NaKTI2, a Kunitz trypsin inhibitor transcriptionally regulated by NaWRKY3 and NaWRKY6, is required for herbivore resistance in Nicotiana attenuata.

KEY MESSAGE: Here, we reported that a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, NaKTI2, is required for herbivore resistance, and transcriptionally regulated mainly by NaWRKY3 and NaWRKY6 but not Jasmonate signaling. Plant protease inhibitor (PI) occurs widely in plant species, and is considered as an important part of plant defense arsenal against herbivores. Transcriptome analysis of Nicotiana attenuata leaves revealed that a Kunitz trypsin inhibitor gene, NaKTI2, was highly elicited after inoculation of Alternaria alternata (tobacco pathotype). However, the roles of NaKTI2 in pathogen- and herbivore resistance and its regulation were unclear. NaKTI2 had typical domains of Kunitz trypsin inhibitors and exhibited a high level of trypsin protease inhibitor activities when transiently over-expressed. The transcripts of NaKTI2 could be induced by A. alternata and Spodoptera litura oral secretions (OS). Silencing NaKTI2 via virus-induced gene silencing technique has no influence on lesion diameters developed on N. attenuata leaves after A. alternata inoculation, but S. litura larvae gained more mass and had higher survivorship on NaKTI2-silenced plants. Meanwhile, the expression of NaPI, a PI gene essential for herbivore resistance previously identified in N. attenuata, was not affected in NaKTI2-silenced plants. Unlike NaPI, which was predominantly regulated by jasmonate (JA) signaling, OS-elicited NaKTI2 transcripts were only slightly reduced in JA-deficient plants, but were dramatically decreased in NaWRKY3- and NaWRKY6- silenced plants, respectively. Further electromobility shift assays indicated that NaWRKY3 and NaWRKY6 could directly bind to the promoter regions of NaKTI2 in vitro. Taken together, our results demonstrate that in addition to NaPI, NaKTI2, a pathogen- and herbivore-induced Kunitz trypsin inhibitor gene, is also required for herbivore resistance, and mainly regulated by NaWRKY3 and NaWRKY6.