Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus

Abstract

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.

Keywords: NLR receptor; Sw-5b; defense signaling; plant innate immunity; tomato spotted wilt virus.

Figure 1

Silencing Sw-5b through VIGS abolished the resistance of Sw-5b-transgenic N. benthamiana to TSWV infection. (A) Sw-5b-transgenic N. benthamiana plants treated with TRV-GUS and TRV-Sw-5b. Agrobacterium harboring TRV1 was mixed equally with Agrobacterium harboring TRV2 with gene fragments of GUS, or Sw-5b, and the mixture was co-infiltrated into 4–6 week old leaves of Sw-5b-transgenic N. benthamiana. The treated plants were photographed at 3 weeks post TRV infection. (B) Analysis of TSWV systemic infection of Sw-5b-transgenic N. benthamiana plants pretreated with TRV-Sw-5b. TRV-GUS treatment was used as the control. The newly emerged leaves of Sw-5b-transgenic N. benthamiana plants silenced for Sw-5b or pretreated with TRV-GUS at 3 weeks post TRV infection were inoculated with crude extract of TSWV-infected tissues. The plants were photographed at 14 days post TSWV inoculation. The systemic infection was evident in all 18 Sw-5b-silenced plants in three repeated experiments. (C) RT–PCR analysis of the expression of TSWV N RNA in systemic leaves of Sw-5b-transgenic N. benthamiana plants pretreated with TRV-GUS or TRV-Sw-5b. The internal reference gene was NbActin. (D) Western blot analysis of TSWV N accumulation in systemic leaves of Sw-5b-transgenic N. benthamiana silenced for Sw-5b and pretreated with TRV-GUS control at 10 dpi post TSWV inoculation. A plant sample expressing empty vector (Vec.) was used as a negative control. Ponceau S staining of Rubisco was used as a protein loading control.

Figure 2

Silencing expression of SGT1, but not RAR1, compromised the Sw-5b-mediated resistance to TSWV. (A) Sw-5b-transgenic N. benthamiana plants infected with TRV-NbSGT1 and TRV-NbRAR1 at 3 weeks post TRV treatment. TRV-GUS treatment was used as a control. Agrobacterium harboring TRV1 was mixed equally with Agrobacterium harboring TRV2-NbSGT1 or TRV2-NbRAR1 and co-infiltrated into leaves of 4–6 week old Sw-5b-transgenic N. benthamiana. The TRV-treated plants were photographed at 3 weeks post agroinfiltration. (B) Quantitative RT-PCR analysis of the expression of NbSGT1 and NbRAR1 mRNA in the newly emerged leaves of TRV-treated transgenic N. benthamiana plants at 3 weeks post agroinfiltration. The NbActin and NbEF1a genes were used as internal reference genes. Asterisks indicate significant differences (Student’s t-test, * p < 0.05). (C) Analysis of TSWV systemic infection of Sw-5b-transgenic N. benthamiana plants silenced for NbSGT1 and NbRAR1. TRV-GUS was used as a control. The gene-silenced newly emerged leaves of Sw-5b-transgenic N. benthamiana plants were inoculated with crude extract of TSWV-infected tissues. The TSWV challenged plants were photographed at 14 days post viral inoculation. The systemic infection was evident in all 18 NbSGT1-silenced plants. (D) RT–PCR detection of TSWV N RNA in systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbSGT1 and NbRAR1 at 14 days post TSWV inoculation. The internal reference gene was NbActin. (E) Western blot analysis of TSWV N protein accumulation in systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbSGT1 and NbRAR1 using a TSWV N-specific antibody. The leaves were collected at 14 days post TSWV inoculation. A plant sample expressed with p2300 empty vector (Vec.) was used as a negative control. Ponceau S staining was used as a protein loading control.

