N6-methyladenosine regulatory genes in common bean (Phaseolus vulgaris): genome-wide investigation, evolution, structure, characterization, and expression patterns during viral infection

doi:10.21203/rs.3.rs-2243840/v1

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N6-methyladenosine regulatory genes in common bean (Phaseolus vulgaris): genome-wide investigation, evolution, structure, characterization, and expression patterns during viral infection

https://doi.org/10.21203/rs.3.rs-2243840/v1

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Background: N6-methyladenosine (m⁶A) is the most abundant modification of the mRNA, which plays multiple roles in the regulation of biological process. The m⁶A regulatory genes have been studied in various plant species. However, comprehensive analysis of m⁶A-related genes in the common bean (Phaseolus vulgaris) are still unperformed.

Results: In our study, a total of 31 m⁶A regulatory genes were identified from common bean genome and grouped into three categories including writers, erasers and readers. The expansion of the m⁶A families in common bean was mostly fueled by segmental duplication or whole genome duplication events. Then, we performed a comprehensive analysis of chromosomal distribution, collinearity relationship, evolutionary selection, gene structure, conserved domain, conserved motif, cis-acting elements, protein-protein interaction with several bioinformatic tools. Moreover, the transcriptomic data exposed that m⁶A related genes were differentially expressed on the primary true leaf and systemic leaf of susceptible and resistant bean varieties. Our RT-qPCR tests revealed that the expression of all m⁶A related genes varied through time upon viral infection.

Conclusion；In this study, a thorough and systematic genome-wide analysis was performed for m⁶A regulatory genes of common bean. Our results will provide evidence for discovery of the functions of the m⁶A components and their family proteins, facilitating further study of the m⁶A-mediated mechanism in common beans during viral infection.

m6A modification

Phaseolus vulgaris

writers

erasers

readers

biotic stress

Methylation is an important regulated modification in proteins and nucleic acids. RNA methylation accounts for more than 60% of all RNA modifications, and N6-methyladenosine (m⁶A) is the most abundant modification in eukaryotic RNA molecules and plays multiple roles in mRNA metabolism, affecting stability, splicing, nuclear export, translation initiation, polyadenylation, nuclear retention of mRNA [1–10]. As a mode of mRNA regulation, m⁶A was firstly reported in poly(A) RNA in 1974, but the interest was not revived until transcriptome-wide m⁶A mapping methods relied on high-throughput sequencing were developed [11, 12]. With the help of m⁶A antibodies to immunoprecipitate modified mRNAs, followed by high-throughput sequencing to identify the methylated transcripts, recent studies revealed that m⁶A is prevalent in the coding sequence (CDS) and 3' untranslated region (3'UTR) with particularly high enrichment near stop codons [10, 12]. Me-RIP-Seq also enabled identification of canonical consensus sequence DRACH (D = G, A or U; R = G or A; H = A, C or U) for m⁶A [10, 12].

In mammals, m⁶A is the pervasive modification in mRNA and affects spermatogonia differentiation, meiosis initiation, brain development and other critical biological processes [13–17]. The core components to achieve its regulatory functions include m⁶A writer, eraser, and reader. The RNA methyltransferases METTL3 and METTL14 are vital components of the multi-subunit "WRITER" complex [18–20]. METTL3 is the catalytic subunit of the complex and is responsible for m⁶A forming in polyadenylated mRNA. METTL14 does not have methylase activity but plays a role in maintaining the integrity of the complex and binding to substrate RNA [21–23]. WTAP, another critical component of the complex, does not possess methylase activity but can interact with METTL3 and enhance the catalytic activity of m⁶A methyltransferase. WTAP is also required to localize METTL3 and METTL14 to the nuclear speckles [24]. m⁶A is a dynamic and reversible process. Previous studies have demonstrated that two Alkbh dioxygenase family proteins, FTO and ALKBH5, were able to catalyze m⁶A removal in vivo and in vitro, which were so-called m⁶A erasers [3, 25]. Both demethylases are α-ketoglutarate (αKG)- and Fe(II)-dependent. Unlike ALKBH5, which explicitly removes the m⁶A modification, FTO also mediates the removal of m⁶Am (N6,2'-O-dimethyladenosine) around 5' UTR of mRNA [26]. m⁶A determines the fate of mRNA and regulates a series of biological processes by recruiting the m⁶A-binding protein "READER". "READER" usually contains a YTH (YT521-B homology) domain [27], and m⁶A exerts its various regulation effect by interacting with different readers. Cytoplasmic protein YTHDF1 could increase mRNA translation efficiency by interacting with translation initiation factors [4]. On the other hand, YTHDF2, another member of the reader family, reduced mRNA stability [28]. YTHDC1 appeared to be the significant player in recognizing nuclear m⁶A modification and enabled regulation of alternative splicing of pre-mRNA [29].

m⁶A modification was firstly identified in the model plant Arabidopsis and a few other crops. In plants, the WRITER complex appears to consist of two MTA-70 family proteins, MTA (METTL3 homologous protein) and MTB (METTL14 homologous protein), together with FIP37 (WTAP homologous protein), VIRILIZER (KIAA1429 homologous protein) and E3 ubiquitin-linked enzyme HAKAI (HAKAI homologous protein) [30–33]. Among the 14 ALKB homologous proteins encoded by the Arabidopsis genome, ALKBH9B was proved to have demethylase activity to remove m⁶A modifications from RNA in vitro, possibly related to mRNA silencing and decay processes [34]. ALKBH10B could oxidatively reverse m⁶A methylation in mRNA in vitro and in vivo, affecting the stability of target transcripts and thereby affecting the flower transition [35]. Thirteen YTH domain-containing proteins have been identified in Arabidopsis, including ECT1 ~ 12 and CPSF30. As a pervasive and conserved molecular regulatory mechanism, m⁶A plays a vital role in plant embryonic development, shoot apical meristem development, flowering, fruit ripening, trichome morphogenesis, and other reproductive processes [30, 35–41]. It is also part of a regulatory network involving plant response to abiotic processes such as salt, heat, and drought [42–46].

