Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium

Summary As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre‐symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual‐mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B−), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria‐dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less‐investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B− plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre‐symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.


Proteins involved in symbiosis are over-produced in mycorrhizal roots in a CaGg-dependent manner
Among AM-related categories enriched only in B+Myc vs NoMyc, the proteome data set revealed two GO terms, namely calcium ion binding (GO:0005509) and calcium-dependent phospholipid binding (GO:0005544). These two categories contained three annexins (Lj4g3v2823370, Lj0g3v0166899, Lj0g3v0203419), proteins which are characterized by their ability to reversibly interact with membranes in a calcium-dependent manner, involving for example cytoskeleton rearrangements and regulation of membrane traffic (Lizarbe et al., 2013), and having specific roles in root symbioses (Kustova et al., 2020). The Medicago truncatula homologue MtAnn1 has been postulated to have a role in cell cycle reactivation and cytoskeleton rearrangements during the early stages of nodulation (de Carvalho Niebel et al., 1998), while MtAnn2 has been proposed to act in connection with membrane traffic and cytoskeleton rearrangements in arbuscule-containing cells (Manthey et al., 2004). Moreover, only in B+Myc a calreticulin (CRT, Lj5g3v1514670), increased. CRT is an endoplasmic reticulum Ca 2+ buffering protein playing relevant cellular functions. CRT and Ca 2+ may regulate the fungal accommodation inside the cortical cells and arbuscule development (Sujkowska-Rybkowska and Znojek, 2018).

Defence-related genes are activated in mycorrhizal L. japonicus plants, irrespective of the fungal endobacteria
The GO:0006950 category contains a large number of stress-responsive genes which are indirectly involved in plant immunity. A U-box domain-containing protein 36-like (Lj4g3v1440680) was the gene with the highest differential expression (log2fold-change>3) within the category in both contrasts, while about 20 different U-box domain-containing genes were present in both B+Myc and B-roots vs the control. As belonging to this family, E3-class ubiquitin ligases were already found as up-regulated upon mycorrhization by Rhizophagus irregularis in both L. japonicus and tomato (Sugimura and Saito, 2017), as well as involved in abiotic stress responses in legume plants (Song et al. 2017). A number of universal stress proteins (USPs) were strongly induced upon G. margarita colonization being more abundant in the B-Myc line vs B+Myc (5 versus 2 genes, respectively). These proteins, which are highly conserved through all kingdoms of life and highly expressed under diverse environmental constraints, are largely unknown and probably acts as molecular chaperones being involved, as demonstrated in Arabidopsis, in the interaction with thioredoxin-h1 modulating ROS concentration (Chi et al., 2019).

Regulation of Lotus japonicus protein turnover and stability in response to mycorrhization by both G. margarita lines
Both transcriptomics and proteomics revealed that protein metabolism is among the most affected by both mycorrhization and CaGg presence. Among the actors involved in protein stability and maintenance, we found that CaGg presence led to a greater abundance of two proteins identified as isomerase-like protein precursors (Lj5g3v1601820, Lj1g3v0841310), enzymes involved in the unfolded and refolded protein response that, in the endoplasmic reticulum, catalyze the formation of disulfide bonds that stabilize protein conformations (Takemoto et al., 2002;Onda and Kobori, 2014;Kimura et al., 2015;Lu and Christopher, 2008;Peng et al., 2017). Interestingly, the chaperone activity of the protein disulfide isomerases is regulated by its redox status (Wang et al., 2013). Additional categories of protein metabolism revealed a specific pattern (Fig. 2a): as in other mycorrhizal plants, L. japonicus cysteine peptidases (GO:0008234) were up-regulated in B+ and Bmycorrhizal roots in both transcriptome and proteome; interestingly serine-type carboxypeptidase activity (GO:0004185), containing several serine carboxypeptidase-like proteins (SCPLs) resulted to be mostly up-regulated in MycB+ vs NoMyc (z-score>0). SCPLs are a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. Their potential role in symbiosis has already been suggested in Medicago truncatula, where a tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development (Rech et al., 2013). On one hand, all these peptidases are considered relevant during the dismantle of the arbuscules and the recycling of fungal proteins (Floss et al., 2017); on the other, they seem to be involved in the tolerance to oxidative stress. OsBISCPL1-overexpressing plants showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stressrelated genes (Liu et al., 2008).

Stigmasterol biosynthesis in L. japonicus is influenced by the presence of CaGg
In our dataset, another finding related to lipid metabolism concerns the plant production of stigmasterol ( Supplementary Fig. 8.6). Its biosynthesis is triggered by ROS, achieved through both terpenoids and lipid biosynthetic pathways, and finalized by the activity of the CYP710A1 (EC1.14.19.41), a cytochrome p450 (Griebel and Zeier, 2010). In A. thaliana, stigmasterol overproduction, and consequent reduction of the β-sitosterol/stigmasterol ratio in plasma membranes, can be forced by pathogens, favoring their proliferation (Griebel and Zeier, 2010). Two mechanisms, which are presumed to be easily translated to other biological systems, were proposed to explain the role of stigmasterol in pathogen infection: on the one side stigmasterol, when compared to β-sitosterol, is poorly efficient in reducing the membrane permeability upon pathogen attack. On the other side, molecular components involved in plant defense are recruited to motile, sphingolipid-rich platforms, or lipid rafts (Ali et al., 2018); the correct functioning of these components highly depends on the motility of their platforms, which can be significantly reduced by the replacement of β-sitosterol with stigmasterol in the surrounding plasma membrane. In the case of L. japonicus -G. margarita system, our dataset indicates that sphingolipid biosynthetic genes were overall induced by both the B+ and B-fungal lines. By contrast, a higher production of stigmasterol was deducted only in B-Myc from the up-regulation of the squalene monoxygenase (EC:1.14.14.17), SMT1 and 2 (EC:2.1.1.143) and CYP710A1.

Putative plant cell-wall degradation processes in B+ and B-symbiotic mycelium
Even if the absence of plant cell wall-degrading enzymes is peculiar of AM fungi genomes, G. margarita possesses several expanded gene families of Carbohydrate-Active Enzymes (CAZymes) (Venice et al., 2020). Some of these enzymes, such as extracellular peroxidases, are auxiliary and may indirectly attack plant cell wall polymers through oxidative damage, explaining the fungal ability to penetrate plant roots without triggering hypersensitive response in host cells (Venice et al., 2020). In the current dataset, an extracellular dihydrogeodin oxidase/laccase was found to be markedly activated in the B+ symbiotic mycelium only (Supplementary Table 7). Additionally, three CAZymes that target chitin (CE4 family) were up-regulated in the B+ symbiotic mycelium only. These deacetylases might be as well involved in elusion of plant responses, as deacetylated chitin is not recognized by plant receptors (Gow et al., 2007).