Rice (Oryza sativa) is the staple food for more than half the world's human population1. However, rice harvests can be threatened by diseases such as bacterial blight, caused by Xanthomonas oryzae, and rice blast, caused by the fungus Magnaporthe oryzae, with the latter responsible for the loss of approximately 30% of annual global rice production2. One way to address this problem is to try to bolster the immune system of rice to lessen its susceptibility to pathogens. In two papers on pages 487 and 491, Xu et al. reveal how plants regulate an immunity-enhancing gene3, and how, using this knowledge, the authors successfully engineered a variety of rice4 that might offer better food security than current varieties.
Plants have an innate immune system that can recognize 'non-self' molecules known as microbe-associated molecular patterns (MAMPs). This recognition process results in the activation of immune-regulator proteins and leads to cellular reprogramming that produces antimicrobial responses. Could immune-regulator genes be used to enhance plant innate immunity? It has proved challenging to engineer a plant that has a strengthened immune response without producing other, undesired effects. A key reason for this is that a defence response is costly. Immune genes engineered to be constantly expressed typically limit fitness by negatively affecting plant growth and reproduction5. Therefore, an improved understanding of immune-response regulation might enable greater success in the engineering of immune regulators in crops.
Immune-related genes are highly regulated through mechanisms, including epigenetic changes that affect the DNA–protein complex called chromatin6, and transcriptional regulation of gene expression7,8. Previous reports have hinted at the possibility of selective turnover7 of messenger RNA, or translational control9 of the synthesis of immune-related proteins from mRNAs. Earlier studies of the thale cress plant Arabidopsis thaliana revealed that the transcription-factor protein TBF1, an immune regulator, is under not only transcriptional control, but also translational control mediated by the 5′ region at the beginning of the mRNA sequence10. Detection of a MAMP called elf18 (a peptide from the bacterial protein translation elongation factor Tu) reverses this translational control, leading to increased synthesis of TBF1 and enhanced immunity10.
Xu and colleagues provide insight into how features of the 5′ region of the TBF1 mRNA mediate its translational control. In this region, there are two previously identified10 short upstream open reading frames (uORFs), sequences that encode short peptides and precede the start of the open reading frame (ORF) that encodes TBF1. The authors identified another feature of interest in this 5′ region — a short adenine-rich nucleotide sequence located just before the two uORFs (Fig. 1). Adenine is a nucleotide known as a purine, and because purines are often designated by the letter 'R', the authors named this sequence the R-motif. They found that, in the absence of pathogen challenge, the uORFs strongly inhibit the ability of the ribosome protein-synthesis machinery to translate the TBF1-encoding ORF located downstream in the mRNA. The R-motif adds to this translational control. When plants were treated with elf18, the authors found that this repression of TBF1 synthesis was rapidly and transiently reversed.
In eukaryotic cells (those that contain membrane-bound organelles such as a nucleus), mRNA uORFs are often associated with modulated translation of a downstream ORF in response to changes in certain cell metabolites11, with the ribosome and uORF-encoded peptide acting as an intracellular metabolite sensor. This translational control can be mediated by ribosome stalling on the uORF for reasons related to the abundance of a specific metabolite. It has been proposed that the uORFs of the TBF1 mRNA control TBF1 synthesis in response to a decline in the amino acid phenylalanine10. Both uORFs encode peptides that contain multiple phenylalanines.
Xu et al. used biochemical assays to investigate the role of the R-motif of TBF1 mRNA and found that it binds members of the polyadenylate-binding protein (PAB) family. Mutational analyses revealed that the presence of the R-motif decreases translation of the downstream ORF and that PAB seems to contribute to this dampening effect. Perhaps relaxation of this translational control involves transient phosphorylation of PAB, which enhances its interaction with factors bound to the 5′ region of an mRNA that promote translation initiation11.
Plants modulate translation to fine-tune responses to numerous external stimuli11. Xu et al. used an approach called ribosome footprinting to precisely map the position of individual ribosomes on mRNAs being translated. This revealed that, in response to elf18 treatment, more than 550 transcripts showed rapid and pronounced changes in the number of ribosomes per mRNA. These observed changes in translation did not seem to be associated with changes in mRNA abundance, suggesting that the expression of many mRNAs is under translational control that is affected by the presence of elf18. Many of these mRNAs contained an R-rich region in their 5′ leader sequence. Further investigation will be needed to understand the role of R-motifs and PAB in elf18-mediated translational regulation in plant cells.
The authors tested whether the advances they had made in understanding the regulation of TBF1 could be used to enable rapid and transient activation of a defence response upon pathogen infection in rice. They used a 'TBF1 cassette' containing the promoter sequence that drives TBF1 expression and the 5′ leader region located before the start of the ORF encoding TBF1. This cassette was effective in regulating the A. thaliana master immune-regulator protein NPR1 in engineered rice.
It had previously been observed5 that high and uncontrolled expression of NPR1 conferred resistance to various pathogens, but was accompanied by undesirable fitness costs, limiting the potential of this approach in an agricultural setting. When Xu and colleagues engineered rice to constantly express NPR1, the rice were stunted in the presence or absence of pathogens. However, when the authors controlled NPR1 expression with the TBF1 cassette, this resulted in a burst of NPR1 accumulation upon infection with X. oryzae, enhancing resistance to bacterial blight without incurring a rice-grain production penalty. Additional tests under various field conditions will be required to determine whether this means of increasing plant innate immunity can result in stable rice production under pressure from a range of pathogens.
These studies highlight the value of using natural regulatory element(s) that provide multiple levels of control to ensure that an engineered gene functions precisely how, when and where it should. Stringent gene control is particularly important for master regulators of stress responses, which often confer fitness costs when expressed globally. The integration of translational-control elements that temper responses on the basis of levels of cellular metabolites or applied small molecules could be combined with cell-specific regulation to express stress-resilience genes more effectively, thereby enhancing crop yields to feed the growing world population.
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Science Signaling (2017)