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. 2018 Jan;19(1):77-89.
doi: 10.1111/mpp.12500. Epub 2016 Dec 4.

Host-induced gene silencing compromises Verticillium wilt in tomato and Arabidopsis

Yin Song  1 , Bart P H J Thomma  1
Affiliations

Host-induced gene silencing compromises Verticillium wilt in tomato and Arabidopsis

Yin Song et al. Mol Plant Pathol. 2018 Jan.

Abstract

Verticillium wilt, caused by soil-borne fungi of the genus Verticillium, is an economically important disease that affects a wide range of host plants. Unfortunately, host resistance against Verticillium wilts is not available for many plant species, and the disease is notoriously difficult to combat. Host-induced gene silencing (HIGS) is an RNA interference (RNAi)-based process in which small RNAs are produced by the host plant to target parasite transcripts. HIGS has emerged as a promising strategy for the improvement of plant resistance against pathogens by silencing genes that are essential for these pathogens. Here, we assessed whether HIGS can be utilized to suppress Verticillium wilt disease by silencing three previously identified virulence genes of V. dahliae (encoding Ave1, Sge1 and NLP1) through the host plants tomato and Arabidopsis. In transient assays, tomato plants were agroinfiltrated with Tobacco rattle virus (TRV) constructs to target V. dahliae transcripts. Subsequent V. dahliae inoculation revealed the suppression of Verticillium wilt disease on treatment with only one of the three TRV constructs. Next, expression of RNAi constructs targeting transcripts of the same three V. dahliae virulence genes was pursued in stable transgenic Arabidopsis thaliana plants. In this host, V. dahliae inoculation revealed reduced Verticillium wilt disease in two of the three targets. Thus, our study suggests that, depending on the target gene chosen, HIGS against V. dahliae is operational in tomato and A. thaliana plants and may be exploited to engineer resistance in Verticillium wilt-susceptible crops.

Keywords: HIGS; RNAi; Verticillium; virulence gene.

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Figures

Figure 1
Figure 1
Schematic organization of the T‐DNA region of the binary vectors used for gene silencing. (A) Schematic representation of the T‐DNA region of the Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) vectors. Verticillium dahliae Ave1, Sge1 and NLP1 DNA fragments were inserted between the double CaMV35S promoter (2×CaMV35Spro) and the nopaline synthase gene terminator (NOSter) in the TRV2 vector to generate the TRV‐based fungal gene silencing vectors TRV::Ave1, TRV::Sge1 and TRV::NLP1, respectively. Control construct TRV::GUS has been described previously (Song et al., 2016). RdRp, RNA‐dependent RNA polymerase; 16K, 16‐kDa cysteine‐rich protein; MP, movement protein; CP, coat protein; Rz, self‐cleaving ribozyme; ccdB, negative selection marker used in bacteria; CmR, chloramphenicol resistance marker; R1 and R2, attR1 and attR2 sites. (B) Schematic diagrams of the T‐DNA region of the binary vectors generated for the production of a hairpin RNA of Verticillium genes Ave1 (pFAST R03_Ave1), NLP1 (pHellsgate 12_NLP1) and Sge1 (pHellsgate 12_Sge1), as well as the green fluorescent protein gene (pFAST R03_GFP and pHellsgate 12_GFP) in transgenic Arabidopsis thaliana plants. CaMV35Spro, CaMV35S promoter; CaMV35Ster, CaMV35S terminator; OCSter, octopine synthase gene terminator; Hygr, hygromycin resistance gene; Kanr, kanamycin resistance gene; B1 and B2, attB1 and attB2 sites. LB and RB, left and right borders of T‐DNA.
Figure 2
Figure 2
Tobacco rattle virus (TRV)‐mediated fungal gene silencing in tomato plants compromises Verticillium dahliae Ave1 expression. (A) On inoculation with the wild‐type race 1 V. dahliae strain JR2, the impairment of Ave1‐triggered immunity in Ve1 tomato plants treated with TRV::Ave1, when compared with the TRV::GUS‐treated plants, is evidenced by stunted Ve1 plants at 14 days post‐inoculation (14 dpi) and fungal outgrowth on plating stem sections on potato dextrose agar (PDA). The Ave1 deletion mutant (V. dahliae JR2 ΔAve1) was used as Verticillium inoculation control. Plants were photographed at 14 dpi. (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Ve1 plants at 14 dpi. Bars represent Verticillium ITS levels relative to tomato actin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in Ve1 tomato plants on TRV::GUS treatment and subsequent inoculation with the wild‐type race 1 V. dahliae strain is set to unity. Different letter labels indicate significant differences (P < 0.05). The data shown are representative of three independent experiments.
Figure 3
Figure 3
Effect of Tobacco rattle virus (TRV)‐mediated NLP1 silencing in Moneymaker tomato plants on Verticillium dahliae inoculation. (A) Agroinfiltration with the TRV::NLP1 construct resulted in the suppression of Verticillium wilt symptoms on tomato plants, whereas no effect on disease development was observed in plants treated with TRV::GUS. The NLP1 deletion mutant (V. dahliae JR2 ΔNLP1) was used as Verticillium inoculation control. Plants were photographed at 14 days post‐inoculation (dpi). (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Moneymaker tomato plants at 14 dpi. Bars represent Verticillium ITS levels relative to tomato actin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in tomato plants on TRV::GUS treatment and subsequent inoculation with the wild‐type V. dahliae strain JR2 is set to 100% (control). Asterisks indicate significant differences when compared with the TRV::GUS‐treated plants on inoculation with the V. dahliae strain JR2 (P < 0.05). The data shown are representative of three independent experiments.
Figure 4
Figure 4
Effect of Tobacco rattle virus (TRV)‐mediated Sge1 silencing in tomato plants on Verticillium dahliae inoculation. (A) On inoculation with the V. dahliae strain JR2, no effect on disease development was observed on TRV::Sge1‐treated plants compared with TRV::GUS‐treated plants. The Sge1 deletion mutant (V. dahliae JR2 ΔSge1) was used as Verticillium inoculation control. Plants were photographed at 14 days post‐inoculation (dpi). (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Moneymaker tomato plants at 14 dpi. Bars represent Verticillium ITS levels relative to tomato actin levels (for equilibration) with standard deviation in a sample of three pooled plants. The fungal biomass in tomato plants on TRV::GUS treatment and subsequent inoculation with the wild‐type V. dahliae strain JR2 is set to 100% (control). Asterisks indicate significant differences when compared with TRV::GUS‐treated plants on inoculation with the V. dahliae strain JR2 (P < 0.05). The data shown are representative of three independent assays. (C) Relative expression level of the Sge1 gene was determined by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) at 14 dpi with the wild‐type V. dahliae strain on TRV::Sge1‐ and TRV::GUS‐treated plants. Bars represent levels of Sge1 transcripts relative to the transcript levels of V. dahliae GAPDH (GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; for normalization) with standard deviation of a sample of three pooled plants. Sge1 expression in V. dahliae in TRV::GUS‐treated plants on inoculation with the wild‐type strain V. dahliae is set to unity. The data shown are representative of three independent experiments.
Figure 5
Figure 5
Analysis of Ve1 Arabidopsis thaliana plants expressing the RNAi Ave1 construct. (A) On inoculation with the race 1 Verticillium dahliae strain JR2, Ve1 plants expressing RNAi Ave1 or the green fluorescent protein (GFP) construct do not show Verticillium wilt symptoms, whereas typical Verticillium wilt symptoms are recorded on plants with or without tomato Ve1 on inoculation with either V. dahliae JR2 or V. dahliae JR2 ΔAve1 at 21 dpi. Col‐0 plants with or without tomato Ve1 were used as controls. The V. dahliae JR2 ΔAve1 strain was used as Verticillium inoculation control. (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Arabidopsis plants at 21 days post‐inoculation (dpi). Bars represent Verticillium ITS levels relative to those of AtRuBisCo (RuBisCo, ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase) (for equilibration) with standard deviation in a sample of five pooled plants. The fungal biomass in Ve1 plants on inoculation with the wild‐type race 1 V. dahliae strain JR2 is set to unity (control). Three independent lines carrying the pFAST R03_Ave1 construct are shown (1, 2 and 3). Different letter labels indicate significant differences (P < 0.05). The data shown are representative of at least three independent experiments.
Figure 6
Figure 6
Arabidopsis thaliana Col‐0 plants expressing the NLP1 RNAi construct show enhanced resistance against Verticillium dahliae. (A) Typical appearance of non‐transgenic A. thaliana and transgenic lines carrying the pHellsgate 12_NLP1 construct to target NLP1 transcripts on mock inoculation or inoculation with V. dahliae strain JR2 or V. dahliae JR2 ΔNLP1 at 21 days post‐inoculation (dpi). (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Arabidopsis plants at 21 dpi. Bars represent Verticillium ITS levels relative to those of AtRuBisCo (RuBisCo, ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase) (for equilibration) with standard deviation in a sample of five pooled plants. The fungal biomass in Col‐0 is set to 100% (control). Three independent lines carrying the pHellsgate 12_NLP1 construct are shown (1, 2 and 3). Asterisks indicate significant differences when compared with Col‐0 (P < 0.05). The data shown are representative of three independent experiments.
Figure 7
Figure 7
Arabidopsis thaliana Col‐0 plants expressing the Sge1 RNAi construct show enhanced resistance against Verticillium dahliae. (A) Typical appearance of non‐transgenic A. thaliana and transgenic lines harbouring the pHellsgate 12_Sge1 construct to target Sge1 transcripts on mock inoculation or inoculation with V. dahliae strain JR2 or V. dahliae JR2 ΔSge1 at 21 days post‐inoculation (dpi). (B) Fungal biomass was determined by quantitative polymerase chain reaction (qPCR) in Verticillium‐inoculated Arabidopsis plants at 21 dpi. Bars represent Verticillium ITS levels relative to those of AtRuBisCo (RuBisCo, ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase) (for equilibration) with standard deviation in a sample of five pooled plants. The fungal biomass in Col‐0 is set to 100% (control). Three independent lines carrying the pHellsgate 12_Sge1 construct are shown (1, 2 and 3). Asterisks indicate significant differences when compared with Col‐0 (P < 0.05). The data shown are representative of three independent experiments.
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