The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to assure that only products that have been safety tested in relation to human health and the environment are marketed. Thus, GMOs must be authorized before use. By complementing more targeted approaches, profiling methods can assess possible unintended effects of transformation. We used microarrays to compare the transcriptome profiles of widely commercialized maize MON810 varieties and their non-GM near-isogenic counterparts. The expression profiles of MON810 seedlings are more similar to those of their corresponding near-isogenic varieties than are the profiles of other lines produced by conventional breeding. However, differential expression of approximately 1.7 and approximately 0.1% of transcripts was identified in two variety pairs (AristisBt/Aristis and PR33P67/PR33P66) that had similar cryIA(b) mRNA levels, demonstrating that commercial varieties of the same event have different similarity levels to their near-isogenic counterparts without the transgene (note that these two pairs also show phenotypic differences). In the tissues, developmental stage and varieties analyzed, we could not identify any gene differentially expressed in all variety-pairs. However, a small set of sequences were differentially expressed in various pairs. Their relation to the transgenesis was not proven, although this is likely to be modulated by the genetic background of each variety.

Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.

Worldwide maize is the second major agricultural commodity and around one-fourth is currently biotech, with significant application of the insect resistant event MON810 particularly in the European Union. Grains are the major commercialized part of the plant, and can be harvested after maturity (for food and feed purposes) or at late milky-starchy stage (for forage uses, with the whole plant). We assessed possible proteomic unintended effects of the MON810 transgene using two-dimensional gel electrophoresis coupled to mass spectrometry. To keep in a realistic scenario we used plants grown in agricultural fields in a region where ~50% of maize was MON810, and analyzed grains at milky-starchy stage. In maize, differential transcripts and metabolites between GM and comparable non-GM varieties tend to be variety specific. Thus, we analyzed two variety pairs, DKC6575/Tietar and PR33P67/PR33P66 which are considered representative of Food and Agriculture Organization 700 and 600 varieties commercially grown in the region. MON810 and non-GM milky-starchy grains had virtually identical proteomic patterns, with a very small number of spots showing fold-variations in the 1-1.8 range. They were all variety specific and had divergent identities and functions. Although 2DE allows the analysis of a limited dataset our results support substantial equivalence between MON810 and comparable non-GM varieties.

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

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 approximately 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.

SummaryBesides the intended effects that give a genetically modified (GM) plant the desired trait, unintended differences between GM and non-GM comparable plants may also occur. Profiling technologies allow their identification, and a number of examples demonstrating that unintended effects are limited and diverse have recently been reported. Both from the food safety aspect and for research purposes, it is important to discern unintended changes produced by the transgene and its expression from those that may be attributed to other factors. Here, we show differential expression of around 0.40% transcriptome between conventional rice var. Senia and Senia-afp constitutively expressing the AFP antifungal protein. Analysis of one-fifth of the regulated sequences showed that around 35% of the unintended effects could be attributed to the process used to produce GM plants, based on in vitro tissue culture techniques. A further 15% were event specific, and their regulation was attributed to host gene disruption and genome rearrangements at the insertion site, and effects on proximal sequences. Thus, only around half the transcriptional unintended effects could be associated to the transgene itself. A significant number of changes in Senia-afp and Senia are part of the plant response to stress conditions, and around half the sequences for which up-regulation was attributed to the transgene were induced in conventional (but not transgenic) plants after wounding. Unintended effects might, as such, putatively result in widening the self-resistance characteristics because of the transgene in GM plants.

Maize is a major food crop and genetically modified (GM) varieties represented 24% of the global production in 2007. Authorized GM organisms have been tested for human and environmental safety. We previously used microarrays to compare the transcriptome profiles of widely used commercial MON810 versus near-isogenic varieties and reported differential expression of a small set of sequences in leaves of in vitro cultured plants of AristisBt/Aristis and PR33P67/PR33P66 (Coll et al. 2008). Here we further assessed the significance of these differential expression patterns in plants grown in a real context, i.e. in the field. Most sequences that were differentially expressed in plants cultured in vitro had the same expression values in MON810 and comparable varieties when grown in the field; and no sequence was found to be differentially regulated in the two variety pairs grown in the field. The differential expression patterns observed between in vitro and field culture were similar between MON810 and comparable varieties, with higher divergence between the two conventional varieties. This further indicates that MON810 and comparable non-GM varieties are equivalent except for the introduced character.

Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.