Identification of the RRM1 gene family in rice (Oryza sativa) and its response to rice blast

Identification and physicochemical properties of RRM1 gene family members in rice

Rice genome sequence, annotation, protein sequence, and gene structure files were downloaded from the Ensembl Plants database (http://plants.ensembl.org/index.html). The HMM (Hidden Markov Model) PF00076.24 (RRM1 domain) of the RRM1 gene family was downloaded from the Pfam database (http://pfam.xfam.org/). We used HMMER3.2 software to search and analyze the database and predict the RRM1 gene family in rice, obtaining an E-value < 1 × 10⁻⁵. Domain analysis of identified RRM1 candidate sequences was performed using conserved RRM1 domain sequences in the Pfam database (PF00076.24) and SMART online analysis software (http://smart.embl.de/). We used the ExPASy (https://www.expasy.org/protparam/) online tools to predict protein isoelectric points, molecular sizes, and the lengths of amino acid protein sequences. Prediction and analysis of protein subcellular locations were performed using the Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) online tool.

Chromosome location of the OsRRM1 gene family and phylogenetic tree construction

The position of the RRM1 gene on the chromosome was analyzed using the rice gene sequence file downloaded from Ensembl Plants, with a chromosome location map drawn using TBtools software. The NJ (neighbor-joining method) phylogenetic tree of the RRM1 protein was constructed using MEGA11.0 (Molecular Evolutionary Genetics Analysis 11.0) with bootstraps set to 1,000 and other parameters left as their defaults. Then, the online software Itol (https://itol.embl.de/) was used to beautify the tree.

Analysis of the conserved domain, gene structure and motif of the OsRRM1 gene family

The conserved domains of the identified gene families were analyzed using the online tool Pfam (http://pfam.xfam.org/) and visualized by TBtools software (Chen et al., 2020).

Rice gene structure annotation files were downloaded from Ensembl Plants (http://plants.ensembl.org/) to identify the structure of the RRM1 gene family, with TBtools software used to draw the genetic structure.

The conserved motif location of the identified RRM1 gene family was predicted using the online tool MEME (https://meme-suite.org/meme/). The parameter settings were: motifs = 10, other parameters = default. The prediction results were plotted using TBtools software.

Interspecies collinearity analysis of the OsRRM1 gene family

Collinearity analysis and prediction of the RRM1 gene in rice and Arabidopsis thaliana were carried out. A collinearity map was drawn using TBtools software.

GO and KEGG analysis of OsRRM1 gene family

GO and KEGG analyses of the OsRRM1 gene family were performed using PlantRegMap (http://plantregmap.gao-lab.org/) and Kobas (https://bio.tools/kobas), respectively. All results were calculated with q < 0.05. Prism8.0 was used to plot the path name as the ordinate and the −log₁₀ (q-value) as the abscissa.

Analysis of presumptive cis-regulatory elements in the promoter region of OsRRM1

We used TBtools to predict cis-regulatory elements in the 2,000 bp upstream gene promoter region of OsRRM1 in the PlantCARE Database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html).

Expression pattern analysis of the RRM1 gene in rice treated with blast fungus

Fungus-inoculated rice seedlings were kept in a dark chamber at 25 °C with 85% humidity for 24 h. They were then maintained in the growth chamber at 26/24 °C under a 14 h light/10 h dark cycle with 85% humidity. Flag leaves were harvested at 24, 36, and 48 h, with three biological replicates collected for each treatment. To study the changes in rice blast gene expression, RNA-seq data were downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/) under accession number GSE157400 (Yang et al., 2020). To present data suitable for clustering display, after data analysis, the absolute FPKM value was obtained by dividing it by the mean of the control group and then performing log₂ transformation. TBtools was used to map the expression patterns of OsRRM1 gene family members identified in rice under blast fungus treatment.

Plant materials and rice blast stress treatment

Dried, mature, well-developed rice seeds were treated with 2% sodium hypochlorite solution for 48 h. The seeds were placed in a hydroponic box and divided into control and treatment groups. The hydroponic box was placed in a growth chamber with a 14/10 h light/dark cycle and 28/24 °C temperature cycle.

The rice seedlings were cultivated in the growth chamber until they reached the two-leaf stage at about 14 days. Finally, suspensions of blast fungus (guy11) with concentrations of 1 × 10⁵ /mL were used as stress treatment. At 0, 12, 24, 36, and 48 h after infection with rice blast (guy11), the young rice leaves were immediately frozen in liquid nitrogen and stored at −80 °C for later use.

Analysis of OsRRM1 gene expression by qRT-PCR

Total RNA was extracted from rice materials according to the instructions of the Total RNA Extraction Kit (Takara) and the experimental method of Du et al. (2021). The first-strand cDNA was synthesized with a PrimeScript First-strand cDNA Synthesis Kit (Takara). Specific primers for four candidate genes were designed (Table 1). Quantitative real-time PCR was performed on a quantitative real-time fluorescent quantitative PCR system (ABI 7500). The relative gene expression was calculated by the 2^−∆∆CT algorithm. For the experimental stress treatment data, the expression levels of each gene at 0 h were standardized to 1, with the expression levels at other time points calculated relative to this.

Subcellular analysis of the OsRRM1 gene

Construction of a subcellular carrier plasmid. The plasmid of the subcellular localization system (1300S-EGFP) was used to construct the vector for this experiment. It was subjected to single-enzyme digestion with Kpn I restriction enzyme to prepare a linearized vector. Specific primers were designed according to the candidate OsRRM1 gene sequence (Table S1), and the rice cDNA was used as a template to amplify the fragment. In this experiment, the homologous recombinant ligase ClonExpress II One Step Cloning Kit (Vazyme) was used to construct the recombinant plasmid.

Instantaneous transformation experiment with tobacco leaves. Several tobacco seeds were sown and grown for 28 d under a 12/12 h light/dark cycle for experimental use. The constructed recombinant plasmid was transferred to Agrobacterium EHA105 by the electrochemical method and cultured at 30 °C for 2 d. Agrobacterium was scraped off the surface of a solid culture dish with an inoculation ring and cultured in 10 mL YEB liquid medium at 170 rpm/min for 1 h. The bacteria were re-suspended with 10 mM MgCl₂ solution. Tobacco plants with good growth conditions were selected, and the lower epidermis of tobacco leaves was injected with a 1 mL syringe and labeled. After injection, the tobacco plants were cultured in low light for 2 d. Tobacco leaves inoculated with Agrobacterium tumefaciens were selected to make slides, and observed and photographed by confocal laser microscopy.

Screening and identification of RRM1 gene family members in rice

In this study, the domains (Pfam: PF00076.24) predicted that 212 RRM1 genes (all with E values < 1 × 10⁻⁵) were identified in the whole rice genome. Their conserved domain was analyzed by Pfam (Fig. 1). The results show that all 212 OsRRM1 genes contained RRM1, but its locations in the genes differed. These genes are named OsRRM1-1–OsRRM1-212 based on their physical location on the chromosome (Table 2). We used Expasy (https://web.expasy.org/protparam/) to analyze the 212 OsRRM1 genes’ molecular weights, lengths, isoelectric points, amino acids, etc. The results show that the lengths of amino acids encoding the 212 rice RRM1 genes ranged from 53 to 1,160 aa, the molecular weights ranged from 5,837 to 127,816 Da, and the theoretical isoelectric point distribution ranged from 3.97 to 12.37. Subcellular localization prediction shows that OsRRM1 was mainly located in the nucleus, followed by the extracellular matrix, mitochondria, chloroplast, cell membrane, and intracytoplasmic matrix. This suggests that these proteins mainly function in the nucleus. These variations suggest potential functional diversity and regulatory mechanisms, but they also pose challenges for researchers aiming to elucidate their roles in rice biology. Understanding how these differences impact protein structure, function, and interaction networks will require comprehensive experimental approaches, including functional assays, protein-protein interaction studies, and computational modeling. Moreover, considering the dynamic nature of gene expression and post-translational modifications, integrating multi-omics data and employing systems biology approaches will be essential for gaining deeper insights into the OsRRM1 gene family’s functions and regulatory networks in rice.

