Chaperones of the endoplasmic reticulum are required for Ve1-mediated resistance to Verticillium

Abstract

The tomato receptor-like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1-enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)-resident chaperones HSP70 binding proteins (BiPs) and a lectin-type calreticulin (CRT). Knock-down of the different BiPs and CRTs in tomato resulted in compromised Ve1-mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP-type Cladosporium fulvum resistance protein Cf-4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf-4 protein. In contrast, knock-down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf-4- and Ve1-mediated immunity.

Figure 1

HSP70 binding proteins (BiPs) of Nicotiana benthamiana co‐purify with Ve1 and Ve1 co‐purifies with tomato lectin‐type calreticulins (CRTs). Ve1‐enhanced green fluorescent protein (eGFP) was expressed in N. benthamiana (A) and Ve1‐haemagglutinin (HA) was co‐expressed with SlCRT2‐eGFP, SlCRT3a‐eGFP or SlCRT3b‐eGFP in N. benthamiana (B). Total protein extracts of the agroinfiltrated leaf tissue were subjected to immunopurification using GFP affinity beads. Total proteins (Input) and immunopurified proteins (IP) were separated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and blotted. Blots were incubated with αGFP antibody to detect immunopurified Ve1‐eGFP (A) or SlCRT‐eGFP (B) fusion proteins. Duplicate blots were incubated with αBiP to detect co‐immunopurifying BiPs (A) or with αHA antibody to detect co‐immunopurifying Ve1‐HA. The Coomassie‐stained blot shows the 50‐kDa Rubisco band present in the input to confirm equal loading. The experiment was performed twice with similar results and a representative example is shown.

Figure 2

Targeting of individual HSP70 binding protein genes (BiPs) by virus‐induced gene silencing (VIGS) results in compromised Ve1‐mediated resistance to Verticillium dahliae. Tomato cultivar Motelle, carrying the Ve1 resistance gene, was subjected to VIGS by inoculation with the indicated TRV:Sl‐BiP constructs. TRV:GFP served as a control. GFP, green fluorescent protein; TRV, Tobacco rattle virus. Two weeks after inoculation with the recombinant viruses, plants were either mock inoculated (left plant in each panel) or inoculated with a race 1 strain of V. dahliae expressing Ave1 (right plant in each panel). Photographs were taken at 10 days after V. dahliae inoculation. Compromised resistance is reflected by the stunted appearance of the V. dahliae‐inoculated plants when compared with the mock‐inoculated plants. Three independent experiments were performed and a representative example is shown.

Figure 3

Targeting of individual lectin‐type calreticulin genes (CRTs) by virus‐induced gene silencing (VIGS) results in compromised Ve1‐mediated resistance to Verticillium dahliae. Tomato cultivar Motelle, carrying the Ve1 resistance gene, was subjected to VIGS by inoculation with the indicated Tobacco rattle virus (TRV) constructs. TRV:GFP (GFP, green fluorescent protein) served as a control. Two weeks after inoculation with the recombinant viruses, plants were either mock inoculated (left plant in each panel) or inoculated with a race 1 strain of V. dahliae expressing Ave1 (right plant in each panel). Photographs were taken at 10 days after V. dahliae inoculation. Compromised resistance is reflected by the stunted appearance of the V. dahliae‐inoculated plants when compared with the mock‐inoculated plants. Three independent experiments were performed and a representative example is shown.

Figure 4

Ve1‐enhanced green fluorescent protein (eGFP) contains complex N‐linked glycans and virus‐induced gene silencing (VIGS) of the different lectin‐type calreticulin genes (CRTs) does not affect Ve1 glycosylation. Ve1‐eGFP was transiently expressed in Nicotiana tabacum (cultivar Samsun) (A) or N. benthamiana (B), silenced for the different CRTs. As a control in (B), Cf‐4‐eGFP was also transiently expressed in N. benthamiana silenced for the different CRTs. The fusion proteins were subsequently immunopurified using GFP affinity beads, run on sodium dodecylsulphate (SDS) gel and blotted. Blots were incubated with αGFP antibody to reveal the total amount of immunopurified Ve1 and Cf‐4 proteins, and duplicate blots were incubated with a horseradish peroxidase antibody (αHRP) to reveal the pool of mature complex glycosylated purified protein. The experiment was performed twice with similar results and a representative example is shown. TRV, Tobacco rattle virus.

Figure 5

Targeting of the individual HSP70 binding protein (BiP) and lectin‐type calreticulin (CRT) genes in Nicotiana tabacum does not lead to a compromised Ve1‐mediated hypersensitive response (HR). Nicotiana tabacum cv. Samsun was subjected to virus‐induced gene silencing (VIGS) by inoculation with the indicated constructs. Three weeks later, Ve1 was transiently co‐expressed with the matching ligand Ave1. Photographs of infiltrated spots were taken another 4 days later. TRV:GUS and TRV:Nb‐SOBIR1/Nb‐SOBIR1 ‐like were included as controls. Three independent experiments were performed and representative examples are shown. TRV, Tobacco rattle virus.