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Comparison of Transcriptome Differences in Soybean Response to Soybean Mosaic Virus under Normal Light and in the Shade

Shading in the intercropping system is a major abiotic factor which influences soybean growth and development, while soybean mosaic virus (SMV) is a biotic factor that limits the yield and quality of soybean. However, little is known about the defense response of soybean to SMV in the shade. Thus, in the current study, both intensity and quality (red:far-red, R:FR) of the light were changed to simulate the shaded environment and comparative transcriptome analysis was performed. Morphologically, plant growth was inhibited by SMV, which decreased 35.93% of plant height and 8.97% of stem diameter in the shade. A total of 3548 and 4319 differentially expressed genes (DEGs) were identified in soybean plants infected with SMV under normal light and in the shade. Enrichment analysis showed that the plant defense-related genes were upregulated under normal light but downregulated in the shade. Pathways that were repressed include plant-pathogen interaction, secondary metabolism, sugar metabolism, and vitamin metabolism. In addition, genes associated with signaling pathways such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) were also downregulated in the shade. A qRT-PCR assay of 15 DEGs was performed to confirm transcriptome results. According to our knowledge, this is the first report on soybean response to dual stress factors. These results provide insights into the molecular mechanisms in which soybean plants were infected with SMV in the shade.


Introduction
Due to the advantages of interspecific complementation, nutrient exchange, marginal effects, and high biodiversity, maize-soybean strip intercropping has become the main cultivation model for soybean in southwestern China [1][2][3]. However, during the symbiosis of maize and soybean, the light environment was changed, as well as disease resistance of soybean [3,4].
Soybean mosaic virus (SMV) is one of the major pathogen causing severe yield loss and is widely distributed worldwide [5]. SMV is a member of the Potyvirus. The genome of SMV is approximately 10 kb in length and encodes 11 mature proteins [6,7]. These proteins work together to successfully attack the plants. After infection with the virus, the leaves of the plant produce symptoms such as mosaic, shrinkage, and deformity. This may be related to damage of chloroplast structure in the host

Plant Material, Virus Inoculation, and Light Treatment
Soybean seeds (Nannong 1138-2, a SMV susceptible variety) were kindly provided by Dr. Kai Li from Nanjing Agricultural University in China. The SMV isolate (YA87) was collected from the field soybean plants in Sichuan Province, China. The bean Phaseolus vulgaris cv. Topcrop was used for local-lesion purification of SMV, and then the virus was propagated on the soybean cv. Nannong 1138-2 [28]. Soybean seeds were surface-sterilized and sown in a mixed matrix containing PINDSTRUP organic soil (Pindstrup Mosebrug A/S, Ryomgaard, Denmark) and vermiculite (v:v, 4:1) in an artificial climate chamber with 25 • C/22 • C day/night temperature, 60% relative humidity and 14 h/10 h of photoperiod. The light condition for seedling growth was PAR of 296.92 µmol m −2 s −1 and R:FR of 5.92. The two unifoliolate leaves of 10-day-old soybean plants were mechanically inoculated with virus inoculum, which was homogenized by SMV-infected leaves in 0.01 mol L −1 K-phosphate buffer (pH 7.4). The mock treatments were inoculated with phosphate buffer without SMV. After inoculation, a part of the plants was transferred to a shaded environment and the other part was kept under normal light. The shading condition was PAR of 50.14 µmol m −2 s −1 and R:FR of 0.55, which was simulated by covering the green filter (type No. 122, Q-MAX, England) and adding far-red light-emitting diode (36 W, light peaking at 735 nm). The following four treatments were applied: Control under normal light (NC), SMV infection under normal light (NS), control in the shade (LC), and SMV infection in the shade (LS).

Sample Collection and Illumina Sequencing
Previous studies have shown that larger changes in transcriptional levels occurred in soybeans infected SMV at 10 dpi [16]. Therefore, we collected the V2 leaves (the second trifoliolate leaf, newly grown) of soybean plants after treatment for 10 dpi. Total RNA was extracted using phenol-chloroform-isoamyl alcohol and lithium chloride, washed using 70% ethanol, and finally checked by Agilent 2100 Bioanalyzer to ensure RIN number > 7.0. After the samples were tested, cDNA libraries were constructed and paired-end sequencing was performed based on the Illumina HiSeq 2500 platform at the Personal Biotechnology Co., Ltd. (Shanghai, China). Three biological replicates were set up for each treatment and a total of 12 independent samples were used for RNA-Seq.