More studies have confirmed the effect of m⁶A modification on biotic stress in plants. AtALKBH9B has been reported to be able to interact with both genomic RNA and the capsid protein of alfalfa mosaic virus (AMV). Knockout mutant of ALKBH9B has been found to exhibit a relative abundance of m⁶A in the AMV genomic RNA, it also impaired systemic infection of AMV, but the infectivity of CMV (cucumber mosaic virus) was not affected [34]. In tobacco infected with tobacco mosaic virus (TMV), m⁶A levels in transcriptome were dramatically decreased [47]. The case studied dynamic changes of m⁶A modification during the interaction between rice and two viruses has revealed the expressions of multiple genes involved in plant major antiviral pathways were regulated by m⁶A modification [48]. The presence of Alkb domain was also confirmed in some plant virus genomes through protein sequence alignments, and this function domain was speculated to participate in repairing methylation damage in virus genomic RNA [49, 50]. These findings deepened our understanding of the important regulatory role of m⁶A modification during interactions between plants and viruses.

Common bean (Phaseolus vulgaris) is an important source of fiber and protein and has been cultivated as a vegetable crop worldwide. Currently, a comprehensive and systematic analysis of the m⁶A gene family in common beans has not yet been performed. Since m⁶A is highly conserved in plant species, the m⁶A components in Arabidopsis were used as a reference to determine homologous proteins in common beans. In this study, the m⁶A gene family in common bean was genome-wide identified, and its evolutionary, structural, cis-acting elements differences, and putative protein interactions were explored. Bean common mosaic virus is one of the most destructive pathogens infection common beans. Here, in this study, BCMV was used to inoculate the bean plants, and the expression patterns of m⁶A gene family after viral infection were studied based on transcriptome data analysis and RT-qPCR. Our study aims to uncover the distinct feature of m⁶A gene family members and provide clues for further research on its biological function involving plant response to biotic stress such as viral infection

2.1 Identification of m⁶A Components in Common Bean

To identify all the m⁶A components in the common bean, we downloaded the annotated sequences of the common bean genome (Phaseolus vulgaris v2.1) from the Phytozome 13 database (DOE-JGI and USDA-NIFA, http://phytozome.jgi.doe.gov/). The amino acid sequences of m⁶A-related proteins reported in Arabidopsis thaliana [51] were obtained from The Arabidopsis Information Resource (TAIR) and used as queries to perform BLASTP with default parameter by TBtools[52] against the common bean genomic sequences. The hidden Markov model (HMM) profiles of corresponding families and domains were used to evaluate the deduced sequences of m⁶A-related protein in P. vulgaris (MT-A70 family (PF05063), WTAP family (PF17098), virilizer domain (PF15912), 2OG-Fe(II) oxygenase superfamily (PF13532), and YTH domain (PF04146))[52]. Then, the conserved domains in each identified sequence were further confirmed using Conserved Domain Database [53]. After filtering the repeated sequences, all 31 putative candidates, the gene IDs, and the full-length amino acid sequences were obtained. Utilizing the ExPaSy tool, the molecular weight (MW) and isoelectric points (pI) were predicted [54].

2.2 Chromosome Location, Synteny Analysis, and Ka/Ks Calculation

The length of each chromosome and the positional information of the m⁶A components and their family genes on the chromosomes were extracted from the common bean GFF file by TBtools. TBtools was used to draw the schematic diagram of the chromosomal length scale and chromosomal locations. The Arabidopsis thaliana genome sequences and annotation files (GFF, FASTA suffix files) were downloaded from the EnsemblPlants (https://plants.ensembl.org/index.html)for the synteny analysis. The gene duplication landscape was obtained using MCScanX[55], and a syntenic map was visualized by TBtools. The putative duplicated genes were highlighted by red lines. The value of Ka and Ks were calculated by "simple Ka/Ks_calculation" in TBtools, and the formula T = Ks/2r(r = 1.5E-8)was used to calculate the divergence time[56].

2.3. Alignment and Phylogenetic Analysis

The selected full-length amino acid sequences were aligned with default parameters using MEGA10.1.7[57]. The secondary structure was annotated in the alignment using the ESPript (https://espript.ibcp.fr) [58]. Alignment results were used to construct a Maximum Likelihood (ML) tree using MEGA10.1.7 with Poisson correction, partial deletion, and 1000 bootstrap replicates. The secondary structures of HsMETTL3 (PDB ID: 5L6D), HsALKBH5 (PDB ID: 4NJ4), and HsYTHDF1(PDB ID: 4RCJ) were downloaded from the NCBI structure database (https://www.ncbi.nlm.nih.gov/structure) [23, 59, 60]. The protein sequences and their identifier (ID) used in this article were supported in Supplementary Materials Table S2.

2.4. Conserved Motif, Conserved Domain, Gene Structure, and Cis-element Analyses

The conserved motifs were analyzed using MEME Suite software [61], and the output file was downloaded. The Batch CD-Search (https://www.ncbi.nlm.nih.gov/cdd/)was used to analyze the conserved domains of multiple full-length protein sequences, and then the output file was downloaded. The information on gene lengths and structure was extracted from the common GFF file and was subsequently visualized by TBtools. Finally, all the downloaded files, together with the common bean GFF file and the phylogenetic tree were submitted to TBtools for visualized analyses. The 2000 bp promoter sequence upstream from the initiation codon site was extracted from the FASTA file of the common bean genome by TBtools. Then, the sequences were submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [62] to identify cis-elements (CREs). A simplified data set of cis-elements was created manually, and then TBtools was used to visualize it.

2.5. GO annotation, sub-cellular localization prediction and the protein-protein interactions

The protein-protein interaction networks of m⁶A regulatory proteins in common bean were constructed using the STRING v11.5 database [63]. The 31 m⁶A regulatory proteins were uploaded to the STRING v11.5, and 30 STRING proteins were found based on the highest bit score. The interaction scores displayed in the network were above 0.900(highest confidence). Meanwhile, functional enrichment of m⁶A-related proteins was exported, including biological process, molecular function and cellular component (Gene Ontology). CELLO2GO was used for predicting the subcellular localization [64].

