Rapid Alkalinization Factor (RALF) gene family genomic structure and transcriptional regulation during host-pathogen crosstalk in Fragaria vesca and Fragaria x ananassa strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals, starting from root elongation, cell growth, pollen tube development and fertilization. Their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF peptides are secreted by different fungal pathogens as effectors during early stages of infections. Campbell and Turner previously identified nine RALF genes in F. vesca v1 genome. Here, based on the recent release of Fragaria x ananassa genome and F. vesca reannotation, we aimed to characterize the genomic organization of the RALF gene family in both type of strawberry species according to tissue specific expression and homology with Arabidopsis . We reveal the presence of 13 RALF genes in F. vesca and 50 in Fragaria x ananassa, showing a non-homogenous localization of genes among the different Fragaria x ananassa subgenomes associated with their different TE element contents and genome remodeling during evolution. Fragaria x ananassa RALF genes expression inducibility upon infection with C. acutatum or B. cinerea was assessed and showed that, among fruit expressed RALF genes, FaRALF3-1 was the only one upregulated after fungal infection. In silico analysis and motif frequency analysis of the putative regulatory elements upstream of the FaRALF3 gene was carried out in order to identify distinct pathogen inducible elements. Agroinfiltration of strawberry fruit with 5’ deletion constructs of the FaRALF3-1 promoter identified a region required for FaRALF3 expression in fruit, but did not identify a region responsible for fungal induced expression.