Fig. 5.
The m6A methyltransferase MTA interacts with MTB in strawberry. a The working model for m6A installations mediated by the methyltransferase complex in mammals. The m6A methyltransferases METTL3 and METTL14 interact and function as the stable catalytic core for internal m6A installations in the form of heterodimer. b Phylogenetic analysis of eukaryotic m6A methyltransferases. The phylogenetic tree was generated by MEGA (version 5.2). Bootstrap values from 1000 replications for each branch are presented. Species names are abbreviated as follows: Hs, Homo sapiens; Ms, Mus musculus; At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays; Sl, Solanum lycopersicum; Nb, Nicotiana benthamiana; Fa, Fragaria × ananassa; Fve, Fragaria vesca. c Transcript levels of the m6A methyltransferase genes MTA and MTB in diploid woodland strawberry at different developmental stages revealed by RNA-seq. S6, the growth stage 6; RS1, the ripening stage 1; RS3, the ripening stage 3. d Representative images of octoploid cultivated strawberry fruit at various developmental stages. SG, small green; Wt, white; IR, initial red; FR, full red. Scale bar = 1 cm. e Transcript levels of MTA and MTB in octoploid strawberry fruit determined by quantitative RT-PCR. The ACTIN gene was used as an internal control. Data are presented as mean ± standard deviation (n = 3). Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test). f Y2H assay revealing the interactions between MTA and MTB. The MTA fused with the activation domain (AD) of GAL4 (AD-MTA) and the MTB fused with the binding domain (BD) of GAL4 (BD-MTB) were co-expressed in yeast. The transformants were grew on SD/-Leu/-Trp (-LW), and further selected on SD/-Leu/-Trp/-His (-LWH) and SD/-Leu/-Trp/-His/-Ade (-LWHA) with or without X-α-gal. g LCI assay revealing the interactions between MTA and MTB. The MTA fused with the N-terminus of LUC (MTA-nLUC) was co-expressed with the MTB or its MT-A70 domain fused with the C-terminus of LUC (cLUC-MTB or cLUC-MTBD) in N. benthamiana leaves. h, i Subcellular localization (h) and colocalization (i) of MTA and MTB. The MTA-mCherry or/and MTB-eGFP fusion proteins were transiently expressed into N. benthamiana leaves. The N. benthamiana leaves expressing eGFP or/and mCherry were used as the negative control. Scale bar = 10 μm