Skip to main content
NCBI home page
Plant Biotechnology Journal logoLink to Plant Biotechnology Journal
. 2021 Nov 17;20(3):511–525. doi: 10.1111/pbi.13733

The m6A reader MhYTP2 regulates MdMLO19 mRNA stability and antioxidant genes translation efficiency conferring powdery mildew resistance in apple

Tianli Guo 1, † , Changhai Liu 1, † , Fanxin Meng 1, Liu Hu 1, Xiaomin Fu 1, Zehua Yang 1, Na Wang 1, Qi Jiang 1, Xiuzhi Zhang 1, Fengwang Ma 1,✉
PMCID: PMC8882777  PMID: 34679252

Summary

N6‐methyladenosine (m6A) reader protein plays an important role in trichome morphology, developmental timing and morphogenesis in Arabidopsis. However, the function of m6A readers in plant‐microbe interaction remains unclear. Here, a Malus YTH‐domain family protein MhYTP2 was initially characterized as an m6A reader. MhYTP2 overexpression increased mRNA m6A modification level and translation efficiency. The m6A in the exon regions appeared to destabilize the mRNAs, whereas m6A in the untranslated regions positively correlated with the associated mRNA abundance. MhYTP2 overexpression enhanced apple powdery mildew resistance, possibly by rapidly degrading the bound mRNAs of MdMLO19 and MdMLO19‐X1 and improving the translation efficiency of the antioxidant genes. To conclude, the results shed light on the apple m6A profile, the effect of MhYTP2 on m6A profile, and the m6A roles in MdMLO19 and MdMLO19‐X1 mRNAs stability and glutamate dehydrogenase 1‐like MdGDH1L mRNA translation efficiency.

Keywords: YTH domain, m6A reader, mRNA stability, translation efficiency, powdery mildew, apple

Introduction

Biotic stresses, including fungal and bacterial diseases, inevitably challenge plant survival and reproduction. Powdery mildew (PM) caused by Podosphaera leucotricha is a devastating disease of apple (Malus × domestica Borkh.), one of the most widely cultivated and economically important fruits worldwide. PM causes white spots on young green tissues, and the infected leaves crinkle, curl and prematurely drop. Blossoms and fruits are not the primary targets of the PM fungi; however, infection of these tissues is also possible (Foulongne et al., 2003; Turechek and Carroll, 2004; Xiao et al., 2001). Subsequently, PM affects apple fruit development and quality, reducing fruit yield and market value.

Plants have evolved several defence mechanisms over time. The plant disease stress responses involve molecular, physiological and cellular adaptations. At the molecular level, plants control gene expression levels by reprogramming their genetic machinery, via which they implement defence mechanisms and increase stress tolerance to minimize the effects of the accompanying biological damage (Fujita et al., 2006). Genetic regulatory networks, including post‐transcriptional control of gene expression, serve as powerful strategies in disease stress responses (Huh and Paek, 2014). For example, apple Mildew Locus O 19 (MdMLO19) plays a pivotal role in PM resistance; its knockdown significantly induced PM resistance (Pessina et al., 2014, 2016). Meanwhile, the absence of Mlo primes the responsiveness for the onset of multiple defence functions in barley (Büschges et al., 1997; Wolter et al., 1993). RNA binding proteins (RBPs), the important components of such regulatory networks that remain active during and after transcription (Chen and Varani, 2013), identify and combine with target RNAs via the RNA binding domains (RBDs). Among the various post‐transcriptional modifications, N6‐methyladenosine (m6A) RNA methylation is one of the most pivotal internal modifications and a conserved post‐transcriptional mechanism that enriches and regulates genetic information in eukaryotes (Yue et al., 2019). The m6A modification accounts for 80% of the RNA base modifications in eukaryotic cells (Kierzek and Kierzek, 2003). Studies have shown that m6A modification in plants influences various growth and development phenomena, including embryo development and seed germination (Vespa et al., 2004; Zhong et al., 2008), apical meristem and flower development (Bodi et al., 2012; Duan et al., 2017; Růžička et al., 2017; Shen et al., 2016), microspore development (Zhang et al., 2019), root development (Chen et al., 2018), leaf surface trichome development (Arribas‐Hernández et al., 2018; Bodi et al., 2012; Chen et al., 2018; Scutenaire et al., 2018; Vespa et al., 2004; Wei et al., 2018), and fruit ripening (Zhou and Tian, 2019).

Plant m6A modification influences stress responses also. Studies have shown that the Arabidopsis m6A reader proteins evolutionarily conserved C‐terminal region1 (ECT1) and evolutionarily conserved C‐terminal region2 (ECT2) interact with stress response protein calcineurin B‐like‐interacting protein kinase1 (CIPK1) (Ok et al., 2005), and ECT2 affects mRNA relocation under heat stress (Scutenaire et al., 2018; Wei et al., 2018). Arabidopsis m6A demethylase ALKBH9B interacts with the viral coat protein and RNA and modulates alfalfa mosaic viral infection (Martínez‐Pérez et al., 2017). Meanwhile, the m6A hypomethylation plays a positive role in drought response in maize (Miao et al., 2020). Studies have demonstrated that these functions of m6A modification in stress responses were achieved through m6A regulating mRNA processing and metabolism, including mRNA transport, degradation (Luo et al., 2014; Meyer and Jaffrey, 2014; Schwartz et al., 2013; Shi and Wei, 2019), stability (Wang et al., 2014), splicing (Zhao et al., 2014) and translation efficiency (Wang et al., 2015; Zhou et al., 2015). The role of methylation depends on the m6A reader protein (Bi et al., 2019; Shi et al., 2019). Arabidopsis ECT2 protein, containing an YTH domain, has been proven to be an m6A reader protein (Scutenaire et al., 2018; Wei et al., 2018). However, the role of apple RBP MhYTP2, a homologue of Arabidopsis ECT2, in the m6A binding function is unknown. Moreover, the m6A methylation machinery and the m6A characteristics and functions responsible for regulating pathological processes of horticultural crops remain largely unknown.

One of the major stress response mechanisms at the physiological level is the increase in the levels of antioxidants, such as ascorbic acid (AsA) and glutathione (GSH), to reduce stress‐induced intracellular ROS accumulation (Gururani and Venkatesh, 2015). The AsA‐GSH cycle, an antioxidant system, plays an important role in scavenging hydrogen peroxide (H2O2) under stress (Wang et al., 2012). GSH, a major endogenous antioxidant, participates in H2O2 detoxification via various glutathione peroxidases (Gu and Chauhan, 2015). Meanwhile, the GSH synthesis regulated by GDH helps plants absorb the excessive NH4+ to produce glutamate, for the first committed step in GSH synthesis (Skopelitis et al., 2006; Tercé‐Laforgue et al., 2013; Yan et al., 2021). However, the direct role of GDH in PM resistance in apple is unclear.

Therefore, the present study aimed at characterizing the Malus YTH domain‐containing RNA binding protein 2 (MhYTP2). The MhYTP2 was cloned from Malus hupehensis (pamp.) Rehd., a PM‐resistant genotype, and overexpressed in Malus domestica cv. ‘Roya Gala’ to explore its function in PM resistance. Further, a global mRNA methylation and transcription analysis of the 35S::MhYTP2 line OE‐2 and the WT plants was performed to examine the influence of MhYTP2 on the overall methylation level. RNA immunoprecipitation (RIP)‐sequencing was conducted to identify the targets of MhYTP2, and ribosome profiling (Ribo‐seq) was performed to analyse the role of MhYTP2 in regulating translation efficiency. Collectively, our work demonstrates that the m6A binding function of MhYTP2 controls PM resistance by binding and affecting the mRNA stability of MdMLO19 and MdMLO19‐X1 while facilitating the translation efficiency of antioxidant genes.

Results

Overexpression of MhYTP2 increases resistance to PM

Three 35S::MhYTP2 lines (OE‐1, OE‐2, OE‐3) showed increased resistance to PM compared with the WT plants in the pots under field conditions (Figure 1a). The 35S::MhYTP2 lines and the WT plants were also tested for their susceptibility to PM under laboratory conditions by artificial inoculation in the following study. Similarly, the 35S::MhYTP2 lines demonstrated lower disease severity than the WT plants 15 days after PM inoculation under laboratory conditions. Although the leaves of the 35S::MhYTP2 lines were partially infected, the spread of spores on the adaxial leaf surface was significantly reduced compared with the WT (Figure 1b). The WGA staining revealed that the pathogen’s spread in the 35S::MhYTP2 lines was significantly reduced compared with the WT (Figure 1c). Besides, the trypan blue staining indicated fewer disease symptoms in 35S::MhYTP2 lines than in the WT (Figure 1d).

