A role for nonsense-mediated mRNA decay in plants: pathogen responses are induced in Arabidopsis thaliana NMD mutants

Abstract

Nonsense-mediated mRNA decay (NMD) is a conserved mechanism that targets aberrant mRNAs for destruction. NMD has also been found to regulate the expression of large numbers of genes in diverse organisms, although the biological role for this is unclear and few evolutionarily conserved targets have been identified. Expression analyses of three Arabidopsis thaliana lines deficient in NMD reveal that the vast majority of NMD-targeted transcripts are associated with response to pathogens. Congruently, NMD mutants, in which these transcripts are elevated, confer partial resistance to Pseudomonas syringae. These findings suggest a biological rationale for the regulation of gene expression by NMD in plants and suggest that manipulation of NMD could offer a new approach for crop protection. Amongst the few non-pathogen responsive NMD-targeted genes, one potential NMD targeted signal, the evolutionarily conserved upstream open reading frame (CuORF), was found to be hugely over-represented, raising the possibility that this feature could be used to target specific physiological mRNAs for control by NMD.

Figure 1. Gene expression in wild-type and NMD mutant Arabidopsis plants.

(A and B) Genes that are up-regulated and down-regulated respectively in NMD mutant plants when compared to wild-type (p<0.05, fold change >1.5). * The overlap of up or down-regulated genes is greater than would be predicted by chance (p<0.05). (C) Condition tree of all genes that are differentially regulated in at least one of the NMD mutants when compared to wild-type (p<0.05, fold change >1.5). Strongly expressed genes are coloured bright yellow and weakly expressed genes are coloured dark blue.

Figure 2. The overlap of KOGS regulated by NMD between Arabidopsis and other organisms.

(A) A Venn diagram of the 42 Arabidopsis (At) NMD-regulated KOGs that are also regulated by NMD in at least one of; Drosophila (Dm), Saccharomyces cerevisiae (Yeast, Sc) and Human HeLa cells (Homo sapiens, Hs). (B) The KOGs represented in the Venn diagram. KOGs are colour coded according to the organisms in which they are NMD-regulated: Blue: Dm and At, yellow: Sc and At, red: Hs and At, green: Dm, Sc and At, purple: Dm, Hs and At, orange Sc, Hs and At and brown: all four organisms.

Figure 3. SMG7 mRNA is stabilised in NMD mutant plants.

(A) Steady state levels of SMG7 mRNA in wild-type, upf1-5 and upf3-1 mutant plants and plants treated with cycloheximide to disrupt NMD, determined by reverse transcriptase PCR. (B) Degradation of SMG7 mRNA in wild-type and upf3-1 mutant plants, determined using real-time qPCR. The error bars represent the standard error of the mean of three technical replicates. The results of two independent treatments are shown. Green bars represent wild-type plants. Blue bars represent upf3-1 mutant plants.

Figure 4. Biological function of NMD-regulated genes.

Biological function gene ontology (GO) terms that are over-represented (p<0.05) amongst the 206 genes that are commonly up-regulated in NMD mutant plants. The horizontal bar indicates the proportion of the 206 NMD genes with the given GO term relative to the proportion of the genome with the same GO term. DRP is defence response to pathogen, SA is salicylic acid and SAR is systemic acquired resistance. GO terms relating to response to stimuli are coloured blue. GO terms relating to auxin metabolism are coloured peach. The remainder are all associated with amino acid catabolism. * p value≤0.01. ** p value≤0.001.

Figure 5. The expression of the 206 ‘common NMD genes’ in response to biotic stimuli.

Green indicates that genes are down-regulated, red indicates up-regulation and black indicates that there was no change. The box surrounds genes that were not pathogen-responsive.

Figure 6. Reduced growth of Pseudomonas syringae DC3000 in NMD mutants.

Growth of Pseudomonas syringae DC3000 in wild-type and NMD mutant Arabidopsis leaves. Numbers of colony forming units were counted 3 days post infection. Bacterial counts are on a log₁₀ scale. Results from independent replicate experiments are shown.

Figure 7. Levels of salicylic acid and coronatine in NMD mutants.

Salicylic acid (SA) content and coronatine content of wild-type and NMD mutant plants. The salicylic acid and coronatine levels of leaves were measured prior to inoculation (NI) and 10 or 20 hours post-infection with Pseudomonas syringae DC3000 (DC10 and DC20 respectively). Wild-type plants are represented by blue bars, upf1-5 mutants by red bars, upf3-1 by green bars and upf3-2 plants by purple bars.