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

Abstract

Abstract Upon insect herbivore attack, 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 NaMLP had no effect on 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 over-expressed 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 over-expressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP over-expressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficent irAOC and ethylene-reduced irACO 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 in a synergistic way.