Figure 1.

Figure 1

Open in a new tab

Overexpression of MhYTP2 increases resistance to powdery mildew (PM) under field and laboratory conditions. (a) Disease severity with PM in the 35S::MhYTP2 lines and the wild type (WT) plants under natural conditions. Bar, 1 cm. (b) Disease severity recorded 15 days after inoculation with PM in the 35S::MhYTP2 lines and the WT plants under laboratory conditions. Bar, 1 cm. (c) Spread of the pathogenic hyphae in the inoculated leaves under laboratory conditions. Bar, 1 mm. (d) Trypan blue staining of the PM inoculated leaves under laboratory conditions. Bar, 1 cm.

Overexpression of MhYTP2 modifies the expression levels of m6A writers and erasers

Arabidopsis ECT2 protein has been reported as an m6A reader (Scutenaire et al., 2018; Wei et al., 2018). Sequence alignment revealed that MhYTP2 is a homolog of Arabidopsis ECT2 protein with a YTH domain (Figure S1). Therefore, we hypothesized that MhYTP2 is also an m6A reader and may affect the mRNA methylation level. The expression levels of several important methyltransferase and demethylase genes (Wei et al., 2018) in the 35S::MhYTP2 lines and WT plants were analysed to investigate the effect of MhYTP2 on the mRNA methylation levels. The quantitative real‐time PCR (RT‐PCR) analysis revealed that the expression levels of the N6‐adenosine‐methyltransferase genes MT‐A70‐like (MdMTA) and non‐catalytic subunit MTB (MdMTB) in the 35S::MhYTP2 lines were slightly lower than that in the WT plants. Meanwhile, the FKBP12‐interacting protein of 37  kDa (MdFIP37) in the 35S::MhYTP2 lines was significantly lower than that in the WT plants (Figure 2a). The transcript levels of demethylases MdALKBH2 and MdALKBH9B were similar in the 35S::MhYTP2 lines and WT plants; however, the transcript level of MdALKBH6 was significantly lower in the 35S::MhYTP2 lines than in the WT (Figure 2b). These observations indicate that MhYTP2 may influence the overall methylation level in apple.

Figure 2.

Figure 2

Open in a new tab

Overexpression of MhYTP2 alters the transcript levels of methyltransferase and demethylase genes. The transcript levels of the (a) methyltransferases MdMTA, MdMTB and MdFIP37 and (b) the demethylases MdALKBH2, MdALKBH6 and MdALKBH9B in the 35S::MhYTP2 lines and the WT plants. Quantitative RT‐PCR was performed to determine the transcript levels using the MdActin gene as the internal control. Data are presented as mean ± standard deviation (n = 4). Different letters indicate significant differences between WT and 35S::MhYTP2 plants, based on one‐way ANOVA and Tukey’s multiple range test (P < 0.05).

Variations in methylome between 35S::MhYTP2 line OE‐2 and WT

Further, m6A‐seq (Dominissini et al., 2013) was performed to assess the transcriptome‐wide m6A methylation in the WT and 35S::MhYTP2 line OE‐2 using leaf samples to investigate the potential influence of MhYTP2 on mRNA methylation. The mRNA samples from WT and 35S::MhYTP2 line OE‐2 were fragmented into approximately 100 nucleotide‐long oligonucleotides (input) before IP using an anti‐m6A affinity purified antibody. Libraries were prepared from input control and IP fragments and subjected to parallel sequencing. The mRNA samples were prepared to perform two independent m6A‐seq experiments. High‐confidence m6A peaks detected in both biological replicates for each line were used for subsequent analysis. A total of 15,225 and 17,303 high‐confidence m6A peaks were identified from the WT and 35S::MhYTP2 line OE‐2 leaves (fold change ≥ 2; P < 0.05), respectively. Here, 12,484 transcripts displayed increased m6A levels, while only 2024 transcripts showed decreased m6A enrichment in 35S::MhYTP2 line OE‐2 compared with the WT plants (fold change ≥ 2; P < 0.05), suggesting a global increase in m6A methylation in 35S::MhYTP2 line OE‐2 compared with the WT plants (Figure 3a) and the influence of MhYTP2 on the overall methylation level. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of m6A‐enriched transcripts differently between 35S::MhYTP2 line OE‐2 and WT was performed to gain functional insights into the role of MhYTP2. The analysis showed that the upregulated transcripts in 35S::MhYTP2 line OE‐2 were enriched in RNA degradation and plant‐pathogen interaction pathways. Downregulated transcripts were mainly enriched in the hormone signal transduction and biosynthesis of flavonoid pathways, implicating the crucial role of MhYTP2 in multiple biological processes (Figure 3b). Further, the distribution of m6A peaks in the whole apple transcriptome was analysed. Each transcript was divided into three non‐overlapping segments based on the reference annotation: 5′ untranslated region (UTR), coding sequence (CDS) and 3′ UTR. As shown in Figure 3c, the meta‐genomic profiles of m6A peaks in all samples indicated that m6A modifications were highly enriched around the CDSs and the 3′ UTRs. Further, the distributions of m6A peaks in the WT and 35S::MhYTP2 line OE‐2 plants were analysed. The percentage of m6A peaks in the CDS regions was lower in the 35S::MhYTP2 line OE‐2 plants (48.30%) than that in the WT plants (53.20%), while those in the 5′ UTRs (12.50% vs. 10.00%) and the 3′ UTRs (39.20% vs. 36.80%) were higher. The m6A modifications around the UTRs in the 35S::MhYTP2 line OE‐2 were highly enriched than in the WT plants (Figure 3c), revealing the effect of MhYTP2 on the stability and translation of the m6A‐modified mRNA (Luo et al., 2020; Wang et al., 2014).

Figure 3.

Figure 3

Open in a new tab

Variations in methylome between 35S::MhYTP2 line OE‐2 and WT. (a) Venn diagrams showing the overlap of m6A peaks identified in two independent m6A‐seq experiments from the leaves of WT and 35S::MhYTP2 line OE‐2. Only the peaks identified in two biological replicates were the confident peaks and used for subsequent data analysis. The number of different m6A‐modified transcripts in the 35S::MhYTP2 line OE‐2 and the WT. (b) KEGG analysis of the m6A‐containing transcripts identified via m6A‐seq. KEGG analysis was performed using the upregulated and downregulated transcripts. (c) Metagenomic profiles of peak summit distributions along with the transcripts composed of three rescaled non‐overlapping segments (5′ UTR, CDS, and 3′ UTR; UTR, untranslated region; CDS, coding sequence). (d) Proportion of the m6A‐modified transcripts containing different m6A peaks and binding motifs identified by HOMER based on the identified MhYTP2‐binding peaks. (e) Diagram showing the correlation between m6A mRNA methylation and gene transcript levels in apple. (f) m6A modification levels of MdMLO19 and MdMLO19‐X1 in the 35S::MhYTP2 line OE‐2 and the WT plants. The arrows indicate the gene direction from 5′ to 3′ end. (g) Lifetime of MdMLO19 and MdMLO19‐X1 mRNAs in the 35S::MhYTP2 line OE‐2 and the WT plants treated with actinomycin D in the transcription inhibition assay. Data are represented as mean ± standard deviation (SD, n = 3 biological replicates * 3 technical replicates). TI, transcription inhibition. (h) MdMLO19 and MdMLO19‐X1 expression levels in the 35S::MhYTP2 lines and the WT plants. Data are represented as mean ± standard deviation (SD, n = 3). Different letters indicate significant differences between WT and 35S::MhYTP2 plants on the same day, based on one‐way ANOVA and Tukey’s multiple range test (P < 0.05).

Among the gene transcripts containing m6A modification, the majority contained one m6A peak (81.04%), followed by a few with two m6A peaks (16.27%); only a few exhibited three or more peaks (2.69%) (Figure 3d), with levels consistent with those in Arabidopsis and tomato (Luo et al., 2014; Wan et al., 2015; Zhou et al., 2019). Besides, hypergeometric optimization of motif enrichment (HOMER; http://homer.ucsd.edu/homer/) was applied (Heinz et al., 2010) to identify the sequence motifs enriched within the m6A peaks in apple. Clustering of the m6A peaks using HOMER identified the “URUAY” sequence motif in apple (Figure 3d), similar to that in Arabidopsis (Wei et al., 2018), tomato (Zhou et al., 2019), and maize (Miao et al., 2020). These data suggest the conservation in m6A modification across plant species.