2.6. Plant Materials and Virus Treatments

The BCMV bean differential seeds of 'DW' and 'RGLC' were provided by National Plant Germplasm System for research purpose and propagated in our lab. Common beans (Phaseolus vulgaris) varieties DW (sensitive to BCMV-C54) and RGLC (resistant to BCMV-C54) were planted in a climate chamber (16 h day, 30°C, 8 h night 24°C), and then mechanically inoculated until the first pair of true leaves were fully expanded (on day 7). The Bean common mosaic virus(C54) used for inoculation was obtained from Jinzhong, Shanxi, and kept in the laboratory. The full-length nucleotide sequence of BCMV-C54 was shown in Supplementary Table S3. The control group was treated with phosphate buffer saline. All plants were placed in a greenhouse (16 h day, 8 h night) for culture. Following that, samples of all the leaves from the treated and healthy plants were taken at 2h, 4h, 8h, 16h, 32h, 64h, 5d, 7d, 14d, and 21d, respectively. Three independent biological replicates were conducted for each sample.

2.7. Expression Profiles of m⁶A Regulatory Genes in Common Bean Based on Transcriptome Data

The local and systemic leaves of the treated group and healthy group after 14dpi were frozen in liquid nitrogen for one hour and sent to the BioMarker(http://www.biomarker.com.cn/) for sequencing. The filtered sequencing results were mapped to the chromosome-level genome of common bean (Phaseolus vulgaris v2.1) and the gene expression level was measured using FPKM (Fragments Per Kilobase of transcript per Million fragments mapped). On the basis of normalized FPKM values, the expression profiles of m⁶A regulatory genes were visualized using TBtools.

2.8. Total RNA Extraction and qPCR Analysis

We ground all the above-mentioned samples in liquid nitrogen and extracted total RNA using Trizol reagent (Biosharp). The RNA was then used to synthesize the first-strand cDNA using the HiScript Ⅲ All-in-one RT SuperMix Perfect for qPCR (Vazyme Biotech Co., Ltd). The quantitative real-time polymerase chain reaction (qRT-PCR) of m⁶A regulatory genes was conducted on the QuantStudio platform using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd). The PCR amplification parameters were as follows:95℃ for 3min, followed by 40 cycles of 95℃ for 10s and 60℃ for 15s. The primers were designed using IDT PrimerQuest Tool (https://sg.idtdna.com/PrimerQuest/Home/Index) and their sequences were listed in Supplementary Table S4. The Act11 gene of common bean was used to normalize the gene expression [65]. All the experiments were performed in two technical replicates. The relative expression level of the selected 28 genes was performed by 2^−ΔΔCT method [66] and presented as mean ± SEM.

3.1. Genome-Wide Identification of m⁶A Regulatory Genes in Phaseolus vulgaris

To identify m⁶A components and their protein families in common bean, the amino acid sequences of m⁶A related proteins reported in Arabidopsis thaliana, including writers, erasers, and readers, were used as queries to perform BLASTP against the bean genomic sequences in TBtools. Following the removal of repeated sequences, 36 putative candidates were retained with their gene identifications.3 of the 36 candidates did not have the 2OG_Fe(II)_Oxy domain (PF13532), and 2 of the 36 candidates did not have the typical YTH domain (PF04146), which were all eventually removed. The remaining 31 candidate genes were renamed based on our subsequent evolutionary analysis. The identified all genes and transcripts ID of common bean were shown in Supplementary Table S1. The amino acid sequence length, relative molecular weights (MWs), and isoelectric points (PIs) of each amino acid were listed in Table 1. In detail, the lengths of the listed proteins ranged from 224 (PvALKBH7) to 2188 (PvVIR) amino acids, and the corresponding range for MWs was 25.44-240.79 KDa. The predicted pI values ranged from 5.25 (PvECT5) to 9.51 (PvECT11). Among these genes, PvFIP37, PvHAKAI and PvVIR, three putative catalytic subunits of the m⁶A methyltransferase complex, only had one copy in the common bean genome, respectively. However, the MT-A70, ALKBH, and YTH domain protein families consisted of multiple members.

Table 1

m⁶A genes detected in the *P.vulgaris* genome.
Gene name	Locus ID	Group	Chr	Protein property
Gene name	Locus ID	Group	Chr	Length(aa)	PI	MW (kDa)
PvMTA	Phvul.010G102500.1	MT-A70	10	761	6.01	84.32
PvMTB	Phvul.007G073300.1		7	1086	6.86	120.67
PvMTC	Phvul.001G016200.1		1	427	8.04	48.64
PvALKBH1A	Phvul.001G262100.1	ALKBH	1	358	6.11	40.36
PvALKBH1B	Phvul.009G262600.1		9	344	9.47	37.40
PvALKBH1C	Phvul.001G131400.1		1	443	8.89	49.26
PvALKBH2A	Phvul.006G137400.1		6	235	9.21	27.06
PvALKBH2B	Phvul.006G137611.1		6	235	9.21	27.02
PvALKBH6	Phvul.004G131600.1		4	259	5.79	29.74
PvALKBH7	Phvul.008G264300.1		8	224	6.36	25.44
PvALKBH8	Phvul.002G123600.1		2	342	7.59	38.29
PvALKBH9A	Phvul.001G044000.2		1	476	5.92	53.60
PvALKBH9B	Phvul.006G214800.1		6	425	8.83	48.48
PvALKBH10A	Phvul.001G147800.1		1	516	5.89	56.99
PvALKBH10B	Phvul.007G168900.1		7	505	6.44	55.72
PvALKBH10C1	Phvul.002G181800.1		2	671	6.36	73.15
PvALKBH10C2	Phvul.003G014200.1		3	691	6.18	74.54
PvECT1	Phvul.001G110200.1	YTH	1	658	7.96	72.22
PvECT2	Phvul.002G247000.1		2	677	6.02	74.03
PvECT3	Phvul.004G080300.1		4	638	5.96	70.15
PvECT5	Phvul.010G165400.1		10	634	5.25	69.57
PvECT6	Phvul.006G121600.1		6	649	5.84	71.55
PvECT7	Phvul.003G119300.1		3	698	7.59	77.24
PvECT8A	Phvul.005G045600.1		5	575	6.76	63.27
PvECT8B	Phvul.004G132700.1		4	231	9.34	26.62
PvECT11	Phvul.006G218800.1		6	557	9.51	62.58
PvECT12	Phvul.002G152600.1		2	379	5.38	42.68
PvCPSF30	Phvul.006G130200.1		6	697	6.28	76.46
PvFIP37	Phvul.002G107400.1		2	337	5.41	38.15
PvVIR	Phvul.007G267500.1		7	2188	5.35	240.79
PvHAKAI	Phvul.008G108800.1		8	436	6.16	47.61

