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

Abstract

20 21 Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They 22 play multiple roles as hormone signals, starting from root elongation, cell growth, pollen tube 23 development and fertilization. Their involvement in host-pathogen crosstalk as negative regulator 24 of immunity in Arabidopsis has also been recognized. In addition, RALF peptides are secreted by 25 different fungal pathogens as effectors during early stages of infections. Campbell and Turner 26 previously identified nine RALF genes in F. vesca v1 genome. Here, based on the recent release of 27 Fragaria x ananassa genome and F. vesca reannotation, we aimed to characterize the genomic 28 organization of the RALF gene family in both type of strawberry species according to tissue specific 29 expression and homology with Arabidopsis. We reveal the presence of 13 RALF genes in F. vesca 30 and 50 in Fragaria x ananassa, showing a non-homogenous localization of genes among the 31 different Fragaria x ananassa subgenomes associated with their different TE element contents 32 and genome remodeling during evolution. Fragaria x ananassa RALF genes expression inducibility 33 upon infection with C. acutatum or B. cinerea was assessed and showed that, among fruit 34 expressed RALF genes, FaRALF3-1 was the only one upregulated after fungal infection. In silico 35 analysis and motif frequency analysis of the putative regulatory elements upstream of the 36FaRALF3 gene was carried out in order to identify distinct pathogen inducible elements. 37 Agroinfiltration of strawberry fruit with 5' deletion constructs of the FaRALF3-1 promoter 38 identified a region required for FaRALF3 expression in fruit, but did not identify a region 39 responsible for fungal induced expression. 40 41 42 3 43 44 123 124 Materials and Methods 125 126 RALF family genes identification, phylogenetic tree analysis, clade determination 127 and chromosome map 128 129 F. vesca RALF genes already reported by Campbell and Turner [6] were implemented 130 through keyword gene search 'RALF' GDR (v4.0.a2) [35]. The output was compared and integrated. 131 Nucleotide sequences of the 13 F. vesca RALF members were used as query for BLASTx on Fragaria 132 x ananassa cv. Camarosa Genome v1.0.a1 proteome [38] to find octoploid RALF homologs. 133 Fragaria x ananassa RALF peptide sequences were aligned by MUSCLE [39] and the phylogeny was 134 inferred using the Maximum Likelihood method and JTT matrix-based model [40]. The tree with 135the highest log-likelihood was chosen. The Initial tree was obtained automatically by applying 136 Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, 137 and then selecting the topology with superior log-likelihood value. Evolutionary analyses were