Studies have reported that m6A deposition influences mRNA abundance (Duan et al., 2017; Luo et al., 2014; Shen et al., 2016; Wan et al., 2015; Wang et al., 2014; Wei et al., 2018; Zhao and Roundtree, 2017). Therefore, RNA‐seq was performed to evaluate the potential correlation between m6A mRNA methylation and gene transcript levels in apple. Comparison of the differentially expressed genes (DEGs; fold change ≥ 2; P < 0.05) identified via RNA‐seq with the transcripts showing altered m6A levels between the 35S::MhYTP2 line OE‐2 and WT plants revealed that the m6A modification in the exon regions appeared to destabilize the mRNAs, whereas m6A in the UTRs was positively correlated with the abundance of associated mRNAs (Figure 3e). MdMLO19, a well‐known S‐gene involved in apple defence against PM (Pessina et al., 2014, 2016), was not identified in the 35S::MhYTP2 line OE‐2; however, the 3′ UTR of MdMLO19 was m6A‐modified in the WT plants. In addition, MdMLO19‐X1 was identified in the 35S::MhYTP2 line OE‐2 with m6A hypermethylation in the exon regions than the WT plants (Figure 3f). MdMLO19‐X1 is an isoform of the previously reported MdMLO19 (Pessina et al., 2014, 2016), with an mRNA sequence similarity of 52.96%, and an amino acid sequence similarity of 63.01% (Table S1). In fact, in addition to MdMLO19 and MdMLO19‐X1, MdMLO1, MdMLO1‐like and MLO11‐like X2 that have not been tested for their function in PM resistance in apple were also found with variations in methylome between 35S::MhYTP2 line OE‐2 and WT.

Furthermore, m6A methylation has been demonstrated to decrease mRNA stability (Duan et al., 2017; Shen et al., 2016; Wang et al., 2014; Zhao et al., 2017). Considering the role of m6A modification in exon regions in mRNA destabilization and that in UTRs in mRNA stability, we hypothesized that MhYTP2 promoted the degradation of MdMLO19 and MdMLO19‐X1 transcripts. Therefore, the lifetime of MdMLO19 and MdMLO19‐X1 transcripts was measured by blocking the transcription with actinomycin D. The MdMLO19 and MdMLO19‐X1 transcripts were degraded rapidly after actinomycin D treatment in the 35S::MhYTP2 line OE‐2 compared with the WT plants (Figure 3g), which suggests that m6A modification promotes MdMLO19 and MdMLO19‐X1 mRNAs degradation. These observations indicate that the site‐specific m6A modification destabilizes MdMLO19 and MdMLO19‐X1 mRNAs. Further quantitative RT‐PCR analysis revealed significantly lower expression levels of MdMLO19 and MdMLO19‐X1 in the 35S::MhYTP2 line OE‐2 than the WT plants after PM inoculation (Figure 3h).

YTH‐domain protein MhYTP2 is an m6A reader and MhYTP2 associates with m6A‐containing MdMLO19 and MdMLO19‐X1

MhYTP2 was identified as a homolog of Arabidopsis ECT2 protein with a YTH domain (Figure S1). Studies have proven ECT2 as an m6A reader protein (Scutenaire et al., 2018; Wei et al., 2018). RIP sequencing was performed using 35S::MhYTP2‐Flag apple calli line MhYTP2‐F2 to determine whether MhYTP2 can bind with m6A‐containing MdMLO19 and MdMLO19‐X1 mRNAs (Figure S2). Unfortunately, MdMLO19 and MdMLO19‐X1 mRNAs were not detected in MhYTP2‐flag IP mRNA samples (Figure 4a). Consequently, the MhYTP2‐His protein from Escherichia coli BL21 was expressed and purified to perform the in vitro RNA electrophoretic mobility shift assay (EMSA) with synthetic 42‐mer RNAs containing either m6A or A. The EMSA showed a direct binding between MhYTP2 and the m6A‐modified MdMLO19 and MdMLO19‐X1 mRNAs, with fourteen and eight “URUAY” motifs in their mRNA sequences, respectively (Appendix S1). (Figure 4b). The result indicated that MhYTP2 binds to RNA transcripts that harbour m6A sites. Thus, MhYTP2, like human and Arabidopsis YTH‐domain family proteins, was confirmed as an m6A reader protein. Therefore, the study’s observations indicate that MhYTP2 binding with and degrading MdMLO19 and MdMLO19‐X1 mRNAs may be a possible route via which MhYTP2 improved apple resistance to PM.

Figure 4.

Figure 4

Open in a new tab

Target genes of MhYTP2 identified by RIP‐seq and EMSA. (a) GO analysis of the MhYTP2‐binding protein transcripts identified in RIP‐seq (n = 3). EMSA validation of the interaction between (b) MhYTP2 and m6A‐modified MdMLO19 mRNA and (c) MhYTP2 and m6A‐modified MdMLO19‐X1 mRNA.

Overexpression of MhYTP2 promotes ribosome occupancy

Gene ontology (GO) analysis of the RIP‐binding transcripts showed that the bound transcripts in 35S::MhYTP2 line OE‐2 compared with the WT plants were enriched for plant‐pathogen interaction and ribosome (Figure 4a). Recent studies showed that m6A modification regulates translation efficiency (Wang et al., 2015; Zhou et al., 2021). Therefore, ribosome profiling was used to assess the ribosome density of each transcript mRNA with or without MhYTP2 overexpression to explore the role of MhYTP2 in mRNA translation. Overexpression of MhYTP2 changed the main distribution range of the translation efficiency data set (Figure 5a). Further analysis based on the abundance ratio of mRNA in the polysomal RNA versus the total RNA (ribosome occupancy) (Merchante et al., 2015) combined with RIP‐seq results revealed that MhYTP2 overexpression enhanced the translation efficiency of MhYTP2 targets and non‐targets (Figure 5b). GO analysis of the different ribosome occupancy transcripts (fold change ≥ 2; P < 0.05) showed that the transcripts were enriched in the antioxidation system (Figure 5c), suggesting the effect of MhYTP2 on the apple antioxidant system.

Figure 5.

Figure 5

Open in a new tab

MhYTP2 enhances the translation of MhYTP2 target and non‐target transcripts. (a) Overexpression of MhYTP2 enhanced translation efficiency. Density distribution of the log10‐fold changes in translation efficiency between the 35S::MhYTP2 line OE‐2 and the WT plants. (b) Overexpression of MhYTP2 enhanced the translation efficiency of MhYTP2 targets and non‐targets. (c) GO analysis of the transcripts with different translation efficiencies identified in Ribo‐seq.

Overexpression of MhYTP2 enhances MdGDH1L translation efficiency

The different ribosome occupancy transcripts in 35S::MhYTP2 line OE‐2 compared with the WT plants were enriched in the antioxidation enzymes (Figure 5c), including MdGDH1L, which affects the synthesis of GSH. The m6A‐seq analysis of the 35S::MhYTP2 line OE‐2 and the WT plants showed that the MdGDH1L mRNA contains m6A sites at 3′UTR. The m6A modification level of MdGDH1L was significantly higher in 35S::MhYTP2 line OE‐2 than that in WT plants (Figure 6a). The RNA immunoprecipitation‐seq conducted using 35S:MhYTP2‐Flag apple calli line MhYTP2‐F2 (Figure S2) showed that M dGDH1L mRNA was enriched in MhYTP2‐flag IP mRNA samples (Figure 6b). Therefore, MhYTP2‐His protein from BL21 Escherichia coli was expressed and purified to perform the in vitro RNA EMSA with synthetic 42‐mer RNAs containing either m6A or A to gain insight into the MdGDH1L m6A binding activity of MhYTP2. The EMSA showed that MhYTP2 bound with m6A‐modified MdGDH1L mRNA (Figure 6b). The MdGDH1L mRNA sequence contains seven “URUAY” motifs (Appendix S1). Furthermore, an overall increase in translation efficiency of the MdGDH1L mRNA was observed by ribosome profiling in 35S::MhYTP2 line OE‐2 (Figure 6c). Meanwhile, the activity of the GDH was significantly higher in 35S::MhYTP2 lines than in the WT (Figure 6d). Further evaluation revealed that GSH and AsA levels in 35S::MhYTP2 lines were higher than the WT plants under normal and PM inoculated conditions (Figure 6e). These findings indicate that MhYTP2 overexpression improved its target MdGDH1L mRNA translation efficiency leading to more MdGDH1L protein and elevated antioxidant activity, resulting in enhanced PM resistance (Figure 6f). These data suggest that critical gene in the antioxidation system undergo m6A‐mediated post‐transcriptional regulation, facilitating translation to increase protein content in response to stress.

Figure 6.

