A single dsRNA spray silences VAMT and shifts habanero pepper fruit metabolism towards capsinoids

Capsaicinoids are synthesized in the placenta of Capsicum fruit, where vanillylamine aminotransferase (VAMT) catalyzes the formation of vanillylamine, the precursor of the pathway. The modulation of pungency has relied on genetic breeding and transgenic approaches, and this pathway has not been addressed by spray-induced gene silencing. The aim of this study was to evaluate whether a single non-invasive spray of double-stranded RNA (dsRNA) targeting VAMT allows the gene to be silenced and capsaicinoid accumulation to be modified in Capsicum chinense fruit. The molecule was designed in silico and applied at 10 days post-anthesis. Pedicel injection reduced the VAMT transcript in a dose-dependent manner, with three levels of inhibition distinguishable from one another. Spraying with surfactant reduced it by 86.2 %, a magnitude statistically indistinguishable from the 90.6 % obtained by injection, and also reduced the Pun1 transcript, a co-regulation previously described only as a difference between cultivars. Analysis by gas chromatography coupled to mass spectrometry showed reductions of 84.4 % in capsaicin and 68.5 % in dihydrocapsaicin, the loss of nonivamide and one further vanillylamine-derived compound, and the detection of capsiate and a second capsinoid, absent in control fruits. The siRNA was detected in non-treated tissues, and a single topical application is therefore sufficient to silence an endogenous biosynthetic gene and shift the metabolic profile of the fruit without genetic modification.