Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses

Abstract

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.

Fig 1

pSYNV-MR reporter gene expression in agroinfiltrated N. benthamiana leaves. (A) Illustration of pSYNV-MR_eGFP-DsRed and pSYNV-MR_rsGFP-CAT with reporter genes substituted between the NheI and BsrGI sites of pSYNV_HRz,ΔRz (see Sections SI to SIV and Fig. S1, S3, and S4 in the supplemental material for construction details). The first reporter in pSYNV-MR_eGFP-DsRed was derived from an enhanced green fluorescent protein (eGFP) gene substituted for the SYNV N gene, and the second reporter was a red fluorescent protein (DsRed) gene replacement of the SYNV P gene. The pSYNV-MR_rsGFP-CAT reporter contained an N gene substitution with a red-shifted green fluorescent protein (rsGFP) gene and a chloramphenicol acetyltransferase (CAT) reporter substitution for the P protein gene. RB and LB, right and left border sequences of pGD, respectively; 35S²P, double CaMV 35S promoter; HRz, = hammerhead ribozyme; , leader sequence; N5′UTR, 5′ untranslated region of N protein mRNA; NPJ/P5′UTR, gene junction separating the N and P protein ORFs and the 5′ untranslated region of the P protein mRNA; L3′UTR, 3′ untranslated region of the L protein mRNA; , SYNV trailer sequence; ΔRz = hepatitis delta virus ribozyme; NOS, pGD plasmid synthetase terminator. (B) Comparisons of GFP expression from the pSYNV-MR_rsGFP-CAT minireplicon and pGD-GFP. Top panels, appearance of punctate GFP foci in N. benthamiana leaf tissue monitored with a Zeiss Lumar epifluorescence microscope for 14 days postinfiltration (dpi) with bacteria harboring the pSYNV-MR_eGFP-DsRed reporter cassette and the SYNV N, P, and L plasmids. The infiltration mixtures also contained bacteria with plasmids encoding the p19 and γb host gene silencing suppressors. The magnification bar represents 100 μm. GFP fluorescence from pSYNV-MR_eGFP-DsRed was evident at 4 to 6 dpi, and the foci continued to express GFP for up to 3 weeks postinfiltration until the infiltrated tissue began to senesce (not shown). Middle panels, micrographs at 10 dpi, showing punctate GFP foci in single cells of N. benthamiana leaf tissue expressing the pSYNV-MR_rsGFP-CAT reporter cassette. Aside from the pSYNV-MR_rsGFP-CAT reporter cassette, the infiltration mixtures were as in the top panels. Different magnifications are shown to illustrate the distribution of the foci and to demonstrate that the foci are confined to single cells. The magnification bar represents 100 μm. Bottom panels, fluorescence from leaf tissue infiltrated with pGD-GFP. Fluorescence was evident by 3 dpi and was evenly distributed throughout infiltrated zones rather than in punctate foci. Fluorescence was most intense at 6 dpi and declined in the 9- and 14-dpi periods. (C) TLC analysis of CAT activity. CAT activities from leaf extracts were analyzed by thin-layer chromatography (TLC) as described in Materials and Methods. M indicates marker chloramphenicol derivatives separated by TLC. CK denotes control leaf tissue infiltrated with pSYNV-MR_rsGFP-CAT alone; 5 dpi and 10 dpi show CAT activity in N. benthamiana leaves harvested at 5 and 10 dpi. Positions of BCAM (nonacetylated fluorescent chloramphenicol substrate) and the CAT products 1Ac-BCAM (fluorescent 1-monoacetylated chloramphenicol), 3Ac-BCAM (fluorescent 3-monoacetylated chloramphenicol), and 1,3Ac-BCAM (fluorescent 1,3-diacetylated chloramphenicol) are indicated.

