A glycosylphosphatidylinositol-anchored protein from Alternaria alternata triggers cell death and negatively modulates immunity responses in chrysanthemum.

key messageGlycosylphosphatidylinositol-anchored protein (GPI-AP) Aa049 works as a key pathogenic factor to assist A. alternata in infecting plants, which is associated with the reactive oxygen species (ROS) pathway. Chrysanthemum black spot disease is a common fungal disease caused by A. alternata, which has severely hindered the development of the chrysanthemum industry. However, there are few reports on pathogenic factors in A. alternata, especially regarding GPI-APs. In this study, we identified a GPI-AP, Aa049, from A. alternata. Bioinformatics predictions suggest the presence of GPI-anchored modification sites at the C-terminus of its amino acid sequence, which is relatively conserved among different Alternaria Nees. Transient overexpression of Aa049 in Nicotiana benthamiana can induce programmed cell death (PCD), and the appearance of necrosis depends on its native signal peptide and GPI-anchored sites. Compared with the wild-type strain, the morphology and growth rate of the colony and mycelia of the ΔAa049-deletion mutants do not change. Still the integrity of the cell wall is damaged, and the virulence of the strain is significantly reduced, indicating that Aa049 plays an essential role as a pathogenic factor in the infection process of A. alternata. Furthermore, the results of quantitative real-time PCR (qRT-PCR) and physiological indicators suggested that the virulence of Aa049 may be exerted through the synthesis and clearance pathways of ROS. This study reveals that GPI-APs in A. alternata can act as virulence factors to aid pathogen invasion, providing a potential target for the development of future biopesticides.