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Title: Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots

Author
item Smigocki, Anna
item PUTOFF, DAVID - FROSTBURG, MARYLAND
item ZUZGA, SABINA - WARSAW, POLAND
item HAYMES, SNEZANA - TOWSON UNIV TOWSON, MD

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2009
Publication Date: 2/12/2009
Citation: Smigocki, A.C., Putoff, D., Zuzga, S., Haymes, S. 2009. Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots. Plant Cell Tissue And Organ Culture. 97(2):167-174. Available doi 10.1007/s11240-009-9512-3.

Interpretive Summary: Insect survival depends on the uptake of nutrients from ingested food; therefore, inhibition of digestive enzymes presents itself as an effective strategy for control of insect pests. To specifically target several major pests of sugar beet, a significant industrial crop producing a third of all sugar consumed in the world, we isolated genetic material from a tobacco plant that has been shown to effectively reduce the population of several insect pests. We report on the introduction of the tobacco genetic material into sugar beet roots in a model laboratory system. Analysis of the sugar beet roots revealed that the tobacco product was being produced at relatively high levels even though the processing of the precursor product to individual active components was either lacking or occurred at a low efficiency. The reduced efficiency may be due to modification of the tobacco product and/or distinct processing machinery that may be operating in sugar beet roots and tobacco. Sugar beet roots that produce the active tobacco product will be used in insect feeding assays to screen for insect resistance. This information will be used by scientists to increase our knowledge of plant insect resistance mechanisms and lead to environmentally safer approaches of insect control by reducing the use of harmful pesticides.

Technical Abstract: Assimilation of dietary proteins is critical to insect survival; therefore, inhibition of digestive proteolytic enzymes presents itself as an effective strategy for control of insect pests. To specifically target proteases of several insect pests of sugar beet, Beta vulgaris, we used PCR and gene specific primers to clone a Nicotiana alata NaPI gene encoding a serine proteinase inhibitor (PI) that has been shown to effectively reduce the population of a number of insect pests. We report on the transformation and expression in sugar beet hairy roots of the NaPI gene. Clonally propagated hairy root lines exhibited relatively high levels of NaPI gene transcripts driven by the constitutive 35S promoter. Using in-gel analysis for PI activity, two protein bands were detected at approximately 24- and/or 28-kDa. Immunoblot analysis with anti-NaPI specific antibody revealed the presence of the expected ~40 kDa precursor, and in some transformants, a ~20 kDa processing intermediate and the mature 6-kDa PIs. In general, processing of the precursor in independently derived transformed clonal lines was either lacking or appeared to occur at a low efficiency. The reduced efficiency of post-translational processing of the N. alata multidomain PI precursor may be attributed to modification and/or altered folding of the recombinant protein or distinct post-translational machinery functioning in sugar beet hairy roots and Nicotiana.