Antonio Leon-Reyes

Networking by small-molecule hormones in plant immunity

Plants live in complex environments in which they intimately interact with a broad range of microbial pathogens with different lifestyles and infection strategies. The evolutionary arms race between plants and their attackers provided plants with a highly sophisticated defense system that, like the animal innate immune system, recognizes pathogen molecules and responds by activating specific defenses that are directed against the invader. Recent advances in plant immunity research have provided exciting new insights into the underlying defense signaling network. Diverse small-molecule hormones play pivotal roles in the regulation of this network. Their signaling pathways cross-communicate in an antagonistic or synergistic manner, providing the plant with a powerful capacity to finely regulate its immune response. Pathogens, on the other hand, can manipulate the plants defense signaling network for their own benefit by affecting phytohormone homeostasis to antagonize the host immune response.

Hormonal Modulation of Plant Immunity

Plant hormones have pivotal roles in the regulation of plant growth, development, and reproduction. Additionally, they emerged as cellular signal molecules with key functions in the regulation of immune responses to microbial pathogens, insect herbivores, and beneficial microbes. Their signaling pathways are interconnected in a complex network, which provides plants with an enormous regulatory potential to rapidly adapt to their biotic environment and to utilize their limited resources for growth and survival in a cost-efficient manner. Plants activate their immune system to counteract attack by pathogens or herbivorous insects. Intriguingly, successful plant enemies evolved ingenious mechanisms to rewire the plants hormone signaling circuitry to suppress or evade host immunity. Evidence is emerging that beneficial root-inhabiting microbes also hijack the hormone-regulated immune signaling network to establish a prolonged mutualistic association, highlighting the central role of plant hormones in the regulation of plant growth and survival.

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.

Ethylene Modulates the Role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in Cross Talk between Salicylate and Jasmonate Signaling

The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play crucial roles in the signaling network that regulates induced defense responses against biotic stresses. Antagonism between SA and JA operates as a mechanism to fine-tune defenses that are activated in response to multiple attackers. In Arabidopsis (Arabidopsis thaliana), NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) was demonstrated to be required for SA-mediated suppression of JA-dependent defenses. Because ET is known to enhance SA/NPR1-dependent defense responses, we investigated the role of ET in the SA-JA signal interaction. Pharmacological experiments with gaseous ET and the ET precursor 1-aminocyclopropane-1-carboxylic acid showed that ET potentiated SA/NPR1-dependent PATHOGENESIS-RELATED1 transcription, while it rendered the antagonistic effect of SA on methyl jasmonate-induced PDF1.2 and VSP2 expression NPR1 independent. This overriding effect of ET on NPR1 function in SA-JA cross talk was absent in the npr1-1/ein2-1 double mutant, demonstrating that it is mediated via ET signaling. Abiotic and biotic induction of the ET response similarly abolished the NPR1 dependency of the SA-JA signal interaction. Furthermore, JA-dependent resistance against biotic attackers was antagonized by SA in an NPR1-dependent fashion only when the plant-attacker combination did not result in the production of high levels of endogenous ET. Hence, the interaction between ET and NPR1 plays an important modulating role in the fine tuning of the defense signaling network that is activated upon pathogen and insect attack. Our results suggest a model in which ET modulates the NPR1 dependency of SA-JA antagonism, possibly to compensate for enhanced allocation of NPR1 to function in SA-dependent activation of PR genes.

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.

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.

Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and Arabidopsis, respectively, was investigated. In vitro analysis showed the ability of TaWRKY78 to bind a –17/+80 region of the wPR4e promoter, containing one cis-acting W-box. Moreover, transient expression analysis performed on both TaWRKY78 and AtWRKY20 showed their ability to recognize the cognate cis-acting elements present in the wPR4e and AtHEL promoters, respectively. Finally, this paper provides evidence that both transcription factors are able to cross-regulate the orthologous PR4 genes with an efficiency slightly lower than that exerted on the cognate promoters. The observation that orthologous genes are subjected to similar transcriptional control by orthologous transcription factors demonstrates that the terminal stages of signal transduction pathways leading to defence are conserved and suggests a fundamental role of PR4 genes in plant defence. Moreover, these results corroborate the hypothesis that gene orthology imply similar gene function and that diversification between monocot and dicot has most likely occurred after the specialization of WRKY function.

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.

Kinome Profiling Reveals an Interaction Between Jasmonate, Salicylate and Light Control of Hyponastic Petiole Growth in Arabidopsis thaliana

Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ∼omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing.