Mol Gen Genet (1992) 235:179-188 © Springer-Verlag 1992 Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene Craig M. Hart*' ***, Bernt Fischer***, Jean-Marc Neuhaus**, and Frederick Meins Jr Friedrich Miescher-Institut,P.O. box 2543, CH-4002 Basel, Switzerland Received March 23, 1992 / AcceptedMay 19, 1992 Summary. The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozy- gous for a chimeric tobacco (Nicotiana tabacum) chitin- ase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-trans- formed plants. Unexpectedly, some transgenic plants ac- cumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, ~25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Si- lencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-de- pendent silencing in which expression was uniform with- in individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was ex- amined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheri- tance. Taken together, the results indicate that silencing [ , results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppres- sion, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated * Present address: Department of Biochemistry, Universityof Gen- eva, CH-1211 Geneva4, Switzerland ** Present address: BotanischesInstitut, Abt. Pflanzenphysiologie, Hebelstrasse 1, CH-4056 Basel, Switzerland *** These authors have both made an equal contribution to this work Correspondence to." F. MeinsJr with cytosine methylation of the transgene at the five restriction sites investigated. Key words: Plant-defense- Gene-silencing Co-suppres- sion - Chitinase - DNA-methylation Introduction Recent studies show that transgenes introduced into plants can interact with homologous host genes leading to decreased expression of both genes (Napoli et al. 1990; van der Krol et al. 1990; Smith et al. 1990). Al- though the inactive state appears to be meiotically trans- mitted in some cases, the phenomenon is stochastic: in- activation is often variable in different parts of the same plant and only some plants in a population show the effect. The present report addresses the problem of the sta- bility and developmental regulation of inhibitory inter- actions between homologous tobacco (Nicotiana taba- cure) and host class I chitinase genes in transgenic Nico- tiana sylvestris. Class I vacuolar chitinases are involved in the induced defense response of plants: they are in- duced by the stress hormone ethylene and by infection with viral, bacterial and fungal pathogens (Boller 1988); they are potent fungicides in vitro, particularly in combi- nation with /%l,3-glucanase (Schlumbaum et al. 1986; Mauch et al. 1988); and transgenic tobacco plants over- expressing the enzyme show decreased susceptibility to certain fungal pathogens (Broglie et al. 1991). To identify possible defense-related functions of this chitinase, we transformed N. sylvestris plants with a chi- meric tobacco gene encoding the enzyme regulated by the 35S RNA expression signals of Cauliflower Mosaic Virus (CaMV) (Neuhaus et al. 1991). Leaves of most plants homozygous for the transgene accumulated chi- tinase at levels up to 120-fold higher than comparable leaves of nontransformed plants. Unexpectedly, some transgenic plants showed very low levels of chitinase, a phenomenon we call silencing. Here we show that si-