Fig. 4: PEBP4 interacted with Raf1 which negatively regulated MAPK phosphorylation and enhanced the virus-induced apoptosis.

a GST pull-down showing the interaction between recombinant His-Raf1 and PE-binding ___domain of GST-PEBP4. b PEBP4 co-immunoprecipitated with Raf1 in viruliferous (V) or nonviruliferous (N) whitefly. c Time-course immunoblots of MAPK pathway phosphorylation. d, e Immunoblots of MAPK pathway phosphorylation in whitefly lysate when co-incubated with a concentration gradient of d PEBP4 and e TYLCV CP. f His-CP and His-Raf1 of different concentrations were co-incubated in whitefly lysate, and co-immunoprecipitated with PEBP4. g Raf1 (blue), PEBP4 (red), and CP (green) were simultaneously labeled in immunofluorescence assays. Colocalization was quantified by ImageJ Coloc2 using Manders’ Colocalization Coefficients (MCC). M1 = PEBP4-Raf1/Total Raf1; M2 = CP-Raf1/Total Raf1. h, i After 24 h feeing on mirdametinib, a phosphorylation inhibitor of MEK, whiteflies were transferred to virus-infected host plants for another 24 h to acquire TYLCV. The midgut and salivary gland were dissected for h TUNEL labeling (green). Nuclei were stained by DAPI (white). Sample sizes (n) for statistical tests indicated in the panels refer to biologically independent whitefly. Values in bar plots represent mean ± SEM. All data were checked for normality by the Wilk-Shapiro test. Two-sided paired t-test was used to separate the means of normally distributed data. i Immunoblot of phosphorylation of MAPK pathway and initiation of apoptosis. j Model of CP-PEBP4 regulation on MAPK cascade in whitefly. CP stabilized Raf1-PEBP4 interaction, which blocked the MAPK phosphorylation cascade.