A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana

Abstract

Nicotiana benthamiana often displays more intense symptoms after infection by RNA viruses than do other Nicotiana species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in N. benthamiana antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in N. benthamiana. One RdRP was similar in sequence to SDE1/SGS2 required for maintenance of transgene silencing, whereas the second, named NbRdRP1m, was >90% identical in sequence to the salicylic acid (SA)-inducible RdRP from Nicotiana tabacum required for defense against viruses. NbRdRP1m expression was induced by SA treatment or challenge with Tobacco mosaic virus, but the gene and transcript sequences differed from those of other SA-inducible RdRPs in that they contained a 72-nt insert with tandem in-frame stop codons in the 5' portion of the ORF. N. benthamiana plants transformed with an SA-inducible RdRP gene from Medicago truncatula were more resistant to infection by Tobacco mosaic virus, Turnip vein-clearing virus, and Sunn hemp mosaic virus (members of Tobamovirus genus), but not to Cucumber mosaic virus and Potato virus X (members of different genera than the tobamoviruses). Our results indicate that N. benthamiana lacks an active SA- and virus-inducible RdRP and thus is hypersusceptible to viruses normally limited in their accumulation by this RdRP. These findings are significant for those studying virus-induced gene silencing, the hypersensitive response and systemic acquired resistance.

Fig. 1.

Analysis of the RdRP content of N. benthamiana. (A) Schematic figure of NbRdRP1m. The length of the ORF (large filled rectangle) and the 5′ and 3′ UTRs (small filled rectangles) are indicated, and the position of a 72-nt insert, beginning at nucleotide 1524, is indicated (cross bars). (B) Sequence alignment of a portion of N. tabacum NtRdRP1 (Nt1; GenBank accession no. AJ011576) with N. benthamiana NbRdRP1m (Nb1m; GenBank accession no. AY574374) showing the position of the 72-nt inserted sequence in NbRdRP1m (opposite the NtRdRP1 sequence marked with dashes). The tandem in-frame stop codons are circled, and nucleotides that differ between the two genes are highlighted. (C) RdRP copy number in N. benthamiana. Genomic DNA was digested with NheI (lane 1), EcoRV (lane 2), NdeI (lane 3), and XhoI (lane 4) and probed with a fragment of NbRdRP1m (nucleotides 1390–2412) representing a partially conserved region within plant RdRPs as determined by sequence alignment with clustalw. The restriction enzymes cleaved 3′ or 5′ of the probed sequence. (D) Analysis of RdRP mRNA sequences for the 72-nt insert. Total RNA from N. benthamiana (lanes 1 and 2) and N. tabacum (lanes 3 and 4) was amplified by using primers specific for NbRdRP1m and NtRdRP1. Lane 5 shows size-marker fragments representing 300, 400, 500, and 600 bp (bottom to top). Only the N. benthamiana samples contained a 72-nt insert.

Fig. 2.

Accumulation of NbRdRP1m transcript in N. benthamiana tissues and after treatment with SA. (A) Relative expression of NbRdRP1m in tissue harvested at early flowering. Expression levels were determined by real-time RT-PCR by using primers specific for NbRdRP1m and EF1α transcript. The expression of NbRdRP1m was normalized to EF1α transcript levels (percentage of EF1α, y axis). Bars represent standard deviations for three replicates. (B) NbRdRP1m transcript levels after SA treatment. Leaves were sprayed with a 2 mM SA solution at the eight-leaf stage. RNA was isolated from sprayed leaves and analyzed as described for A for NbRdRP1m transcript. Bars represent standard deviations for three replicates per time point.

Fig. 3.

MtRdRP1 transcript levels in transgenic N. benthamiana. Transcript levels from leaves of T0 plants transformed with a vector containing (lines designated R) or not containing (lines designated V) MtRdRP1. MtRdRP1 transcript levels were determined by Northern analysis using a probe complementary to MtRdRP1 (nucleotides 1–2135) (A) or real-time RT-PCR with gene-specific primers (conditions and controls as described for Fig. 2 A) (B). For the Northern analysis, lanes contained 20 μg of total RNA, and the same membrane was stripped and probed for 18S rRNA (18S). Each lane represents extract from independent plants.

Fig. 4.

Viral RNA accumulation in T0 cuttings of N. benthamiana expressing MtRdRP1 after challenge with TMV M^IC1,3,6. RNA was isolated from systemic leaves 6 (A)or13(B) dpi with TMV M^IC1,3,6 (1 μg/ml). Total RNA (10 μg) from MtRdRP1-expressing (lines R1-1 and R15-1), vector control (line V16-2), or healthy (H) plants was hybridized with probe complementary to the TMV coat protein ORF or identical to the 5′ 1.5 kb of the TMV genome, respectively, for sense or minus-sense genomic viral RNA determinations (+gTMV or -gTMV). The same membrane was probed for 18S rRNA (18S) at each date. Each lane represents extract from one plant.

Fig. 5.

Symptom suppression and decreased viral RNA accumulation displayed by T1 progeny of N. benthamiana expressing MtRdRP1 after challenge with TMV, SHMV, TVCV, or CMV. For A–D (images), the plant on the left did not contain MtRdRP1 (line V19-1), and the plant on the right expressed MtRdRP1 (line R1-1 for TMV, SHMV, or CMV and R15-1 for TVCV). (A) Symptoms at 21 dpi with TMV U1. (B) Symptoms at 21 dpi with SHMV. (C) Symptoms at 21 dpi with TVCV. (D) Symptoms at 14 dpi with CMV. (E) Accumulation of TVCV RNA in plants inoculated with TVCV. Leaves were harvested at 10 dpi, and total RNA (3 μg per sample) from MtRdRP1-expressing (lines R1-1 and R15-1), vector control (line V19-1), and healthy (H) plants was hybridized with a probe complementary to the 3′ end of the TVCV genome (nucleotides 5455–6311; +gT). The same membrane was probed for 18S rRNA (18S). Each group of three lanes represents results from developmentally matched plants. (F) Accumulation of CMV RNA in plants inoculated with CMV. Leaves were harvested at 13 dpi and total RNA (2 μg per sample) from MtRdRP1-expressing (lines R1-1 and R15-1), vector control (line V19-1), and healthy (H) plants was hybridized with a probe complementary to CMV RNA 2 (nucleotides 367–840; +gC). Ethidium-bromide-stained 18S rRNA (18S) was visualized on the membrane. Each group of three lanes represents results from developmentally matched plants.

Fig. 6.

Symptoms induced on N. benthamiana after VIGS with NbRdRP1m. (A) Schematic diagram showing the insertion site within a PVX vector of two fragments of NbRdRP1m (5′, nucleotides 1200–2239; 3′, nucleotides 2600–3174) in either sense (PVX-5s and PVX-3s) or antisense (PVX-5as and PVX-3as) orientation. (B and C) Symptoms induced on leaves at 20 dpi with PVX containing an NbRdRP1m insert (PVX-3s) (B) or not containing an insert (PVX) (C). Symptoms were enhanced (leaf necrosis) when the RdRP sequence was present in PVX.