Figure 6

Open in a new tab

MhYTP2 promotes the translation efficiency of MdGDH1L. (a) m6A modification levels of MdGDH1L in the 35S:: MhYTP2 line OE‐2 and the WT plants. The arrows indicate the gene direction from the 5′ to 3′ end. (b) RIP‐seq and EMSA validation of the interaction between MhYTP2 and m6A‐modified MdGDH1L mRNA. MhYTP2‐F2 represents transgenic 35S::MhYTP2‐Flag apple callus line 2. (c) Translation efficiency of MdGDH1L in the 35S::MhYTP2 line OE‐2 and the WT plants. (d) Changes in GDH enzyme activities in apple leaves under normal and infected conditions. (e) Changes in GSH and AsA accumulation. Parameters were measured at the start and 15 days after inoculating PM. Data are represented as mean ± standard deviation (SD, n = 3). Different letters indicate significant differences between WT and 35S::MhYTP2 plants on the same day, based on one‐way ANOVA and Tukey’s multiple range test (P < 0.05) in (d) and (e). FW, fresh weight. (f) MdGDH1L protein expression in the 35S::MhYTP2 lines and the WT plants at the start and 15 days after inoculating PM. Asterisks indicate significant differences (***P < 0.001).

MhYTP2 modulates genes encoding transcription elongation factors and translation initiation factor

Further, the effect of MhYTP2 on the translation efficiency of other genes involved in the antioxidation system was evaluated based on the abundance ratio of mRNA in the polysomal RNA versus the total RNA (Merchante et al., 2015). The genes L‐ascorbate peroxidase (APX) and peroxidase (PER) exhibited significant changes in translation efficiency when MhYTP2 was overexpressed (Figure 7a). Besides, the activities of APX and POD were significantly higher in 35S::MhYTP2 lines than in the WT (Figure S3). This change could not be due to m6A deposition because the transcripts of these genes were not m6A‐modified according to the m6A‐seq data sets. Therefore, we speculated that MhYTP2 might regulate the translation efficiency of numerous transcripts beyond direct m6A modification. As expected, MhYTP2 overexpression altered the translation efficiency of mRNAs of several antioxidation‐related genes, such as APX2, PER3, PER42 and PER47, which do not contain m6A modification.

Figure 7.

Figure 7

Open in a new tab

MhYTP2 modulates genes encoding transcription elongation factors and translation initiation factor and indirectly enhances translation efficiency of several antioxidant genes. (a) Translation efficiency of MdAPX2, MdPER3, MdPER42, and MdPER47 in the 35S::MhYTP2 line OE‐2 and the WT plants. (b) m6A modification levels of MD17G1038800, MD14G1104300 and MD10G1126600 in the 35S::MhYTP2 line OE‐2 and the WT plants. The arrows indicate the gene direction from the 5' to 3' end. (c) RIP‐seq validation of the MhYTP2‐binding m6A‐modified MD17G1038800, MD14G1104300, and MD10G1126600 mRNAs. MhYTP2‐F2 represents transgenic 35S::MhYTP2‐Flag apple callus line 2. Asterisks indicate significant differences (***P < 0.001; **P < 0.01; *P < 0.05).

Detailed analysis of the m6A‐seq and RIP‐seq data revealed that the transcripts of genes encoding transcription elongation factors (MD17G1038800 and MD14G1104300) and translation initiation factor (MD10G1126600) (Table S2), which play pivotal roles in facilitating protein synthesis by promoting the elongation of mRNA transcription and initiation of mRNA translation, respectively, exhibited m6A hypermethylation in the UTRs in 35S::MhYTP2 line OE‐2 (Figure 7b). RIP analysis showed direct interactions between MhYTP2 and the transcripts of MD17G1038800, MD14G1104300 and MD10G1126600 (Figure 7c). Taken together, MhYTP2 may modulate the translation efficiency of its non‐targets indirectly by regulating m6A‐modified mRNAs of transcription elongation factors and translation initiation factor in addition to modulation of translation efficiency via direct binding with the m6A‐modified target, such as MdGDH1L mRNA. The results collectively indicate that MhYTP2 increased PM resistance by rapidly degrading MdMLO19 and MdMLO19‐X1 mRNAs and increasing the antioxidant genes translation efficiency. Based on these results, a model has been proposed for the MhYTP2 function in apple PM disease resistance (Figure 8).

Figure 8.

Figure 8

Open in a new tab

m6A reader protein MhYTP2 regulates apple resistance to PM.

Discussion

Several researchers have investigated m6A of mammalians, and the discovery of m6A demethylases and the mapping of the m6A methylomes in the mammalian systems have indicated that m6A methylation of mRNA is a reversible and dynamic process with regulatory functions (Dominissini et al., 2012; Fu et al., 2014; Jia and Fu, 2013; Jia et al., 2011; Meyer et al., 2012; Nilsen, 2014; Zheng et al., 2013). Further characterization of the mammalian reader protein YTHDF2 that recognizes m6A modifications and subsequently affects mRNA stability indicated the importance of m6A in post‐transcriptional regulation of gene expression (Fu et al., 2014). Studies have shown that YTHDF1 mediates translation and promotes translation efficiency (Wang et al., 2015) and YTHDF3 plays a significant role in modulating the translation of m6A‐modified mRNAs (Li et al., 2017). Although the data available is scarce, the situation in plants seems to be similar. The Arabidopsis YTH‐domain m6A reader protein ECT2 accelerated the degradation of three transcripts related to trichome morphogenesis, thereby affecting trichome branching (Scutenaire et al., 2018; Wei et al., 2018). Meanwhile, the present study focused on the m6A binding properties of MhYTP2 and its effect on mRNA and confirmed that the apple YTH‐domain protein MhYTP2 is an m6A reader.

Overexpression of MhYTP2 in apple modified the expression levels of genes encoding m6A writers and erasers. The m6A writers with methyltransferase function for MdFIP37and that with demethylase function for MdALKBH6 were significantly downregulated in the 35S::MhYTP2 lines. Based on the finding that MhYTP2 modified the expression levels of genes encoding m6A writers and erasers, the transcriptome‐wide m6A sequencing was conducted. Transcriptome‐wide m6A distributions in the 35S::MhYTP2 line OE‐2 and WT apple plants indicated that the methylation sites are highly conserved compared with that in Arabidopsis and tomato (Scutenaire et al., 2018; Wei et al., 2018; Zhou et al., 2019), indicating a fundamental function of m6A in plants.

Further, the relationship between m6A distribution and gene expression was analysed. Importantly, the discovered features of the m6A distribution associated with gene expression in apple mRNAs were found different from Arabidopsis. The distinct enrichment of m6A in the exon regions was correlated with the overall upregulation of mRNA expression level in Arabidopsis (Scutenaire et al., 2018; Wei et al., 2018), while that in the apple was correlated with the overall downregulation of mRNA expression level in this study. Noticeably, m6A methylation at UTRs was positively correlated with gene expression. These observations indicated that the correlation between methylation level and gene expression depends on the modification site. Moreover, the m6A modifications in the 35S::MhYTP2 line OE‐2 plants were highly enriched around the UTRs than that in the WT plants, which reveals that MhYTP2 may mainly affect the stability and translation of the m6A‐modified mRNA (Anderson et al., 2018; Meyer et al., 2015). We speculate that the role MhYTP2 plays on the stability and translation of the m6A‐modified mRNAs associated with disease resistance may affect apple defence against PM.

Studies have shown that the natural and artificial loss‐of‐function mutations of MLO S‐genes reduce susceptibility to PM pathogens (Büschges et al., 1997). MdMLO19 induced by PM is a functional S‐gene in apple, and its knockdown substantially reduced PM susceptibility (Pessina et al., 2016). Nevertheless, the regulatory mechanisms underlying the MdMLO19 signalling pathway, especially the regulation of the MdMLO19 gene at the post‐transcriptional level, remain largely unknown. In this work, MdMLO19 and MdMLO19‐X1, an isoform of MdMLO19, underwent m6A‐mediated post‐transcriptional regulation, which showed significant decrease in m6A modification levels of MdMLO19 in the 3′ UTR and significant induction in m6A modification levels of MdMLO19‐X1 in the exon regions, respectively, in the 35S::MhYTP2 line OE‐2 compared with the WT plants. The changed m6A modification levels of MdMLO19 and MdMLO19‐X1 mRNAs accelerated their degradation in 35S::MhYTP2 line OE‐2. These findings provide a novel information that MdMLO19 and MdMLO19‐X1 mRNAs are post‐transcriptionally regulated via m6A‐modification.

