Functional Analysis Of A Kunitz Type Protease Inhibitor Gene NaKTI2 In Nicotiana Attenuata Resistance To Pathogens And Herbivores

Plant protease inhibitor(PI)occurs widely in plant species and is considered as an important part of plant defense arsenal.Our previous transcriptome analysis of Nicotiana attenuata leaves reveals that a kunitz trypsin inhibitor 2 gene(NaKTI2)is highly elicited after inoculation of Alternaria alternata(tobacco pathotype).However,the roles of NaKTI2 in pathogen and herbivore resistance and its regulation are unclear.Protein sequences analysis revealed that NaKTI2 had typical domains of kunitz protease inhibitors and exhibited a high level of trypsin protease inhibitor activities when transiently over-expression in the leaves of Nicotiana benthamiana.Disrupted NaKTI2 expression via virus induced gene silencing(VIGS)technique had no influence on the lesion diameters developed on N.attenuata leaves after A.alternata inoculation,suggesting that NaKTI2 was not essential for A.alternata resistance.Interestingly,the transcripts of NaKTI2 could be highly induced by wounding,and amplified when Spodoptera litura oral secretions(OS)were added to the wounds.Further evidence indicated that NaKTI2 played a role in herbivore resistance as S.litura larvae gained more mass on NaKTI2-silenced plants than those grown on plants transformed with empty vector,which was consistent with the finding that lower level of trypsin protease inhibitor activities were detected in NaKTI2-silenced plants.In addition,OS-elicited NaKTI2 transcripts were slightly reduced in JA-deficient plants and significantly decreased in NaWRKY3-and NaWRKY6-slienced plants,respectively.Further electrophoretic mobility shift assay(EMSA)indicated that NaWRKY3 and NaWRKY6 could directly bind to the promoter regions of NaKTI2 in vitro.Taken together,our results demonstrate that NaKTI2,a pathogen-and herbivore-induced kunitz protease inhibitor gene,is required for resistance to insect herbivore S.litura but not fungal pathogen A.alternate and is transcriptionally regulated by NaWRKY3 and NaWRKY6.