Chromosome localization and phylogenetic tree analysis of the OsRRM1 gene family

The positions of 212 OsRRM1 genes on chromosomes were mapped using TBtools software (Fig. 2). There were 212 OsRRM1 genes distributed on all 12 chromosomes, among which the 31 OsRRM1 genes on chromosome 3 were the most distributed, and only eight OsRRM1 genes were on chromosome 10, being the least distributed. Distinct gene clusters were formed on chromosomes 1, 2, and 3.

Phylogenetic trees of the 212 OsRRM1 proteins were constructed (Fig. 3). By analyzing the structure of the evolutionary tree, these proteins could be divided into five classes. The group V contained the highest number of OsRRM1 proteins (61). The group III contained 58 RRM1 proteins, while the group II and group IV contained 33 and 53 RRM1 proteins, respectively. The group I had the lowest number of RRM1 proteins (7). These groupings reflect the correlation and kinship between OsRRM1 protein sequences. By studying the differences and similarities between these groups, we can better understand the evolution and functions of OsRRM1 proteins.

Motif analysis and gene structure analysis of the OsRRM1 gene family

Gene structures and conserved motifs are one of the conserved expression modes of gene families. To better understand the structure of the OsRRM1 gene, the exon intron structure of the OsRRM1 gene was analyzed using annotated information from the rice reference genome (Fig. S1). The results show that the number, type, and order of conserved motifs in the OsRRM1 gene family were different. The sequence length and gene structure were also very different. This may be the result of replication of these sequences. However, according to the cluster analysis, the conserved motifs and gene structures of each category had similar distributions, which proves that the classification results are reliable.

The MEME online prediction tool was used to identify the conserved motifs of rice OsRRM1 proteins. Multiple motifs existed in the 212 OsRRM1 protein sequences (Fig. S1), and the types and numbers of motifs were highly overlapping. In addition, gene families within the same subfamily in the evolutionary tree were composed similarly on the motif. The gene families of the same subfamily in the phylogenetic tree were similar in motif composition, reflecting the sharing or similarity of their functions.

Evolutionary analysis of the OsRRM1 gene family and collinearity analysis between rice and Arabidopsis

A phylogenetic tree was constructed by comparing 212 and 230 AtRRM1—a total of 442 members. According to the topological structure of the evolutionary tree, the RRM1 proteins of the two species can be divided into five groups (Figs. S2–S7). Most of the RRM1 protein members of rice and Arabidopsis do not cluster into their own clades. Each subfamily contains members of the RRM1 family of Arabidopsis and rice, and the members of each subfamily may have similar functions and domains. According to the phylogenetic relationship of the protein sequences, the function of the OsRRM1 protein can be predicted by the function of the plant RRM1 protein, the function of which is known.

To further explore the evolutionary relationships of the OsRRM1 gene family, collinearity analysis between rice and Arabidopsis was conducted. The results (Fig. 4) show that 20 pairs of RRM1 genes in the two species were collinear, while no collinearity was found on chromosomes 8, 9, 10, 11, and 12 of rice or chromosome 4 of Arabidopsis. The analysis suggests that 20 pairs of RRM1 genes may have similar functions in rice and Arabidopsis, and were conserved during evolution.

