The salicylic acid dependent and independent effects of NMD in plants

In eukaryotes, nonsense-mediated mRNA decay (NMD) targets aberrant and selected non-aberrant mRNAs for destruction. A recent screen for mRNAs showing increased abundance in Arabidopsis NMD-deficient mutants revealed that most are associated with the salicylic acid (SA)-mediated defense pathway. mRNAs with conserved peptide upstream open reading frames (CpuORFs or CuORFs) are hugely overrepresented among the smaller class of NMD-regulated transcripts not associated with SA. Here we show that the common phenotypes observed in Arabidopsis NMD mutants are SA-dependent, whereas the upregulation of CpuORF-containing transcripts in NMD mutants is independent of SA. We speculate that CpuORFs could allow the conditional targeting of mRNAs for destruction using the NMD pathway

A Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in Arabidopsis thaliana NMD Mutants

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

Adaptive and Specialised Transcriptional Responses to Xenobiotic Stress in <i>Caenorhabditis elegans</i> Are Regulated by Nuclear Hormone Receptors

<div><p>Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive molecules. Consequently, we have investigated the induced responses of the model nematode <i>Caenorhabditis elegans</i> to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals <i>ahr-1, hif-1, mdt-15</i> and <i>nhr-8</i> were not required for any of these inducible responses and <i>skn-1</i> regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on <i>nhr-176</i> we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole.</p></div

GFP expression of reporter lines when NHR-176 was knocked down by RNAi prior to chemical induction.

<p>GFP expression in <i>cyp-35d1::GFP</i> adult stage upon induction by 0.25 mM thiabendazole when fed on <i>E. coli</i> HT115 (DE3) containing <i>pl4440</i> (control; A and C) and when fed on <i>E. coli</i> HT115 (DE3) containing <i>pl4440::nhr-176</i> (B and D). Low magnification images showing GFP expression in <i>cyp-35d1::GFP</i> adult stage under UV illumination (E) upon induction by 0.5% DMSO when fed on <i>E. coli</i> HT115 (DE3) containing <i>pl4440</i> (F) upon induction by 0.25 mM thiabendazole when fed on <i>E. coli</i> HT115 (DE3) containing <i>pl4440</i> (G) upon induction by 0.25 mM thiabendazole when fed on <i>E. coli</i> HT115 (DE3) containing <i>pl4440::nhr-176</i> (F). Scale bar represents 100 µm in A-D.</p

Expression of GFP in a selection of reporter strains driven by promoters for genes encoding CYP, GST and UGTs.

<p>Images with UV and visible illumination of <i>cyp-35b3::GFP</i>, <i>cyp-35d1::GFP</i>, <i>gst-31::GFP</i> and <i>ugt-25::GFP</i> reporter strains in adult stage under exposure to 1.0 mM chloroquine, 0.25 mM thiabendazole, 0.5 mM dazomet and 2.0 mM imidacloprid, respectively. An example of each of the spatial expression patterns observed is shown; intestine (<i>cyp-35b3::GFP</i> and <i>cyp-35d1::GFP</i>) pharynx/hypodermis/vulva (<i>gst-31::GFP</i>) and hypodermis/vulva (<i>ugt-25::GFP</i>). Scale bar represents 100 µm in all images.</p

The response of GFP reporter strains under xenobiotic exposure.

<p>Low magnification images with UV illumination of <i>cyp-35b3::GFP</i>, <i>cyp-35d1::GFP</i>, <i>gst-31::GFP</i> and <i>ugt-25::GFP</i> reporter strains in adult stage under exposure to 0.25 mM beta-napthoflavone, 1.0 mM chloroquine, 0.5 mM dazomet, 0.25 mM juglone, 2.0 mM imidacloprid and 0.25 mM thiabendazole. Scale bar represents 500 µm in all images.</p

GFP expression upon xenobiotic induction in reporter strains driven by promoters for A) <i>gst-31</i> B) <i>ugt-13</i> C) <i>cyp-35b3</i> D) <i>cyp-35d1</i> E) <i>ugt-25</i> following exposure to dsRNA to knockdown transcription factors.

<p>Xenobiotic exposures were A) and B) 0.5 mM dazomet, C) 1.0 mM chloroquine, D) 0.25 mM thiabendazole and E) 2.0 mM imidacloprid. The mean of three biological replicates is plotted with standard error of the mean. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 from one-way Analysis of Variance.</p

GFP expression in reporter strains driven by promoters for genes significantly upregulated (p≤0.01) under xenobiotic exposure in qPCR analyses.

<p>(0 = no expression, 5 = strong expression).</p

Total number of eggs laid by nematodes grown under RNAi and thiabendazole exposure from L1 stage for four days.

<p>The mean of three biological replicates is plotted with standard error of the mean. *p<0.05, **p<0.01 and ***p<0.001 from one-way Analysis of Variance.</p