The Tomato spotted wilt virus cell-to-cell movement protein (NSM ) triggers a hypersensitive response in Sw-5-containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy

Abstract

Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum 'SR1' plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM ) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N. benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM ) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.

Keywords: NSM; Sw-5; TSWV; avirulence; cell-to-cell movement; resistance gene; tospovirus.

Figure 1

Challenge of untransformed and Sw‐5b‐transformed Nicotiana benthamiana with resistance‐inducing Tomato spotted wilt virus (TSWV) BR‐01 and resistance‐breaking TSWV GRAU isolates. Photographs from all leaves were taken at 5–7 days post‐inoculation (dpi), with the exception of those shown in (E). (A) Untransformed N. benthamiana plant challenged with TSWV BR‐01 and showing typical chlorotic lesions on locally infected leaves. (B) Enlarged view of a locally infected leaf from (A). (C) Nicotiana benthamiana Sw‐5b challenged with TSWV BR‐01. (D) Enlarged view of the virus‐challenged leaf from (C). (E) Nicotiana benthamiana Sw‐5b challenged with TSWV BR‐01 showing the challenged leaf at 15 dpi. (F) Nicotiana benthamiana Sw‐5b challenged with TSWV GRAU. (G) Enlarged view of the virus‐challenged leaf from (F). (H) Reverse transcription‐polymerase chain reaction (RT‐PCR) for monitoring systemic infection in wild‐type (wt) and Sw‐5b N. benthamiana plants inoculated with TSWV (BR‐01 and GRAU), Impatiens necrotic spot virus (INSV) and Melon yellow spot virus (MYSV). Total RNA of non‐inoculated upper leaves was used as a template for N (nucleocapsid) gene amplification at 10 dpi. M, GeneRuler 100‐bp Plus DNA ladder.

Figure 2

Schematic representation of the expression vector constructs used to challenge Nicotiana benthamiana transformants containing Sw‐5b. (A) Tomato spotted wilt virus (TSWV) genes (NS_M of BR‐01 and GRAU isolates; N, NS_S and the glycoprotein precursor (GP) from BR‐01) cloned individually into the pEAQ‐HT vector. AttR1/R2, Gateway recombination sites. (B) Tomato spotted wilt virus (TSWV) genes (NS_M of BR‐01 and GRAU isolate, N, NS_S) cloned individually in the appropriated restriction sites (ClaI and SalI) of the pGR107 vector (Lovato et al., 2008). LB, left border; RB, right border; 35S, Cauliflower mosaic virus promoter region; RdRP, Potato virus X (PVX) RNA‐dependent RNA polymerase; 25K, 12K and 8K, PVX movement proteins (triple gene block); CP promoter, PVX coat protein subgenomic RNA promoter; CP, PVX coat protein; NOS, nopaline synthase terminator.

Figure 3

Response of transgenic Nicotiana benthamiana (Sw‐5b) leaves infiltrated with pEAQ‐HT and Potato virus X (PVX) constructs expressing NS_M from isolates BR‐01 and GRAU, respectively. Photographs of leaves shown in (A) and (B) were taken 5–6 days post‐inoculation (dpi). In both panels, leaves were submitted to treatment with ethanol (destaining) for removal of chlorophyll to facilitate hypersensitive response (HR) visualization. (A) Individual leaf simultaneously infiltrated with three constructs pEAQ‐HT‐NS_{M ‐ BR ‐01}, pEAQ‐HT‐NS_{M ‐ GRAU} and pEAQ‐HT empty vector (left side) and with Potato virus X (PVX)‐Gw‐NS_{M ‐ BR ‐01}, PVX‐Gw‐NS_{M ‐ GRAU} and PVX‐Gw empty vector (right side), showing the visual response on infiltration. (B) Whole leaves infiltrated with pEAQ‐HT‐NS_{M ‐ BR ‐01}, pEAQ‐HT‐NS_{M ‐ GRAU} and pEAQ‐HT empty vector (left side) and with PVX‐Gw‐NS_{M ‐ BR ‐01}, PVX‐Gw‐NS_{M ‐ GRAU} and PVX‐Gw empty vector (right side), showing the visual response on infiltration. (C) Western immunoblot analysis with specific polyclonal antiserum against NS_M protein (34 kDa) of Tomato spotted wilt virus (TWSV). 34 kDa, molecular weight marker protein. Samples were taken from N. benthamiana (Sw‐5b) plants inoculated with the wild‐type TSWV virus and infiltrated with the constructs pEAQ‐HT and PVX‐Gw containing NS_M BR‐01 and NS_M GRAU. Extract from healthy plants was used as a negative control.

Figure 4

Response of susceptible and resistant (harbouring the Sw‐5 gene) tomato near‐isogenic line leaves infiltrated with individual pEAQ‐HT constructs expressing NS_M from isolate BR‐01 or GRAU. Photographs of leaves shown were taken 4 days post‐inoculation (dpi). In both cases, leaves were submitted to treatment with ethanol (destaining) for removal of chlorophyll to facilitate hypersensitive response (HR) visualization. Individual leaves from the resistant line (left side) and susceptible isoline (right side) were simultaneously infected with three constructs pEAQ‐HTNS_{M ‐ BR ‐01}, pEAQ‐HT‐NS_{M ‐ GRAU} and pEAQ‐HT empty vector. Leaves of the resistant line showed the visual necrotic response (HR lesions) on infiltration.