Virus-Inducible Activation of Hypersensitive Response Confers Broad-Spectrum Resistance to Diverse Viruses in Plants

Abstract

The hypersensitive response (HR) is a powerful plant defence mechanism that restricts pathogen spread through localised cell death. However, constitutive activation of HR can impair growth and development, limiting its utility in crop breeding and protection. In the study, we developed a virus-inducible HR system by coupling an Avirulence/Resistance (Avr/R) gene pair, Avr4/Cf-4 effector-resistance gene pair, with a promoter (Solyc04g076730) specifically activated by geminivirus infection. We demonstrate that co-expression of Avr4 and Cf-4 in Nicotiana benthamiana triggers robust HR and significantly reduces accumulation of multiple geminiviruses, including TYLCV, TYLCCNV, SLCMV and BSCTV. Transcriptomic analysis of virus-infected tomato identified Solyc04g076730 as a virus-responsive promoter with minimal basal activity. Transient assays and stable transformation in both N. benthamiana and tomato revealed that this promoter effectively drives HR only upon viral infection, resulting in reduced viral loads, attenuated symptoms and improved plant vigour. Importantly, transgenic plants expressing the Solyc04g076730::Avr4/Cf-4 construct maintained normal growth under non-infected conditions. Our findings offer a promising strategy for engineering broad-spectrum resistance to viral pathogens in crops.

Keywords: R gene; geminivirus; hypersensitive response; inducible promoter; virus resistance.

FIGURE 1

Necrosis in leaves induced by Avr4/Cf‐4 and Avr9/Cf‐9. (a) Leaves of Nicotiana benthamiana plants that were transiently agro‐infiltrated with Avr4 (A4), Cf‐4 (C4) (white circle) and Avr4/Cf‐4 (A4/C4). Scale bars = 1 cm. (b) Leaves of N. benthamiana plants that were transiently agro‐infiltrated with Avr9 (A9), Cf‐9 (C9) (white circle) and Avr9/Cf‐9 (A9/C9). The hypersensitive responses are indicated by red circles. Images were taken at 7 days post‐infiltration.

FIGURE 2

The Avr4/Cf‐4 gene pair effectively alleviates viral systemic spread by triggering the hypersensitive response in inoculated leaves. (a, d, g and j) Phenotypes of inoculated leaves and systemically infected leaves of Nicotiana benthamiana plants that were transiently agro‐infiltrated with different virus and gene combinations. Virus group: agro‐infiltrated only with virus; virus + Avr4 group: agro‐infiltrated with virus and Avr4; virus + Cf‐4 group: agro‐infiltrated with virus and Cf‐4; virus + Avr4 + Cf‐4: agro‐infiltrated with virus, Avr4, and Cf‐4. Images were taken at 14 days post‐infiltration. Scale bars = 1 cm. (b, e, h and k) Systemically infected leaves of N. benthamiana plants that were transiently agro‐infiltrated with different virus and gene combinations. Virus group: agro‐infiltrated only with virus; virus + Avr4 group: agro‐infiltrated with virus and Avr4; virus + Cf‐4 group: agro‐infiltrated with virus and Cf‐4; virus + Avr4 + Cf‐4: agro‐infiltrated with virus, Avr4, and Cf‐4. Images were taken at 14 days post‐infiltration. (c, f, i and l) Quantification of viruses in N. benthamiana plants that were inoculated with the four virus and gene combinations after 14 days (NbActin acts as a reference). Data are presented as mean ± SEM (n = 20). Different letters indicate significant differences (p < 0.05) based on one‐way ANOVA followed by Tukey's multiple comparison test.

FIGURE 3

A virus‐inducible promoter from Solyc04g076730. (a) Expression levels of five genes (Solyc04g076730, Solyc06g068890, Solyc01g066910, Solyc11g066980 and Solyc12g011090) in tomato plants were promoted at 14 days post‐inoculation (dpi) with TYLCV (SlActin acts as a reference). (b) Expression levels of five genes (Solyc04g076730, Solyc06g068890, Solyc01g066910, Solyc11g066980 and Solyc12g011090) in tomato plants were promoted at 14 dpi with TYLCCNV (SlActin acts as a reference). Data are presented as mean ± SEM (n = 6), ns, no significant difference, ***p < 0.001 based on Student's t test. (c–g) Staining for β‐glucuronidase (GUS) activity under the promoters of the five genes in Nicotiana benthamiana leaves at 14 dpi with pBINplus (negative control (c), TYLCV (d), TYLCCNV (e), SLCMV (f) or BSCTV (g)). Scale bars = 1 cm.