Fig 2

SYNV core protein requirements and gene silencing suppressor effects on GFP expression. (A) GFP foci in N. benthamiana leaves at 6 dpi with bacteria harboring the pSYNV-MR_eGFP-DsRed reporter cassette. The pGD panel shows a control pGD plasmid not encoding GFP. The −N, −P, and −L panels illustrate tissue infiltrated with pSYNV-MR_eGFP-DsRed and SYNV core protein mixtures lacking the N, P, or L plasmid, respectively. The +NPL panel shows fluorescence observed as single-cell foci scattered throughout the infiltrated regions of N. benthamiana leaves agroinfiltrated with mixtures containing the pSYNV-MR_eGFP-DsRed reporter cassette and the SYNV N, P and L plasmids. All experiments were conducted in the presence of p19 and γb gene silencing suppressors. The bar in the pGD panel represents 100 μm, and the remaining panels are the same magnification. (B) Western blot analysis showing GFP and SYNV N and P protein expression. Protein extracts recovered from leaf tissue were detected by immunoblotting with primary mouse GFP antibodies and secondary goat anti-mouse–horseradish peroxidase (HRP) antibodies or with rabbit antibodies elicited against the SYNV N and P proteins and secondary goat anti-rabbit–HRP antibodies. Lanes indicate uninfiltrated tissue (U), pGD-infiltrated tissue (pGD), and tissue infiltrated with pSYNV-MR_eGFP-DsRed lacking N (−N), P (−P), or L (−L), or pSYNV-MR_eGFP-DsRed containing N, P, and L (+NPL). mN, N protein monomer; dN, N protein dimer; other designations are as in Fig. 1. (C) Requirement of the N, P, and L proteins for CAT expression from SYNV plasmids in N. benthamiana tissue. Leaves were infiltrated with mixtures of bacteria containing various combinations of the N, P, and L plasmids for core protein expression, pSYNV-MR_rsGFP-CAT, and the TBSV p19 and BSMV γb gene silencing suppressors. Designations along the side of the TLC plate are as in Fig. 1. (D) Effects of viral gene silencing suppressors on GFP expression from the pSYNV-MR_eGFP-DsRed reporter. N. benthamiana leaves were agroinfiltrated with plasmids for expression of the N, P, and L proteins and pSYNV-MR_eGFP-DsRed in the absence or presence of various combinations of the gene silencing suppressors TBSV p19, BSMV γb, and TEV HC-Pro. The numbers of GFP foci were substantially lower in the absence of gene silencing suppressors (−Suppressor) than in their presence, and a synergistic increase in the numbers of fluorescent cells occurred upon addition of two or more suppressors. The bar in the −Suppressor panel represents 100 μm, and the remaining panels are the same magnification.

Fig 3

RNA blot analysis of pSYNV-MR transcription and replication products. (A) Northern blot detection of genomic-sense MR RNAs hybridizing to a positive-sense rsGFP probe. Total RNA extracts from N. benthamiana leaves at 10 dpi with bacteria harboring pSYNV-MR_rsGFP-CAT, combinations of the N, P, and L proteins, plus the p19 and γb suppressors were used. RNA from mock-infiltrated tissue (pGD) was used as a negative control. A genomic sense RNA corresponding in size to the 1,791-nt genomic-sense MR (gMR) size marker hybridized to the probe only in tissue infiltrated with derivatives harboring pSYNV-MR_rsGFP-CAT and plasmids for expression of SYNV N, P, and L proteins. Sample loading in all panels was evaluated by hybridization with an 18S rRNA probe (bottom). The 720-nt negative-sense complement to the GFP mRNA (−GFP) and the gMR size markers are identical in panels A, B, and C. (B) Presence of gRNAs hybridizing to the positive-sense leader () probe. (C) Hybridization of genomic-sense RNAs in N. benthamiana extracts to a positive-sense trailer () probe complementary to the 3′ end of the gRNAs. (D) Antigenomic RNAs hybridizing to a negative-sense rsGFP probe. A hybridizing RNA appearing slightly above the 720-nt GFP ORF size marker (GFP mRNA) was observed only in N. benthamiana leaves infiltrated with a complete mixture of plasmids expressing pSYNV-MR_rsGFP-CAT and the SYNV N, P, and L proteins. The location of a marker corresponding in size to the 1,791-nt “positive-sense” (antigenomic) RNA is shown along the side of the panel. Only minor amounts of a hybridizing band corresponding to this RNA were detected in any of the hybridizations, and these are variable, suggesting that progeny agRNA transcripts are not amplified. (E) Detection of SYNV-MR agRNAs hybridizing to the negative-sense CAT probe. A 660-nt hybridizing band, corresponding to the CAT mRNA, hybridized to the probe only in tissue infiltrated with derivatives harboring pSYNV-MR and plasmids for expression of the N, P, and L proteins. (F) Analysis of SYNV agRNAs hybridizing to a negative-sense leader () probe. The intense hybridization corresponding to the 144-nt RNA was present only in tissue infiltrated with derivatives harboring the pSYNV MR and plasmids for expression of the core proteins.

