Construction of expression vectors harboring the full-length or partial SVBV promoters
The full-length SVBV promoter from the Chinese SVBV isolate (GenBank accession number HE681085) was amplified by PCR. Sequence analysis using the Plant CARE program [22] showed that the full-length SVBV promoter contained several typical cis-acting elements, such as a TATA box, CAAT box, and potential cis-regulatory elements, including GATA motifs and TC-rich repeats. In previous research, important domains of viral promoters were generally incorporated to generate different mutants, and the driven activities were assessed in tobacco and other plants [23]. According to this prediction, a full-length SVBV promoter from the Chinese isolate (SP1) and three deletion mutants (SP2, SP3, and SP4) were constructed (Fig. 1A). The position of the SP1 promoter in the SVBV genome is shown. The mutant SP2 promoter contained a fragment of SP1, ranging from nucleotides − 324 to + 1 bp, and this mutant retained only the downstream core promoter region, which harbored a GA motif and two CAAT boxes. The mutant SP3 promoter had a deletion from nucleotide positions − 984 to − 819 bp and thus represented the upstream regulatory elements plus the core promoter region. The mutant SP4 promoter contained the full-length promoter sequence, except the 30 nucleotides upstream of the transcription initiation site (+ 1). Full-length and mutant SVBV promoters were individually used to replace the 35S promoter in the pCHF3 or pINT121 expression vector to produce pCHF-SP1, pCHF-SP2, pCHF-SP3, pCHF-SP4, pINT-SP1, pINT-SP2, pINT-SP3, and pINT-SP4. The expression vectors pCHF3 and pINT121 without their original 35S promoter (pCHF3-35SΔ and pINT121-35SΔ) served as negative control vectors. In this study, the full-length promoter of the SVBV isolate from the USA (SP-US) was also amplified by PCR and used to replace the 35S promoter in pCHF3 and pINT121 to generate pCHF-FLt-US and pINT-FLt-US, respectively. These two vectors were also used for comparisons in this study (Fig. 1B, C).
The full-length SVBV SP1 promoter showed potential for transient expression of exogenous genes in plants
To detect the SP1 promoter activity in transient expression, Agrobacterium harboring expression vector pCHF-SP1, pCHF-SP2, pCHF-SP3, pCHF-SP4, pCHF-FLt-US or pCHF3-35SΔ was injected into N. benthamiana stems. The injected stems were harvested at 64 hpi, and freehand-cut cross-sections from these stems were examined under a Leica DC300 stereomicroscope. The results of the study showed that GFP-derived green fluorescence appeared exclusively in the vascular tissues of stem sections injected with the expression vectors pCHF-SP1, pCHF-SP2, pCHF-SP3, pCHF-SP4, and pCHF-FLt-US. The fluorescence intensity of GFP driven by the SP1 promoter was significantly stronger than that of the other mutants and was also stronger than that of GFP driven by the CaMV 35S promoter. No green fluorescence was observed in sections from the plants injected with Agrobacterium harboring pCHF3-35SΔ (Fig. 2A).
GUS genes were then transiently expressed in the stem sections inoculated by Agrobacterium harboring the expression vectors pINT-SP1, pINT-SP2, pINT-SP3, pINT-SP4, pINT-FLt-US, and pINT-35SΔ, following injection into the stems of N. benthamiana plants. Histochemical staining of sections of paraffin-embedded stem tissues followed by light microscopy showed that the blue staining signal, representing GUS gene expression, was mainly localized in the vascular tissues and in some cells in the cortex of all pINT-SP1-, pINT-SP2-, pINT-SP3-, pINT-SP4-, or pINT-FLt-US-injected stem sections. GUS intensity in cells harboring the SP1 promoter was significantly stronger than that of other mutants and stronger than that of 35S-driven GUS. No blue staining was observed in sections from stems injected with Agrobacterium harboring the pINT-35SΔ vector (Fig. 2B).
GUS activity was then analyzed in tissues using fluorometric assays. Agrobacterium harboring vectors pINT-SP1, pINT-SP2, pINT-SP3, pINT-SP4, pINT-FLt-US, and pINT-35SΔ were inoculated into N. benthamiana leaves, and the inoculated leaves were harvested at 3 days postinoculation and analyzed for GUS activity using fluorometric assays. The results showed that the average GUS activity in leaves inoculated with Agrobacterium harboring the pINT-SP1 vector was approximately 3.2- and 1.8-fold greater than those in leaves inoculated with Agrobacterium harboring the pINT121 and pINT-FLt-US vectors, respectively (Fig. 2C). The mean GUS activity in leaves inoculated with Agrobacterium harboring the mutant pINT-SP4 vector was approximately 1.4-fold greater than that in leaves inoculated with Agrobacterium harboring the pINT121 vector, but was only approximately 75% that in leaves inoculated with Agrobacterium harboring the pINT-FLt-US vector. The mean GUS activity in leaves inoculated with Agrobacterium harboring the mutant pINT-SP2 or pINT-SP3 vector was lower than that in leaves inoculated with Agrobacterium harboring the pINT121 vector.