Read Alignment and Expression Analysis
The reads number, Q30, N (%), Q20 (%), and Q30 (%) of raw data were counted. After removing reads containing sequencing adapters and reads of low quality, the clean data were mapped to the reference genome of Glycine max (Glyma2.0) using Bowtie2 and Tophat2. The reads mapped to exon region were also counted. HTSeq (Version 0.11) was used to calculate the read count mapped to each gene as the initial expression level of the gene. Gene expression levels were normalized using the RPKM (reads per kb per million reads) method. Differential expression analysis between treatments was identified by DESeq2 with screening parameters of log 2 FC (fold change) > 1 and p-adj (adjusted p-value) < 0.05.

Functional Enrichment Analysis of DEGs
The latest genomic reference information of Glycine max was obtained from the Soybase (www. soybase.org), including Gene Ontology (GO) annotations for each gene. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were obtained from the KEGG database. A hypergeometric test was used to find out the GO terms and KEGG pathways that were significantly enriched by DEGs. The enrichment analyses of GO and KEGG were performed using the OmicShare online website (www.omicshare.com/tools).

Validation of Gene Expression by qRT-PCR
To verify the accuracy and reproducibility of the RNA-Seq data, qRT-PCR assays were conducted with gene specific primers. Total RNA from the same treated samples were extracted. Reverse transcription was performed using 5× All-In-One RT Master Mix kit (AccuRT Genomic DNA Removal Kit, ABM, Vancouver, Canada). In addition, 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used and Eppendorf Mastercycler ep realplex (Eppendorf, Hamburg, Germany) instrument was used for the qRT-PCR experiment. Each treatment contained three independent biological replicates and three technical replicates. The expression level of soybean β-actin gene was used as an internal reference. The fold change value of gene expression was calculated using the 2 −∆∆Ct method. The sequences of specific primers were listed in Table S1.

Plant Phenotypes and Virus Detection after Inoculation
Soybean seedlings of 10 days were mechanically inoculated with SMV and subsequently grown under normal light (PAR, 296.92 µmol m −2 s −1 and R:FR, 5.92) and in the shade light (PAR, 50.14 µmol m −2 s −1 and R:FR, 0.55), respectively. After 10 days of treatment, the growth of the plants was affected by the viruses and light conditions, and the leaves showed typical mosaic symptoms. The plant height and petiole length of the plants were significantly elongated in the shade ( Figure 1A). However, after SMV infection, the development of the plant was impaired, and both the plant height and stem diameter were reduced ( Figure 1C,D). RT-PCR demonstrated that the virus-inoculated plants were SMV-positive ( Figure 1B). Plant growth and defense are often negatively correlated because growth and development are affected by the infection of the pathogen [29]. The phenomenon of viral infection leading to dwarfing of plants is often observed [16,30,31]. In our experiments, soybean seedling growth and development was impaired after infection with SMV. In the shade, plant growth is more severely damaged, with a reduction of 35.93% of plant height and 8.97% of stem diameter. Similarly, Díaz-Cruz et al. studied soybean infected with SMV for 10 dpi and found that the average plant height was reduced to 26% as compared to the control (CK) [16]. This may be an important factor in the reduction of yield in the shade, that is, the growth and development of plants are seriously affected by the virus.

Evaluation of RNA-Seq Data
To investigate the changes in the levels of gene expression in response to SMV infection under normal light and in the shade, we performed transcriptome sequencing of soybean plants. A total of 12 independent cDNA libraries were generated, which included triplicates of four treatments: SMV infection (NS) and control (NC) under normal light, SMV infection (LS), and control (LC) in the shade. The raw data are shown in Table S2. The Q20 percentages of raw data were above 95.62%. Both clean reads and clean base percentages were greater than 98% (Table S3). Overall, the reads total mapped on the reference genome were more than 83%, and the uniquely mapped reads were over 93% (Table S4). It should be noted that the mapping percentage of LS2 was lower compared to other samples, which might be affected by sample preparation or sequencing. Statistics on mapping to genomic regions showed that the proportion of mapping to exon regions accounted for more than 97%, including LS2 (Table S4). These data indicated that reliable transcriptome data was available for subsequent differential analyses.