3.2. Chromosomal Location and Collinearity Analysis of m⁶A Regulatory genes in common bean

All 31 genes were distributed on ten chromosomes in the common bean (a total of 11 chromosomes), and they were primarily located at the proximal or distal ends of the chromosomes. The MT-A70, ALKBH, and YTH family genes were highlighted in different colors (Fig. 1A). Among these genes, PvALKBH2A and PvALKBH2B were adjacent on chr06, however, no tandem duplication event was identified. Gene family expansion in common bean driven by duplication was searched using genome-wide synteny analysis, and three gene pairs, PvALKBH10A- PvALKBH10B, PvALKBH10C1-PvALKBH10C2 and PvECT6-PvECT7 were identified as segmental duplication/whole genome duplication (WGD) (Fig. 1B). Therefore, segmental duplication/WGD event appeared to be the primary factor involved in expanding the common bean m⁶A gene family. Ka/Ks calculation was used to evaluate the extent and type of selective pressure of each gene pair. All three gene pairs were under purifying selection, and the earliest differentiation (PvALKBH10A-PvALKBH10B) occurred 28.42 million years ago (Table 2). To further investigate the phylogenetic mechanisms of bean m⁶A components and their protein families, a synteny analysis between common bean and Arabidopsis was constructed. Eighteen orthologous pairs consisting of 14 common bean genes and 13 Arabidopsis genes were identified (Fig. 1C), indicating these orthologous pairs' existence before the divergence of Arabidopsis and common bean.

Table 2

༎The evolutionary analysis of duplicated m⁶A regulatory genes and estimated divergence times.
GeneⅠ	Location	GeneⅡ	Location	Type of duplication	K_a	K_s	K_a/K_s	T = K_s/2r(MYA)
PvALKBH10A	1	PvALKBH10B	7	Segmental/WGD	0.126348392	0.852865956	0.148145662	28.42
PvECT6	6	PvECT7	3	Segmental/WGD	0.174929236	0.683184232	0.256049874	22.77
PvALKBH10C1	2	PvALKBH10C2	3	Segmental/WGD	0.189372849	0.669639206	0.28279833	22.32

3.3. Evolutionary and functional key residue Analyses of m⁶A components of common bean

To analyze the evolutionary relationship among bean MT-A70 family proteins, a phylogenetic tree was constructed using common bean, Arabidopsis MT-A70 sequences, and human reference sequences (Table S2). Phylogenetic analysis suggested that the MT- A70 proteins of common bean could be divided into three clades: METTL3 subfamily (PvMTA), METTL14 subfamily (PvMTB), and METTL4 subfamily (PvMTC) (Fig. 2A). Compared to Arabidopsis and humans, no orthologs were found in MT- A70 proteins of common bean, suggesting little functional diversity or redundancy. A further multiple sequence alignment showed that many functional sites, including residues involved in AdoMet interactions and RNA binding, were conserved (Fig. 2B), indicating that a core heterodimer catalyzing mechanism might be similar among common bean, human, and Arabidopsis.

To analyze the evolutionary relationship among ALKBH family proteins in common bean, a phylogenetic tree was constructed using common bean, Arabidopsis ALKBH sequences, and human reference sequences (Table S2). ALKBH proteins of common bean could be divided into seven subfamilies (Fig. 3A). In bean and Arabidopsis, ALKBH9 and ALKBH10 subfamily proteins were orthologs of the m⁶A methylase HsALKBH5. Further multiple sequence alignment of HsALKBH5 and ALKBH9 subfamily proteins showed that many functional sites, including residues involved in 2OG and metal-binding, were conserved (Fig. 3B), suggesting that PvALKBH9 subfamily proteins may have a similar catalyzing mechanism for methyl group removal as human HsALKBH5.

To analyze the evolutionary relationship among YTH family proteins in common bean, a phylogenetic tree was constructed using the reference sequences of common bean, Arabidopsis YTH sequences, and humans (Table S2). The YTH proteins of common bean could be divided into YTHDF and YTHDC subfamilies, and the YTHDC subfamily comprised two subclades: PvYTHDC and PvCPSF30 (Fig. 4A). Similar to Arabidopsis, two YTH proteins belonged to the YTHDC subfamily in the common bean (also two in Arabidopsis). In contrast, fewer belonged to the YTHDF subfamily (nine in bean and 11 in Arabidopsis). Moreover, two copies of AtECT8 orthologs were found in common beans, suggesting functional diversity or redundancy. Additional multiple sequence alignment of YTHDF and YTHDC subfamily proteins displayed that many functional sites, including residues involved in the aromatic cage, contact with m⁶A, and RNA binding, were conserved, suggesting that PvYTHDFs might share a similar m⁶A binding structure and have a similar read mechanism to human and Arabidopsis YTHDF proteins. (Fig. 4B). Additionally, multiple sequence alignment of YTHDC subfamily proteins also displayed a conserved aromatic cage, suggesting the ability of the m⁶A read mechanism (Figure S1).