FIGURE 4

The Avr4/Cf‐4 gene pair under the virus‐inducible promoter of Solyc04g076730, SlVIP‐HR, responds to multiple viruses and induces hypersensitive response when transiently expressed in Nicotiana benthamiana. (a) Schematic of the T‐DNA region in the recombinant construct, p1300‐Solyc04g076730‐pr‐Avr4‐3×FLAG‐P2A‐Cf4‐EGFP. (b) and (c) Trypan blue staining in N. benthamiana leaves showing virus responses of the recombinant construct, p1300‐Solyc04g076730‐pr‐Avr4‐3×FLAG‐P2A‐Cf4‐EGFP, at 14 days post‐inoculation with TYLCV, TYLCCNV, SLCMV or BSCTV. Scale bars = 1 cm.

FIGURE 5

Transiently agro‐infiltrated SlVIP‐HR strongly relieves multiple virus infected symptoms in systemic leaves of Nicotiana benthamiana at 14 days post‐inoculation (dpi). (a, c, e and g) Phenotypes of N. benthamiana leaves systemically infected with SlVIP‐HR and a virus (TYLCV, TYLCCNV, SLCMV or BSCTV) at 14 dpi. The p1300‐GFP: Empty vector acted as a negative control. Scale bars = 1 cm. (b, d, f and h) Quantification of viruses in leaves of N. benthamiana plants at 14 dpi with TYLCV, TYLCCNV, SLCMV or BSCTV (NbActin acted as a reference). Data are presented as mean ± SEM (n = 30), ***p < 0.001 based on Student's t test.

FIGURE 6

SlVIP‐HR confers Nicotiana benthamiana resistance against viral infections. (a, c, e and g) Phenotypes of systemically infected N. benthamiana leaves at 14 days post‐inoculation (dpi) with TYLCV, TYLCCNV, SLCMV or BSCTV. The top panel of each image shows the phenotype of the whole plant; the bottom panel of each image shows the phenotype of systemic leaves. Scale bars = 1 cm. (b, d, f and h) Quantification of viruses in systemically infected N. benthamiana leaves of plants at 7, 14 and 21 dpi with TYLCV, TYLCCNV, SLCMV or BSCTV (NbActin acted as a reference). Data are presented as mean ± SEM (n = 30), ***p < 0.001 based on Student's t test.

FIGURE 7

SlVIP‐HR confers tomato resistance against TYLCV infection. (a) Phenotypes of whole plants and systemically infected leaves of wild‐type tomato and the transgenic tomato line SlVIP‐HR at 21 days post‐inoculation (dpi) with TYLCV. The top panel of the image shows the phenotype of the whole plant from a front view; the bottom panel of the image shows the phenotype of the whole plant from a top view. Scale bars = 1 cm. (b) Plant height at 21 dpi with TYLCV. (c) Representative images of systemically infected leaves of tomato plants at 21 dpi with TYLCV. (d) Quantification of TYLCV in systemically infected tomato leaves at 21 dpi (SlActin acts as a reference). Data are presented as mean ± SEM (n = 30), ***p < 0.001 based on one‐way ANOVA with Student's t test.

FIGURE 8

SlVIP‐HR confers tomato resistance against TYCCNV infection. (a) Phenotypes of whole plants and systemically infected leaves of wild‐type tomato and the transgenic tomato line SlVIP‐HR at 21 days post‐inoculation (dpi) with TYLCCNV. The top panel of the image shows the phenotype of the whole plant from a front view; the bottom panel of the image shows the phenotype of the whole plant from a top view. Scale bars = 1 cm. (b) Quantification of TYLCCNV in systemically infected tomato leaves at 21 dpi (SlActin acted as a reference). (c) Representative images of systemically infected leaves of tomato plants at 21 dpi with TYLCCNV. (d) Quantification of TYLCCNV in systemically infected tomato leaves at 21 dpi (SlActin acts as a reference). Data are presented as mean ± SEM (n = 30), ***p < 0.001 based on Student's t test.

FIGURE 9

SlVIP‐HR confers tomato resistance against whitefly‐transmitted TYLCV infection. (a) Phenotypes of whole plants and systemically infected leaves in wild‐type tomato and the transgenic tomato line SlVIP‐HR at 6 weeks post‐transmission of TYLCV by whiteflies. Scale bars = 1 cm. (b) and (c) Representative images of systemically infected leaves of tomato plants at 6 weeks post‐whitefly‐transmitted TYLCV infection. (d) Quantification of TYLCV in systemically infected tomato leaves at 6 weeks post‐viral infection (SlActin acted as a reference). Data are presented as mean ± SEM (n = 30), ***p < 0.001 based on Student's t test.