Annemart Koornneef

NPR1 Modulates Cross-Talk between Salicylate- and Jasmonate-Dependent Defense Pathways through a Novel Function in the Cytosol

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Cross Talk in Defense Signaling

Plants are equipped with an array of defense mechanisms to protect themselves against attack by herbivorous insects and microbial pathogens. Some of these defense mechanisms are preexisting, whereas others are only activated upon insect or pathogen invasion. Induced defense responses entail fitness

Kinetics of Salicylate-Mediated Suppression of Jasmonate Signaling Reveal a Role for Redox Modulation

Cross talk between salicylic acid (SA) and jasmonic acid (JA) signaling pathways plays an important role in the regulation and fine tuning of induced defenses that are activated upon pathogen or insect attack. Pharmacological experiments revealed that transcription of JA-responsive marker genes, such as PDF1.2 and VSP2, is highly sensitive to suppression by SA. This antagonistic effect of SA on JA signaling was also observed when the JA pathway was biologically activated by necrotrophic pathogens or insect herbivores, and when the SA pathway was triggered by a biotrophic pathogen. Furthermore, all 18 Arabidopsis (Arabidopsis thaliana) accessions tested displayed SA-mediated suppression of JA-responsive gene expression, highlighting the potential significance of this phenomenon in induced plant defenses in nature. During plant-attacker interactions, the kinetics of SA and JA signaling are highly dynamic. Mimicking this dynamic response by applying SA and methyl jasmonate (MeJA) at different concentrations and time intervals revealed that PDF1.2 transcription is readily suppressed when the SA response was activated at or after the onset of the JA response, and that this SA-JA antagonism is long lasting. However, when SA was applied more than 30 h prior to the onset of the JA response, the suppressive effect of SA was completely absent. The window of opportunity of SA to suppress MeJA-induced PDF1.2 transcription coincided with a transient increase in glutathione levels. The glutathione biosynthesis inhibitor l-buthionine-sulfoximine strongly reduced PDF1.2 suppression by SA, suggesting that SA-mediated redox modulation plays an important role in the SA-mediated attenuation of the JA signaling pathway.

Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59

Interactions between the plant hormones salicylic acid and jasmonic acid play an important role in the regulation of plant defense responses against pathogens and insects. This work provides mechanistic insight into this hormonal crosstalk by showing that salicylic acid antagonizes jasmonic acid–dependent defenses by targeting the transcriptional activator ORA59. Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59.

Herbivore-Induced Resistance against Microbial Pathogens in Arabidopsis

Caterpillars of the herbivore Pieris rapae stimulate the production of jasmonic acid (JA) and ethylene (ET) in Arabidopsis (Arabidopsis thaliana) and trigger a defense response that affects insect performance on systemic tissues. To investigate the spectrum of effectiveness of P. rapae-induced resistance, we examined the level of resistance against different pathogens. Although the necrotrophic fungus Alternaria brassicicola is sensitive to JA-dependent defenses, herbivore-induced resistance was not effective against this pathogen. By contrast, caterpillar feeding significantly reduced disease caused by the bacterial pathogens Pseudomonas syringae pv tomato and Xanthomonas campestris pv armoraciae. However, this effect was apparent only locally in caterpillar-damaged tissue. Arabidopsis mutants jar1, coi1, ein2, sid2, eds5, and npr1 showed wild-type levels of P. rapae-induced protection against P. syringae pv tomato, suggesting that this local, herbivore-induced defense response does not depend exclusively on either JA, ET, or salicylic acid (SA). Resistance against the biotroph Turnip crinkle virus (TCV) requires SA, but not JA and ET. Nevertheless, herbivore feeding strongly affected TCV multiplication and TCV lesion formation, also in systemic tissues. Wounding alone was not effective, but application of P. rapae regurgitate onto the wounds induced a similar level of protection. Analysis of SA-induced PATHOGENESIS RELATED-1 (PR-1) expression revealed that P. rapae grazing primed Arabidopsis leaves for augmented expression of SA-dependent defenses. Pharmacological experiments showed that ET acts synergistically on SA-induced PR-1, suggesting that the increased production of ET upon herbivore feeding sensitizes the tissue to respond faster to SA, thereby contributing to an enhanced defensive capacity toward pathogens, such as TCV, that trigger SA-dependent defenses upon infection.

Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic acid

Cross-talk between jasmonate (JA), ethylene (ET), and Salicylic acid (SA) signaling is thought to operate as a mechanism to fine-tune induced defenses that are activated in response to multiple attackers. Here, 43 Arabidopsis genotypes impaired in hormone signaling or defense-related processes were screened for their ability to express SA-mediated suppression of JA-responsive gene expression. Mutant cev1, which displays constitutive expression of JA and ET responses, appeared to be insensitive to SA-mediated suppression of the JA-responsive marker genes PDF1.2 and VSP2. Accordingly, strong activation of JA and ET responses by the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola prior to SA treatment counteracted the ability of SA to suppress the JA response. Pharmacological assays, mutant analysis, and studies with the ET-signaling inhibitor 1-methylcyclopropene revealed that ET signaling renders the JA response insensitive to subsequent suppression by SA. The APETALA2/ETHYLENE RESPONSE FACTOR transcription factor ORA59, which regulates JA/ET-responsive genes such as PDF1.2, emerged as a potential mediator in this process. Collectively, our results point to a model in which simultaneous induction of the JA and ET pathway renders the plant insensitive to future SA-mediated suppression of JA-dependent defenses, which may prioritize the JA/ET pathway over the SA pathway during multi-attacker interactions.

Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis

The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.

Apolipoprotein B Knockdown by AAV-delivered shRNA Lowers Plasma Cholesterol in Mice

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.

Histone modifications do not play a major role in salicylate-mediated suppression of jasmonate-induced PDF1.2 gene expression

Cross-talk between salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. We investigated the putative role of histone modifications in the regulation of SA-mediated suppression of PDF1.2 transcription. Chromatin immunoprecipitation analysis using an antibody directed against acetylated histone H3 revealed that SA does not affect the association of this histone modification at the PDF1.2 promoter, suggesting that chromatin remodeling does not play a major role in SA/JA cross-talk.

Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.