Alternaria solani Effector AsCEP20, Essential for Virulence, Targets Potato StFtsH4 Protein to Suppress Plant Disease Resistance

Abstract

Alternaria solani is an important necrotrophic pathogen causing potato early blight. However, the pathogenic molecular mechanisms of A. solani remain unclear. Previous work identified a specific effector AsCEP20 in A. solani through multi-omics analysis. AsCEP20 is required for the full virulence of A. solani and targets the host chloroplasts. In this study, we screened out 46 candidate proteins that potentially interact with AsCEP20 in Nicotiana benthamiana using co-immunoprecipitation followed by liquid chromatography-tandem mass spectrometry analysis. We identified a candidate target protein in potato, filamentation temperature-sensitive H4 (StFtsH4), which is located in chloroplasts, based on homologous alignment and subcellular localisation analysis. The interaction between AsCEP20 and StFtsH4 was further confirmed by co-immunoprecipitation, yeast two-hybrid assay and bimolecular fluorescence complementation assays. The interaction site between AsCEP20 and StFtsH4 is also the chloroplast. Silencing the potato StFtsH4 gene resulted in suppressed pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) bursts, and defence-related genes were significantly downregulated. These results suggest that StFtsH4 positively regulates plant immunity. Therefore, AsCEP20 targets the chloroplast protein StFtsH4 to promote pathogen infection. AsCEP20 attenuates the efficiency of light energy utilisation in photosynthesis by targeting StFtsH4. These results suggest that AsCEP20 suppresses StFtsH4-mediated potato disease resistance to A. solani. With the increase of light intensity, ROS continued to accumulate in the chloroplast of StFtsH4-silenced plant leaves, while defence-related genes significantly decreased. Our findings reveal that the impaired StFtsH4 function limits plant photosynthesis, thereby affecting immune signalling.

Keywords: Alternaria solani; chloroplast; effectors; plant–pathogen interaction.

FIGURE 1

Screening of effector AsCEP20 interacting proteins. (a) Histogram showing the relative content of candidate target proteins in Nicotiana benthamiana compared to the control group. (b) Histogram showing the number of proteins present in the AsCEP20 immunoprecipitation group but absent in the IgG control group. (c) Histogram illustrating candidate target proteins based on their localisation in plant cells. (d) Amino acid sequence characteristics pattern map of candidate interacting protein StFtsH4 (Favorita), StFtsH4 (Agria) and NbFtsH in N. benthamiana. (e) Subcellular localisation of StFtsH4 (Favorita) in N. benthamiana leaves. N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens GV3101 carrying StFtsH4‐YFP or YFP plasmid constructs. The upper panels show the localisation of free YFP as a control. The bottom panels show the localisation of StFtsH4‐YFP. Bars, 20 μm.

FIGURE 2

Interaction between AsCEP20 and StFtsH4 in chloroplasts. (a) Co‐immunoprecipitation assay showed that AsCEP20 associates with StFtsH4 in planta. Constructs for StFtsH4‐GFP and AsCEP20‐MYC were transiently expressed in Nicotiana benthamiana leaves. Immunoprecipitations were performed with anti‐GFP agarose (GFP‐IP), and StFtsH4 was detected in the immunoprecipitates via anti‐GFP antibody. The sizes of AsCEP20‐MYC bands are marked with asterisk. The protein marker is labelled on the right. (b) Yeast two‐hybrid assay indicated that AsCEP20 interacts with StFtsH4 as either the bait or the prey. (c) Bimolecular fluorescence complementation assay confirmed that the interaction between AsCEP20 and StFtsH4 occurs in chloroplasts. N. benthamiana leaves were agroinfiltrated with a mixture of Agrobacterium tumefaciens strains harbouring constructs StFtsH4‐nYFP and AsCEP20‐cYFP (top panel), and the negative control StFtsH4‐nYFP and cYFP, nYFP and AsCEP20‐cYFP, nYFP and cYFP. YFP fluorescence was monitored at 2 days post‐agroinfiltration using confocal laser scanning microscope. Bar, 25 μm.