The full-length SVBV SP1 promoter showed potential for stable expression of exogenous genes in plants
The strength of promoter activity determines the expression levels of transgenes in plants. To compare the strength of promoter activity among the SVBV SP1, SVBV USA (FLt-US), and CaMV 35S (35S) promoters, tobacco plants were stably transformed with pINT-SP1, pINT-FLt-US, pINT121, or pINT-35SΔ vector, and transgenic tobacco seedlings or leaves were harvested and analyzed for promoter expression using histochemical staining. The results showed that both SVBV SP1 and FLt-US promoters conferred stronger GUS gene expression in transgenic tobacco seedlings and expand throughout the whole leaves compared with that in pINT121-transformed tobacco (Fig. 3A, B). Histochemical staining also showed that GUS gene expression was mainly observed in vascular bundles and that all transgenic tobacco plants showed GUS gene expression in the elongation zone of the roots (Fig. 3C). Furthermore, in stem cross-sections from transgenic plants, GUS gene expression was mainly present in the epidermal layer, pith, cortex, and vascular cells (Fig. 3D). These results suggested that the expression strength of the SVBV SP1 promoter was greater than that of the SVBV FLt-US and CaMV 35S promoters.
Analysis of GUS activity and accumulation of GUS mRNA in transgenic plants
To confirm that the activity of the SVBV SP1 promoter was stronger than that of the SVBV FLt-US promoter or CaMV 35S promoter, leaves were harvested from plants transformed with the pINT-SP1, pINT-FLt-US, or pINT121 vector and analyzed for GUS activity using fluorometric assays. The results showed that the average GUS activity in leaves harvested from plants transformed with the pINT-SP1 vector was approximately 1.7- and 3.1-fold greater than that in leaves harvested from plants transformed with the pINT-FLt-US or pINT121 vector (Fig. 4A). This finding was consistent with our GUS staining results (Fig. 3) and suggested that the promoter of the Chinese SVBV isolate may be used to generate stable transgenic plants with significantly higher levels of transgene expression than that in the plants transformed with a vector harboring the SVBV FLt-US or CaMV 35S promoter.
To confirm the GUS activity observed in Fig. 4A, we next assessed GUS mRNA levels in different transgenic plants using RT-qPCR of total RNA isolated from tobacco seedlings transformed with the pINT-SP1, pINT-FLt-US, pINT121, and pINT-35SΔ vectors. The results showed that GUS mRNA accumulated to a significantly higher level in pINT-SP1-transformed tobacco seedlings than in pFLt-US- or pINT121-transformed seedlings (Fig. 4B). This result was consistent with the results of GUS activity assays (Fig. 4A) and indicated that the transcriptional activity of the SVBV SP1 promoter was indeed stronger than that of the SVBV FLt-US or CaMV 35S promoter.
Influence of other SVBV-encoded proteins on SP1 promoter activity
SVBV gene-specific primers (Table 1) were used to amplify full-length ORFs I, II, III, IV, V, and VI from the Chinese SVBV isolate, yielding PCR products with lengths of 986, 485, 320, 1415, 2099, and 1556 bp, respectively. The resulting PCR fragments were individually cloned into the expression vector pBIN438, harboring the 35S promoter, to generate pBIN-ORFI, pBIN-ORFII, pBIN-ORFIII, pBIN-ORFIV, pBIN-ORFV, and pBIN-ORFVI, respectively. These expression vectors were then individually transformed into Agrobacterium and co-inoculated with Agrobacterium harboring pINT-SP1 into N. benthamiana leaves. Leaves co-inoculated with Agrobacterium harboring pINT-SP1 and pBIN438 were used as controls. The results showed that the GUS activities in leaves co-inoculated with Agrobacterium harboring pINT-SP1 and pBIN-ORFV or Agrobacterium harboring pINT-SP1 and pBIN-ORFVI were 2.7- and 2.4-fold greater than those in leaves co-inoculated with Agrobacterium harboring pINT-SP1 and pBIN438 (Fig. 5A). Furthermore, GUS activities in leaves co-inoculated with Agrobacterium harboring pINT-SP1 and pBIN-ORFI, pINT-SP1 and pBIN-ORFII, pINT-SP1, pBIN-ORFIII, pINT-SP1, and pBIN-ORFIV were similar to those in the leaves co-inoculated with Agrobacterium harboring pINT-SP1 and pBIN438. These results indicated that SVBV ORF V and VI could enhance foreign gene expression driven by the SVBV SP1 promoter, whereas other SVBV ORFs had no significant effect on foreign gene expression driven by this promoter. We further assessed GUS mRNA levels in different transgenic plants using RT-qPCR of total RNA isolated from N. benthamiana leaves co-inoculated with the pBIN-ORFV, pBIN-ORFVI, and pINT-121. Leaves co-inoculated with Agrobacterium harboring pBIN438 and pINT-121 were used as controls. The results showed that GUS mRNA accumulated to a significantly higher level in pBIN-ORF/pINT-121 and pBIN-ORFVI/pINT-121 than those in leaves co-inoculated with Agrobacterium harboringin pBIN438/pINT-121 (Fig. 5B).