Post inoculation phenotype in resistant and susceptible lines
The phenotype was studied to define the SMV strain SC3-inoculated susceptible and resistant lines. As expected, SMV symptoms were not visible in mock-inoculated resistant and susceptible lines. The susceptible line resulted in yellow-green mosaic and slightly curling symptoms on the leaves. The onset of symptoms was observed 7 dpi onwards, and the mosaic and severe curling symptoms were at 14 dpi. In contrast, no visual disease symptoms were observed in the resistant line (Fig. 1). The plant height of the susceptible line was significantly lower than that of the control, while there was no significant difference seen in the resistant line and its control (Fig. 2A-B). The SMV-CP transcripts were quantified by qRT-PCR analysis. Resistant lines showed low accumulation of the SMV-CP transcripts at 7 and 14 dpi compared to that of the susceptible line. SMV inoculation in the susceptible line resulted in a rise in an SMV-CP transcripts accumulation from 7 dpi onwards, which is highly associated with the phenotype changes after virus infection. Further, it was confirmed by DAS-ELISA analysis (Fig. 2C-D). These results indicate that the virus can gradually expand to the upper leaves in the susceptible line and can inhibit the normal growth of the plant, while the development of the virus in the resistant line is blocked, allowing the plant to grow normally.
Summary of small RNA libraries data sets
This study constructed 18 small RNA libraries from resistant and susceptible lines at 0 dpi, 7 dpi, and 14 dpi of SMV and generated 231.41 million raw reads ranging from 10.46 to 18.73 million reads. After removing the low-quality reads, a total of 225.42 million clean reads were obtained. Each sample was not less than 10.26 million clean reads, and the Q30 value of all samples was >95% (Table 1). Next, we filtered out sRNAs such as rRNA, tRNA, snRNA, snoRNA, and other ncRNAs and repetitive sequences in the clean reads to obtain unannotated reads containing miRNAs. Among all the reads in each sample, the total amount of rRNA and unannotated reads accounts for about 95% or more. Notably, scRNAs were not detected in all the samples (Additional file 1: Figure S1). After comparing unannotated reads with the soybean reference genome (Wm82.a2. v1), it is found that the ratio of the sequence in the alignment ranges from 27.83 to 53.80%, most of which are around 40%. The number of clean reads compared to the positive chain for each sample is higher than the number of clean reads compared to the negative chain (Additional file 2: Table S1, Additional file 3: Figure S2).
Identification of known and novel miRNA
A total of 1,092 miRNAs, including 608 known miRNAs and 484 novel miRNAs, were detected from all the libraries [35]. For known miRNAs, the number of miRNAs with a 21 nt length was the largest, followed by 22 nt and 24 nt. The proportion of miRNAs with a 20-23 nt length in which the first base of the 5’ end was U exceeds 50% (Fig. 3A-C). For the novel miRNAs, the number of miRNAs with a length of 21 nt was the largest, followed by 24 nt and 20 nt. The preference for U at the 5’end of these novel miRNAs was weaker than that of known miRNAs (Fig. 3D-F). At the known miRNA level, 509 known miRNAs, which belong to 108 families. Of these, the MiR166 family having the highest number of miRNAs (18), followed by MiR169_2 (13), MiR159 (11), and MiR482 (11). At the novel miRNA level, 196 miRNAs in 80 families, of these, the MiR4352 family had the largest number of miRNAs (17), followed by MiR4378 (12) and MiR4374 (11) (Additional file 4: Table S2).
Differentially expressed miRNAs after SMV infection
To identify the differentially expressed miRNAs among the different groups, we analyzed the miRNAs expression by volcano plot. It revealed that the number of differentially expressed miRNAs in the susceptible line from 0 dpi to 7 dpi, 7 dpi to 14 dpi, and 0 dpi to 14 dpi was higher compared to the resistant line. When the two lines were compared at the same time point, the number of differentially expressed miRNAs at 7 dpi and 14 dpi was significantly higher than 0 dpi (Additional file 5: Figure S3). By comparing the two lines 0 dpi with 7 dpi with the Wayne diagram, we found that 4 miRNAs were individually up-regulated while 1 miRNA was individually down-regulated in the resistant line. On the other hand, 295 and 119 miRNAs were individually up-regulated and down-regulated, respectively, in the susceptible line. Of these, two up-regulated miRNAs (gma-miR5761b, novel-miR-173) and four down-regulated miRNAs (gma-miR5037c,gma-miR5371-3p,gma-miR5371-5p,novel-miR-297) were common in both lines (Fig. 4A-F). Next, we compared the two lines 0 dpi with 14 dpi. It revealed that 8 miRNAs were individually up-regulated in the resistant line, and 3 were individually down-regulated. In the susceptible line, 400 miRNAs were individually up-regulated, and 98 were individually down-regulated. One up-regulated miRNA (gma-miR391-5p) and four down-regulated (gma-miR5371-5p novel-miR-101,novel-miR-224,novel-miR-317) were common in both lines (Fig. 4A-F). Collectively, these results indicate that different miRNAs may be involved in the process of virus-plant interaction by responding to SMV infection at different stages of the resistant and susceptible lines.