3.4. Phylogenetic and gene structure Analyses of m⁶A components of common bean

To further explore the structure and sequence characteristics of MT-A70 family genes in common beans, a maximum likelihood phylogenetic tree (Fig. 5A) was constructed using full-length amino acid sequences to classify visualized analyses. In the conserved motif analysis, compared to PvMTA and PvMTB, PvMTC lacked some motifs (3,4,8,9), whereas PvMTA and PvMTB basically had the same motifs of the same arrangement (Fig. 5A). Meanwhile, except for the MT-A70 protein domain (CDD: pfam05063), or MT-A70 superfamily domain (CDD: pfam05063), PvMTB had extra PRK12678 superfamily (CDD: PRK12678) and Med15 superfamily (CDD: pfam09606) domains (Fig. 5A), indicating that PvMTB may participate in different regulatory pathways. A number of studies have shown that structural similarity and diversity play an important role in gene family evolution. Using the TBtools, we generated an exon-intron map for the MT-A70 gene family. Figure 5A showed a detailed representation of the gene's coding region, introns and untranslated regions. A key component of eukaryotic genomes, introns are actively involved in genetic recombination that results in gene rearrangements and evolution of the genome. The MT-A70 family genes have 5 to 8 introns.

To further explore the structure and sequence characteristics of ALKBH genes in common beans, a maximum likelihood phylogenetic tree (Fig. 5B) was constructed using full-length amino acid sequences to classify visualized analyses. In the conserved motif analysis, the PvALKBH9 and PvALKBH10 subfamilies had the same motifs 1–3,5,8 and motif 10 with the same arrangement, and PvALKBH10 subfamily had two additional motifs, 4 and 7. The PvALKBH2 subfamily had two different motifs 6 and 9. PvALKBH8 had three motifs 1, 4 and 8.PvALKBH6 had three motifs and possessed a extra motif 1 than PvALKBH7 (Fig. 5B).All of the above suggested a potential loss of function or functional differentiation of these proteins. Among the PvALKBH proteins, all had a conserved domain of either 2OG- Fe(II)_Oxy, 2OG- Fe(II)_Oxy2, or 2OG-Fe(II)_Oxy superfamily. PvALKBH10C1 and PvALKBH10C2 had a different domain of PHA03378, PvALKBH8 had an extra domain of RRM_SF superfamily and PvALKBH10B had a special domain of MSCRAMM_ClfB superfamily (Fig. 5B). PvALKBH genes displayed relatively different gene structures, including the number and position of exons and introns (Fig. 5B). The ALKBH family genes have 1 to 8 introns.

To further explore the structure and sequence characteristics of YTH genes in common beans, a maximum likelihood phylogenetic tree (Fig. 5C) was constructed using full-length amino acid sequences to classify visualized analyses. Most PvYTHDF subclade proteins exhibited the conserved motifs 1–7, except PvECT11 lacked motifs 5–6, and PvECT8B lacked motif 1 and motif 6. PvECT1-3 and PvECT5 had an extra conserved motif 8, whereas PvECT8A had an extra motif 10, and PvECT6-7 had two additional motifs 9 and 10. PvYTHDC subclade proteins all had conserved motifs 2–3, whereas PvCPSF30 had an extra motif 5. Together, these results indicated that the YTH family proteins were highly conserved in common beans and that there was a slight evolutionary divergency in the subfamily or subclade. All PvYTHs had a typical YTH conserved domain (CDD: pfam04146) of similar length. Additionally, PvCPSF30 had the YTH1 superfamily domain (CDD: COG5084). PvYTH genes clustered in the same clade shared a similar gene structure, including the number and position of exons and introns (Fig. 5C).

3.5. Cis-acting element Analyses of MT-A70 Family of common bean

To better study the responsive regulation and potential biological functions of m⁶A-related genes from common bean plants, the PlantCare database was used to analyze the cis-acting elements in 31 m⁶A regulatory gene promoter regions (Fig. 6). In m⁶A regulatory components, light responsive, and phytohormone responsive and stress responsive elements are more common than plant growth and development related elements.

In the light responsive category, Box 4 was identified in all MT-A70, ALKBH, and YTH family members except PvCPSF30 and encompassed the highest proportion (39.4%,32.6%,36.4%), followed by the G-box (10.6%,22.3%,15.5%). The most prevalent cis-element in the phytohormone group was ABRE, which was responsible for abscisic acid responsiveness, followed by CGTCA and TGACG motifs associated with methyl jasmonic acid (MeJA) responsiveness. Several stress-responsive elements were identified in the promoter regions of m⁶A regulatory genes. The most prevalent cis-element in this group was ARE in response to anaerobic induction. GC motif in response to anoxic specific inducibility was only present in the promotor region of PvCPSF30. The plant growth and development response related cis-elements like CAT-box, circadian, GCN4 motif, HD-Zip 1, O2-site and RY-element were revealed (Fig. 7), which are linked with meristem expression, circadian control, endosperm expression, differentiation of palisade mesophyll cells, zein metabolism regulation and seed-specific regulation. More plant growth and development responsive elements were identified in the promoter regions of ALKBH family members than in the upstream region of other m⁶A regulatory components.

