The dynamics of N6-methyladenine RNA modification in interactions between rice and plant viruses

Abstract

Background: N⁶-methyladenosine (m⁶A) is the most common RNA modification in eukaryotes and has been implicated as a novel epigenetic marker that is involved in various biological processes. The pattern and functional dissection of m⁶A in the regulation of several major human viral diseases have already been reported. However, the patterns and functions of m⁶A distribution in plant disease bursting remain largely unknown.

Results: We analyse the high-quality m⁶A methylomes in rice plants infected with two devastating viruses. We find that the m⁶A methylation is mainly associated with genes that are not actively expressed in virus-infected rice plants. We also detect different m⁶A peak distributions on the same gene, which may contribute to different antiviral modes between rice stripe virus or rice black-stripe dwarf virus infection. Interestingly, we observe increased levels of m⁶A methylation in rice plant response to virus infection. Several antiviral pathway-related genes, such as RNA silencing-, resistance-, and fundamental antiviral phytohormone metabolic-related genes, are also m⁶A methylated. The level of m⁶A methylation is tightly associated with its relative expression levels.

Conclusions: We revealed the dynamics of m⁶A modification during the interaction between rice and viruses, which may act as a main regulatory strategy in gene expression. Our investigations highlight the significance of m⁶A modifications in interactions between plant and viruses, especially in regulating the expression of genes involved in key pathways.

Keywords: Interactions; N 6-methyladenosine; Plant viruses; Rice.

Fig. 1

Flow chart of the investigation of m⁶A methylation during infection of rice by RBSDV or RSV. A Symptoms of RBSDV- and RSV-infected rice plants in plastic buckets at 60 days post infection (dpt). The left plant is a mock-treated plant, the middle two plants are infected with RBSDV, and the plant on the right is infected with RSV. B RT-PCR and western blot (WB) detection of RSV using a specific pair of primers corresponding to RdRp and anti-NS3 specific antiserum. Total proteins were stained with Coomassie brilliant blue (CBB), which was treated as the loading control. C RT-PCR and WB were performed to detect RBSDV in rice using a specific pair of primers corresponding to CP. Anti-p10 specific antiserum was carried out for the WB. D Experimental flow chart of m⁶A-IP-seq and RNA-seq using RBSDV- and RSV-infected plants. NGS, next-generation sequencing. MeRIP, methylated RNA immunoprecipitation

Fig. 2

Circos plots of the m⁶A methylome in rice plants infected with RBSDV or RSV. The six rings from the outside to the inside show the genomic positions (1st), gene density (2nd), peak density of mock-treated rice plants (3rd), peak density of RSV-treated rice plants (4th), peak density of RBSDV-treated rice plants (5th), and the common peak density of RBSDV- and RSV-infected rice plants (6th)

Fig. 3

Circos plots of the m⁶A methylome in RBSDV and RSV genomic RNAs. A Distribution of m⁶A methylated reads on the ten RBSDV genomic RNAs. Six rings from outside to inside show genomic positions (1st), reads distribution of RBSDV_1_Input (2nd), reads distribution of RBSDV_1_IP (3rd), reads distribution of RBSDV_2_Input (4th), reads distribution of RBSDV_2_IP (5th), and the GC content of the genomic RNA (6th). B Distribution of m⁶A methylated reads on the four RSV genomic RNAs. Six outer rings were indicated similarly to RBSDV above. C m⁶A methylation peaks in the full-length RBSDV segment 5 (upper panel), 6 (middle panel), and 9 (bottom panel). The detail peaks regions and viral gene annotation are shown in the Additional file 2: Table S6. Top numbers show the full length of the analysed RNA segments, and bp is the short name of base-pair. Blue colour marked line shows the m⁶A peak region on viral genome, and number 1 and 2 mean the two replicate of the m⁶A-IP-sequencing. D Distribution of m⁶A peaks on the RSV genomic RNA1 (upper panel), RNA2 (middle panel), and RNA4 (bottom panel). The detail peak regions and viral gene annotation are shown in the Additional file 2: Table S6. Top numbers show the full-length of the analysed genomic RNAs, and the nt means nucleotide. Other marks are similar with Fig. 3C

Fig. 4

Activation of rice m⁶A methylation by virus infection. A Histograms show the number of unique peak in mock-treated and RBSDV- and RSV-infected rice plants. The Y-axis represents the peak number, and the X-axis represents the treatments. B Significant distribution analysis of the different peaks in mock-treated and RBSDV- and RSV-infected rice samples. The Y-axis represents the peak number, and the X-axis represents the negative value of the logarithm of the p- value base-10. C Histogram showing the different regulated numbers of m⁶A methylated genes in RBSDV- and RSV-infected rice compared with mock-treated rice samples. D Comparisons of the fold change of the different regulated peaks in RBSDV- and RSV-infected rice plants. The Y-axis represents the logarithmic of peak folded-enrichment base-2. E Significant distribution analysis of the different peaks in RBSDV- and RSV-infected rice samples. The Y-axis represents the numbers of different regulated peaks, and the X-axis represents the negative value of the logarithmic value of the p- value base-10