Fig 4

Analysis of the polarity of reporter gene expression from SYNV MR derivatives. (A) Schematic illustrations of SYNV MR derivatives expressing either eGFP, DsRed, or CAT reporter genes. For each pair of MRs, the constructs differ only in the orders of reporter genes relative to each other. (B to D) Expression of eGFP and CAT in leaves infiltrated with pSYNV-MR_eGFP-CAT or pSYNV-MR_CAT-eGFP, along with equal mixtures of agrobacteria harboring N, P, or L plasmids and gene silencing suppressors. (B) GFP expression monitored at 5, 7, and 10 dpi under an epifluorescence microscope. (C) Tissue extracts prepared from infiltrated leaves shown in panel B at 10 dpi were blotted with anti-GFP antibody for Western blots. Coomassie blue stains of the small subunit of Rubisco (Rubisco L) shown in panels C and F were used as controls to assess protein loading. The designations mGFP and dGFP indicate the monomer and dimer forms of GFP protein, respectively. (D) CAT activities analyzed by TLC. CAT products are as designated in Fig. 1. (E and F) Simultaneous visualization of eGFP and DsRed and evaluation of their relative expression levels. (E) Leaves infiltrated with pSYNV-MR_eGFP-DsRed or pSYNV-MR_DsRed-eGFP as described for panel B. The expression of fluorescent reporters was monitored at 7 dpi under a Zeiss LSM 780 confocal microscope. The same microscopy setting was used for both constructs to facilitate equal evaluations of the relative fluorescence levels. (F) Levels of eGFP and DsRed expression in infiltrated leaves at 5 and 10 dpi were analyzed in Western blots with specific antibodies.

Fig 5

Reporter gene expression to assess the functions of N protein mutants and cis-acting effects of MR mutations. (A) Effects of SYNV N protein mutants on GFP expression from pSYNV-MR_eGFP-DsRed. Expression of GFP at 6 dpi in leaves infiltrated with mixtures of bacteria containing plasmids for expression of pSYNV-MR_eGFP-DsRed, the P and L proteins, the p19 and γb suppressors, and various N protein mutants. Western blots show GFP (top) and N protein accumulation. N_wt, wild-type N protein; N_F42A and N_Y51A, proteins with site-specific mutations in the helix-loop-helix region of the SYNV N gene; N_YIY, triple mutant N protein containing alanine substitutions for tyrosine, isoleucine, and tyrosine at residues 48, 50, and 51, respectively. The N_KKRR mutant contains alanine substitutions targeting lysine and arginine residues 469, 470, 480, and 481 that destroy the bipartite nuclear localization signal and disrupt nuclear localization. Immunoblotting of GFP elicited by the N protein derivatives is shown at the bottom right. See the text for various effects that these mutations have on N and P protein interactions and N and P protein colocalization in subnuclear foci. The designations along the side of the blot identify positions of the GFP monomers (mGFP) and dimer (dGFP) and the N protein monomers and dimers (mN and dN, respectively). N_KKRR forms a high-molecular-weight multimer that does not migrate into the depicted region of the gel. (B) cis-acting effects of minireplicon mutants on GFP expression in infiltrated tissue. pGD, pGD lacking GFP; GFP, pGD encoding GFP; MR_wt, pSYNV-MR_GFP-DsRed; Δ, 18-nt deletion at the 5′ terminus of the sequence of pSYNV-MR_eGFP-DsRed; Δ, 18-nt deletion at the 3′ terminus of pSYNV-MR_GFP-DsRed; ΔHRz, HRz deletion preceding pSYNV-MR_eGFP-DsRed to prevent transcript processing. The lower panel depicts a Western blot probe of GFP accumulation in tissue infiltrated with the derivatives shown in the other panels.