3.6. Go annotation and putative protein-protein interaction Analyses of MT-A70 Family of common bean

To predict the localization and potential functions of m⁶A regulatory protein of common bean, subcellular localization prediction and Go ontology annotation analysis was conducted. A total of 31 proteins were categorized into 55 functional groups, and they were divided into four categories: Biological process, Molecular function, Cellular component, and Subcellular localization (Fig. 8). About the molecular function, we found that approximately 18.99% of annotated proteins function in the activity of binding, followed by nucleic acid binding (11.39%), RNA-binding (10.76%) and metal ion binding (9.49%). In the biological process, m⁶A members involved in the nucleic acid metabolic processes (8.96%) and RNA metabolic processes (7.96%), macromolecule modification (7.46%), regulation of gene expression (6.47%), followed by negative regulation of biological process (5.97%), RNA modification (5.97%), positive regulation of cellular process (5.47%) and negative regulation of gene expression (5.47%). Subsequently, predicted m⁶A proteins are also annotated with regulation of mRNA metabolic process (5.47%), negative regulation of cellular process (4.98%), mRNA metabolic process (4.48%), mRNA destabilization (4.48%), methylation, macromolecule methylation, RNA processing and RNA methylation (< 4%) in biological process annotation. In the cellular component, the m⁶A proteins were annotated with the cellular anatomical entity and intracellular (both 19.35%), intracellular organelles (15.48%), cytoplasm and nucleus (both 14.84%). Furthermore, in terms of subcellular localization, our results demonstrated that the proportion of m⁶A proteins located in nuclear was approximately 89.66%, whereas the extracellular, cytoplasmic, and plasma membranes contained the same amount (3.45%).

A total of 31 m⁶A regulatory proteins including writers, erasers, and readers were aligned to the thirty STRING proteins, which were used to construct the interaction networks. All m⁶A writer components, with the exception of PvMTC, had significant connections, suggesting that they might have contributed in m⁶A modification by building protein complexes. In addition, we found that putative homologs of ALKBH1A and ALKBH2A were frequently mentioned together in other organisms, which could be explained by their comparable functions in removing m⁶A modification and lowering the overall m⁶A level (Fig. 9).

3.7. Transcriptomic analysis

To discover the potential functions of m⁶A pathway genes in response to viral infection, the expression profiles of 31 genes in true primary leaf and systemic leaf were obtained from our transcriptome datasets (Fig. 10). Among six m⁶A writer genes, we found that almost all of the m⁶A regulatory genes displayed high expression in control and treatment (FPKM > 10). On the other hand, PvMTC expressed at moderate levels (FPKM > 1 and FPKM < 10) in eight groups of samples. Among the ALKBH family genes, PvALKBH1B and PvALKBH1C showed extremely low expression levels(FPKM < 1), suggesting that they were nonfunctional or had temporal and spatial-specific expression patterns. In the resistant cultivars, the expression of PvALKBH9B increased significantly after viral infection. In the sensitive varieties, the expression level of PvALKBH10A in the local leaves decreased significantly after viral infection.PvECT2 showed predominant expression among all 31 genes in all groups of samples(FPKM> 100), and its expression level in RGLC primary true leaf was significantly higher than that in systemic leaf, which could also be found in PvECT8A.Besides, in the YTH family, only PvECT8B was basically not expressed(FPKM < 1). Cluster analysis of all samples showed that the samples from different parts of the susceptible cultivar infected by the virus were clustered together, while the healthy control of the resistant cultivar were clustered together with the samples from the same parts treated by the BCMV.

3.8. Expression analysis

It has shown that m⁶A modification is involved in plant response to various biotic stresses. In this study, bean common mosaic virus was used as a biotic stress factor to study the reaction of m⁶A regulatory genes in healthy and treated plants at 2/4/8/16/32/64h and 5/7/14/21 d after viral inoculation (Fig. 11). The expression patterns of 28 genes were diverse. The components of m⁶A writer complex (PvMTA, PvMTB, PvHAKAI and PvVIR) exhibited generally the same trend of expression, and these genes in virus-treated plants were basically upregulated compared with healthy controls before 16 hours, and significantly downregulated at 64 h and 5 days. In the ALKBH family, except for PvALKBH9B and PvALKBH10C1/C2, all other members were highly expressed in the early stages of viral infection (2–4 hours). The ALKBH1 subfamily was significantly upregulated at 32/64 h and then downregulated at 14/21d. PvALKBH9A was upregulated for one day after viral stimulation, after that its expression was not substantially different from that of the control. The expression of PvALKBH10A and PvALKBH10B fluctuated after viral infection, but the changing pattern of the two was quite similar. The expression of PvALKBH10C1-2 was enhanced in the early stage of BCMV infection, and basically downregulated in the later stage, but they were significantly upregulated at 21 days. Compared with other genes, PvALKBH9B showed a unique expression trend. It was slightly downregulated within 8 hours after viral inoculation, and then this gene was continuously upregulated, with the highest expression observed at 7 dpi. With the exception of ECT5, all identified reading proteins were downregulated at 5d. PvECT1 was significantly upregulated within the first four hours of viral inoculation, with little change in most later periods. The expression profile of PvECT2 is relatively unique, exhibited sustained descending expression at all time points. The expression of PvECT3/5/8B fluctuated slightly in the early stage of viral infection and was basically downregulated after 5 days. The expression of PvECT6 was significantly downregulated in 2 h after viral infection and was significantly upregulated at 21 days. The expression of PvECT11 and PvCPSF30 increased within four hours of viral infection and was downregulated in the later stage. The expression variation of genes involved in m⁶A modification in common beans were complex and diverse, which may be closely related to their biological functions when responding to viral infection in plants.

m⁶A regulatory genes have been described in some plant species, such as Arabidopsis, tomato, tea, and poplar [51, 67–69]. Various functions of a few m⁶A related proteins have been confirmed and provided new insights into the study of this pervasive mRNA regulation mode. Completion of common bean genome sequencing facilitates investigation of the identity of m⁶A gene family at the whole genome level. In this study, we initially identified 36 putative m⁶A regulatory genes of common bean using BLASTP analysis. Five genes, including three putative eraser genes and two putative reader genes, were removed due to the absence of the corresponding conserved domain in their full-length protein sequences with both HMMER and CDD-search. The remaining 31 genes were renamed and characterized using various bioinformatic and quantitative methods, which would serve as the basis for more functional research on the m⁶A regulatory members. The number of m⁶A genes varied among species, 33, 24, and 34 m⁶A proteins have been identified in A. thaliana, tomato, and tea, respectively, in contrast, poplar possessed 61 m⁶A proteins, and in the case of wheat and upland cotton [51], the number dropped to A. thaliana and tomato. No direct correlation has been found between the number of m⁶A genes and the genome size of different plant species, and the abundance of m⁶A genes in many species, including common bean, appeared to be a result of gene duplications. In this study, three duplicated gene pairs were identified in m⁶A family genes of common bean, with all three involving segmental duplication or WGD. Notably, no pattern of tandem duplication was detected from the m⁶A gene family, which suggested that segmental duplication/WGD, rather than tandem duplication, were crucial for the development of m⁶A regulatory genes. All duplicated m⁶A gene pairs were subjected to Ka/Ks analysis, and the ratios revealed that all these genes have experienced strong purifying selection and evolved in a highly conserved manner.