The m6A modification has also been demonstrated to affect translation efficiency beyond mRNA stability (Wang et al., 2015). The 35S::MhYTP2 line OE‐2 of this study showed a global increase in m6A modification levels (Figure 3a) and translation efficiency (Figure 5b). Upregulation of GDH genes has been observed under stress conditions (Fontaine et al., 2012; Lehmann and Skrok, 2010). In this study, the m6A modification at 3′ UTR and translation efficiency of MhYTP2‐binding MdGDH1L were significantly higher in the 35S::MhYTP2 line OE‐2 than in the WT plants (Figures 6a, c). Further, the changes in translation efficiency appeared to be directly regulated by m6A deposition on the transcripts of MdGDH1L or indirectly by m6A‐mediated regulation of transcription elongation factors and translation initiation factor for MdAPX2, MdPER3, MdPER42, and MdPER47. The improved translation efficiency of MdGDH1L made the transgenic plants resistant to PM. These findings collectively reveal a novel layer of gene regulation in the antioxidation system signalling pathway and establish a link between the m6A‐mediated antioxidation system and apple disease resistance.

The present study identifying the function of the apple m6A reader MhYTP2 in mRNA stability and translation sheds light on the mechanisms through which m6A functions in RNA metabolism and plant disease resistance. Considering the multiple roles of MhYTP2 on m6A metabolism, the regulation identified may have an essential function in many biological processes.

Methods

WT and 35S::MhYTP2 plant treatment

The WT plants and the 35S::MhYTP2 plants of the Malus domestica cv. ‘Roya Gala’ were used in this study (Liu et al., 2019). The WT and 35S::MhYTP2 plants generated by tissue culture were initially grown on MS agar containing 0.2 mg/L 6‐benzylaminopurine (6‐BA) and 0.2 mg/L indoleacetic acid (IAA) at 23 °C, 60 µmol/m2/s, and 14 h photoperiod. After 45 days of growth on MS agar, the plantlets were transferred to the rooting MS agar media containing 0.5 mg/L indole butyric acid and 0.5 mg/L IAA and maintained for another 45 days. The WT and 35S::MhYTP2 plantlets with similar size were transferred to small plastic pots (8.5 cm × 8.5 cm × 7.5 cm) containing a mixture of soil and perlite (1 : 1, v : v). After 60 days of growth in a growth chamber under 50% relative humidity, 28 °C, and a long photoperiod (16 h:8 h, light:dark), plants of similar size were selected to test the response to PM inoculation.

The inoculation experiments following the dry‐brushing of healthy leaves with P. leucotricha obtained from the diseased leaves were carried out in a growth chamber at 50% relative humidity and 28 °C in the dark for 24 h. Disease severity on all inoculated leaves was visually assessed 15 days after the inoculation. Six leaves per plant were harvested before and after inoculation and immediately frozen in liquid nitrogen. All frozen samples were stored at −80 °C. The phenotype recording and trypan blue and leaf fungal hyphae WGA staining were carried out 15 days after inoculation. In addition, the disease severity under field conditions was determined manually based on the size of white spots on leaves.

Microscopy

WGA‐AF 488 (Molecular Probes, Karlsruhe, Germany) was used to stain the fungal hyphae, and propidium iodide (Sigma, Shanghai, China) to stain the plant cells. Samples were infiltrated with a staining solution (1 µg/L propidium iodide, 10 µg/L WGA‐AF 488; 0.02% Tween 20 in PBS pH 7.4), incubated for 30 min, and washed in 1× PBS (137 mm NaCl, 2.7 mmKCl, 12 mm phosphate buffer, pH 7.4). Images were recorded on a TCS‐SP5 confocal microscope (Leica, Bensheim, Germany), at an excitation wavelength of 488 nm and a detection wavelength of 500–540 nm. The mCherry fluorescence (an excitation wavelength of 561 nm and a detection wavelength of 580–630 nm) was used for live‐cell imaging of fungal hyphae in apple leaves. Green‐fluorescence protein was excited with a 488 nm laser, and the emission was detected at 495–530 nm for fungal hyphae. The samples were observed, and images were recorded using a CoolSNAP‐HQ charge‐coupled device camera (Photometrics, Shanghai, China) controlled with the imaging software MetaMorph (Universal Imaging, Los Angeles, CA, USA).

GDH activity assay and antioxidant metabolite analysis

GDH activity and the GSH and AsA content of apple leaves were determined using the specific kits (Sangon Biotech, Shanghai, China), following the manufacturer’s instructions.

Ribosome profiling

Ribo‐seq using apple callus was performed at lcsciences (LC‐Bio Corporation, Hangzhou, China; https://www.lcsciences.com/), according to the protocol described by Zhou et al. (2018).

RNA extraction and quantification and gene expression analysis

Total RNA was extracted from the collected samples using a spin column plant total RNA purification kit (Sangon Biotech), according to the manufacturer’s instructions. The cDNA was reverse transcribed from the total RNA using a PrimeScript ® RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Further, quantitative RT‐PCR was conducted on a LightCycler® (Roche, Basel, Switzerland) 96 real‐time PCR detection system (Roche) using the ChamQ SYBR quantitative PCR Master Mix (Vazyme Biotech, Nanjing, China), according to the manufacturer’s instructions. The apple MdActin (XM_008344381) was used as the internal control gene. The relative expression level of the target gene transcript was determined by the 2‐ΔΔCt method (Kim et al., 2009; Livak and Schmittgen, 2001). All primers are listed in Table S3.

RIP‐seq and EMSA

RNA immunoprecipitation was performed as previously described by Zhou et al. (2019). The 35S::MhYTP2‐Flag ‘Orin’ cultivar’s calli were flash‐frozen and crushed in liquid nitrogen, and the frozen powder was homogenized in 15.75 mL of lysis buffer (100 mm Tris‐HCl, pH 7.5, 150 mm NaCl, 0.5% IGEPAL, and 1% plant protease inhibitor cocktail from Sigma). The crude extract was clarified by centrifugation at 3000 g for 10 min at 4 °C. The immunoprecipitation was performed using 11.25 mL of the crude extract incubated for 1.5 h at 4 °C on a rotating wheel with 120 µL of Flag‐trap magnetic beads (Chromotek, Munich, Germany). RNA was eluted from the beads with 400 µL of guanidium extraction buffer and precipitated overnight at −22 °C with 800 µL of pure ethanol. RNA was precipitated by centrifugation, resuspended in 350 µL of RTL buffer (Qiagen, Hilden, Germany), purified according to the manufacturer’s instructions, and eluted in 15 µL of RNase‐free water. The RNA solution was concentrated to 6 µL using the RNA clean and concentrator kit (Zymo Research, Irvine, CA, USA. Code: R1015). Similarly, RNA was extracted from 200 µL of crude extract to monitor RNA in the input fraction. RNA samples from both the eluate and input fractions were treated with DNase as part of the Qiagen RNEasy purification procedure. The beads were resuspended in 15 µL of Laemmli buffer and incubated for 5 min at 95 °C, and the supernatant was collected from the beads using a magnetic device. Sequencing was done on an Illumina HiSeq machine with 2 × 100 cycles of Solexa paired‐end sequencing. The experiment was repeated three times. Whole RIP and protein immunoprecipitation experiments were conducted using the freshly prepared and harvested calli for each replicate.

Electrophoretic mobility shift assay was performed following a previously reported method (Wang et al., 2014; Wei et al., 2018) with minor modifications. The digoxin‐labelled RNA oligonucleotides used to assay the binding affinity of MhYTP2‐His are listed in Appendix S2. The RNA probe was used at 4 nmol concentration. The concentration of MhYTP2‐His ranged from 0 to 2000 nm.

mRNA purification and m6A‐seq

Total RNA was extracted from the collected samples using a spin column plant total RNA purification kit (Sangon Biotech). Intact mRNA was purified from total RNA using Dynabeads mRNA purification kit (Ambion, Austin, TX, USA. Code: 61006).

The m6A IP was carried out following a previously reported procedure with slight modifications (Chen et al., 2015; Dominissini et al., 2013). The purified mRNA samples from the WT and 35S::MhYTP2 line OE‐2 plants were digested using DNase I to generate 100 nucleotide‐long fragments by incubating at 94 °C for 5 min in RNA fragmentation buffer (10 mm ZnCl2, 10 mm, Tris‐HCl, pH 7.0). The reaction was stopped using 0.05 m EDTA (Ambion Code: AM8740), followed by phenol‐chloroform extraction and ethanol precipitation. For the m6A‐seq, anti‐m6A polyclonal antibody (10 µg antibody for 5 µg mRNAs; Synaptic Systems (Chromotek. Code: 202003)) was incubated in IP buffer containing 150 mm NaCl, 0.1% NP‐40 (v/v), 10 mm Tris‐HCl (pH 7.4), and 300 U/mL RNase inhibitor (Promega, Madison, WI, USA. Code: N2112S) for 2 h at 4 °C. The mixture was IP by incubating with 50 µL Protein A beads (Sigma Code: P9424) at 4 °C for another 2 h. After washing twice with high‐salt buffer consisting of 50 mm Tris‐HCl (pH 7.4), 1 m NaCl, 1 mm EDTA, 1% NP‐40 (v/v), and 0.1% SDS (w/v) and twice with IP buffer, bound mRNAs were eluted from the beads by incubating with 6.7 mm N6‐methyladenosine (Sigma Code: M2780) in IP buffer and recovered via phenol‐chloroform extraction and ethanol precipitation. Then, 50 ng of the IP mRNA or pre‐IP mRNA (input control) was used for library construction with NEBNext ultra RNA library prep kit for Illumina (NEB, Beijing, China. Code: E7530). High‐throughput sequencing was performed on the Illumina HiSeq X sequencer with a paired‐end read length of 150 bp according to the standard protocol (Zhou et al., 2021). The sequencing was carried out with two independent biological replicates, and each RNA sample was prepared from a mix of at least 10 apple leaves.

