Quantitative detection of relative expression levels of the whole genome of Southern rice black-streaked dwarf virus and its replication in different hosts

A disease caused by Southern rice black-streaked dwarf virus (SRBSDV), a notorious member of Fijivirus, was first detected in 2001 in Yangjiang City, Guangdong Province, P.R. China and has caused serious loss in rice production in recent years [1‐6]. SRBSDV is a plant virus that can infect plants of the Poaceae family, such as rice (Oryza sativa) and corn (Zea mays) [7‐9]. SRBSDV is also an insect virus that affects the white-backed planthopper (WBPH) Sogatella furcifera Horváth, the main insect vector species that transmits SRBSDV in a persistent propagative manner [5, 7, 9, 10]. This disease possesses a long latent period and is difficult to detect at its early stages. However, symptoms readily develop it’s the later stages of infection and include dwarfing of plants, stiff leaves, witches’ brooming of leaves, branching at upward rootlets in the nodes, and the development of small waxy swellings on the stems [11, 12]. Thus, understanding the interaction between SRBSDV and its hosts is crucial to develop a better understanding of the biology and management of this disease. Xu et al. (2012) constructed two transcriptome groups (viruliferous and non-viruliferous WBPH) and further analyzed potential interaction genes. The genes involved in primary metabolism, ubiquitin-proteasome, cytoskeleton dynamics, and immune responses were up-regulated in viruliferous WBPH [13]. However, the molecular mechanism through which SRBSDV successfully infects and replicates in both plant and insect hosts remains unclear. Thus, whole-gene expression analysis of SRBSDV in the various hosts, including rice, corn, and WBPH is vital for gaining insights into the infection and replication process.

SRBSDV, a double-band RNA virus, is most closely related to but distinct from rice black-streaked dwarf virus (RBSDV), which is also a Fijivirus member [11, 14]. Comparison of the different genomic segments of SRBSDV with their counterparts in RBSDV suggests that SRBSDV encodes thirteen open reading frames (ORFs) and possesses at least six putative structural proteins (P1, P2, P3, P4, P8, and P10) and five putative nonstructural proteins (P6, P7-1, P7-2, P9-1, and P9-2) [11]. However, the functions of these thirteen genes have rarely been studied. P7-1 induces the formation of tubular structures in heterologous Lepidoptera Sf9 cells and has been proposed to transport SRBSDV particles [15]. Immunofluorescence staining of P9-1 in Sf9 cells has shown that P9-1 is sufficient to induce the formation of viroplasm-like structures [16]. The P6 protein has been identified as an RNA silencing suppressor [17]. However, no reports are available to date for other ORFs. The putative function of the translated proteins can only be postulated based on their RBSDV homologs. P1, P2, and P4 are putative RNA-dependent RNA polymerase (RdRp), a core protein, and an outer-shell B-spike protein, respectively [14, 18]. P3, P8, and P10 are an inner shell protein, a putative core, and a major outer capsid protein, respectively [14, 18, 19].

At present, the primary methods for detecting SRBSDV major are reverse transcription-polymerase chain reaction (RT-PCR) [12, 20‐23] and dot enzyme-linked immunosorbent assay [20, 24, 25]. However, both methods are only qualitative and not quantitative, and the sensitivity of these assays is limited. SYBR Green I-based real-time quantitative PCR is a reliable, accurate, and effective method that has been extensively applied to detect mRNA levels. Real-time PCR has been recently used in the detection of plant viruses, such as Epstein‐Barr virus[26], Pseudorabies virus[27], cauliflower mosaic virus[28], Tenuivirus[29], rice tungro bacilliform virus, and rice tungro spherical virus[30]. Recently, two papers have reported on the quantitative PCR (qPCR) detection of SRBSDV based on a single gene. Zhang et al. (2013) used the P4 gene as a detection gene to conduct qPCR for the simultaneous detection and differentiation of RBSDV and SRBSDV [31]. SRBSDV titer in rice plants was determined using qPCR based on P10 [32]. However, simultaneous detection of the real-time mRNA expression level of the whole genes in hosts has not been conducted so far. In this study, a qPCR method was adopted to simultaneously analyze the whole-gene expression levels of SRBSDV in distinct hosts (rice, corn, and WBPH) and monitor changes in their expression in real-time in rice. The results of this study are crucial for understanding the interaction between SRBSDV and its hosts. And further to provide molecular information for investigating the function of viral genes. Moreover, this study may serve as a basis for future investigations on interactions between SRBSDV and its major plant hosts and insect vector.

It has been previously reported that SRBSDV is a dsRNA virus whose genome lacks poly-A [7, 11]. In this study, Oligo-(dT)18 primers were used as anchor primers to simultaneously synthesize the single-stranded cDNA templates of the whole thirteen genes of SRBSDV. We have identified that in SRBSDV-infected Z. may, poly-A is added to P10 and P7 mRNA (Additional file 1). We propose that in the processing of primary RNA transcripts to produce mature mRNA molecules, the viral mRNA uses poly-A polymerase of the host to add poly-A to increase the stability of the molecule and avoid degradation by the ribozyme in host cells. Further research should clarify whether poly-A generally exists in all SRBSDV genes and investigate the molecular mechanism by which this occurs.

