Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c

Abstract

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candidate, CSEP0055, contributes to the aggressiveness of the fungus. This result suggests that CSEP0055 is involved in the suppression of plant defence. A yeast two-hybrid screen indicated that CSEP0055 interacts with members of the barley pathogenesis-related protein families, PR1 and PR17. Interaction with PR17c was confirmed by bimolecular fluorescence complementation analyses. Down-regulation and over-expression of PR17c in epidermal cells of barley confirmed that this protein is important for penetration resistance against the powdery mildew fungus. In line with this, PR17c was found to be apoplastic, localizing to the papillae formed in response to this fungus. The CSEP0055 transcript did not start to accumulate until 24 h after inoculation. This suggests that this gene is expressed too late to influence primary penetration events, but rather sustains the fungus at sites of secondary penetration, where PR17c appears to be able to accumulate.

Figure 1

Host‐induced gene silencing indicates an effector role for CSEP0055. Relative haustorial entry, calculated on the basis of the number of β‐glucuronidase (GUS)‐expressing barley epidermal cells with and without haustoria (top), after transformation with the 35S promoter‐driven CSEP0055 RNA interference (RNAi) constructs. Data shown are mean values; n = 5; 100% relative entry equals 24.4% absolute entry in empty vector control; a minimum of 100 transformed cells was counted in each repeat. Error bars are ± standard deviation (SD). **P < 0.01 (Student's t‐test). Bars, 25 μm.

Figure 2

Yeast two‐hybrid (Y2H) identification of PR17c as an interactor of CSEP0055. Growth on solid minimal (SD) medium with 50 mm 3‐amino‐1,2,4‐triazole (3‐AT) and without histidine (H), and β‐galactosidase (X‐gal) activity on colony lifts made from clones grown on SD with histidine, indicate positive protein–protein interaction. SNF1/SNF4 serves as positive control. CSEP0443/PR17c serves as negative control. This experiment was repeated at least three times with similar results.

Figure 3

CSEP0055–PR17c interaction visualized by bimolecular fluorescence complementation (BiFC) in tobacco leaves. (A) Confocal image of epidermal leaf cells from Nicotiana benthamiana infiltrated with a mixture of Agrobacterium clones harbouring T‐DNA constructs for 35S promoter‐driven expression of cCFP‐PR17c and CSEP0055‐nYFP. (B) Positive control. (C, D) Negative controls. This experiment was repeated at least three times with similar results. Bars, 50 μm.

Figure 4

Transient induced gene silencing and transient over‐expression suggest that barley PR17c plays a role in defence against Blumeria graminis f.sp. hordei (Bgh). Relative haustorial entry, calculated on the basis of the number of β‐glucuronidase (GUS)‐expressing barley epidermal cells with and without haustoria (see Fig. 1) after transformation with 35S promoter‐driven RNAi constructs that covered the 3’ and 5’ parts of the coding region of PR17c (A) and after transformation with a ubiquitin promoter‐driven construct encoding full‐length PR17c (B). Data shown are mean values; n = 4 (A) and n = 3 (B); 100% relative entry equals 15.8% (A) and 18.0% (B) absolute entry in empty vector controls; a minimum of 100 (A) and 200 (B) transformed cells were counted in each repeat. Error bars are ± standard deviation (SD). *P < 0.05; **P < 0.01 (Student's t‐test).

Figure 5

PR17c localizes to Blumeria graminis f.sp. hordei (Bgh)‐induced papillae in barley epidermal cells. (A) Confocal image of cell of noninoculated control leaf transiently transformed with constructs for ubiquitin promoter‐driven expression of PR17c‐mCherry and cyan fluorescent protein (CFP). The PR17c‐mCherry signal is present at the cell margin and the CFP signal is cytosolic. The merged image shows that PR17c‐mCherry and CFP are nonoverlapping and separated by a distance (arrow in inset 1), and that PR17c‐mCherry spreads to the apoplastic space between nontransformed adjacent epidermal cells (arrowhead in inset 2). (B) Confocal image of cell transiently transformed with the PR17c‐mCherry construct, recorded 24 h after inoculation. PR17c‐mCherry accumulates at the site of attempted penetration (arrowhead) by a Bgh appressorium (arrow). (C) Confocal image of cell transiently transformed with a construct for 35S promoter‐driven expression of mCherry, recorded 24 h after inoculation. mCherry is cytosolic and not focused at sites of attempted penetration (arrows). Bars, 10 μm. Stippled line silhouettes indicate spores and appressorial hyphae.

Figure 6

CSEP0055 expression coincides with PR17c accumulation during secondary penetration events. (A) Quantitative polymerase chain reaction (PCR) of the CSEP0055 transcript in the fungal material on the leaf surface. The BghGAPDH transcript (GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase) was used as reference, and the quantification was made relative to 0 h after inoculation. Each data point is based on three biological and two technical repeats. (B) Confocal image of cell transiently transformed with a construct for ubiquitin promoter‐driven expression of PR17c‐mCherry, recorded 48 h after inoculation. PR17c‐mCherry accumulates at three sites of attempted penetration (arrowhead) from a Bgh secondary hypha, outlined by a stippled line silhouette. Bars, 10 μm.