Figure 3

Sw-5b-mediated resistance to TSWV was independent of EDS1 and NDR1. (A) Sw-5b-transgenic N. benthamiana plants treated with TRV-NbEDS1 and TRV-NbNDR1. TRV-GUS was used as a control. Agrobacterium harboring TRV1 was mixed equally with Agrobacterium harboring TRV-NbEDS1 or TRV-NbNDR1 and co-infiltrated into leaves of 4–6 week old Sw-5b-transgenic N. benthamiana. The TRV-treated plants were photographed at 3 weeks post agroinfiltration. (B) qRT-PCR analysis of expression of NbEDS1 and NbNDR1 mRNA in newly emerged leaves of Sw-5b-transgenic N. benthamiana treated with TRV-NbEDS1, TRV-NbNDR1, or TRV-GUS. Samples were collected at 3 weeks post TRV treatment. Values were normalized using NbActin and NbEF1a genes as a reference. Student’s t-test was used for statistical analysis (* p < 0.05). (C) Analysis of TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants silenced for NbEDS1 and NbNDR1. TRV-GUS was used as the control. The gene-silenced newly emerged leaves of Sw-5b-transgenic N. benthamiana plants were inoculated with crude extract of TSWV-infected tissues. The plants challenged with TSWV were photographed at 14 days post inoculation. (D) RT–PCR analysis of expression of TSWV N RNA in systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbSGT1 and NbRAR1 at 14 days post TSWV inoculation. A TSWV-infected N. benthamiana sample was used as a positive control. The reference gene was NbActin. (E) Western blot analysis of TSWV N protein accumulation in systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbSGT1 and NbRAR1 in panel C using specific antibodies against N. A plant sample expressed with p2300 empty vector (Vec.) was used as a negative control. Ponceau S staining was used as a protein loading control.

Figure 4

Analysis of the requirement for NPR1 in Sw-5b-mediated resistance to TSWV. (A) TRV-NbNPR1- and TRV-GUS-treated Sw-5b transgenic N. benthamiana plants at 3 weeks post TRV agroinfiltration. (B) qRT-PCR analysis of the expression of NbNPR1 mRNA in newly emerged leaves of Sw-5b transgenic N. benthamiana plant treated with NPR1-silenced transgenic plants. The NbActin and NbEF1a genes were used as internal controls. Student’s t-test was used for statistical analysis (* p < 0.05). (C) Analysis of systemic infection of TSWV in Sw-5b-transgenic N. benthamiana plants silenced for NbNPR1. TRV-GUS plants were used as a control. The gene-silenced newly emerged leaves of Sw-5b-transgenic N. benthamiana plants were inoculated with TSWV. The photographs of treated plants were taken at 14 days post TSWV inoculation. (D) RT–PCR detection of TSWV N RNA in systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbNPR1 at 14 days post TSWV inoculation. A sample from TSWV-infected N. benthamiana tissues was used as a positive control. The NbActin gene was used as an internal reference gene. (E) Western blot analysis of TSWV N protein accumulation in the systemic leaves of Sw-5b-transgenic N. benthamiana plants silenced for NbNPR1 in panel C using specific antibodies against TSWV N. A plant sample expressed with p2300 empty vector (Vec.) was used as a negative control. Ponceau S staining was used as a protein loading control.

Figure 5

Requirement for the helper NLRs NRC2/3/4, NRG1, ADR1, and NRG1/ADR1 in Sw-5b-mediated resistance to TSWV infection. (A) TRV-based NbNRC2/3/4, NbNRG1, NbADR1, and NbNRG1/NbADR1 gene-silenced plants at 3 weeks post TRV treatment. (B) qRT-PCR analysis of the expression of NbNRC2a, 2b, 3, and 4 or NbNRG1 and NbADR1 mRNA transgenic plants treated with TRV-NbNRC2/3/4, TRV-NbNRG1, TRV-NbADR1, and TRV-NbNRG1/NbADR1. The NbActin and NbEF1a genes were used as reference genes. Student’s t-test was used for statistical analysis (* p < 0.05). (C) Analysis of systemic TSWV infection of Sw-5b-transgenic N. benthamiana plants silenced for NbNRC2/3/4, NbNRG1, NbADR1, or NbNRG1/NbADR1. TRV-GUS-treated plants were used as a control. The gene-silenced newly emerged leaves of Sw-5b-transgenic N. benthamiana plants were inoculated with TSWV. The photographs of TSWV-challenged plants were taken at 14 days post TSWV inoculation. The systemic infection was evident in all 18 NbNRC2/3/4-silenced plants in three repeated experiments. (D) RT–PCR detection of TSWV N RNA in systemic leaves of plants in panel C at 14 days post TSWV infection. A sample from a TRV-GUS-treated plant was used as a negative control. The NbActin gene was used as an internal reference gene. (E) Western blot analysis of the accumulation of TSWV N protein in systemic leaves of plants in panel C using N-specific antibodies. The samples were harvested at 14 dpi. A plant leaf sample expressed with p2300 empty vector (Vec.) was used as a negative control. Ponceau S staining was used as a protein loading control.