A Modular Fusion Protein Strategy Confers Broad-Spectrum Resistance Against Phytophthora infestans, Alternaria solani, and Ralstonia solanacearum

Abstract

Potato production is severely threatened by the oomycete Phytophthora infestans, the fungus Alternaria solani, and the bacterium Ralstonia solanacearum; however, strategies capable of simultaneously managing these three pathogen classes remain relatively scarce. In this study, we designed a series of signal peptide (SP)-fused recombinant constructs containing four functional modules with distinct antimicrobial and immune-inducing properties: GAFP1-2×FYVE, an oomycete-inhibiting antimicrobial protein; BbAFP1-ErBD, a fungal-suppressive protein fragment; the pathogen-associated molecular pattern (PAMP) csp22 derived from R. solanacearum, which elicits effective anti-bacterial immunity; and the microbe-associated molecular pattern (MAMP) PpEli2 identified from Pythium periplocum, which triggers broad-spectrum plant defense responses. Through systematic evaluation of six module order rearrangements, we identified SP-cBG as the optimal multi-domain combination. Further optimization via the introduction of a rigid alpha-helical linker (HL4) yielded SP-cBG-HL4, which tended to show superior resistance against all three pathogens, as reflected in smaller lesion diameters and decreased bacterial titers. qRT-PCR analyses revealed that SP-cBG-HL4 significantly upregulated PTI (CYP71D20, PTI5), SA (PR1, PR2), and JA/ET (PR3, PR4) defense marker genes. Collectively, these findings show that modular domain assembly combined with linker-mediated modular combinatorial optimization represents a powerful engineering strategy for achieving broad-spectrum disease resistance, offering a promising approach to simultaneously control oomycete, fungal, and bacterial pathogens of plants.