Background: The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results: Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions: Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to ensure that only safety-tested products are marketed. Over the last few years, targeted approaches have been complemented by profiling methods to assess possible unintended effects of transformation. Here we used a commercial (Affymertix) microarray platform (i.e. allowing assessing the expression of ~1/3 of the genes of maize) to evaluate transcriptional differences between commercial MON810 GM maize and non-transgenic crops in real agricultural conditions, in a region where about 70% of the maize grown was MON810. To consider natural variation in gene expression in relation to biotech plants we took two common MON810/non-GM variety pairs as examples, and two farming practices (conventional and low-nitrogen fertilization). MON810 and comparable non-GM varieties grown in the field have very low numbers of sequences with differential expression, and their identity differs among varieties. Furthermore, we show that the differences between a given MON810 variety and the non-GM counterpart do not appear to depend to any major extent on the assayed cultural conditions, even though these differences may slightly vary between the conditions. In our study, natural variation explained most of the variability in gene expression among the samples. Up to 37.4% was dependent upon the variety (obtained by conventional breeding) and 31.9% a result of the fertilization treatment. In contrast, the MON810 GM character had a very minor effect (9.7%) on gene expression in the analyzed varieties and conditions, even though similar cryIA(b) expression levels were detected in the two MON810 varieties and nitrogen treatments. This indicates that transcriptional differences of conventionally-bred varieties and under different environmental conditions should be taken into account in safety assessment studies of GM plants.