The m6A‐seq data were analysed as previously described by Zhou et al. (2019). Here, the apple GDDH13 (https://iris.angers.inra.fr/gddh13/the‐apple‐genome‐downloads.html) was used as the reference genome. The m6A peaks were visualized using Integrated Genome Viewer (IGV, 2.8.0; http://www.igv.org).

RNA‐seq

The input reads of the m6A‐seq were used for RNA‐seq analysis as previously described (Trapnell et al., 2010). The uniquely mapped reads of each sample were assembled by Cufflinks. Gene expression level was calculated as fragments per kilobase of exon per million mapped fragments (FPKM) using Cuffdiff, which provides statistical routines for assessing the differential gene expression levels (Trapnell et al., 2010). Differentially expressed genes were defined based on a cut‐off criterion of FPKM fold change ≥2 and P < 0.05.

GO and KEGG analyses

The clean reads obtained after removing the adapter and low‐quality reads were aligned to the apple reference genome GDDH13 by HISAT2 (Kim and Langmead, 2015). The featureCounts (Liao and Smyth, 2014) was used to count the reads mapped to each gene. The DEGs of Ribo‐seq and RIP‐seq were further subjected to GO analysis on the agriGO database (version 2.0; http://systemsbiology.cau.edu.cn/agriGOv2/). Meanwhile, the DEGs of m6A‐seq were subjected to KEGG (http://www.genome.jp/kegg/) analysis.

mRNA stability assay

The mRNA stability was assessed as previously described (Duan et al., 2017) with minor modification. Briefly, tissue‐cultured WT and 3 5S::MhYTP2 line OE‐2 plants were grown on modified MS agar with 0.2 mm actinomycin D (transcription inhibitor), as described in the section on WT and 35S::MhYTP2 plant treatment. The tissues were collected 0, 5, 15 and 20 days after adding transcription inhibitor and immediately frozen in liquid nitrogen. The tissues were stored at −80 °C until further gene expression analysis.

Western blot analysis

The proteins were separated by 10% SDS‐PAGE and transferred to an Immobilon‐P PVDF membrane for western blotting. The membrane was blocked with 5% non‐fat milk in PBST buffer for 2 h at room temperature. The immunoblotting was conducted by incubating with anti‐GDH1L antibody (1 : 10 000) at room temperature for 2 h, followed by incubation with HRP‐conjugated anti‐rabbit IgG secondary antibody (1 : 10 000) at room temperature for another 2 h. The immunoreactive bands were visualized using the enhanced chemiluminescence detection kit, as mentioned earlier. The antibodies were synthesized from the Wuhan Institute of Biotechnology (China) (www.atagenix.com).

Statistical analysis

All data were analysed using IBM SPSS Statistics 21 (IBM Corp., Chicago, IL) and graphed with Sigma Plot 12.0 software (Systat Software, CA). Data are presented as the mean ± standard error (SE) of the mean of triplicates for each measurement. Data were analysed using an independent t test (P < 0.05) or subjected to one‐way analysis of variance (ANOVA).

Conflicts of interest

The authors declare no competing interests.

Author contributions

GTL and LCH conceived the project, designed and implemented the experiments, and wrote the paper. MFX, HL, FXM, YZH, WN, JQ and ZXZ implemented and assisted the experiments. LCH and MFW conceived the project, designed the experiments, discussed the results and edited the paper.

Supporting information

Appendix S1 m6A domain analysis of mRNA sequences.

Appendix S2 Digoxin labelled RNA oligonucleotides of MdMLO19, MdMLO19‐X1 and MdGDH1L for EMSA.

Figure S1 MhYTP2 conserved motifs analysis of YTH‐domain family proteins in Arabidopsis and human.

Figure S2 Confirmation of transgenic 35S:: MhYTP2‐Flag apple callus lines MhYTP2‐F1 and MhYTP2‐F2.

Figure S3 Changes in APX and POD enzyme activities in apple leaves under normal and infected conditions.

Table S1 The amino acid sequence alignment of MdMLO19‐X1 identified by us and MdMLO family reported in the literature (Pessina et al., 2014).

Table S2 Related comment information of transcription elongation factors (MD17G1038800, MD14G1104300) and translation initiation factors (MD10G1126600).

Table S3 Sequences of primers for quantitative real‐time PCR.

PBI-20-511-s005.pdf (546.7KB, pdf)

Table S4 Differential m6A peaks and differentially expressed genes between the 35S::MhYTP2 line OE‐2 and the WT plants.

PBI-20-511-s002.xlsx (3.9MB, xlsx)

Table S5 m6A‐containing transcripts identified via m6A‐seq in the WT plants.

PBI-20-511-s006.xlsx (4.8MB, xlsx)

Table S6 m6A‐containing transcripts identified via m6A‐seq in the 35S::MhYTP2 line OE‐2.

PBI-20-511-s001.xlsx (5.4MB, xlsx)

Table S7 Target genes of MhYTP2 identified by RIP‐seq.

PBI-20-511-s003.xls (826.2KB, xls)

Table S8 Genes with differential translation efficiency and expression levels between the 35S::MhYTP2 line OE‐2 and the WT plants.

PBI-20-511-s004.xlsx (7.8MB, xlsx)

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFD1000300), the Major Science and Technology Projects in Shaanxi Province (2020zdzx03‐01‐01), the China Agriculture Research System of MOF and MARA (CARS‐27), the Fundamental Research Funds for the Central Universities (2452019050) and China Postdoctoral Science Foundation (2017M610657, 2018T111108).

Guo, T. , Liu, C. , Meng, F. , Hu, L. , Fu, X. , Yang, Z. , Wang, N. , Jiang, Q. , Zhang, X. and Ma, F. (2022) The m6A reader MhYTP2 regulates MdMLO19 mRNA stability and antioxidant genes translation efficiency conferring powdery mildew resistance in apple. Plant Biotechnol. J., 10.1111/pbi.13733

