a few NM bryophyte lineages points to a case of neofunctionalization in NM species of a gene required for AM symbiosis.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
This project investigated the repeated loss of the ability to establish symbiotic associations with AM fungi in lineages of vascular and non-vascular plants, which is an overlooked problem in plant biology. Using a comparative genomic approach, it aimed at identifying genes involved in adaptations that compensate for the loss of symbiosis. The first contribution of this work was conceptual, and emerged from the comprehensive genomic database that was built for the project. The results generated here indicate that ancient, species-rich and cosmopolitan plant clades such as the Caryophyllales and Cyperaceae are exclusively composed of nonmycorrhizal species, which suggests that the most recent common ancestor of each of these clades was already unable to establish AM symbiosis. This supports the idea that the nonsymbiotic status of some plant lineages can be an evolutionary stable strategy.
Using the model liverwort Marchantia, experimental approaches were used to investigate the function of candidate genes revealed by genomic analyses. The experimental validation of candidate genes is still ongoing work, and is expected to advance our understanding on the biological functions of two genes, a membrane receptor and a gene putatively involved in phosphate metabolism.
Finally, the pathogen assays conducted with M. polymorpha mutants led to the accidental discovery of a new fungi strain that protects Marchantia plants against infection by the pathogen Colletotrichum nymphaeae. This new strain was isolated from an individual that did not show disease symptoms even after 17 days post-inoculation, when all other plants were clearly infected and dead. This protective effect was later confirmed in controlled experiments, conducted with both M. polymorpha and M. paleacea. The fungus C. nymphaeae is a major pathogen of strawberry plants causing the anthracnose disease, and for this reason we are currently conducting an experiment using strawberry plants to determine whether the same protective effect can be observed. The patentability of this discovery is currently being evaluated by the technology transfer office at the host university.
Using the model liverwort Marchantia, experimental approaches were used to investigate the function of candidate genes revealed by genomic analyses. The experimental validation of candidate genes is still ongoing work, and is expected to advance our understanding on the biological functions of two genes, a membrane receptor and a gene putatively involved in phosphate metabolism.
Finally, the pathogen assays conducted with M. polymorpha mutants led to the accidental discovery of a new fungi strain that protects Marchantia plants against infection by the pathogen Colletotrichum nymphaeae. This new strain was isolated from an individual that did not show disease symptoms even after 17 days post-inoculation, when all other plants were clearly infected and dead. This protective effect was later confirmed in controlled experiments, conducted with both M. polymorpha and M. paleacea. The fungus C. nymphaeae is a major pathogen of strawberry plants causing the anthracnose disease, and for this reason we are currently conducting an experiment using strawberry plants to determine whether the same protective effect can be observed. The patentability of this discovery is currently being evaluated by the technology transfer office at the host university.