One of the advantages of working with bacteria is that genes can be clustered together under control of a single promoter (known as an operon). Multiple protein products can therefore be generated from a single messenger RNA strand (the mRNA is said to be polycistronic (Fig. 1)). As a result entire metabolic pathways can be created using a single promoter.
In eukaryotic systems this is generally not the case. mRNA in eukaryotes is typically monocistronic (there are a few exceptions but these undergo post-transcriptional processing and are not viewed as truly polycistronic e.g. lower box Fig. 1). This limitation means more DNA cloning/synthesis, repeated rounds of transformation and increased difficulty in controlling the expression levels of different genes – which can be important for metabolic engineering, for example.
A new report in Plant Physiology from Ilan Sela’s lab. therefore caught my attention: “Expression of an entire bacterial operon in plants”. The authors used a disarmed virus system that they had previously described in 2007 for gene silencing. Both papers are open access.
The virus (a disarmed and modified Tomato yellow leaf curl virus) hosts the researchers gene sequences rather than those which cause pathogenicity in plants. The modified virus is able to spread through the plant, without genome integration, and uses the hosts molecular machinery to produce either antisense RNA for silencing or reporter gene constructs.
This technology also has the built-in benefits that it is not reliant on genetic modification of the plant, it can be applied to ‘hard-to-transform species’ such as wheat, pepper, grapevine, citrus, and olive and is unable to be transmitted through whitefly vectors.
In the current paper they expand the number of genes expressed using this system through the introduction of a bacterial operon. The operon chosen was the PRN operon from Pseudomonas fluorescens (for those that are interested, bases 4,157,074 to 4,162,815). PRN (pyrronitrin) is an anti-fungal, anti-bacterial compound that inhibits the growth of some plant pathogens. The operon contains four genes coding for the enzymes responsible for PRN biosynthesis.
The researchers were able to capitalise on the fact that the organelles of eukaryotes (mitochondria and chloroplasts) are, evolutionarily speaking, symbiotic bacteria. Though they have been with us a long time and much of their genome has been transferred to the host nucleus, the inner working of such organelles are still very much bacterial: in other words, they know how to process polycistronic mRNA.
The result is that the organelles were able to process the polycistronic mRNA (carried by the viral vector and expressed using the host’s transcriptional machinery) to produce all four proteins required for PRN biosynthesis. Within two days of infection with the modified Tomato yellow leaf curl virus PRN production was evident. The researchers also found that plants that produced PRN were far less susceptible to fungal infection.
Whilst I am not clear on the mechanism by which the polycistronic mRNA is targeted/imported into the organelles (and indeed which organelles are involved – though it looks like the chloroplasts certainly are), the outcome is pretty clear. Given the speed of generating and assembling synthetic DNA sequences, this holds the potential to be a rapid means of screening different combinations of plant metabolic engineering strategies, without the need for full plant transformation procedures.