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Title: CHARACTERIZATION OF NOVEL SESQUITERPENOID BIOSYNTHESIS IN TOBACCO EXPRESSING A FUNGAL SESQUITERPENE SYNTHASE

Author
item ZOOK, MICHAEL - DEPT BOTANY, MSU
item Hohn, Thomas
item BONNEN, ALICE - DEPT BOTANY, MSU
item TSUJI, JUN - DEPT BOTANY, MSU
item HAMMERSCHMIDT, RAYMOND - DEPT BOTANY, MSU

Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/6/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plants produce anti-microbial compounds called phytoalexins as part of their chemical defense against disease causing microorganisms. In tobacco and related plants, the most prominent phytoalexins are members of a group of compounds called sesquiterpenoids. To better understand the regulation and importance of phytoalexin production we have introduced a fungal gene for sesquiterpenoid production into tobacco. Cell cultures containing the fungal gene were found to produce a novel sesquiterpenoid compound. These results have implications for efforts to alter plant chemical defenses through genetic engineering and may facilitate improvements in plant disease resistance.

Technical Abstract: The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabaccum). Cell homogenates from transformant cell suspension cultures had sesquiterpene synthase activities equal to or greater than that of cellulase-treated wild- type cell suspension cultures. Trichodiene was the sole sesquiterpene synthase product in reactions containing cell homogenates from unelicited transformants while both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor presentation. Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase in those transformant cell lines with unelicited sesquiterpene synthase activity. Trichodiene was identified in transformant cell suspension cultures by GC/MS analysis. Incorporation of [3H]mevalonate into trichodiene was correlated with the levels of trichodiene synthase activity found in transformants. No trichodiene was observed in extracts from the wild-type cell line or transformant TH10 which lacks an intact copy of the trichodiene synthase gene. In vivo labeling with [3H]mevalonate and [3H]trichodiene revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell suspension cultures. These results demonstrate that increases in sesquiterpenoid biosynthesis following elicitation are likely due to increases in the production of FPP and that the resulting FPP can be efficiently utilized by foreign sesquiterpene synthases to produce high levels of novel sesquiterpenoids.