Camalexin (3-thiazol-2'-yl-indole) is the major phytoalexin found in Arabidopsis thaliana.Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments.Camalexin is formed when indole-3-acetonitrile (IAN)is catalyzed by the cytochrome P450 monooxygenase CYP71A13.Here,we demonstrate that the Arabidopsis GH3.5 protein,a multifunctional acetyl-amido synthetase,is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes.Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection.The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro.In support of the in vitro reaction,feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D.Dihydrocamalexic acid (DHCA),the precursor of camalexin and the substrate for PAD3,was accumulated in gh3.5-1Dlpad3-1,suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis.Furthermore,expression of the major camalexin biosynthesis genes CYP79B2,CYP71A12,CYP71A13 and PAD3 was strongly induced in gh3.5-1D.Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys),and upregulation of the major biosynthetic pathway genes.