NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata.

key messageWe identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Upon insect herbivore attack, wild tobacco Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing or overexpression of NaMLP had no effect on the lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP overexpressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling pathways in a synergistic way.