Additional file 1 of A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation
Creators: name: Alhusayni, Sultan
name: Roswanjaya, Yuda Purwana
name: Rutten, Luuk
name: Huisman, Rik
name: Bertram, Simon
name: Sharma, Trupti
name: Schon, Michael
name: Kohlen, Wouter
name: Klein, Joël
name: Geurts, Rene
Publisher: name: figshare
Year: 2024
Subjects: subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1. Figure S1. Root architecture of Trema orientalis accession RG33 and Parasponia andersonii accession WU1 differs. (A) Primary root length 21 days post-germination. (B) Growth of primary root 1-21 days post-germination (mm/day) (C) The average number of lateral roots per plant, and (D) lateral root density (cm-1 main root) 21 days post-germination (E) Average lateral root length in five selected root 21 days post-germination (mm). n=5 +/- s.e. Different letters above the bars indicate statistical significance (p &lt; 0.05) as determined by student t-test. Tor, red: T. orientalis RG33, Pan, blue: P. andersonii WU1. Plants were grown in vitro on ½ strength modified Hoagland medium in 12 cm square plates. Figure S2. Phylogenetic reconstruction of SYMRK orthologs. Phylogeny was reconstructed on an alignment of SYMRK proteins from 51 plant species belonging to the Nitrogen fixation clade and two species belonging to the Solanales and two species of the Poales. In addition, Trema and Parasponia SYMRK homologous proteins were added to show that these groups are outside the SYMRK clade. Branch support is indicated by posterior probabilities. Lineages are labelled by species name and gene identifier. Figure S3. Parasponia andersoniisymrk CRISPR-Cas9 mutant alleles. (A) Structure of Pansymrk gene spanning 7,280 bp and possessing 15 exons and 14 introns. Indicated are the positions of two sgRNAs (purple arrowheads) in exons 4 and 5. (B) Sequence alignment of the fourth and fifth exons of PanSYMRK in wild type (WT) and the three mutants Pansymrk-4, Pansymrk-5, and Pansymrk-6. Note:Pansymrk-4 is a homozygote mutant possessing a 303 bp deletion whereas line 5 and 6 are bi-allelic. In the bi-allelic mutant lines, both alleles (A and B) are shown. Highlighted in blue and red are the sgRNA target sites and PAM sequences, respectively. Figure S4. Parasponia symrk-5 mutant trans-complementation assay of mycorrhization. (A) Representative image of Pansymrk-5 A. rhizogenes transformed root with empty vector (EV). (B) complementation with pPanSYMRK:PanSYMRK gene and (C) trans-complementation with pPanSYMRK:PanSYMRKGA gene.  Visualization of Rhizophagus irregularis infection 6 weeks post-inoculation. Figure S5. Analysis of SYMRK Intron 12 splice variant expression in T. orientalis and P. andersonii roots. (A) Mapping of root RNA sequence reads to SYMRK gene models of T. orientalis and P. andersonii showing a ~ 300 bp region around intron 12. (B) Difference in intron retention of SYMRK intron 12 and detected by qRT-PCR in P. andersonii and T. orientalis RG33. The barplot represents the means of three biological replicates ± SD. Table S1. Frequency of GA-AG intron splice sites in four plant species. Frequency of GA-AG intron splice sites in annotated gene models of Parasponia andersonii, Trema orientalis, Lotus japonicus, and Medicago trancatula genomes. Total number of introns with GA-AG splice sites are based on available annotations. Table S2. T. orientalis individuals collected in Malaysia, Sabah possess a GA donor splice site at intron 12. Twenty-eight Trema orientalis individuals collected at 5 distinct locations in Malaysia, Sabah all possess a non-canonical GA donor splice site at intron 12. T. orientalis plants collected from locations outside Malaysia were found to possess a GC donor splice site at intron 12. Table S3. List of Golden Gate constructs used in this study. Each construct is identified by a unique number, an ID, and a brief description. The level of Golden Gate assembly for each vector is indicated, as well as the plasmid backbone into which the constructs were cloned. Table S4. Primers used in this study. List of primers, their purpose, and their sequence that were used in various applications in this study.