A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X

Abstract

Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

Figure 1. Expression of terpenoid synthase genes after PVX infection.

Relative expression levels of different terpenoid synthase genes in third day/fifth day after treatment (3^rd D/5^th D) N. benthamiana. Plants were infiltrated with Agrobacterium carrying Potato Virus X (PVX) plasmid or pGreen empty vector alone (Control). Values are mean ± SE (n = 6). Letters indicate significant differences among different treatments (P < 0.05, Duncan's multiple-range test).

Figure 2. Silencing of NbTPS1 and NbEAH decreases plant resistance against PVX.

sTRV1 and sTRV2 vectors were used for N. benthamiana virus-induced gene silencing (VIGS). Ten days after inoculation, plants were further infiltrated with Agrobacterium containing PVX-GFP. (A) Relative expression level of NbTPS1 gene in control and NbTPS1-silenced N. benthamiana plants. Values are mean ±SE (n = 6). (B) Relative expression level of NbEAH gene in control and NbEAH-silenced N. benthamiana plants. Values are mean ± SE (n = 6). (C) GFP imaging was performed under UV illumination 6 days after PVX-GFP infection. Mock, infiltrated with Agrobacterium only; Control, infiltrated with Agrobacterium containing sTRV1 and empty sTRV2 vector. Bar: 20 mm. (D) The amount of GFP in injected leaves. 1, Control plant; 2, TPS1 VIGS plant; 3, EAH VIGS plant. (E) The amount of GFP in systemic leaves. 4, Control plant; 5, TPS1 VIGS plant; 6, EAH VIGS plant. The large subunit of Rubisco (rbcL) is shown as a protein loading control. The experiment was repeated at least three times with similar results. Full-length blots/gels are presented in Figure S2.

Figure 3. Altered volatile and non-volatile organic compounds by PVX infection.

(A) Chromatogram of non-volatile compounds produced by N. benthamiana leaves that were infiltrated with Agrobacterium only. (B) Chromatogram of non-volatile compounds produced by N. benthamiana leaves that were infiltrated with Agrobacterium containing PVX. Chromatogram of volatile compounds emitted from N. benthamiana plants (C) and PVX-infected N. benthamiana plants (D) that were treated with methyl jasmonate (MeJA). IS, internal standard (camphor); 1, α-pinene; 2, β-pinene; 3, D-limonene; 4, linalool; 5, α-bergamotene; 6, epi-aristolochene.

Figure 4. Capsidiol 3-acetate is synthesized by NbTPS1 and NbEAH.

(A) Subcellular localization of NbTPS1 and NbEAH. N. benthamiana were transformed with Agrobacterium carrying either YFP, NbTPS1-YFP or NbEAH-YFP and ER Marker-CFP. After 48 h incubation, the transformed cells were observed under a confocal microscope. N, nucleus. Scale bar, 50 μm. (B) In vitro enzymatic assays of NbTPS1. Chromatogram of the products obtained by incubating (E,E)-FPP with recombinant proteins HIS-SUMO or HIS-NbTPS1. (C) In vivo enzymatic assays of NbTPS1. N. benthamiana leaves were co-infiltrated with sTRV1 and sTRV2-EAH to obtain EAH VIGS plants, while plant co-infiltrated with sTRV1 and sTRV2 served as a control. Chromatogram of the products in control N. benthamiana leaf that were infiltrated with Agrobacterium containing YFP (Upper) or TPS1-YFP (Middle) or in EAH-silenced plant leaves that were infiltrated with Agrobacterium containing TPS1-YFP (Below). IS, internal standard (camphor). (D) Relative amount of capsidiol 3-acetate in TPS1-, EAH-silenced and vector control plants. (E) Models of capsidiol 3-acetate biosynthesis in N. benthamiana.

Figure 5. JA pathway is involved in the production of PVX-induced capsidiol 3-acetate.

Relative NbTPS1 (A) and NbEAH (B) expression levels (mean ± SE, n = 5) in N. benthamiana treated with MeJA or 0.01% Tween-20 (Control). (C) Relative expression level of NbCOI1 gene in control and NbCOI1-silenced N. benthamiana plants. Values are mean ± SE (n = 6). (D) GFP imaging was performed under UV illumination 6 days after PVX-GFP infection. Mock, infiltrated with Agrobacterium only; Control, infiltrated with Agrobacterium containing sTRV1 and empty sTRV2 vector. Bar: 20 mm. (E) The amount of GFP in injected leaves (1, 2) and systemic leaves (3, 4) 1, Control plant; 2, COI1 VIGS plant; 3, Control plant. 4, COI1 VIGS plant. The large subunit of Rubisco (rbcL) is shown as a protein loading control. The experiment was repeated at least three times with similar results. Full-length blots/gels are presented in Figure S7. Relative NbTPS1 (F) and NbEAH (G) expression levels (mean ± SE, n = 5) in COI1 VIGS and control N. benthamiana in sixth day after PVX infection. (H) Relative amount of capsidiol 3-acetate in COI1 VIGS and control plants. Values are mean ± SE (n = 6). Asterisks indicate significant differences between different treatments. (*, P < 0.05; **, P < 0.01; Student's t-test).