Statistics on the Number of DEGs
DEGs were screened out between the treatments with log 2 FC (fold change) > 1 and p-adj (adjusted p-value) < 0.05. Under the normal light, a total of 3548 DEGs were identified between soybean leaves infected with SMV and CK, of which 2228 were upregulated and 1320 were downregulated. Under shading treatment, a total of 4319 genes were affected, including 2167 upregulated and 2152 downregulated (Figure 2A). This suggested that a large number of genes were downregulated when the soybean was infected with SMV in the shade. In order to clearly observe the effect of light on DEGs, we performed overlapping analysis on genes that were upregulated and downregulated, respectively. Overall, 380 genes (Nu-Lu, Figure 2B) were upregulated and 66 genes (Nd-Ld, Figure 2C) were downregulated under two light treatments. However, 225 genes were downregulated under normal light but upregulated in the shaded light (Nd-Lu, Figure 2B). In contrast, 490 genes were upregulated under normal light but downregulated in the shade light (Nu-Ld, Figure 2C). This indicated that the normally activated genes of the plant were suppressed when the light was insufficient.

GO Function Enrichment Analysis of DEGs
Gene Ontology (GO) enrichment analysis was used to determine the functional classification of DEGs between different treatments. Genes were divided into three categories: Biological process, molecular function, and cellular component. Among them, the enrichment terms of the biological process were the most common. The functional classification of DEGs after viral infection was very similar, in both normal light ( Figure 3A) and shade ( Figure 3B). For example, the cellular process, single-organism process, metabolic process, response to stimulus, and biological regulation in biological process, the binding and catalytic activity in molecular function, and cell and cell part in cellular component. The difference was observed in the shade, where the number of genes that were downregulated was greater ( Figure 3B). Obviously, in response to stimulus, signaling and immune system process, DEGs were mainly upregulated under normal light. However, in the shade, the numbers of downregulated genes were increased dramatically, even more than the number of upregulated genes. This suggested that light had a positive regulatory effect on the expression of immune-related genes.

KEGG Pathway Enrichment Analysis of DEGs
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed to determine changes in metabolic pathways following the viral infection and light treatment. Figure 4 showed the top 15 enrichment pathways for each group of DEGs. Under normal light, the most significant phenylpropanoid biosynthesis was induced, followed by plant-pathogen interaction, linolenic acid metabolism, and phenylalanine metabolism, while the protein processing in endoplasmic reticulum was repressed, as well as sugar metabolism (starch and sucrose metabolism, glycosaminoglycan degradation, galactose metabolism, and fructose and mannose metabolism), nitrogen metabolism, and vitamin metabolism (thiamine metabolism and vitamin B6 metabolism) ( Figure 4A,B). In the shade, Linoleic acid metabolism, other glycan degradation, and fatty acid biosynthesis were induced. However, the plant-pathogen interaction, protein processing in endoplasmic reticulum, and secondary metabolism (flavonoid biosynthesis, zeatin biosynthesis, isoflavonoid biosynthesis, and flavone and flavonol biosynthesis) were repressed ( Figure 4C,D). Like GO enrichment, plant immune-related pathways of soybean plants were activated in normal light but inhibited in shading conditions, indicating the positive regulation of light on plant defense mechanism.
It was described that the genes related to metabolism, proteins with binding function, development, and defense were differentially expressed when the soybean was infected with SMV [14]. Also, in previous studies, it was shown that at 10 days after infection with SMV, defense response, and signal transduction related genes in soybean were significantly induced, while energy and photosynthesis genes were repressed [16]. These studies are consistent with the results of our study. Phenylalanine is a precursor of a series of secondary metabolites. It plays an important role in response to stress. Defensive pathways including plant-pathogen interaction were greatly activated under the normal light. However, in the shade, the plant-pathogen interaction was significantly inhibited, along with secondary metabolism and vitamin metabolism. The secondary metabolites, such as flavonoid and isoflavonoid, have been documented to be important in stressed resistance [32,33]. Vitamins, some small molecular compounds, play an important role in the integrity of biological functions. Previous studies have shown that after sweet potato was infected by virus, the vitamin biosynthesis process was repressed [34].
The genes involved in plant immunity and pathogen infection constitute the plant-pathogen interaction pathways [35]. Effective regulation of plant defense systems is the basis for successful resistance to pathogens. WRKY and MYB are transcription factors involved in plant stress resistance under both biological and abiotic stresses [36][37][38]. An R2R3-MYB transcription factor has effect on tomato yellow leaf curl virus infection in tomato [39]. CaM is a Ca 2+ -binding protein that plays a role in developmental and stress responses. When tobacco was infected with TMV, NtCaM1, NtCaM2, and NtCaM13 were accumulated before induction of PR1 and PR3 expression [40]. EDS1, indispensable in the SA defense path, has been reported [41,42]. This series of regulatory genes was downregulated when plants were infected with SMV in the shade, suggesting that the plant's defense network cannot be activated in the shade.