FIGURE 3

StFtsH4 positively regulates the pathogen‐associated molecular pattern‐triggered immunity (PTI) plant response. (a) The silencing efficiency in potato leaves was assessed using reverse transcription‐quantitative PCR (RT‐qPCR) in StFtsH4 knockdown plants. StActin was used as a reference gene in potato. Error bars represent means ± SD from three biological replicates and two technical repetitions. Differences were evaluated using the two‐tailed Student's t tests (***p < 0.001). (b) Quantification of reactive oxygen species production in the leaf discs of TRV:StFtsH4 and TRV:00 plants. At least five leaf discs per treatment were measured using a luminol‐based chemiluminescence assay. RLU, relative luminescence units. Data are presented as mean ± SEM (n = 5). (c) RT‐qPCR analysis showing the expression of defence‐related genes StFRK1 and StWRKY7 in TRV:StFtsH4 gene silenced potato plants. Total RNA was extracted from leaves. StActin was used as a reference gene in potato. Error bars represent means ± SD from three biological replicates and two technical repetitions. Differences were evaluated using the two‐tailed Student's t tests (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 4

Virulence assay of Alternaria solani wild type (WT) and ΔAsCEP20 mutants on TRV:StFtsH4 and TRV:00 (a), and the sizes of necrotic lesions were measured at 5 days post‐inoculation (b). Detached leaves were inoculated with conidial suspensions of the WT strain and the AsCEP20 deletion mutant. Statistical significance determined by one‐way ANOVA with Bonferroni p value adjustment.

FIGURE 5

AsCEP20 attenuates plant photosynthesis by targeting the protein StFtsH4. Fv/Fm (a) and NPQ (b) of TRV:00 and TRV:StFtsH4 potato leaves were measured. Mean values and standard deviations were assessed from three biological replications. Asterisk indicated the significant difference (*p < 0.05, **p < 0.01, ns means not significant, two‐tailed Student's t tests). The colour images are the chlorophyll fluorescence parameter images of the leaves. Fv/Fm (c), NPQ (d) and qL (e) of potatoes inoculated with wild type (WT) and ΔAsCEP20 mutant strains of Alternaria solani were measured. Mean values and standard deviation were assessed from three biological replications. Asterisk indicated the significant difference (*p < 0.05, ns means not significant, two‐tailed Student's t tests). The colour images show the chlorophyll fluorescence parameter images of the leaves.

FIGURE 6

StFtsH4 regulates chloroplast homeostasis to enhance plant susceptibility. (a) Light response curve of TRV:00 and TRV:StFtsH4 plants. Differences were evaluated using the two‐tailed Student's t tests (*p < 0.05, **p < 0.01). Data are means ± SD (n = 3). (b) Quantification of reactive oxygen species production in chloroplasts at different light intensities. RLU, relative luminescence units. Differences were evaluated using the two‐tailed Student's t tests (*p < 0.05, ***p < 0.001, ns means not significant). Data are presented as mean ± SD (n = 6). (c) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis showing the expression of defence‐related gene StFRK1 at different light intensities. (d) RT‐qPCR analysis showing the expression of defence‐related gene StWRKY7 at different light intensities. StActin was used as a reference gene in potato. Error bars represent means ± SD from three biological replicates and two technical repetitions. Differences were evaluated using the two‐tailed Student's t tests (*p < 0.05, **p < 0.01, ns means not significant).

FIGURE 7

Model of AsCEP20 targets StFtsH4 to regulate plant disease resistance. The conidia of Alternaria solani germinate and extend into hyphae, which directly invade the epidermal cells of the host plant. The A. solani effector AsCEP20 targets StFtsH4 to inhibit reactive oxygen species bursts, the expression of the immune‐related genes and attenuate the efficiency of light energy utilisation in plant photosynthesis, leading to enhanced plant susceptibility. ROS, reactive oxygen species.