Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

Abstract

Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

Keywords: Globodera; NB-LRR proteins; PAMP-triggered immunity (PTI); SPRYSEC; cyst nematodes; effector proteins; plant-parasitic nematode.

Figure 1

GrSPRYSEC-15 induces a hypersensitive response in N. tabacum and chlorosis in N. benthamiana. (A) N. tabacum leaves were infiltrated with Agrobacterium carrying pEAQ35S expressing AtRx, INF1, GrSPRYSEC-15, or GFP as indicated. Leaves were photographed at 4 DPI. (B) N. tabacum inoculated by agroinfiltration with PVX-GFP vector or (C) PVX-GrSPRYSEC-15. Pictures were taken at 14 DPI. Insets show close ups of leaves displaying typical PVX symptoms and the sites of agroinfiltration of PVX-GrSPRYSEC-15 are seen as yellowish spots in (C). (D) N. benthamiana inoculated with PVX-GFP vector or (E) PVX-GrSPRYSEC-15 or (F) PVX-GrSPRYSEC-5. Plants were photographed at 21 DPI.

Figure 2

Suppression of cell death induced by NB-LRR and PiNPP proteins by GrSPRYSECs in N. benthamiana and N. tabacum. (A) N. benthamiana leaves were co-infiltrated with Agrobacterium carrying binary vectors expressing Rx, CP and P38 together with either empty vector (EV, left hand side) or the indicated effectors expressed from pEAQ35S (right hand side). Three days DPI, leaves were decolorized with methanol to highlight cell death reactions. (B) N. tabacum leaves were co-infiltrated with Agrobacterium containing binary vectors expressing AtRx and P38 together with either empty vector (EV, left hand side) or the indicated effectors expressed from pEAQ35S (right hand side). (C,D) N. tabacum leaves were co-infiltrated with Agrobacterium carrying expression vectors for PiNPP and P38 together with either empty vector (EV, left hand side) or the indicated effectors (right hand side). Effectors were expressed from a PVX expression vector in (C,D). Cell death symptoms were scored at 3-5 DPI and the pictures were taken at 5 DPI.

Figure 3

GrSPRYSECs suppress Rx-mediated resistance to PVX. N. benthamiana leaves were co-infiltrated with Agrobacterium carrying binary vectors expressing PVX-GFP and Rx together with (A) 1, GrSPRYSEC-5; 2, empty vector; 3, GrSPRYSEC-8; 4, empty vector; 5 GrSPRYSEC-15; 6, empty vector; 7, GrSPRYSEC-19; 8, empty vector; and Rx replaced with empty vector. (B) 1, GrSPRYSEC-18; 2, empty vector; 3, GrSPRYSEC-4; 4, empty vector; GFP expression was visualized and photographed under UV illumination at 4 DPI. (C,D) Anti GFP immune blotting was performed on total protein samples taken at 4 DPI from infiltrated N. benthamiana leaf patches expressing the different construct combinations as described in (A,B). Numbering corresponds to the number on the leaf above each blot. Ponceau staining (lower panel) was used to show equal loading.

Figure 4

GrSPRYSECs proteins suppress virus resistance mediated by the N gene. N. benthamiana leaves were co-infiltrated with Agrobacterium carrying binary vectors expressing PVX-GFP, N, and P50 together with (A) 1, GrSPRYSEC-5; 2, empty vector; 3, GrSPRYSEC-8; 4, empty vector; 5, GrSPRYSEC-15; 6, empty vector; 7, P0; 8, P38. (B) 1, GrSPRYSEC-19; 2, empty vector; 3, GrSPRYSEC-4; 4, empty vector; 5, P0; 6, P38. GFP expression was visualized under UV illumination at 4 DPI. (C,D) Anti GFP immune blotting was performed on total protein samples taken at 4 DPI from N. benthamiana leaf patches co-expressing the combinations of constructs described in (A,B). The number on the blot corresponds to the number on the leaf above each blot. Ponceau staining (lower panel) was used to show equal loading.