A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation

Abstract

Background: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK.

Results: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare.

Conclusion: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Keywords: Arbuscular mycorrhizal symbiosis; Common symbiosis signalling pathway; LRR-type transmembrane receptor kinase; Mutualistic endosymbiosis; Nitrogen-fixing nodulation symbiosis; Non-canonical splice site; Parasponia andersonii; Plant evolution; SYMRK; Trema orientalis.

Fig. 1

Trema orientalis accession RG33 and Parasponia andersonii accession WU1 differ in mycorrhizal colonisation. A Comparison of mycorrhization efficiency in the root system of P. andersonii WU1 (blue) and T. orientalis RG33 (red) at 2, 4 and 6 weeks post-inoculation with Rhizophagus irregularis DOAM197198. F%: The frequency of mycorrhiza in the root system. M%: the intensity of mycorrhizal colonisation in the root system. A%: Arbuscule abundance in the root system. B a%: Averaged arbuscule abundance detected in 50 randomly selected 1 cm infected segments of a root system. Error bars represent the SE of 10 biological replicates for each 50 × 1 cm root segment that has been analysed. Analysis was done according to Trouvelot et al. (1986) [26]. C Toluidine blue-stained P. andersonii and D T. orientalis root segment visualising R. irregulates arbuscules 6 weeks post-inoculation. Size bar = 10 μm

Fig. 2

Parasponia andersonii SYMRK is essential for mycorrhization and nodulation. A Nodule numbers formed in P. andersonii empty vector control line (EV) and three Pansymrk mutant lines, 6 weeks post-inoculation with Mesorhizobium plurifarium BOR2. B mycorrhization efficiency in the root system of P. andersonii EV-control and three independent Pansymrk mutant lines 6 weeks post-inoculation with Rhizophagus irregularis DOAM197198. F%: The frequency of mycorrhiza in the infected root system. M%: the intensity of mycorrhizal colonisation in the infected root system. A%: Arbuscule abundance in the infected root system. a%: Averaged arbuscule abundance detected in 50 randomly selected 1 cm segments of a root system. Error bars represent the SE of 10 biological replicates, for each 50 × 1 cm root segment that has been analyzed. Analysis was done according to Trouvelot et al. (1986) [26] (C-F): Toluidine blue-stained P. andersonii EV-control C, Pansymrk-4 D, Pansymrk-5 E, and Pansymrk-6 F root segment visualizing R. irregulates infections 6 weeks post-inoculation. Size bar = 10 μm

Fig. 3

PanSYMRK ectopic expression induces spontaneous nodulation in Parasponia andersonii. (A, B) Bright-field A and green fluorescent image B of P. andersonii A. rhizogenes-transformed roots expressing GFP and PanSYMRK under control of the pLjUBI1 promoter showing spontaneously formed nodule-like structures (6 weeks post planting). C Relative gene expression of PanSYMRK in P. andersonii A. rhizogenes-transformed roots containing an empty vector (EV) or pLjUBI1:PanSYMRK (n = 3). D Longitudinal section of a spontaneously formed nodule-like structure visualizing cortical and pericycle cell divisions

Fig. 4

Parasponia symrk-5 mutant trans-complementation of root nodule symbiosis. A Schematic representation of P. andersonii SYMRK gene structure. Arrowhead points to the location of the introduced GA mutation in PanSYMRK at the 5’-donor splice site of intron 12. B Nodule number per plant formed on Pansymrk-5 A. rhizogenes transformed root with pPanSYMRK:PanSYMRK gene (n = 13). (C-E) Nodule number per plant (n = 5) C, representative image of green flurescence protein (GFP) nodule D and a section through a mature nodule E of Pansymrk-5 A. rhizogenes transformed root with pPanSYMRK:PanSYMRK^GA carrying a GA mutation at the 5’-donor splice site of intron 12. Nodules were harvested and analysed at 8 weeks post inoculation with Mesorhizobium plurifarium BOR2 (OD600 = 0.025)

Fig. 5

Phylogeny of SYMRK including the splice site dinucleotide motifs for intron 12. Phylogeny was reconstructed based on an alignment of SYMRK orthologous proteins from 19 species. Leaves are labelled by their respective species, gene name if available) and gene identifier. The non-canonical GC donor splice site is common in SYMRK intron 12 of dicotyledon species, except in Glycine max SYMRKβ and Pisum sativum SYM19, where GC is substituted by GT. In contrast, only Trema orientalis RG33 possesses a GA motif in this position (highlighted in red)

Fig. 6

SYMRK intron 12 unique non-canonical donor splice site occurs in a Trema orientalis population endogenous to Sabah, Malaysia. A Locations of 28 Trema orientalis specimens collected in Malaysian Borneo, province of Sabah. 1: Sayap, 2: Poring, 3: Mahua, 4: Gunung Alab, and 5: Inobong. Plants were collected in 2012 as described in Merckx et al. (2015) [31] (see also Table S2). Map data © 2023 Google. B The ‘GA’ donor splice site of intron 12 is unique to Trema orientalis of Malaysia, Sabah, whereas related accessions and species possess a non-canonical ‘GC’ at this position in SYMRK