Genomic structure and transcript analysis of the Rapid Alkalinization Factor (RALF) gene family during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

Rapid Alkalinization Factors (RALFs) are cysteine-rich peptides ubiquitous within plant kingdom. They play multiple roles as hormonal signals in diverse processes, including root elongation, cell growth, pollen tube development, and fertilization. Their involvement in host-pathogen crosstalk as negative regulators of immunity in Arabidopsis has also been recognized. In addition, peptides homologous to RALF are secreted by different fungal pathogens as effectors during early stages of infection. Previous studies have identified nine RALF genes in the diploid strawberry (Fragaria vesca) genome. This work describes the genomic organization of the RALF gene families in commercial octoploid strawberry (Fragaria × ananassa) and the re-annotated genome of F. vesca, and then compares findings with orthologs in Arabidopsis thaliana. We reveal the presence of 15 RALF genes in F. vesca genotype Hawaii 4 and 50 in Fragaria x ananassa cv. Camarosa, showing a non-homogenous localization of genes among the different Fragaria x ananassa subgenomes. Expression analysis of Fragaria x ananassa RALF genes upon infection with Colletotrichum acutatum or Botrytis cinerea showed that FanRALF3-1 was the only fruit RALF gene upregulated after fungal infection. In silico analysis was used to identify distinct pathogen inducible elements upstream of the FanRALF3-1 gene. Agroinfiltration of strawberry fruit with deletion constructs of the FanRALF3-1 promoter identified a 5’ region required for FanRALF3-1 expression in fruit, but failed to identify a region responsible for fungal induced expression.