Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing

Abstract

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Keywords: Beet necrotic yellow vein virus; genome editing; guide RNA delivery; multiple-genes expression vector; sugar beet.

Figure 1

Testing the infectivity of BNYVV full‐length cDNA clones in Nicotiana benthamiana and Beta macrocarpa. (a) Schematic representation of the construction of BNYVV full‐length infectious clones. BNYVV RNA1, RNA2, RNA3, RNA4 and RNA5 were cloned between the double CaMV 35S promoter (2 × 35S) and a ribozyme sequence (Rz) followed by a Nos terminator (Nos) in the pCB301 plasmid. (b) Systemic symptoms on N. benthamiana and B. macrocarpa agroinfiltrated with Agrobacterium tumefaciens cells harbouring the pCB‐BN1, pCB‐BN2, pCB‐BN3, pCB‐BN4 and pCB‐BN5 (pCB‐BN12345), or the pCB301 empty vector. (c) and (d): RT‐PCR Detection of BNYVV RNAs 2, 3, 4 and 5 from infiltrated and systemically infected leaves of N. benthamiana (c) and B. macrocarpa (d). The Mock and BN12345 DNAs were obtained by RT‐PCR from pCB301 and pCB‐BN12345‐infiltrated plants, respectively. N. benthamiana EF1A and B. macrocarpa Actin genes were used as loading controls. (e) and (f) Western blotting analysis of BNYVV CP and p42 on both of the infiltrated and systemically infected leaves of N. benthamiana (e) and B. macrocarpa (f). Coomassie brilliant blue (Coom.) staining is shown as a loading control.

Figure 2

Development of BNYVV RNA2 and RNA4 expression vectors. (a) Schematic representation of the construction of BNYVV RNA2 and RNA4‐based expression vectors. The RTD region from nt 1565 to nt 1872 of RNA2 was replaced by the sGFP. GUS sequence was fused to the C‐terminal of RNA4 encoded p31 or the N‐terminal 27 amino acids of p31. (b) Systematic symptoms (under visible light) and GFP expression pattern (under UV light) of Nicotiana benthamiana agroinfiltrated by pCB301 empty vector and pCB‐BN1/2G at 11 dpi, respectively. (c) Immunodetection using antibodies against BNYVV CP and sGFP in N. benthamiana systemically infected leaves at 11 dpi using the corresponding antibodies. Coomassie brilliant blue staining (Coom.) is shown as a loading control. (d) Systemic expression of a large protein by the BNYVV RNA4‐based vector after agroinfiltration of N. benthamiana plants for expression of pCB‐BN1/2/4p31N27aa‐GUS or pCB‐BN1/2/4‐GUS. GUS activity in the entire plants was detected by histochemical staining with X‐Gluc at 11 dpi. The empty vector pCB301 was used as a negative control.

Figure 3

RNA2 and RNA4‐based vectors elicit co‐expression of two proteins in whole plants. (a) Schematic representation of the construction of pCB‐BN2‐sGFP and pCB‐BN4‐p31‐P2A‐mCherry expression vectors. P2A: The “self‐cleaving” 2A sequence of porcine teschovirus‐1. (b) Immunodetection of BNYVV CP, sGFP and mCherry in infiltrated leaves, systemically infected leaves, and roots of Nicotiana benthamiana infected with pCB‐BN1/2G/4 m. Nicotiana benthamiana plants agroinfiltrated with pCB301 empty vector were used as a negative control. Equal loading was verified by staining the gel with Coom. (c) Confocal microscopy of different tissues of systemically infected N. benthamiana. Images of infiltrated leaves were taken at 5 dpi, while those of stem, roots and systemically infected leaves were taken at 14 dpi.

Figure 4

Simultaneous expression of four recombinant proteins from BNYVV‐based vectors in Nicotiana benthamiana. (a) Schematic representation of the construction of pCB‐BN2‐sGFP, pCB‐BN3‐mCherry, pCB‐BN4‐p31GUS and pCB‐BN5‐eCFP. The p25 sequence of RNA3 was replaced by mCherry and the eCFP‐HA sequence was fused to the C‐terminus of p26 encoded by RNA5 (b) Symptom expression (Bright), fluorescence observation (UV‐light) and GUS activity (GUS) were detected in N. benthamiana rdr6i leaves inoculated with pCB‐BN1/2G/3mC/4GUS/5eC at 5 dpi. The wild type BNYVV infectious clones pCB‐BN12345 was used as a negative control. (c) Immunodetection with corresponding antibodies against BNYVV eCFP‐HA, CP, sGFP and mCherry in infiltrated leaves of N. benthamiana infected with pCB‐BN1/2G/3mC/4GUS/5eC. The mock control resulted from pCB‐BN12345 infected plants. The arrow indicates the expected band and the asterisk indicates nonspecific band. Equal loading was verified by staining the gel with Coom. (d) Confocal microscopy of N. benthamiana leaves infiltrated with pCB‐BN1/2G/3mC/4GUS/5eC at 5 dpi. Fluorescent signals were not observed in the N. benthamiana cells infiltrated with pCB‐BN12345, Bars = 10 μm.