References

  1. Anderson, S.J. , Kramer, M.C. , Gosai, S.J. , Yu, X. , Vandivier, L.E. , Nelson, A.D.L. , Anderson, Z.D. et al. (2018) N6‐methyladenosine inhibits local ribonucleolytic cleavage to stabilize mRNAs in Arabidopsis . Cell Rep. 25, 1146–1157. [DOI] [PubMed] [Google Scholar]
  2. Arribas‐Hernández, L. , Bressendorff, S. , Hansen, M.H. , Poulsen, C. , Erdmann, S. and Brodersen, P. (2018) An m6A‐YTH module controls developmental timing and morphogenesis in Arabidopsis . Plant Cell, 30, 952–967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bi, Z. , Liu, Y.H. , Zhao, Y.L. , Yao, Y.X. , Wu, R.F. , Liu, Q. , Wang, Y.Z. et al. (2019) A dynamic reversible RNA N6‐methyladenosine modification: current status and perspectives. J. Cell Physiol. 234, 7948–7956. [DOI] [PubMed] [Google Scholar]
  4. Bodi, Z. , Zhong, S. , Mehra, S. , Song, J. , Graham, N. , Li, H.Y. , May, S. et al. (2012) Adenosine methylation in Arabidopsis mRNA is associated with the 3' end and reduced levels cause developmental defects. Front. Plant Sci. 3, 48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Büschges, R. , Hollricher, K. , Panstruga, R. , Simons, G. , Wolter, M. , Frijters, A. , van Daelen, R. et al. (1997) The barley Mlo gene: a novel control element of plant pathogen resistance. Cell, 88(5), 695–705. [DOI] [PubMed] [Google Scholar]
  6. Chen, M. , Urs, M.J. , Sánchez‐González, I. , Olayioye, M.A. , Herde, M. and Witte, C.P. (2018) m6A RNA degradation products are catabolized by an evolutionarily conserved N6‐Methyl‐AMP deaminase in plant and mammalian cells. Plant Cell, 30, 1511–1522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen, T. , Hao, Y.J. , Zhang, Y. , Li, M.M. , Wang, M. , Han, W. , Wu, Y. et al. (2015) m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell, 16, 289–301. [DOI] [PubMed] [Google Scholar]
  8. Chen, Y. and Varani, G. (2013) Engineering RNA‐binding proteins for biology. FEBS J. 280, 3734–3754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dominissini, D. , Moshitch‐Moshkovitz, S. , Schwartz, S. , Salmon‐Divon, M. , Ungar, L. , Osenberg, S. , Cesarkas, K. et al. (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A‐seq. Nature, 485, 201–206. [DOI] [PubMed] [Google Scholar]
  10. Dominissini, D. , Moshitch‐Moshkovitz, S. , Salmon‐Divon, M. , Amariglio, N. and Rechavi, G. (2013) Transcriptome‐wide mapping of N 6 ‐methyladenosine by m6A‐seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189. [DOI] [PubMed] [Google Scholar]
  11. Duan, H.C. , Wei, L.H. , Zhang, C. , Wang, Y. , Chen, L. , Lu, Z. , Chen, P.R. et al. (2017) ALKBH10B is an RNA N6‐methyladenosine demethylase affecting Arabidopsis floral transition. Plant Cell, 29, 2995–3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fontaine, J.X. , Tercé‐Laforgue, T. , Armengaud, P. , Clément, G. , Renou, J.P. , Pelletier, S. , Catterou, M. et al. (2012) Characterization of a NADH‐dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism. Plant Cell, 24, 4044–4065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foulongne, M. , Pascal, T. , Pfeiffer, F. and Kervella, J. (2003) QTLs for powdery mildew resistance in peach × Prunus davidiana crosses: consistency across generations and environments. Mol. Breed. 12, 33–50. [Google Scholar]
  14. Fu, Y. , Dominissini, D. , Rechavi, G. and He, C. (2014) Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306. [DOI] [PubMed] [Google Scholar]
  15. Fujita, M. , Fujita, Y. , Noutoshi, Y. , Takahashi, F. , Narusaka, Y. , Yamaguchi‐Shinozaki, K. and Shinozaki, K. (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr. Opin. Plant Biol. 9, 436–442. [DOI] [PubMed] [Google Scholar]
  16. Gu, F. , Chauhan, V. and Chauhan, A. (2015) Glutathione redox imbalance in brain disorders. Curr. Opin. Clin. Nutr. Metab. Care, 18, 89–95. [DOI] [PubMed] [Google Scholar]
  17. Gururani, M.A. , Venkatesh, J. and Tran, L.S. (2015) Regulation of photosynthesis during abiotic stress‐induced photoinhibition. Mol. Plant, 8, 1304–1320. [DOI] [PubMed] [Google Scholar]
  18. Heinz, S. , Benner, C. , Spann, N. , Bertolino, E. , Lin, Y.C. , Laslo, P. , Cheng, J.X. et al. (2010) Simple combinations of lineage‐determining transcription factors prime cis‐regulatory elements required for macrophage and B cell identities. Mol. Cell, 38, 576–589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huh, S.U. and Paek, K.H. (2014) APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression. BMC. Plant Biol. 14, 75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jia, G.F. , Fu, Y. , Zhao, X. , Dai, Q. , Zheng, G.Q. , Yang, Y. , Yi, C.Q. et al. (2011) N6‐methyladenosine in nuclear RNA is a major substrate of the obesity‐associated FTO. Nat. Chem. Biol. 7, 885–887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jia, G.F. , Fu, Y. and He, C. (2013) Reversible RNA adenosine methylation in biological regulation. Trends Genet. 29, 108–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kierzek, E. and Kierzek, R. (2003) The thermodynamic stability of RNA duplexes and hairpins containing N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines. Nucleic. Acids. Res. 31, 4472–4480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim, D. , Langmead, B. and Salzberg, S.L. (2015) HISAT: a fast spliced aligner with low memory requirements. Nat. Methods, 12, 357–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim, K.M. , Cho, H. , Choi, K. , Kim, J. , Kim, B.‐W. , Ko, Y.‐G. , Jang, S.K. et al. (2009) A new MIF4G domain‐containing protein, CTIF, directs nuclear cap‐binding protein CBP80/20‐dependent translation. Genes Dev. 23, 2033–2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lehmann, T. , Skrok, A. and Dabert, M. (2010) Stress‐induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos. Physiol. Plant. 138, 35–47. [DOI] [PubMed] [Google Scholar]
  26. Li, A. , Chen, Y.‐S. , Ping, X.‐L. , Yang, X. , Xiao, W. , Yang, Y. , Sun, H.‐Y. et al. (2017) Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 27, 444–447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liao, Y. , Smyth, G.K. and Shi, W. (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30, 923–930. [DOI] [PubMed] [Google Scholar]
  28. Liu, C.H. , Guo, T.L. , Wang, N. , Wang, Q. , Xue, Y.C. , Zhan, M.H. , Guan, Q.M. et al. (2019) Overexpression of MhYTP2 enhances apple water‐use efficiency by activating ABA and ethylene signaling. Environ. Exp. Bot. 157, 260–268. [Google Scholar]
  29. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real‐time quantitative PCR and the 2(‐Delta Delta C(T)) method. Methods, 25, 402–408. [DOI] [PubMed] [Google Scholar]
  30. Luo, G.Z. , MacQueen, A. , Zheng, G. , Duan, H. , Dore, L.C. , Lu, Z. , Liu, J. et al. (2014) Unique features of the m6A methylome in Arabidopsis thaliana . Nat. Commun. 5, 5630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luo, J.H. , Wang, Y. , Wang, M. , Zhang, L.Y. , Peng, H.R. , Zhou, Y.Y. , Jia, G.F. et al. (2020) Natural variation in RNA m6A methylation and its relationship with translational status. Plant Physiol. 182, 332–344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martínez‐Pérez, M. , Aparicio, F. , López‐Gresa, M.P. , Bellés, J.M. , Sánchez‐Navarro, J.A. and Pallás, V. (2017) Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs. Proc. Natl Acad. Sci. USA, 114, 10755–10760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Merchante, C. , Brumos, J. , Yun, J. , Hu, Q. , Spencer, K. , Enríquez, P. , Binder, B. et al. (2015) Gene‐specific translation regulation mediated by the hormone‐signaling molecule EIN2. Cell, 163, 684–697. [DOI] [PubMed] [Google Scholar]
  34. Meyer, K.D. and Jaffrey, S.R. (2014) The dynamic epitranscriptome: N6‐methyladenosine and gene expression control. Nat. Rev. Mol. Cell Biol. 15, 313–326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Meyer, K. , Patil, D. , Zhou, J. , Zinoviev, A. , Skabkin, M. , Elemento, O. , Pestova, T. et al. (2015) 5' UTR m6A promotes cap‐independent translation. Cell, 163, 999–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meyer, K.D. , Saletore, Y. , Zumbo, P. , Elemento, O. , Mason, C.E. and Jaffrey, S.R. (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′UTRs and near stop codons. Cell, 149, 1635–1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miao, Z.Y. , Zhang, T. , Qi, Y.H. , Song, J. , Han, Z.X. and Ma, C. (2020) Evolution of the RNA N6‐methyladenosine methylome mediated by genomic duplication. Plant Physiol. 182, 345–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nilsen, T.W. (2014) Molecular biology. Internal mRNA methylation finally finds functions. Science, 343, 1207–1208. [DOI] [PubMed] [Google Scholar]
  39. Ok, S.H. , Jeong, H.J. , Bae, J.M. , Shin, J.S. , Luan, S. and Kim, K.N. (2005) Novel CIPK1‐associated proteins in Arabidopsis contain an evolutionarily conserved C‐terminal region that mediates nuclear localization. Plant Physiol. 139, 138–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pessina, S. , Angeli, D. , Martens, S. , Visser, R.G. , Bai, Y. , Salamini, F. , Velasco, R. et al. (2016) The knock‐down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica). Plant Biotechnol. J. 14, 2033–2044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pessina, S. , Pavan, S. , Catalano, D. , Gallotta, A. , Visser, R.G. , Bai, Y. , Malnoy, M. et al. (2014) Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica . BMC Genom. 15, 618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Růžička, K. , Zhang, M. , Campilho, A. , Bodi, Z. , Kashif, M. , Saleh, M. , Eeckhout, D. et al. (2017) Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol. 215, 157–172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schwartz, S. , Agarwala, S. , Mumbach, M. , Jovanovic, M. , Mertins, P. , Shishkin, A. , Tabach, Y. et al. (2013) High‐resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell, 155, 1409–1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Scutenaire, J. , Deragon, J.M. , Jean, V. , Benhamed, M. , Raynaud, C. , Favory, J.J. , Merret, R. et al. (2018) The YTH domain protein ECT2 is an m6A reader required for normal trichome branching in Arabidopsis . Plant Cell, 30, 986–1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shen, L. , Liang, Z. , Gu, X. , Chen, Y. , Teo, Z.W. , Hou, X. , Cai, W.M. et al. (2016) N6‐methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis . Dev. Cell, 38, 186–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shi, H.L. , Wei, J.B. and He, C. (2019) Where, when, and how: context‐dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell, 74, 640–650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Skopelitis, D.S. , Paranychianakis, N.V. , Paschalidis, K.A. , Pliakonis, E.D. , Delis, I.D. , Yakoumakis, D.I. , Kouvarakis, A. et al. (2006) Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell, 18, 2767–2781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tercé‐Laforgue, T. , Bedu, M. , Dargel‐Grafin, C. , Dubois, F. , Gibon, Y. , Restivo, F.M. and Hirel, B. (2013) Resolving the role of plant glutamate dehydrogenase: II. Physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously. Plant Cell Physiol. 54, 1635–1647. [DOI] [PubMed] [Google Scholar]
  49. Trapnell, C. , Williams, B.A. , Pertea, G. , Mortazavi, A. , Kwan, G. , van‐Baren, M. J. , Salzberg, S. L. et al. (2010) Transcript assembly and quantification by RNA‐Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Turechek, W.W. , Carroll, J.E. and Rosenberger, D.A. (2004) Powdery mildew of apple. New York State Integrated Pest Management Program: Cornell Universit. [Google Scholar]
  51. Vespa, L. , Vachon, G. , Berger, F. , Perazza, D. , Faure, J.D. and Herzog, M. (2004) The immunophilin‐interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication. Plant Physiol. 134, 1283–1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wan, Y.Z. , Tang, K. , Zhang, D.Y. , Xie, S.J. , Zhu, X.H. , Wang, Z.G. and Lang, Z.B. (2015) Transcriptome‐wide high‐throughput deep m6A‐seq reveals unique differential m6A methylation patterns between three organs in Arabidopsis thaliana . Genome Biol. 16, 272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang, P. , Yin, L.H. , Liang, D. , Li, C. , Ma, F.W. and Yue, Z.Y. (2012) Delayed senescence of apple leaves by exogenous melatonin treatment: toward regulating the ascorbate‐glutathione cycle. J. Pineal Res. 53, 11–20. [DOI] [PubMed] [Google Scholar]
  54. Wang, X. , Lu, Z. , Gomez, A. , Hon, G.C. , Yue, Y. , Han, D. , Fu, Y.E. et al. (2014) N6‐methyladenosine‐dependent regulation of messenger RNA stability. Nature, 505, 117–120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wang, X. , Zhao, B. , Roundtree, I. , Lu, Z. , Han, D. , Ma, H. , Weng, X. et al. (2015) N6‐methyladenosine modulates messenger RNA translation efficiency. Cell, 161, 1388–1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wei, L.H. , Song, P. , Wang, Y. , Lu, Z. , Tang, Q. , Yu, Q. , Xiao, Y. et al. (2018) The m6A reader ECT2 controls trichome morphology by affecting mRNA stability in Arabidopsis . Plant Cell, 30, 968–985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wolter, M. , Hollricher, K. , Salamini, F. and Schulze‐Lefert, P. (1993) The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype. Mol. Gen. Genet. 239(1–2), 122–128. [DOI] [PubMed] [Google Scholar]
  58. Xiao, C.L. , Chandler, C.K. , Price, J.F. , Duval, J.R. , Mertely, J.C. and Legard, D.E. (2001) Comparison of epidemics of botrytis fruit rot and powdery mildew of strawberry in large plastic tunnel and field production systems. Plant Dis. 85, 901–909. [DOI] [PubMed] [Google Scholar]
  59. Yan, L.U. , Gong, Y. , Luo, Q. , Dai, G.‐X. , Teng, Z. , He, Y. , Wu, X. et al. (2021) Heterologous expression of fungal AcGDH alleviates ammonium toxicity and suppresses photorespiration, thereby improving drought tolerance in rice. Plant Sci. 305, 110769. [DOI] [PubMed] [Google Scholar]
  60. Yue, H. , Nie, X.J. , Yan, Z.G. and Weining, S. (2019) N6‐methyladenosine regulatory machinery in plants: composition, function and evolution. Plant Biotechnol. J. 17, 1194–1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhang, F. , Zhang, Y.‐C. , Liao, J.‐Y. , Yu, Y. , Zhou, Y.‐F. , Feng, Y.‐Z. , Yang, Y.‐W. et al. (2019) The subunit of RNA N6‐methyladenosine methyltransferase OsFIP regulates early degeneration of microspores in rice. PLoS. Genet. 15, e1008120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhao, B.S. , Roundtree, I.A. and He, C. (2017) Post‐transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhao, X.U. , Yang, Y. , Sun, B.‐F. , Shi, Y. , Yang, X. , Xiao, W. , Hao, Y.‐J. et al. (2014) FTO‐dependent demethylation of N6‐methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 24, 1403–1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zheng, G. , Dahl, J.A. , Niu, Y. , Fedorcsak, P. , Huang, C.M. , Li, C.J. , Vågbø, C.B. et al. (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell. 49, 18–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhong, S. , Li, H. , Bodi, Z. , Button, J. , Vespa, L. , Herzog, M. and Fray, R.G. (2008) MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex‐specific splicing factor. Plant Cell, 20, 1278–1288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhou, J. , Wan, J. , Gao, X. , Zhang, X. , Jaffrey, S.R. and Qian, S.B. (2015) Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature, 526, 591–594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhou, J. , Wan, J. , Shu, X.E. , Mao, Y.H. , Liu, X.M. , Yuan, X. , Zhang, X.Q. et al. (2018) N6‐methyladenosine guides mRNA alternative translation during integrated stress response. Mol. Cell, 69, 636–647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhou, L.L. , Tang, R.K. , Li, X.J. , Tian, S.P. , Li, B.B. and Qin, G.Z. (2021) N6‐methyladenosine RNA modification regulates strawberry fruit ripening in an ABA‐dependent manner. Genome Biol. 22, 168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zhou, L.L. , Tian, S.P. and Qin, G.Z. (2019) RNA methylomes reveal the m6A‐mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening. Genome. Biol. 20, 156. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1 m6A domain analysis of mRNA sequences.

