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

Ali S, Magne M, Chen S, Obradovic N, Jamshaid L, Wang X, Bélair G, Moffett P - Front Plant Sci (2015)

Bottom Line: Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts.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.

View Article: PubMed Central - PubMed

Affiliation: Département de Biologie, Université de Sherbrooke Sherbrooke, QC, Canada ; Horticulture R & D Centre, Agriculture and Agri-Food Canada St-Jean-sur-Richelieu, QC, Canada ; Division of Biological and Environmental Sciences and Engineering, Center for Desert Agriculture, King Abdullah University of Science and Technology Thuwal, Saudi Arabia.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Although a number of effectors have been shown to repress defense-related cell death, cell death is not an absolute requirement for halting pathogen proliferation in plants, suggesting that additional mechanisms contribute to immunity, which may or may not be repressed by a given effector. We investigated whether the SPRYSEC proteins could also suppress disease resistance mediated by the Rx and N proteins, which confer resistance to viruses without inducing cell death (Bendahmane et al., 1999; Bhattacharjee et al., 2009). To test this, PVX-GFP was agroinfiltrated in N. benthamiana leaves with the Rx gene along with either empty vector as a control or the SPRYSEC effectors. GFP was then visualized by UV illumination and immuno-blotting 4 days later as a proxy for virus accumulation in N. benthamiana leaves. In the leaf patches co-agroinfiltrated with PVX-GFP, Rx, and empty vector, little, or no GFP was observed, whereas all SPRYSEC effectors allowed significant accumulation of GFP in the infiltrated areas as observed visually and by anti-GFP immune-blotting (Figure 3). As a further demonstration that the SPRYSEC proteins can inhibit defense responses other than cell death, we used an assay based on the N gene, which confers resistance to Tobacco mosaic virus (TMV) through the recognition of the P50 subunit of the viral replicase (Bhattacharjee et al., 2009). The co-expression of N and P50 in N. benthamiana leaves inhibits the accumulation of PVX-GFP in the absence of cell death (Bhattacharjee et al., 2009). We co-expressed N and P50 with PVX-GFP together with either empty vector or the SPRYSEC effectors and monitored the accumulation of PVX-GFP visually and by immuno-blotting. The P0 protein from polerovirus was used as a positive control in this assay as it has been shown to inhibit N-mediated anti-viral defense responses (Bhattacharjee et al., 2009). P0 and all tested SPRYSEC effectors inhibited the ability of N to suppress PVX-GFP accumulation in this assay (Figure 4) indicating that they are able to inhibit the cell death-independent defense pathways induced by N.


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

Ali S, Magne M, Chen S, Obradovic N, Jamshaid L, Wang X, Bélair G, Moffett P - Front Plant Sci (2015)

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.
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Related In: Results  -  Collection

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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.
Mentions: Although a number of effectors have been shown to repress defense-related cell death, cell death is not an absolute requirement for halting pathogen proliferation in plants, suggesting that additional mechanisms contribute to immunity, which may or may not be repressed by a given effector. We investigated whether the SPRYSEC proteins could also suppress disease resistance mediated by the Rx and N proteins, which confer resistance to viruses without inducing cell death (Bendahmane et al., 1999; Bhattacharjee et al., 2009). To test this, PVX-GFP was agroinfiltrated in N. benthamiana leaves with the Rx gene along with either empty vector as a control or the SPRYSEC effectors. GFP was then visualized by UV illumination and immuno-blotting 4 days later as a proxy for virus accumulation in N. benthamiana leaves. In the leaf patches co-agroinfiltrated with PVX-GFP, Rx, and empty vector, little, or no GFP was observed, whereas all SPRYSEC effectors allowed significant accumulation of GFP in the infiltrated areas as observed visually and by anti-GFP immune-blotting (Figure 3). As a further demonstration that the SPRYSEC proteins can inhibit defense responses other than cell death, we used an assay based on the N gene, which confers resistance to Tobacco mosaic virus (TMV) through the recognition of the P50 subunit of the viral replicase (Bhattacharjee et al., 2009). The co-expression of N and P50 in N. benthamiana leaves inhibits the accumulation of PVX-GFP in the absence of cell death (Bhattacharjee et al., 2009). We co-expressed N and P50 with PVX-GFP together with either empty vector or the SPRYSEC effectors and monitored the accumulation of PVX-GFP visually and by immuno-blotting. The P0 protein from polerovirus was used as a positive control in this assay as it has been shown to inhibit N-mediated anti-viral defense responses (Bhattacharjee et al., 2009). P0 and all tested SPRYSEC effectors inhibited the ability of N to suppress PVX-GFP accumulation in this assay (Figure 4) indicating that they are able to inhibit the cell death-independent defense pathways induced by N.

Bottom Line: Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts.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.

View Article: PubMed Central - PubMed

Affiliation: Département de Biologie, Université de Sherbrooke Sherbrooke, QC, Canada ; Horticulture R & D Centre, Agriculture and Agri-Food Canada St-Jean-sur-Richelieu, QC, Canada ; Division of Biological and Environmental Sciences and Engineering, Center for Desert Agriculture, King Abdullah University of Science and Technology Thuwal, Saudi Arabia.

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.

No MeSH data available.


Related in: MedlinePlus