Figure 5

BNYVV‐based vectors for expression and subcellular co‐localization of multiple genes in Beta vulgaris. (a) Symptom expression (Bright), fluorescence observation (UV‐light) and GUS activity (GUS) were detected in B. vulgaris cv TY‐309 leaves inoculated with pCB‐BN1/2G/3mC/4GUS/5eC at 10 dpi. Wild type BNYVV infectious clones pCB‐BN12345 were used as a negative control. (b) Confocal microscopy of B. vulgaris leaves infiltrated with pCB‐BN1/2G/3mC/4GUS/5eC at 8 dpi. Fluorescence was not observed in B. vulgaris cells infiltrated with pCB‐BN12345, Bars = 10 μm. (c) Immunodetection using antibodies against BNYVV eCFP‐HA, CP, sGFP and mCherry in infiltrated leaves of B. vulgaris. The mock infected plants were agroinfiltrated for expression of the pCB‐BN12345 plasmid. The arrow indicates the expected band and the asterisk indicated a nonspecific band. Equal loading was verified by staining the gel with Coom. (d) Schematic representation of the construction of the pCB‐BN3‐GFP‐p14 and the pCB‐BN4‐RFP‐Fib2 expression vector. The p25 sequence of RNA3 was replaced by the GFP‐p14 sequence. The P2A‐RFP‐NbFib2 sequence was fused to the C‐terminus of RNA4 encoded p31. (e) Confocal images at 10 dpi. showing subcellular distribution of GFP‐p14 and RFP‐Fib2 expressed in B. vulgaris cells infiltrated with pCB‐BN1/2/3GFP‐p14/4RFP‐Fib2. Fluorescent signals were not detected in B. vulgaris cells infected with the wild type BNYVV pCB‐BN12345 infectious clones. Bars = 10 μm.

Figure 6

BNYVV‐based genome editing using the CRISPR/Cas9 system in N. benthamiana. (a) Schematic representation of the construction of BNYVV‐based genome editing vectors. The NbPDS3‐targeting and gRNA scaffold sequences displayed in scale were fused to the C‐terminus of the RNA4 p31 sequence. (b) Photobleached phenotype of transgenic Cas9‐expressing N. benthamiana (KQ334) plants inoculated with pCB‐BN1/2/4gRNA::NbPDS (right). Lack of photobleaching after agroinfiltration with pCB‐BN1/2/4 (left). Pictures were taken at 5 weeks post‐infiltration (wpi). (c) The infectivity of BNYVV and the expression of gRNA::NbPDS were detected in systemic infected leaves of KQ334 plants infiltrated with pCB‐BN1/2/4 or pCB‐BN1/2/4gRNA::NbPDS at 5 wpi. NbEF1A was used as the control. (d) BNYVV‐based genome editing mutations of NbPDS in N. benthamiana. The NbPDS sequence was amplified by genomic DNA PCR from pCB‐BN1/2/4‐infiltrated control plants or from photobleached leaves of pCB‐BN1/2/4gRNA::NbPDS ‐infiltrated KQ334 plants. Purified PCR products were digested with NcoI and separated on a 2% agarose gel. Lane 2, pCB‐BN1/2/4 infiltrated, line 3, pCB‐BN1/2/4gRNA::NbPDS infiltrated. The mutation rate was counted by the software Image J. The arrowheads indicated restriction products. (e): Sanger sequencing of wild type (WT) and mutant versions of NbPDS from photobleached leaves area after BNYVV‐based NbPDS editing. The target/mutated sequences were shown in red. The protospacer‐associated motif (PAM) is shown in green and the NcoI site is shown in blue over the site. Different deletions were indicated by numbers to the right of the sequence (‐ means deletions of n nucleotides).