Functional Characterization of Effector Protein Pi07555 from Phytophthora infestans and Screening for Its Host Targets

doi:10.13560/j.cnki.biotech.bull.1985.2026-0041

Abstract

Abstract:

Objective To elucidate the biological functions and host target proteins of the effector Pi07555 from Phytophthora infestans. Methods The secretory activity of the signal peptide of effector Pi07555 was verified using the SignalP 6.0 online server and the yeast invertase secretion system. The virulence function of the Pi07555 gene was analyzed via combined assays including quantitative real-time PCR (RT-qPCR), Agrobacterium-mediated transient gene expression, and in vitro leaf inoculation. The host target protein of Pi07555 and the function of its encoding gene were identified through yeast two-hybrid (Y2H) assay, luciferase complementation imaging (LCI) assay, and virus-induced gene silencing (VIGS) technology. Results The Pi07555 gene encodes 170 amino acids, and the N-terminal signal peptide has secretory activity. The Pi07555 gene is upregulated at the early stage of pathogenic Phytophthora infection and is localized to the plant nucleus, cytoplasm, and cell membrane. The transient expression of Pi07555 promotes pathogenic Phytophthora infection but does not inhibit BAX and INF1-mediated hypersensitive cell death. The yeast two-hybrid and luciferase complementation imaging assays demonstrated that potato hypersensitive-induced reaction 1 (StHIR1) interacts with Pi07555. The StHIR1 gene is upregulated 48 hours after pathogenic Phytophthora infection, and silencing its homolog significantly increased the resistance of Nicotiana benthamiana to pathogenic Phytophthora. Conclusion Pi07555 is a secreted virulence effector that localizes to the plant nucleus, cytoplasm, and plasma membrane.

Key words: potato, Phytophthora infestans, effector, Pi07555, protein interaction

WANG Hui-jie, DAO Wen-jing, ZHANG Bei-ni, FU Yan-hong, HUANG ya-nan, WANG Hong-yang. Functional Characterization of Effector Protein Pi07555 from Phytophthora infestans and Screening for Its Host Targets[J]. Biotechnology Bulletin, 2026, 42(6): 139-148.

Figures/Tables 9

Table 1 Sequence alignment results of candidate target genes

Clone ID	Gene ID	Gene annotation
AD-12	XM_006364913.2	Solanum tuberosum transcription factor bHLH93-like， StbHLH93
AD-39	NM_001318700.1	Solanum tuberosum glucan endo-1，3-beta-glucosidase， StGLU
AD-51	XM_006367576.2	Solanum tuberosum 21 kD protein-like， St21kDa
AD-109	XM_006354735.2	Solanum tuberosum hypersensitive-induced response protein 1， StHIR1

Fig. 1 Validation of the signal peptide secretory function of effector Pi07555Negative control: pSUC2 and YTK12 strains; positive control: pSUC2-PsAvr1b; experimental group: pSUC2-Pi07555

Fig. 2 Effector Pi07555 promotes pathogen colonizationA: Expression dynamic of Pi07555 in potato ‘Cooperation-88’ during P. infestans infection; B: phenotypes of N. benthamiana leaves at 6 days post-inoculation (dpi) with P. infestans after transient expression of Pi07555; C: lesion areas of N. benthamiana leaves at 6 dpi with P. infestans after transient expression of Pi07555. ** P0.01

Fig. 3 Analysis of cell death suppression by effector Pi07555A and B: Analysis of the immunosuppressive function of Pi07555 on INF1; C and D: Analysis of the immunosuppressive function of Pi07555 on BAX; positive control: INF1/BAX; negative control: INF1/BAX+GFP; experimental groups: Pi07555 alone, and INF1/BAX+Pi07555

Fig. 4 Subcellular localization and Western blot analysis of effector Pi07555Merge: Merged field; GFP: Green fluorescence field; Bright: Bright field; DAPI: Blue fluorescent field; FM4-64: Red fluorescent field. Scale bar=50 μm; CBB staining represents Coomassie brilliant blue staining; * represents the target protein

Fig. 5 Toxicity and autoactivation assays of the bait protein Pi07555A: Toxicity test; B: transcriptional autoactivation test

Fig. 6 Screening for interacting proteins of effector Pi07555

Fig. 7 Verification of candidate protein interactions by luciferase complementation imaging assay

Fig. 8 Expression pattern and functional analysis of the HIR1 geneA: Expression pattern of StHIR1 in potato ‘Cooperation 88’ during P. infestans infection; B: silencing efficiency assay for NbHIR1; C: disease phenotypes of NbHIR1-silenced N. benthamiana after inoculation with strain 88069; D: quantification of lesion areas on NbHIR1-silenced N. benthamiana inoculated with strain 88069. * and ** indicate significant differences at P0.05 and P0.01, respectively

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