AM fungi play a crucial role in sustainable agriculture due to their numerous benefits. However, a comprehensive exploration of the whole transcriptome and the effectome repertoire of the AM fungus F. mosseae has not yet been performed. Uncovering the transcript structure of F. mosseae `could yield valuable insights into the gene repertoires and molecular mechanisms that govern the interaction of AM fungi with various crop plant species and the resulting agricultural advantages.
In comparison to other arbuscular mycorrhizal (AM) fungal species, F. mosseae demonstrates the presence of a wide array of carbohydrate-active enzymes (CAZymes) (Table S1) and protein kinases (Table S2). In-depth analysis of CAZymes shows the presence of a diverse range of carbohydrate metabolism enzymes in F. mosseae that are important in nutrient recycling and establishing associations with the host. Strikingly, F. mosseae exhibits limited activity across most kinase categories, with the highest abundance in the AGC and CAMK clusters. AGC kinases play a range of roles in fungi, including cell growth, viability, metabolism, programmed cell death, stress resistance, cell wall integrity maintenance, appressoria formation and many more [25,26], while CAMK kinases play a part in calcium-driven signaling pathways, including the MAPK pathway, and are involved in various cellular processes important in the establishment of symbiosis, such as modulation by rhizobia and mycorrhizal infection. The dominance of these kinase groups implies that F. mosseae may have developed specific adaptations and control mechanisms that are highly dependent on AGC and CAMK signaling pathways. In comparison to other species, F. mosseae has fewer kinases under the TK, TKL, and PIKK groups. It also entirely lacks PDHK and RIO kinases. Such distinctions indicate that F. mosseae may employ distinct signaling and regulatory approaches compared to its counterparts.
A diverse set of functional domains have been identified in the F. mosseae transcriptome (Table S3), indicating their involvement in signal transduction and stress response pathways. These domains, such as Acetyltransferases, Actin, Aldo/keto reductase, Crinkler effector protein, HMG box, Kinesins, Methyltransferases, Major Facilitator Superfamily, Nucleotide-sugar transporters, NUDIX domain, and Piwi proteins, play crucial roles in various aspects of mycorrhizal symbiosis. They contribute to protein modification, fungal growth and penetration, detoxification, immune suppression, gene expression regulation, nutrient uptake, cell wall synthesis, and defense responses [19,27,28,29,30,31,32,33].
Chemical cross-talk originating from fungal and plant partners has been reported to regulate interactions between AM fungi and plants. The development of the mycorrhizal relationship has been associated with the high-level expression of these chemical signals via genetic and/or metabolic regulation between both partners. When AM fungi first interact with a host plant root, plant immune responses are reported to be influenced both locally and systemically [33]. The production of strigolactone by plant roots has been suggested as the first stimulus from plants towards initiating endo-symbiotic interactions. The functions of plant-expressed signaling molecules during interactions with AM fungi are being widely explored, and their roles have been well elucidated [34]. However, the molecular signals relayed by AM fungi to establish an association with an enormously large variety of plants remain to be completely deciphered.
Effector proteins expressed by AM fungi are known to regulate defense mechanisms in the host in favor of the symbiotic association. In this study, the effectors expressed by F. mosseae that could be important in establishing the symbiotic association were identified, and their expression was also validated at 3 time points (20 days, 1 month, and 6 months). The observed expression of the HOG1_1, HOG1_2, and KexB effector genes in both hyphae and spore samples sheds light on the active role of these genes in the lifecycle of F. mosseae. Notably, the differential expression of HOG1_1 in spores proximal to host roots underscores the potential influence of spatial positioning on gene activation. This finding aligns with Boon et al. [35], which suggested that spores and hyphae in closer proximity to host roots exhibit heightened symbiotic responses. Such responses might be attributed to the varied temporal molecular signaling depending on the distance between the host root and the spore location. Moreover, the decreased expression of the HOG1_2 effector in the spores from older cultures compared to those from younger cultures provides intriguing insight into the possible age-related modulation of this gene. Interestingly, the lack of expression in the qPCR analysis for the cokel2 effector identified in transcriptome sequences in all examined conditions indicates that its role may be more context-specific or that it might operate under unique environmental or developmental circumstances not covered in this study. This absence warrants further investigation to pinpoint the specific conditions or triggers for cokel2 expression, which could reveal deeper insights into its function and importance in the symbiotic relationship.
With regard to F. mosseae effectors, the HOG1 proteins (HOG1_1 and HOG1_2) are proposed to act within the mitogen-activated protein kinase pathway (MAP kinase) related to the protein kinase (SPS1) family. This pathway plays a cardinal role in cellular functions ranging from proliferation to transcriptional regulation [36]. Upon activation, this kinase migrates to the cell nucleus, influencing nuclear targets [37]. Phosphorylation mechanisms by kinases, exemplified in the MAPK pathway, are vital for signal transduction, metabolism, and other fungal activities [38] and similarly in higher eukaryotes [36]. These pathways also oversee stress responses, immune defense, and modulation of various cellular functions [39,40,41].
