The mutant STAY-GREEN (Cssgr) in cucumber interacts with the CSEP30 protein to elicit a defense response against Podosphaera xanthii

Abstract

Disease-resistant plants activate immune responses by specifically recognition Candidate Secreted Effector Proteins (CSEPs) through resistance (R) proteins. In research on cucumber powdery mildew resistance breeding, several R genes and CSEPs have been identified; however, the specific interactions between R proteins and CSEPs are still largely unexplored. In this study, we used a luciferase reporter assay to identify six CSEPs from Podosphaera xanthii that potentially induce cell death in cucumber. Subsequent yeast two-hybrid analysis revealed that only the mature form of CSEP30 (CSEP30^∆SP) interacted with the cucumber mutant STAY-GREEN (Cssgr), a gene previously recognized for its broad-spectrum resistance in genetic studies. This interaction was confirmed using pull-down and co-immunoprecipitation assays. Additionally, to determine if the interaction leads to phenotypic changes, Cssgr and CSEP30^∆SP were transiently expressed in tobacco leaves. The infiltration of Cssgr in tobacco resulted in reduced chlorosis compared to the wild-type CsSGR. Co-infiltration of Cssgr with CSEP30^∆SP induced distinct dry necrotic lesions, contrasting the effects observed when Cssgr and CSEP30^∆SP were infiltrated separately. Additionally, after P. xanthii infection in moderately powdery mildew-resistant Gy14 cucumber, similar necrotic lesions and specific expression of Cssgr, as along with defense response-related genes (CsPR1 and CsLecRK6.1), were observed. This study suggests that the interaction between Cssgr and CSEP30^∆SP could trigger cell death and defense response, offering new insights into the molecular function of Cssgr in disease resistance in Gy14 cucumber.

Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01504-6.

Keywords: Candidate secreted effector proteins; Cucumis sativus; Podosphaera xanthii; Powdery mildew; STAY-GREEN.

Fig. 1

Relative luciferase (LUC) activity 1 day after co-expression of candidate secreted effector proteins (CSEP) and a LUC reporter in cucumber cotyledons. The mock was injected with infiltration solution. The empty vector was used as a positive control, with LUC activity from co-expressing pCambia vector and the LUC reporter gene normalized to 1. Asterisks denote significant differences between the positive control and CSEP-treated groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars represent the standard deviation. All experiments were conducted in triplicate and yielded consistent results

Fig. 2

Yeast two-hybrid (Y2H) assay shows the interaction between CSEP30^∆SP and Cssgr. a The assay results illustrate the interaction between bait proteins (pGBKT7-CsSGR, pGBKT7-Cssgr) and prey proteins (CSEP30, CSEP47, and CSEP48,). Blue colonies of yeast cells on SD/–Ade/–His/–Leu/–Trp/X-α-Gal/Aba (QDO/X/A) medium represent positive interaction. b The mature form CSEP30^∆SP was used as prey construct. The presence of blue yeasts colonies confirms a positive interaction between CSEP30^∆SP and Cssgr proteins. Lack of growth on QDO/X/A plates indicates no interaction

Fig. 3

Glutathione S-transferase (GST) pull-down and Co-immunoprecipitation (Co-IP) analyses confirm the interaction between CSEP30^∆SP and Cssgr. a Protein interaction between Cssgr and CSEP30^∆SP is demonstrated by the GST pull-down assay. b Co-IP assay showing the interaction between Cssgr and CSEP30^∆SP in vivo. Proteins isolated from N. benthamiana tissues co-infiltrated with the indicated plasmid combinations were extracted and immunoprecipitated using anti-GFP or anti-HA antibodies. The experiments were repeated three times with consistent results

Fig. 4

Subcellular localization of Cssgr and CSEP30^∆SP in N. benthamiana cells. Fluorescent images of N. benthamiana leaves were captures 48 h post-infiltration with GFP, Cssgr-GFP, and CSEP30^∆SP-GFP individually. Chlorophyll (Chl) autofluorescence is shown in red in the second column. The merge of Cssgr-GFP and CSEP30^∆SP-GFP fluorescence with Chl fluorescence appears in yellow-orange tones, indicating localization to the chloroplast. Scale bar = 20 μm

Fig. 5

Phenotypic assessment of individual and combined infiltration of CsSGR, Cssgr, and CSEP30^∆SP. a Phenotype of CsSGR and Cssgr at 12, 24, 36, and 48 h post-infiltration. b Chlorophyll content at the same time points. Statistically significant differences are indicated by different asterisks (^** represents P < 0.01,). c Cssgr co-infiltrated with CSEP30^∆SP results in dry necrotic lesions on abaxial leaf at 3 days post-infiltration. Scale Bar = 1 cm

Fig. 6

Comparison of powdery mildew (PM) disease symptom and defense-related gene pression between S6 and Gy14. a Disease symptom in Gy14 and S6 leaves at 15 days post-inoculation of P. xanthii. Gy 14 leaves exhibit yellowing on the adaxial side and dry necrotic lesions on the abaxial side. b Relative expression levels of CsSGR, CsPR1, CsPR3, CsPAL, and CsLecRK6.1 genes in cucumber Gy14 and S6 in response to P. xanthii inoculation, as determined by qRT-PCR. Statistically significant differences at 15 days-post inoculation are indicated by different asterisks (ns, P > 0.05; *, P < 0.05; ***, P < 0.001)