In distinct hosts (rice, corn, and WBPH), the thirteen SRBSDV genes displayed some similarities in expression levels suggesting that SRBSDV uses the same strategy to infect and replicate in these hosts. P3, P7-1 and P9-2 were the dominantly expressed genes in rice and WBPH. These genes also displayed relatively high expression levels in corn, suggesting that they have important roles during infection of hosts by SRBSDV. The P7-1 protein in SRBSDV is a virus movement protein that induces the formation of tubular structures in insect cells [15]. The observed high expression of P7-1 suggests that the formation of tubular structures is important for SRBSDV infection after the host cell is invaded. P3 and P9-2 are thought to encode an inner shell protein and a non-structure protein, respectively [11]. However, the molecular functions of these two genes have not yet been characterized in detail. The observed high expressions of P3 and P9-2 suggest that they have important roles in SRBSDV infection. Further studies should verify the significance of their functions. The P6 gene, a suppressor of SRBSDV, was found to be expressed at a relatively low level. The NS3 protein, a suppressor of the rice stripe virus, displayed the highest expression levels in the rice plant and insect vector [29]. The high expression level of P6 may indicate that it is sufficient for suppression of immune responses in plant and insect hosts. Notably, several interesting differences can be considered. Expression levels of most genes relative to P10 were higher in rice and WBPH than in corn. Thus, corn may exhibit stronger immune response against SRBSDV infection. Although P9-1 displayed high expression level in corn, but it showed low expression level in rice and WBPH. P9-1 has been characterized to be involved in viroplasm formation and viral replication inside the infected cell [16]. The high expression level of P9-1 may indicate that SRBSDV hastens its replication against the strong immune response of corn. Furthermore, most genes showed higher abundance in WBPH than in rice and corn. This finding may be attributed to more intensive replication and assembly of SRBSDV in insect hosts than in plant hosts. These high-expression genes could be used as target genes because of their greater sensitivity than other genes (P10 is a common gene for SRBSDV detection) in disease diagnostics and molecular biology studies of SRBSDV. To verify this speculation, a RT-PCR method was adopted for the detection of single SRBSDV-infected WBPH using P9-2 and P10 primers. SRBSDV-infected WBPH was successfully detected by using the P9-2 primers. However, no detection was evident in two parallel tests when using the P10 primers (Additional file 2).

A qPCR method was used to trace the expression levels of the SRBSDV genes in SRBSDV-infected rice on days 14, 20, 30, 40 and 50. On day 20, only P1 (putative RdRp) displayed a down-regulation among the 13 genes. On day 30, expression levels of the other 12 genes were reduced to 5.10, 8.20, 15.15, 4.46, 3.25, 6.93, 3.80, 3.09, 4.92, 5.40, 6.71 and 75.16% of day 20, respectively. Reduction rate of the 12 genes was approximately 90%, except for P10, which had a reduction rate of approximately 24%, close to that of P1 on day 20. The increase rate of P1 on day 30 was 46.75%, which was very close to the average increase rate (56.02%) of the other 12 genes. Additionally, except those of P1, The expression levels of the other 12 proteins decreased on 30 days. It was proposed that the mechanism of changing of virus amount at plant host was deemed to relate with plant immunity [33]. In many plant virus, like as Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Tomato spot wilt virus (TSWV), Tobacco mosaic virus (TMV) and so on, the concentration of virus will reach at high peak at 7–10 days after infecting plant host, then virus concentration will be decreased at a lower levels [34]. At present, many proteins, genes, materials and signal pathways were found to possess the activity of inhibition replication of virus under the stress of virus infection, for instance, pathogenesis-related proteins, N gene, phytoalexin and SA signal pathway [35]. Moreover, P5-1 and P5-2, P7-1 and P7-2, as well as P9-1 and P9-2 are in the same segment of SRBSDV dsRNA. However, significant differences in expression levels were found even between ORFs in the same segment. In addition, expression level of P9-1 was higher than that of P9-2, except on day 14. P9-2 was suggested to be more important during the early stages of SRBSDV infection because of its dominate expression. Future studies should investigate the function of these genes and the factors leading to difference in their expression levels.

These results provide a basis for further studies on the infection mechanism and interaction rules between SRBSDV and its hosts. A SYBR Green I-based real-time qPCR method was used to simultaneously analyze the expression levels of 13 SRBSDV genes in distinct hosts (rice, corn, and WBPH) and to monitor real-time changes in their expression levels in rice. P7-1, P9-2, and P3 were the most dominantly expressed genes in the three hosts, suggesting that they have significant roles in the SRBSDV infection process and that they could be targets for SRBSDV diagnosis. Moreover, similar expression patterns of SRBSDV were observed in distinct hosts, which suggested that the same strategy was taken by SRBSDV to infect different hosts. Real-time changes in SRBSDV gene expression levels in rice were analyzed at different time points after infection. Four different expression patterns were found in 13 genes and the time point corresponding to P1 down-regulation was also identified.