Appendix S2 Digoxin labelled RNA oligonucleotides of MdMLO19, MdMLO19‐X1 and MdGDH1L for EMSA.

Figure S1 MhYTP2 conserved motifs analysis of YTH‐domain family proteins in Arabidopsis and human.

Figure S2 Confirmation of transgenic 35S:: MhYTP2‐Flag apple callus lines MhYTP2‐F1 and MhYTP2‐F2.

Figure S3 Changes in APX and POD enzyme activities in apple leaves under normal and infected conditions.

Table S1 The amino acid sequence alignment of MdMLO19‐X1 identified by us and MdMLO family reported in the literature (Pessina et al., 2014).

Table S2 Related comment information of transcription elongation factors (MD17G1038800, MD14G1104300) and translation initiation factors (MD10G1126600).

Table S3 Sequences of primers for quantitative real‐time PCR.

PBI-20-511-s005.pdf (546.7KB, pdf)

Table S4 Differential m6A peaks and differentially expressed genes between the 35S::MhYTP2 line OE‐2 and the WT plants.

PBI-20-511-s002.xlsx (3.9MB, xlsx)

Table S5 m6A‐containing transcripts identified via m6A‐seq in the WT plants.

PBI-20-511-s006.xlsx (4.8MB, xlsx)

Table S6 m6A‐containing transcripts identified via m6A‐seq in the 35S::MhYTP2 line OE‐2.

PBI-20-511-s001.xlsx (5.4MB, xlsx)

Table S7 Target genes of MhYTP2 identified by RIP‐seq.

PBI-20-511-s003.xls (826.2KB, xls)

Table S8 Genes with differential translation efficiency and expression levels between the 35S::MhYTP2 line OE‐2 and the WT plants.

PBI-20-511-s004.xlsx (7.8MB, xlsx)

Articles from Plant Biotechnology Journal are provided here courtesy of Society for Experimental Biology (SEB) and the Association of Applied Biologists (AAB) and John Wiley and Sons, Ltd

RESOURCES