Functional characterisation of the EDS1–MYC2 regulatory node in Arabidopsis

Plants rely on a multi-layered, cell autonomous immune system to combat a plethora of pathogens. While a first, broad defence response is sufficient to restrict growth of most invaders, some pathogens have evolved means to overcome this. In an evolutionary arms race plants have evolved intracellular receptors that recognise host-adapted pathogens and initiate a sustained and potent immune response call ETI (effector-triggered immunity). EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and its signalling partners PAD4 (PHYTOALEXIN DEFICIENT 4) or SAG101 (SENESCENCE-ASSOCIATED GENE 101) form a central convergence point for those intracellular receptors and act as a decision- making node for SA (salicylic acid) dependent and SA independent transcriptional reprogramming. Despite our increasing knowledge about plant immunity the molecular function of the EDS1/PAD4 complex and how these proteins are regulated remains unclear. Recent work established an antagonistic regulation between the JA (jasmonic acid) key TF (transcription factor) MYC2 and EDS1. While MYC2 enhances bacterial virulence by repressing the EDS1 promoter, ETI activated EDS1 represses MYC2 signalling and dampens pathogen growth. This cross-regulation represents an intersection of ETI and JA signalling and allows fine-tuning of the plant’s immune response. How EDS1 controls MYC2 accumulation and activity is not known. Here, I show regulation of MYC2 abundance and MYC2 transactivation activity by EDS1 family proteins. Further, I present evidence for the underlying molecular mechanisms of this regulation and identify new components in this pathway. Specifically, PAD4 and SAG101 but not EDS1, stabilise MYC2 protein while EDS1 but not PAD4 or SAG101, promote MYC2 transactivation activity. Thus, protein accumulation is not indicative of MYC2 transcriptional output. MYC2 activity promotion is lost in a JAZ repressor uncoupled MYC2 variant (MYC2s) or when co-expressed with the bacterial virulence protein avrRPS4, indicating i) regulation of JAZ proteins by EDS1 and ii) immunity context dependent regulation of MYC2. Functional characterisation of this regulation shows that besides JAZ repression MYC2 is phosphorylated in an EDS1 dependent manner. In eds1-2 plants MYC2S123 is phosphorylated, suggesting that EDS1 either represses a protein kinase or activates a protein phosphatase. I show interaction of the protein kinase EDR1 (ENHANCED DISEASE RESISTANCE 1), a negative regulator of plant immunity, with PAD4 and with MYC2 in Arabidopsis (Arabidopsis thaliana) protoplasts. Whether EDS1 regulates MYC2 via EDR1 or another, so far unknown, component remains to be tested.Results presented in this work provide insights into the regulatory relationship of EDS1 and MYC2. EDS1 promotes MYC2 transactivation activity as shown by enhanced MYC2 target gene expression. Molecularly, EDS1 regulates MYC2 via JAZ proteins and likely via MYC2 phosphorylation. More detailed analysis will be necessary to address this entirely. Ultimately, the impact of the presented data depends on the functional relevance of this regulation. For this, in planta experiments in pathogen challenged and unchallenged conditions will be key

An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana

Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way

Temporal Control of Leaf Complexity by miRNA-Regulated Licensing of Protein Complexes

The tremendous diversity of leaf shapes has caught the attention of naturalists for centuries. In addition to interspecific and intraspecific differences, leaf morphologies may differ in single plants according to age, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the progression from the juvenile to the adult phase is characterized by increased leaf serration. A similar trend is seen in species with more complex leaves, such as the A. thaliana relative Cardamine hirsuta, in which the number of leaflets per leaf increases with age. Although the genetic changes that led to the overall simpler leaf architecture in A. thaliana are increasingly well understood, less is known about the events underlying age-dependent changes within single plants, in either A. thaliana or C. hirsuta. Here, we describe a conserved miRNA transcription factor regulon responsible for an age-dependent increase in leaf complexity. In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana and of leaflets in C. hirsuta. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs. These findings point to posttranslational interaction between unrelated miRNA-targeted transcription factors as a core feature of these regulatory circuits.European Molecular Biology Organization (EMBO) fellowship; Fundação para a Ciência e a Tecnologia fellowships; EMBO Installation Grant; National Natural Science Foundation of China grants: (31222029, 912173023); State Key Basic Research Program of China grant: (2013CB127000); Deutsche Forschungsgemeinschaft grants: (SPP1530 and a Gottfried Wilhelm Leibniz Award); Max Planck Society

