Exploration of disease suppression function of LsGRP1C on crucifer anthracnose and its antifungal mechanism

Plants are sessile organisms and unable to avoid pathogen attack by moving ability. Plants may protect themselves by generating biochemical products with antimicrobial activity. LsGRP1 is a glycine-rich defense-related protein of Lilium cv. Stargazer, with a C-terminal portion (LsGRP1C) comprised of 38 amino acids rich in cysteine residue. Colletotrichum higginsianum causing crucifer anthracnose is a hemibiotrophic fungal pathogen. In vitro assays with the synthetic peptide of LsGRP1C showed inhibition of spore germination, appressorium formation and mycelial growth of C. higginsianum. Immunofluorescence staining showed that LsGRP1C bound to the outer layer of C. higginsianum mycelia. SYTOX Green staining assay demonstrated that LsGRP1C caused membrane permeabilization of C. higginsianum and its suppression by cations. Accordingly, disruption of fungal membrane integrity by LsGRP1C due to its net positive charge was presumed. On the other hand, DAPI, H2DCFDA and TUNEL staining assays showed that LsGRP1C caused apoptosis-like programmed cell death phenomena in C. higginsianum, including cytoplasm shrinkage, intracellular accumulation of reactive oxygen species, chromatin condensation and nuclear DNA cleavage etc. Transient expression of LsGRP1, LsGRP1ΔNΔG, LsGRP1ΔC or LsGRP1C in the leaves of Arabidopsis thaliana followed by inoculation with C. higginsianum demonstrated that expression of LsGRP1, LsGRP1ΔNΔG, and LsGRP1C could suppress symptom development and sporulation, and the secondary hyphae were not present as indicated by staining and microscopic examinations. Thus, the primary hyphal cell death of C. higginsianum caused by the expression of LsGRP1, LsGRP1ΔNΔG and LsGRP1C in planta was presumed.