Fig. 5

Analyses of the relationship between m⁶A methylation with expression levels of the target gene in rice under plant virus infection. A Euclidian distance (ED) coefficients among gene expression profiles generated by RNA-seq analysis of the two biological replicates of the three treatments. RNA-seq was performed simultaneously with m⁶A-IP-seq with total RNA extracted at 60 dpt. A lower value means a closer ED of the two compared objects, and with higher reproducibility of the two replicates. B The percentage of rice m⁶A methylated and un-methylated genes at a defined FPKM levels (< 1, 1–5, and > 5). Different colour densities indicate different percentages of the corresponding gene. C Comparisons of number of non-m⁶A methylated genes and number of m⁶A methylated genes in their gene bodies with high (FPKM > 1) and low (FPKM > 1) expression levels in the three treatments. Relationships between gene expression and number were tested using the chi-square test. “*” indicate p- value < 0.05, and “**” means p- value < 0.01. D Box plot comparing FPKM expression levels between non-m⁶A methylated genes and m⁶A methylated genes in the three treatments. A two-tailed unpaired Student’s t -test was performed to calculate the p- values of these three treatments

Fig. 6

GO and KEGG analyses of different m⁶A methylated genes in RBSDV- and RSV-infected rice plants. A Percentages of the different gene bodies (5′-UTR, 3′-UTR, 1st exon, and other exons) in different m⁶A methylated genes in rice infected with RBSDV alone, RSV alone, or with both. B GO analysis of the different m⁶A methylated genes in rice infected with RBSDV alone, RSV alone, or with both compared with mock-treated rice plant. C KEGG analysis of the different m⁶A methylated genes in rice infected with RBSDV alone, RSV alone, or with both

Fig. 7

Identification of predominant consensus motifs containing m⁶A methylation sites in mock-treated and RBSDV- and RSV-infected rice plant using DREME and MEME suites. A Sequences logo representations of the consensus motifs containing m⁶A sites in mock-treated rice samples. B The most enriched consensus motif in RBSDV-infected samples. C The most enriched consensus motif in RSV-infected samples

Fig. 8

Rice m⁶A methylation levels are positively associated with the expression of key genes involved in antiviral RNA silencing pathways and plant hormone signals. A Comparisons of m⁶A methylation levels of the respect mock-treated and RBSDV- and RSV-infected rice plants at 0, 1, 2, 4, 8, and 16 dpi by LC-MS/MS. Error bars indicate mean ± SD, with three biological replicates. B Dot-blot analysis of m⁶A levels in extracted total RNA from samples at 16 dpi using the specific anti-m⁶A antibodies. The left side of the membrane depicts the amount of loaded mRNA from mock-treated and RBSDV- and RSV-infected rice plants, respectively. C qRT-PCR analysis of the relative expression of OsAGO18 in mock-treated and RBSDV- and RSV-infected rice plants at 0, 1, 2, 4, 8, and 16 dpi. D Relative expression of the OsSLRL1 in the three treatments at 0, 1, 2, 4, 8, and 16 dpi. E Analysis of m⁶A methylation levels on different fragments of OsAGO18 by m⁶A-IP-qPCR. The upper panel indicates the gene structures of OsAGO18 labelled with fragments amplified in the m⁶A-IP-qPCR assay. The results of positions 1 and 12 were chosen for figure exhibition. F m⁶A-IP-qPCR assay of the m⁶A methylation levels of different fragments on OsSLRL1. Similarly, the upper panel represents the gene structures labelled with fragments amplified in the m⁶A-IP-qPCR analyses. Results of positions 2, 3, and 4 were selected for the display in the figure. Error bars denote mean ± SD, n = 3 biological replicates in all qRT-PCR assays

Fig. 9

Integrated analyses of the main components of m⁶A methylation machinery in rice with m⁶A methylation modifications and gene expression in plants infected with viruses. A Relative expression levels of five “WRITER” components in rice plants by qRT-PCR analyses. B qRT-PCR analysis of the relative expression of five “ERASER” components in rice plants. C Relative expression levels of twelve “READER” component genes were determined by qRT-PCR. D Relative expression levels of five methyl “DONER” synthesis genes were analysed by qRT-PCR. E Rice m⁶A methylation pathways and related m⁶A methylated genes under plant virus infections. Blue coloured letters indicate the m⁶A methylated genes of certain treatments. For instance, RBSDV: OsMTA3, means the OsMTA3 gene was methylated in RBSDV-infected sample. All qRT-PCR assays were performed with three biological replicates, and the error bars denote the mean ± SD

Fig. 10

Integrated analyses of main antiviral RNA silencing pathway-related genes with m⁶A methylation modifications and gene expression levels in rice infected with viruses. A Relative expression levels of nine OsDCL genes in mock-treated and RBSDV- and RSV-infected samples. B Relative expression levels of five OsRDR genes in mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. C Relative expression levels of 17 OsAGO genes were determined with respect to mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. D Relative expression levels of nine resistance genes, including seven OsGDI genes, one OsSOT1 gene, and one OsStvb-i were determined in mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. E Rice antiviral RNA silencing pathways and the related m⁶A methylated genes in virus-infected plants. Blue coloured letters depict the m⁶A methylated genes of a certain treatment, as detailed in Fig. 9E. All qRT-PCR assays were performed with three biological replicates. The error bars denote the mean ± SD