The constitution of the writer complex appears to be conserved between mammals and plants, similar to Arabidopsis, the components of the m⁶A writer complex in common bean included the orthologs of MTA, MTB, FIP37, VIR, and HAKAI (Table 1). Notably, only one copy of MTA, MTB and MTC existed in common bean, whereas several copies of MTB have been reported in other evaluated plant species [51]. A total of 14 ALKBH proteins have been identified in common beans, and these proteins were distributed in seven clades based on phylogenetic analysis. Resembling human ALKBH1, AtALKBH1D was found to localize partially in chloroplasts which indicated it might play a role in alkylation lesion repair in the organellar system. ALKBH1 has also been reported to be required for efficient mitochondrial translation and respiratory complex activities through the mediation of tRNA modification [70, 71]. AtALKBH2, an orthologue of mammalian ALKBH2, displayed enzyme activities to repair methylation damage in DNA [72]. AtALKBH6 has been proven to have RNA binding activities and was able to interact with both m⁶A and m5c modified RNAs. alkbh6 mutant had a downregulated expression profile of ABA signaling-related genes and a lower survival rate under abiotic stresses, and both suggested ALKBH6 in Arabidopsis was involved in plant growth and abiotic stress response [73]. Similar to mammalian ALKBH8, AtALKBH8 served as a dioxygenase and enabled hydroxylation of mcm5U to (S)-mchm5U in tRNA, which could facilitate further protein translation [74]. ALKBH9B and ALKBH10 have been identified as major m⁶A demethylases in Arabidopsis, which participated in the regulation of flower transition and viral infection through modification of m⁶A level in target mRNA [34, 35]. ALKB homologues identified in common bean may have similar functions as described in mammalians and model plant Arabidopsis, and members in ALKBH9 and ALKBH10 subfamilies, which were classified as orthologous to AtALKBH9 and AtALKBH10, might be the most potential m⁶A demethylases in common bean. Most readers have a YTH domain, and more reader proteins identified in plants than in other eukaryotes suggested functional redundancy or m⁶A may exert its effect through different regulatory mechanisms [8]. Previous studies confirmed cytoplasmic ECT2, ECT3 and ECT4 in Arabidopsis were able to associate with m⁶A sites and were required for normal leaf emergence and morphology [75]. Another distinct protein functioning as an m⁶A reader is CPSF30, which was localized in the nucleus and could regulate genes participating in the salicylic acid pathway through mRNA splicing [76]. Orthologous to YTH-containing proteins of Arabidopsis have been identified in common beans through phylogenetic analysis (Fig. 4). Theoretically, these proteins may function similar to their orthologues in Arabidopsis. Whether they act as real m⁶A readers in common bean has yet to be determined.

The cis-acting elements present in the promoter regions strongly suggested that m⁶A could be turned on by various molecular switches and participated in the regulation of an extensive network of genes involved in response to light, phytohormone, stress and developmental processes. The emergence of a large number of light responsive cis-regulatory elements in the promoter region of m⁶A related genes indicated that m⁶A might play a unique role in the photosynthesis of common beans. The screening of prominent cis-regulatory elements associated with phytohormone suggested the putative involvement of the respective genes in ABA and JA signal pathways. The presence of stress and plant growth and development-related cis-acting elements indicated that m⁶A was likely to have important functions that affect host response to anaerobic induction and meristem expression, circadian control, endosperm expression, differentiation of palisade mesophyll cells, zein metabolism regulation and seed-specific regulation in common bean. The protein-protein interaction analysis showed components of ‘WRITER’ complex could interact with each other, the high score (> 0.900, highest confidence) displayed in the network and the similar scenario found in Arabidopsis suggested this prediction was reliable.

Exploring the roles of genes can be facilitated greatly by gene expression patterns. In this study, RNA-seq data was used to perform expression analysis on viral infected local and systemic bean leaves. Most putative m⁶A components showed a broad expression pattern upon viral infection in bean plants, suggesting that they played a comprehensive and essential regulatory role in common bean response to BCMV infection. PvMTA and PvMTB had similar expression patterns, which was consistent with the hypothesis that both components form a writer complex to catalyze the formation of m⁶A. However, PvMTC showed a relatively lower expression compared with other methylases, suggesting that PvMTC might mediate a more specific regulatory pathway.

In the PvALKBH family, two genes in the ALKBH9 subclade displayed significantly higher expression than the other branches. The expression of PvALKBH9B increased significantly after viral infection in resistant varieties, indicating that PvALKBH9B may be related to the series response of resistant varieties to viral infection. In the YTH family, PvECT2 showed predominant expression among all the m⁶A regulatory genes including the reader genes. Meanwhile. Our RT-qPCR tests revealed that the expression of all m⁶A related genes varied through time after viral infection. The expression of most genes was induced rapidly and persisted at high expression level at the early stage of viral infection (0-32hrs), and the expression pattern after that varied diversely. Notably, the two genes in the ALKBH9 clade showed opposite trends, likely performing different functions during viral infection, PvALKBH9A was upregulated at early stages, and PvALKBH9B was upregulated at a later stage which expression reached the highest point at 7-14d. Together, our results will provide evidence for discovery of the functions of the m⁶A regulatory genes, highlighting further study of the biological role of m⁶A in responding to viral infection.

