Characterization of a putative poplar E3 RING-H2 ubiquitin ligase that when over-expressed is correlated with negative impacts on feeding and development of the whitemarked tussock moth, Orgyia leucostigma

From a forward genetics screen of activation-tagged Populus tremula x P. alba 717-IB4 (Pt x Pa) trees we identified the mutant E8-16 as resistant to Orgyia leucostigma larvae feeding.  Previous choice and no-choice bioassays have confirmed that insect feeding and development were impaired on E8-16 compared to wild-type (WT) trees.  We have also identified the mutation in E8-16 as a T-DNA insertion on chromosome 10 resulting in the activation of 10s12800, an E3 RING-H2 ubiquitin ligase.  E3 ligases serve an important role in protein turnover by tagging proteins with an ubiquitin label, signaling for the target protein to be degraded by the 26S proteasome complex.  To recapitulate the phenotype 10s12800 was inserted into the pCAMBIA-S1300 vector and introduced into the Pt x Pa and P. canadensis x P. grandidentata (Pc x Pg) genetic backgrounds. Molecular and bioassay evaluation of 22 transgenic lines identified one line, TL4, with elevated expression of the transgene (2 fold higher, p<0.01) and 27% less feeding in a choice bioassay (p<0.0001) compared to a vector control but did not show a difference in no-choice assays.  A 4x44K Agilent microarray comparing global transcriptomes of E8-16 and WT trees revealed 22 differentially expressed probes including 10s12800, MADS-BOX transcription factors, and endochitinases.  A separate validation of the microarray has confirmed the over-expression of MADS-BOX and bZIP transcription factors (p<0.001) and we are currently evaluating several endochitinases.  We are also currently using an auto-ubiquitinylation kit to confirm the predicted enzyme function of 10s12800 and using GFP tagged 10s12800 with fluorescent microscopy to identify protein localization within transiently transfected protoplasts.