DEGs Involved in Plant Hormone Signal Transduction
Plant hormones play an important role in plant growth and development. Some of these hormones are essential for plant immunity [43]. It is well-known that salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) play a leading role in disease resistance. We analyzed the DEGs of these three hormone pathways in the current study. Under normal light, the plants activated genes from three pathways after 10 days of SMV infection as show in Figure S3. The induction of ethylene-responsive transcription factor ERF (GLYMA_10G186800) and PR1 (GLYMA_15G062300) were the largest among others (Table 2). However, DEGs were mainly suppressed in the shade conditions ( Figure S4). The most downregulated ERF (GLYMA_02G006200) had a log 2 FC value of −6.71, followed by PR1, with a Log 2 FC value of −5.91. The MYC and JAR of the JA pathway were also significantly suppressed. These results indicated that the defense hormone regulatory network was inhibited when soybean plants was infected with SMV in the shade.
SA is an important hormone involved in the regulation of resistance to biotrophic and hemibiotrophic pathogens such as plant viruses [44]. In the current study, the SA pathway was repressed when soybean plants were infected with SMV in the shade conditions. It has been studied that light is an important factor for the regulation of plant defense signaling pathways [21]. Both light intensity and light quality can affect the expression of the PR gene in plant-pathogen interaction systems [24]. Phytochromes positively regulated Nicotiana tabacum against CMV [26] and ChiVMV [27] via a salicylic acid-dependent pathway. In addition, changes in the light conditions can also affect the JA pathway, which modulates the plant resistance to pathogens, such as Podosphaera xanthii [45], Pseudomonas syringae [46], Magnaporthe grisea [25], and Botrytis cinerea [23]. In this study, JA signal pathway of soybean plants was repressed in the shade. Previous studies have reported that ETH may act as a regulator of SA and JA pathway [47]. There are few reports on the effects of light on the ETH pathway. In this study, many ERF genes were found downregulated, suggesting that the ETH may also have an important role in regulating the defense in shade.
Here, we found that the expression of some genes was susceptible to light change, such as GLYMA_05G119500, GLYMA_14G032200, GLYMA_15G062700, and GLYMA_20G200500. The expression of those genes has changed dramatically in soybean in the shade (LC). However, infection with SMV has less effect on its gene expression. For other genes, their expression changed significantly after viral infection, but not sensitive to light change, such as GLYMA_01G046600 and GLYMA_15G140000. It suggested that there were differences between the effects of light and virus on soybean. Overall, qRT-PCR and RNA-Seq showed consistent expression patterns ( Figure 5). The correlation coefficients between qRT-PCR and RNA-Seq were more than 0.9, except for GLYMA_16G026900 and GLYMA_20G090000. Minor expression differences might be due to differences in sensitivity between the two methods. These results indicated that the RNA-Seq data was reliable.

Conclusions
In this study, we simulated the shading environment by changing the light intensity and light quality. The response of soybean plants infected with SMV after 10 days under normal light and shade was studied. SMV infection severely limits the growth and development of soybeans, and the effects in the shade were more serious. At the transcriptional level, SMV infecting soybeans under normal light activates plant defense-related pathways, while expression of many genes was repressed in the shade, especially immune-related genes. Our study revealed that the defense response of soybean to SMV cannot be effectively activated in the shade and provided a basis for further study of the molecular mechanism of soybean and SMV interaction in the shade.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/9/793/s1, Figure S1. Plant pathogen interaction pathway map between NC vs NS. Figure S2. Plant pathogen interaction pathway map between LC vs LS. Figure S3. Plant defense hormone signal pathway map between NC vs NS. Figure S4. Plant defense hormone signal pathway map between LC vs LS. Table S1. Primers used for qRT-PCR validation. Table S2. Statistics of raw data. Table S3. Statistics of clean reads. Table S4. Statistics of RNA-Seq map. Table S5. Differentially expressed genes involved in plant-pathogen interaction (ko04626) pathway between NC and NS. Table S6. Differentially expressed genes involved in plant-pathogen interaction (ko04626) pathway between LC and LS.