In our study, a thorough and systematic genome-wide analysis was performed for m⁶A regulatory genes of common bean. A total of 31 genes were ultimately identified and categorized into three families including m⁶A writers, erasers, and readers. The expansion of the m⁶A regulatory genes in common bean was mostly fueled by segmental duplication/WGD events. Furthermore, we performed a comprehensive analysis of chromosomal distribution, collinearity relationship, evolutionary selection, gene structure, conserved domain, conserved motif, cis-acting elements, protein-protein interaction, and transcriptomic profiling. Subsequently, we detected the expression pattern of the identified m⁶A related genes of common bean upon viral infection. Genome-wide study of m⁶A pathway genes gives a deeper insight into the evolutionary selection and provides a foundation for understanding the m⁶A-mediated regulatory mechanism in the common bean.

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Ethics approval and consent to participate

No specific permits were required, plant materials were obtained under the supervision and permission of Shanxi Agricultural University guidelines and according to national guidelines and all authors comply with all the local and national guidelines. All the experiments were carried out according to the Shanxi Agricultural University lab rules.

Availability of data and materials

The Phaseolus vulgaris m⁶A protein sequences were collected from Phytozome 13 database (DOE-JGI and USDA-NIFA, http://phytozome.jgi.doe.gov/). The Arabidopsis m⁶A protein sequences were downloaded from TAIR database (http://www.arabidopsis.org). All RNA-Seq data were submitted to the NCBI SRA database (http://www.ncbi.nlm.nih.gov/sra/) under the following accession number: SRR22266475, SRR22266474, SRR22266463, SRR22266458, SRR22266457, SRR22266456, SRR22266455, SRR22266454, SRR22266453, SRR22266452, SRR22266473, SRR22266472, SRR22266471, SRR22266470, SRR22266469, SRR22266468, SRR22266467, SRR22266466, SRR22266465, SRR22266464, SRR22266462, SRR22266461, SRR22266460, SRR22266459(BioProject accession number:PRJNA900512).The GenBank accession number of BCMV-C54 nucleotide sequence was OP828732.

Funding

This work was supported by Shanxi Province Returned Overseas Talents Program (grant no. 2020-063), National Natural Science Foundation of China (grant no. 32202247) and Natural Science Foundation of Shanxi of China (grant no. 20210302123367).

Authors’ contributions

Jingya Niu and Muning Tang conceived and designed the experiments; Guyue Wang, Xinhua Wang, Sifan Huo, Wenyan Wu, Xingrui Liang and Xinqi Huang contributed reagents/materials/analysis tools; Xue Feng and Chenchen Jing provided guidance on the whole manuscript. Jingya Niu and Muning Tang wrote the article. All authors read and approved the final manuscript.

Acknowledgements

We appreciate Pro. Dr. Xue Feng and Dr. Chenchen Jing for their effort in revising and provided guidance on the whole manuscript.

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No competing interests reported.

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N6-methyladenosine regulatory genes in common bean (Phaseolus vulgaris): genome-wide investigation, evolution, structure, characterization, and expression patterns during viral infection

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Identification of m⁶A Components in Common Bean

2.2 Chromosome Location, Synteny Analysis, and Ka/Ks Calculation

2.3. Alignment and Phylogenetic Analysis

2.4. Conserved Motif, Conserved Domain, Gene Structure, and Cis-element Analyses

2.5. GO annotation, sub-cellular localization prediction and the protein-protein interactions

2.6. Plant Materials and Virus Treatments

2.7. Expression Profiles of m⁶A Regulatory Genes in Common Bean Based on Transcriptome Data

2.8. Total RNA Extraction and qPCR Analysis

3. Results

3.1. Genome-Wide Identification of m⁶A Regulatory Genes in Phaseolus vulgaris

3.2. Chromosomal Location and Collinearity Analysis of m⁶A Regulatory genes in common bean

3.3. Evolutionary and functional key residue Analyses of m⁶A components of common bean

3.4. Phylogenetic and gene structure Analyses of m⁶A components of common bean

3.5. Cis-acting element Analyses of MT-A70 Family of common bean

3.6. Go annotation and putative protein-protein interaction Analyses of MT-A70 Family of common bean

3.7. Transcriptomic analysis

3.8. Expression analysis

4. Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

N6-methyladenosine regulatory genes in common bean (Phaseolus vulgaris): genome-wide investigation, evolution, structure, characterization, and expression patterns during viral infection

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Identification of m6A Components in Common Bean

2.2 Chromosome Location, Synteny Analysis, and Ka/Ks Calculation

2.3. Alignment and Phylogenetic Analysis

2.4. Conserved Motif, Conserved Domain, Gene Structure, and Cis-element Analyses

2.5. GO annotation, sub-cellular localization prediction and the protein-protein interactions

2.6. Plant Materials and Virus Treatments

2.7. Expression Profiles of m6A Regulatory Genes in Common Bean Based on Transcriptome Data

2.8. Total RNA Extraction and qPCR Analysis

3. Results

3.1. Genome-Wide Identification of m6A Regulatory Genes in Phaseolus vulgaris

3.2. Chromosomal Location and Collinearity Analysis of m6A Regulatory genes in common bean

3.3. Evolutionary and functional key residue Analyses of m6A components of common bean

3.4. Phylogenetic and gene structure Analyses of m6A components of common bean

3.5. Cis-acting element Analyses of MT-A70 Family of common bean

3.6. Go annotation and putative protein-protein interaction Analyses of MT-A70 Family of common bean

3.7. Transcriptomic analysis

3.8. Expression analysis

4. Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.1 Identification of m⁶A Components in Common Bean

2.7. Expression Profiles of m⁶A Regulatory Genes in Common Bean Based on Transcriptome Data

3.1. Genome-Wide Identification of m⁶A Regulatory Genes in Phaseolus vulgaris

3.2. Chromosomal Location and Collinearity Analysis of m⁶A Regulatory genes in common bean

3.3. Evolutionary and functional key residue Analyses of m⁶A components of common bean

3.4. Phylogenetic and gene structure Analyses of m⁶A components of common bean