GO and KEGG analysis of the OsRRM1 gene family

GO annotation results (Fig. 5A) show that the OsRRM1 gene family plays an important role in the alternative mRNA splicing, pre-spliceosome, RNA splicing, U2-type spliceosomal complex, defense response, immune response, immune system process, innate immune response. KEGG analysis (Fig. 5B) showed that the OsRRM1 gene family plays an important role in spliceosome, RNA processing and transport. For example, RRM1-185 (Os09g0565200) enhances rice resistance. By regulating the stability of chloroplast mRNA, it is important for the mRNA stability of NAD (P) H dehydrogenase (NDH) complex (Bang et al., 2021). RRM1-212 (Os12g0632000) is related to mRNA splice and ribosome synthesis. Under heat shock conditions, it may be involved in regulating RNA transport from specific stress-related genes to the nucleus, maintaining RNA stability and protein translation, etc. Overdosage of RRM1-212 can improve its tolerance to high temperature stress (Sahi et al., 2007). RRM1-113 (Os06g0112400), named PigmR, promotes the accumulation of PIBP in the nucleus to activate immunity (Zhai et al., 2019). As a transcription factor, PIBP1 can directly bind to the promoters of defense genes OsWAK14 and OsPAL1 to activate their expression. When PIBP1 and Os06g02240 were knocked out, blast resistance was greatly reduced (Zhai et al., 2019). Overexpression of RRM1-208 (Os12g0572400) (OsSCL30-OE) decreased the resistance of rice to low temperature, drought and salt stress, and led to the accumulation of reactive oxygen species (Zhang et al., 2022). These results further confirm the reported function of the OsRRM1 gene.

Characterization of presumptive cis-regulatory elements in the promoter region of the OsRRM1 gene

In plants’ response to stress, gene expression is closely related to cis-regulatory elements in the promoter region. Using the PlantCARE online analysis tool, the promoter of the rice OsRRM1 gene was predicted in the 2,000 bp region, and five stress-response regulatory elements were found, including the TGACG motif (involved in the JA response), CGTCA motif (involved in the MeJA response), ABRE motif (involved in abscisic acid stress), TCA element (involved in salicylic acid reactivity) and WUN motif (wound response element). In the OsRRM1 gene family, the element associated with the largest number of stress response elements was ABRE (Fig. S8), and ABA was synthesized mainly in response to blast stress. These results show that the stress-related response elements of the OsRRM1 gene were relatively intact, suggesting that it might be involved in regulation of the stress response in rice. However, the types and amounts of stress-related elements contained in each OsRRM1 gene promoter were different, which also indicates that each OsRRM1 had different responses to different stresses.

Expression pattern of the RRM1 gene family in rice after treatment with blast fungus

Using RNA-seq data, heat maps of the 212 OsRRM1 genes represented by log₂-fold-change values were constructed at different time periods after infection with rice blast (Fig. 6). All OsRRM1 genes were expressed, and three major clusters of expression patterns were distinguished according to the expression specificity at different time periods after treatment. The RRM1 gene in two clusters showed an obvious up-regulation trend.

Expression analysis of the OsRRM1 gene in response to biological stress

To further investigate the expression changes of OsRRM1 gene under biological stress, we selected eight OsRRM1 genes from five phylogenetic groups by analyzing GO and KEGG results and expression heat maps. The transcription level of eight OsRRM1 genes was measured by qRT-PCR. Rice seedlings were cultured in an artificial growth chamber until the two-leaf stage (about 14 d), and qRT-PCR was performed at 0, 12, 24, 36 and 48 h after infection with P. oryzae. Disease spots were observed on rice leaves after 7 days (Fig. S9). As shown in the figure (Fig. 7), there was no significant difference in the expression level of RRM1-193 (group I) under blast stress. The expression level of RRM1-79 (group II) was slightly down-regulated under blast stress, but the difference was not obvious. There was no significant difference in the expression of RRM1-10 (group III) under blast stress. The expression level of RRM1-37 (group IV) decreased significantly under blast stress. However, the expression levels of RRM1-15, RRM1-61, RRM1-76 and RRM1-207 (group V) were significantly increased under blast stress. Therefore, it is speculated that the RRM1 gene in group V plays a role in rice blast response.

Subcellular analysis of OsRRM1 genes

To further explore the subcellular localization of the OsRRM1 protein, transient expression of OsRRM1-15 and OsRRM1-207 was performed in tobacco epidermal cells. Confocal laser microscopy revealed green fluorescent protein signals of OsRRM1-15 on the nucleus and cell membrane (Fig. 8) and of OsRRM1-207 on the nucleus, cell membrane, and cytoplasm, indicating the locations of these genes.