Furthermore, the Kex effectors in F. mosseae warrant attention given their significant implications in fungal growth and effector secretion. The literature documents the indispensable role of Kex proteases, e.g., KexB, in effector processing in various fungal species, including pathogens such as Candida albicans [42]. KexB is linked with essential infection-related activities, from hyphal formation to proteinase secretion. In Aspergillus fumigatus, KexB plays a role in N-glycan processing, signifying its influence on fungal cell wall modulation and overall development [43]. Such associations suggest that Kex effectors in AM fungi might be pivotal in fostering and sustaining fungus‒plant root symbiosis.
The CoKEL2 domain in another F. mosseae effector suggests its role in fungal signaling recognition, signalling pathway activation, and mycorrhizal symbiosis-related gene modulation [44]. Kelch domain proteins, as found in Gigaspora rosea, have been verified to participate in protein‒protein interactions [45]. Moreover, Thkel1 gene-produced Kelch domain proteins in Trichoderma harzianum T34 support Arabidopsis thaliana plants against salt stress [46]. Furthermore, Kelch domain protein overexpression in Brassicaceae plants instigates defense responses against pathogens and boosts root colonization, consequently driving plant productivity [39]. In summary, these effectors in F. mosseae underline their centrality in fungal-plant communications, cellular process activations, and mycorrhizal symbiosis-related gene modulation.
Furthermore, to decipher the function of AM fungal effectors in modulating the molecular mechanisms in plants towards symbiotic associations, protein‒protein interaction studies may be important [17,47]. In the current study, the F. mosseae effectors showing potential hits in the PHI database were predicted to have effective protein‒protein interactions with previously identified F. mosseae-induced S. lycopersicum plant proteins [31,41]. Protein‒protein docking revealed positive interactions between F. mosseae’s effector proteins with the HOG domain and the host plant’s defensins, aquaporins, bluecopper, and PTO proteins. F. mosseae effectors with the HOG domain seem to be key mediators in its symbiotic relationship with the host plant. Their interaction with defensins, small, cysteine-rich peptides that play a role in plant defense against pathogens [48], suggests a possible modulation of host immune responses, allowing for effective fungal colonization. However, there is limited information available on the role of host effectors with HOG domains in plant defense mechanisms, and further research is needed to explore the role of effectors with HOG domains and their potential involvement in symbiotic interactions. The interactions with aquaporins, which are pivotal for water and nutrient transport [49], hint at a mechanism where the fungus might influence the plant's hydration and nutrient uptake dynamics, likely enhancing the symbiotic efficiency. The interaction of the HOG domain-containing protein with Bluecopper proteins may indicate potential involvement in electron transport processes or even oxidative stress modulation [50]. Last, by interacting with the PTO protein, a known disease-resistance pathway component, the effector might attempt to modulate plant signaling, balancing defense and symbiosis to benefit both partners [51].
Furthermore, F. mosseae’s effector with the Kex domain showed a positive interaction with plant aquaporin proteins. The observed interaction distinctly highlights the pivotal role of hydration and nutrient conveyance within the symbiotic framework. Alterations introduced by the Kex domain on the aquaporin's functionality may profoundly influence the equilibrium of water and nutrient transfer dynamics. It is conceivable that this effector orchestrates the plant's hydration mechanisms to establish a milieu optimal for fungal proliferation and reciprocal nutrient distribution, underscoring the complex molecular interplay inherent to such a symbiotic association.
Positive interactions were also observed between F. mosseae effectors with the CoKel2 domain and the plant cytochrome P450 proteins. Given the diverse activities of cytochrome P450 enzymes in many biosynthetic pathways [52], this interaction could have an impact on a variety of plant metabolic processes. Whether it is the regulation of hormone production affecting growth and response dynamics or the manipulation of defensive component synthesis, such as phytoalexins, the CoKel2 domain could be a key in determining the biochemical landscape of the host during symbiosis.
Drawing from the insights of this study, it is evident that F. mosseae has evolved sophisticated molecular strategies to foster and sustain symbiotic ties with plant hosts. This mutual relationship, characterized by a plethora of protein interactions, effector functions, and signaling pathways, mirrors the long evolutionary journey and mutual reliance between the two biological realms. The range of effectors in F. mosseae, with their distinctive domains interacting with particular host proteins, depicts a synchronized adjustment of plant activities, from defense responses to the dynamics of nutrient and water absorption.
Additionally, even though the full role of each effector and its contribution to symbiosis demands more research, the data presented here pave the way for subsequent studies. Building on these findings, further research can delve into the molecular details, which might eventually pave the way for refining farming techniques that capitalize on fortified plant-fungi alliances.