Notably, among the 295 miRNAs individually up-regulated at 0 dpi to 7 dpi in the susceptible line, 7 miRNAs including novel-miR-50, novel-miR-70, novel-miR-449, novel-miR-466; gma-miR2118a-3p, gma-miR5041-5p, and gma-miR10440 were targeting the NBS-LRR genes. Besides, 1 miRNA (novel-miR-49) that was individually up-regulated from 0 dpi to 7 dpi in the resistant line also targeted the NBS-LRR gene. Moreover, among the 400 miRNAs were individually up-regulated from 0 dpi to 14 dpi in the susceptible line, 7 miRNAs were targeting NBS-LRR genes, 6 of which are the same as before, and the remaining one is gma-miR390d (Table 2), indicating that the up-regulation of miRNAs targeting NBS-LRR resistance genes in the susceptible line could lead to the down-regulation of corresponding target genes, which may potentially affect the disease resistance of plants.
Target gene prediction and annotation of all miRNAs and differentially expressed miRNAs
A total of 20925 potential target genes were obtained. Five hundred eighty-one known miRNAs predicted 9240 target genes, and 439 novel miRNAs predicted 15,256 target genes (Table 3, Additional file 6: Table S3). Out of 20925 target genes, 20,913 genes were annotated (Table 4). The number of target genes annotated by differentially expressed miRNAs between different samples were analysed, and it was found that the minimum was 373 target genes at 0 dpi between the resistant and susceptible lines, and the maximum was 15,522 target genes in susceptible line from 0dpi to 14 dpi (Additional file 7: Table S4). The Gene Ontology (GO) analysis showed that: (1) From 0 dpi to 7 dpi, the metabolic process, cell part, and binding were the most enriched terms in both lines under the biological process, cellular component, and molecular function categories (Fig. 5A, Additional file 8: Figure S4A). (2) From 0 dpi to 14 dpi, the metabolic process cell and catalytic activity were the most enriched terms in the resistant line, respectively, while metabolic process cell, cell part binding, and catalytic activity were the most enriched terms in the susceptible line (Fig. 5B, Additional file 8: Figure S4B). It revealed that genes associated with different pathways in the two lines are likely to contribute to plants' resistance and susceptibility.
The Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that under the cellular processes, environment information processing, genetic information processing, metabolism, organismal systems categories: (1) 0 dpi to 7 dpi, the number of genes annotated to RNA degradation (11, 9.40 %) and biosynthesis of amino acids (12, 10.26 %) accounted for a higher proportion of the total annotated genes in the resistant line. The proportion of genes annotated to endocytosis (124, 5.09 %), plant hormone signal transduction (133, 5.46 %), and plant-pathogen interaction (132, 5.41 %) pathways were higher in the susceptible line (Fig. 6A, Additional file 9: Figure S5A). (2) From 0 dpi to 14 dpi, RNA degradation (5, 8.93%) and glutathione metabolism (5, 8.93%) annotated in the resistant line had a high proportion of genes in the pathway, while endocytosis (124, 5.09%), plant hormone signal transduction (133, 5.46%), biosynthesis of amino acids (109, 4.47%) and plant-pathogen interaction (132, 5.41%) annotated in the susceptible line had a high proportion of genes in the pathway (Fig. 6B, Additional file 9: Figure S5B). The results of the KEGG analysis also showed that the plant resistance response was a complex process. The genes involved in RNA degradation may play an important role in the resistant line, while the genes involved in endocytosis and plant hormone signal transduction may promote susceptibility in the susceptible line.
Validations of miRNA expressions by stem-loop RT-PCR and qRT-PCR
Two known miRNAs (gma-miR1507a and gma-miR390d) and two novel miRNAs (novel-miR49 and novel-miR70) have contrast expression patterns and targeting NBS-LRR genes in the resistant and susceptible lines were validated by stem-loop RT-PCR and qRT-PCR [36]. By stem-loop reverse transcription and RT-PCR, about 65 bp bands were found at 0 dpi, 7 dpi, and 14 dpi in both lines, indicating that these selected miRNAs were real in soybeans, and the figures showed the first six bands of each miRNA cropped from full-length gels (Fig. 7A-D, Additional file 10: Figure S6). Then qRT-PCR showed that these four miRNAs’ expression patterns were almost the same as the sequencing results (Fig. 7E-F). From 0 dpi to 7 dpi, the expression of novel-miR49 was down-regulated in the resistant line but not significantly changed in the susceptible line. The novel-miR70 did not show significant changes in the resistant line. There was no significant difference of miR1507a in the resistant line, but it was significantly down-regulated in the susceptible line. From 0 dpi to 14 dpi, novel-miR49 showed no significant difference in the resistant line.