An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in Arabidopsis thaliana

Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way

An artificial miRNA system reveals that relative contribution of translational inhibition to miRNA-mediated regulation depends on environmental and developmental factors in <i>Arabidopsis thaliana</i>

<div><p>Development and fitness of any organism rely on properly controlled gene expression. This is especially true for plants, as their development is determined by both internal and external cues. MicroRNAs (miRNAs) are embedded in the genetic cascades that integrate and translate those cues into developmental programs. miRNAs negatively regulate their target genes mainly post-transcriptionally through two co-existing mechanisms; mRNA cleavage and translational inhibition. Despite our increasing knowledge about the genetic and biochemical processes involved in those concurrent mechanisms, little is known about their relative contributions to the overall miRNA-mediated regulation. Here we show that co-existence of cleavage and translational inhibition is dependent on growth temperature and developmental stage. We found that efficiency of an artificial miRNA-mediated (amiRNA) gene silencing declines with age during vegetative development in a temperature-dependent manner. That decline is mainly due to a reduction on the contribution from translational inhibition. Both, temperature and developmental stage were also found to affect mature amiRNA accumulation and the expression patterns of the core players involved in miRNA biogenesis and action. Therefore, that suggests that each miRNA family specifically regulates their respective targets, while temperature and growth might influence the performance of miRNA-dependent regulation in a more general way.</p></div

miRNA mode of action is developmentally and temperature-dependent.

<p><i>(A) LUC mRNA expression levels assayed by qRT-PCR normalized to LUC mRNA in rLUC control plants (red dotted line)</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates</i>. <i>(B) LUC protein levels</i>. <i>Black dots represent one biological replicate each calculated from two technical replicates</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates normalized to LUC protein levels in rLUC control plants (red dotted line)</i>. <i>(C) Coexistence index is the ratio of average protein levels by average mRNA levels from each sample and condition</i>. <i>(D) AGO1 expression levels assayed by qRT-PCR</i>. <i>Black dots represent one biological replicate each calculated from two technical replicates</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates</i>. <i>(E) AGO10 expression levels assayed by qRT-PCR</i>. <i>Black dots represent one biological replicate each calculated from two technical replicates</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates</i>. <i>(A-E) “Inflores” stands for inflorescences</i>. * shows tissues in which temperature has a significant effect in a pairwise comparison (p<0.05). Letters and lines show significant differences between tissues in ANOVA-test after Tukey correction (adjusted p<0.05).</p

AmiR-LUC accumulation is developmentally and temperature-dependent.

<p><i>(A) Mature amiR-LUC accumulation assayed by qRT-PCR</i>. <i>Black dots represent one biological replicate each calculated from two technical replicates</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates</i>. <i>“Inflores” stands for inflorescences</i>. <i>(B) Representative sRNA blot for amiR-LUC accumulation</i>. * shows tissues in which temperature has a significant effect in a pairwise comparison (p<0.05). Letters and lines show significant differences between tissues in ANOVA-test after Tukey correction (adjusted p<0.05).</p

Effect of development and temperature on the expression of miRNA biogenesis factors.

<p><i>(A) DCL1</i>. <i>(B) HYL1</i>. <i>(C) DRB2</i>. <i>(D) SE</i>. <i>(E) CPL1</i>. <i>Black dots represent one biological replicate each calculated from two technical replicates</i>. <i>Lines</i>, <i>(blue = 16</i>°<i>C</i>, <i>green = 23</i>°<i>C) represent the average between two biological replicates</i>. <i>“Inflores” stands for inflorescences</i>. * shows tissues in which temperature has a significant effect in a pairwise comparison (p<0.05). Letters and lines show significant differences between tissues in ANOVA-test after Tukey correction (adjusted p<0.05).</p