Study of Rapid Alkalinization Factor (RALF)genes as plant susceptibility agents during plant-pathogen interaction in strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals and recently their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF homologue peptides are secreted by different fungal pathogens as effectors during early stages of infections. The aim of this work was to characterize RALF genes as susceptibility factors during plant pathogen interaction in strawberry. For this, the genomic organization of the RALF gene families in the octoploid strawberry (Fragaria × ananassa) and the re-annotated genome of Fragaria vesca were described , identifying 13 member in F. vesca (FvRALF) and 50 members in F. x ananassa (FaRALF). The changes in expression of fruit FaRALF genes was investigated upon infection with C.acutatum and B. cinerea showing that, among RALF genes expressed in fruit, FaRALF3 was the only one upregulated by fungal infection in the ripe stage. A role of FaRALF3 as susceptibility gene was then assessed trough Agrobacterium-mediated transient FaRALF3 overexpression and silencing in fruits, revealing that FaRALF3 expression promotes fungal growth and hyphae penetration in host tissues. In silico analysis was used to identify distinct pathogen inducible elements upstream of the FaRALF3 gene. Agroinfiltration of strawberry fruit with deletion constructs of the FaRALF3 promoter identified a 5’ region required for FaRALF3 expression in fruit, but failed to identify a region responsible for fungal induced expression. Furthermore, FaRALF3 and strawberry receptor FERONIA (FaMRLK47) were heterologously expressed in E. coli in order to purify active proteins forms and study RALF-FERONIA interaction in strawberry. However, it was not possible to obtain pure and active proteins. Finally RNAi transgenic plants silenced for the FvRALF13 gene were genotypically and phenotypically characterized suggesting a role of FvRALF13 in flowering time regulation and reproductive organs development.