Calcium channel CNGC20 and calmodulin 7 regulate melatonin-induced calcium signaling and aphid resistance in watermelon

Melatonin plays a crucial role in regulating plant defense against various environmental stresses. However, its role and the underlying mechanism in enhancing plant resistance to aphid infestation remains elusive. In this research, we discovered that aphid infestation notably upregulated the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (ClCOMT1) and increased melatonin levels in watermelon (Citrullus lanatus). The overexpression of ClCOMT1 enhanced defensive responses and resistance to aphids in watermelon. This was accompanied by the accumulation of cytosolic free calcium ([Ca2+]cyt), upregulation of the Ca2+-permeable channel gene cyclic nucleotide-gated ion channel 20 (ClCNGC20), and downregulation of calmodulin 7 (ClCaM7). ClCaM7 interacts with ClCNGC20 and suppresses its channel activity. In contrast, the knockout of ClCOMT1 showed opposite effects compared with its overexpression. Pretreatment with LaCl3 (a Ca2+ influx channel inhibitor) or EGTA (a Ca2+ chelator) compromised ClCOMT1 overexpression-induced aphid resistance; whereas, CaCl2 or the Ca2+ ionophore A23187 effectively restored aphid resistance in clcomt1 mutant. Moreover, silencing ClCNGC20 impaired ClCOMT1 overexpression-induced [Ca2+]cyt accumulation and aphid resistance; conversely, silencing ClCaM7 restored [Ca2+]cyt accumulation and aphid resistance in the clcomt1 mutant. Overexpression of watermelon ClCNGC20 elevated [Ca2+]cyt levels and improved aphid resistance in transgenic Arabidopsis plants, whereas overexpression of watermelon ClCaM7 had the opposite effect. Collectively, these findings imply the crucial role of ClCNGC20 and ClCaM7 in mediating melatonin-induced [Ca2+]cyt elevation, subsequently activating defensive responses in plants. This study offers insights into the molecular mechanisms underlying melatonin-mediated plant aphid resistance, with potential applications in the breeding or engineering of aphid-resistant cucurbit varieties.