Over-expression of heterologous PI genes in crop plants has resulted in enhanced resistance to a wide spectrum of insect pests. However, the ongoing challenge of the PI based insect control strategy is the need to discover and characterize new and novel PIs to address the inherent and induced XAV939 complexity of the insect gut proteases. PIs such as those derived from non-host plants to which the targeted insect has had minimal or no prior exposure can generally be most useful for enhancing insect resistance in engineered plants. In a study of sugar beet root defense responses, a single serine PI gene was identified among the more than 150 sugar beet genes whose expression was found to be modulated by a dipteran pest of sugar beet, the root maggot. Expression of the BvSTI gene was determined to be up-regulated by mechanical- and insect-wounding in sugar beet lines used in breeding for root maggot resistance. The observed lack or reduced accumulation and activity of BvSTI PI in tissues of susceptible and less resistant lines emphasized the potentially important role of the BvSTI PI in insect pest defense mechanisms. In this study, the prospect of over-expressing the sugar beet BvSTI gene for control of lepidopteran insect pests in genetically modified N. benthamiana was investigated. Serine proteases that include trypsin-, chymotrypsin- and elastase-like have been well-documented as comprising the major midgut proteolytic activities in lepidopteran insects. Homozygous T2 populations of transgenic N. benthamiana plants carrying a single copy of the BvSTI transgene construct exhibited phenotypes that were similar to the normal untransformed plants. Elevated levels of BvSTI gene transcripts driven by the constitutive CaMV35S promoter were detected in all analyzed T2 homozygous plants. Presence of the recombinant BvSTI proteins in the T2 transformants was confirmed on SCH727965 Western blots with BvSTIspecific antibody that cross-reacted with low quantities of peptides in the range of 22�C25 kDa and 30 kDa that was previously observed in sugar beet. These finding suggests that processing and modification of the recombinant BvSTI protein may be different in the tobacco background as compared to its regulation in sugar beet. Detection of low levels of recombinant PI protein has been reported by others. Independently derived apple transformants with increased resistance to the light-brown apple moth had low levels of the recombinant PI protein. It has also been shown that feeding inhibition did not necessarily increase beyond that observed with low protein concentrations in studies where recombinant PI proteins were fed to larvae. Because the detected signal on Western blots was weak this suggests a possible high turnover and/or modification of the BvSTI protein in N. benthamiana irrespective of the high transcript and trypsin protein activity levels in the BvSTI transformants. A unique and distinct clear zone of at about 30 kDa was detected in all five homozygous BvSTI transformants by an in gel trypsin activity assay. Two additional activity zones corresponding to proteins of approximately 28 and 26 kDa were also visible in the transformed lines but not in the untransformed control plants. No apparent cross reactivity of the BvSTI specific antibody with the 28 and 26 kDa trypsin inhibitory proteins was observed on Western blots although one can argue that the 26 kDa protein is similar to that in the range of 21�C25 kDa.