Establishment of heritable transposon-like transgene silencing in Arabidopsis is disrupted in large multigenic T-DNAs with GC-rich coding sequences

Variability and eventual loss of transgene expression due to silencing is a significant challenge for plant genetic engineering, especially for large, complex transgene insertions. We have characterized in detail the silencing mechanisms that occur when a suite of transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase system is introduced into Arabidopsis. Certain transgene constructs using coding sequences (CDS) with a plant codon bias underwent aggressive silencing, similar to that of active transposable elements (TEs). Silencing was mitigated by diversifying regulatory sequences and was prevented when GC-rich native microalgal CDS were substituted for canola-biased transgenes. This was accompanied by reduced accumulation of 21/22-nt siRNAs, low levels of 24-nt siRNAs and sparse (gene-body-like) CDS DNA methylation, indicating that the transition from modest transcript degradation to heritable silencing was suppressed by using high GC transgenes. These observations provide a strategy for countering transgene silencing in genetic engineering of complex plant traits.