Poster: Signaling, cell-to-cell

Abs # 442: Silencing of tomato mitogen-activated protein kinases (MAPKs) by virus-induced gene silencing

Presenter:	Ranf, Stefanie , steffi@biol.sc.edu
Authors	Ranf, Stefanie  (A)   Stratmann, Johannes W (A)
Affiliations:	(A): University of South Carolina, Department of Biological Sciences

Wounding and the wound signaling peptide systemin activate two distinct groups of genes, signaling genes and effector genes in tomato leaves. They also result in the activation of two or three mitogen-activated protein kinases (MAPKs), respectively (Holley et al., 2003). To elucidate the function of the three tomato MAPKs (LeMPKs) for wounding/systemin-responsive gene expression, we are currently applying a loss-of-function approach using tobacco rattle virus (TRV) induced gene silencing. This system has been optimized for tomato plants (Liu et al., 2002). LeMPK sequences will be cloned into a T-DNA expression cassette of the pTRV2 vector. As TRV is a bipartite RNA virus, Agrobacterium tumefaciens will be transformed with either pTRV1 or pTRV2. A mix of both cultures will then be infiltrated into lower leaves of two-week-old tomato seedlings. The T-DNA inserts into the plant genome and is transcribed to form infectious TRV virions. As a result, silencing of the viral RNA occurs along with silencing of the target genes. Each LeMPK will be silenced alone. In addition, LeMPK1 and 2 will also be silenced together since they are 95% identical at the amino acid level and activated by the same stress signals. We hypothesize that these two MAPKs are functionally redundant. For silencing of single MAP kinases we will use the 3‘UTRs. Double silencing of LeMPK1 and 2 will be attempted by using a sequence fragment of the coding region with a high percentage of identical sequence stretches. Infected plants will be screened for silencing by real time PCR and MAPK activity assays. Determining transcript and protein levels of signaling genes and effector genes in the silenced plants is expected to unravel the functions of the three MAPKs for gene expression.