The strawberry Fra a 1 allergen is a homolog of the major birch pollen allergen Bet v 1.It is synthesized by red ripe fruits of Fragaria x ananassa while white fruits of a mutant genotype,which is known to be tolerated by individuals affected by allergy,are devoid of it.Proteomic analyses have shown that Fra a 1 is down-regulated in the tolerated whitefruited genotype along with enzymes of the anthocyanin pigment pathway.In this study,we report the spatial and temporal expression of three Fra a genes that encode different isoforms,and the transient RNAi-mediated silencing of the Fra a genes in strawberry fruits of the red-fruited cultivar Elsanta with an ihpRNA construct.As a consequence of reduced levels of Ira a mRNAs,fruits were obtained that produced significantly decreased levels of anthocyanins and upstream metabolites.This effect is consistent with the parallel down-regulation of the phenylalanine ammonia lyase(FaPAL) and to a lesser extent of the chalcone synthase(FaCHS) transcript levels also found in these fruits.In naturally occurring whitefruited genotypes of F.chiloensis and F.vesca,Fra a transcript levels are higher than those of the red-fruited varieties,likely to compensate for the low expression levels of FaPAL and FaCHS in these mutant genotypes.The results demonstrate that Fra a expression is directly linked to flavonoid biosynthesis and show that the Fra a allergen has an essential biological function in pigment formation in strawberry fruit.