The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 ( basic leucine zipper 60 ) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro , the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta . Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.
more »
« less
Probing natural variation of IRE1 expression and endoplasmic reticulum stress responses in Arabidopsis accessions
Abstract The environmental effects shape genetic changes in the individuals within plant populations, which in turn contribute to the enhanced genetic diversity of the population as a whole. Thus, individuals within the same species can acquire and accumulate genetic differences in their genomes depending on their local environment and evolutionary history. IRE1 is a universal endoplasmic reticulum (ER) stress sensor that activates an evolutionarily conserved signalling cascade in response to biotic and abiotic stresses. Here, we selected nine different Arabidopsis accessions along with the reference ecotype Columbia-0, based on their geographical origins and differential endogenous IRE1 expression under steady-state conditions to investigate the natural variation of ER stress responses. We cloned and analysed selected upstream regulatory regions of IRE1a and IRE1b , which revealed differential levels of their inducibility. We also subjected these accessions to an array of biotic and abiotic stresses including heat, ER stress-inducing chemical tunicamycin, phytohormone salicylic acid, and pathogen infection. We measured IRE1-mediated splicing of its evolutionarily conserved downstream client as well as transcript accumulation of ER-resident chaperones and co-chaperones. Collectively, our results illustrate the expression polymorphism of a major plant stress receptor and its relationship with molecular and physiological ER stress sensitivity.
more »
« less
- Award ID(s):
- 1350244
- NSF-PAR ID:
- 10201615
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Malik, Harmit S. (Ed.)The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.more » « less
-
more » « less
Abstract Phenotypic plasticity is the primary mechanism of organismal resilience to abiotic and biotic stress, and genetic differentiation in plasticity can evolve if stresses differ among populations. Inducible defence is a common form of adaptive phenotypic plasticity, and long‐standing theory predicts that its evolution is shaped by costs of the defensive traits, costs of plasticity and a trade‐off in allocation to constitutive versus induced traits. We used a common garden to study the evolution of defence in two native populations of wild arugula
Eruca sativa (Brassicaceae) from contrasting desert and Mediterranean habitats that differ in attack by caterpillars and aphids. We report genetic differentiation and additive genetic variance for phenology, growth and three defensive traits (toxic glucosinolates, anti‐nutritive protease inhibitors and physical trichome barriers) as well their inducibility in response to the plant hormone jasmonic acid. The two populations were strongly differentiated for plasticity in nearly all traits. There was little evidence for costs of defence or plasticity, but constitutive and induced traits showed a consistent additive genetic trade‐off within each population for the three defensive traits. We conclude that these populations have evolutionarily diverged in inducible defence and retain ample potential for the future evolution of phenotypic plasticity in defence. -
Abstract Like other eukaryotes, fungi use MAP kinase (MAPK) pathways to mediate cellular changes responding to external stimuli. In the past two decades, three well-conserved MAP kinase pathways have been characterized in various plant pathogenic fungi for regulating responses and adaptations to a variety of biotic and abiotic stresses encountered during plant infection or survival in nature. The invasive growth (IG) pathway is homologous to the yeast pheromone response and filamentation pathways. In plant pathogens, the IG pathway often is essential for pathogenesis by regulating infection-related morphogenesis, such as appressorium formation, penetration, and invasive growth. The cell wall integrity (CWI) pathway also is important for plant infection although the infection processes it regulates vary among fungal pathogens. Besides its universal function in cell wall integrity, it often plays a minor role in responses to oxidative and cell wall stresses. Both the IG and CWI pathways are involved in regulating known virulence factors as well as effector genes during plant infection and mediating defenses against mycoviruses, bacteria, and other fungi. In contrast, the high osmolarity growth (HOG) pathway is dispensable for virulence in some fungi although it is essential for plant infection in others. It regulates osmoregulation in hyphae and is dispensable for appressorium turgor generation. The HOG pathway also plays a major role for responding to oxidative, heat, and other environmental stresses and is overstimulated by phenylpyrrole fungicides. Moreover, these three MAPK pathways crosstalk and coordinately regulate responses to various biotic and abiotic stresses. The IG and CWI pathways, particularly the latter, also are involved in responding to abiotic stresses to various degrees in different fungal pathogens, and the HOG pathway also plays a role in interactions with other microbes or fungi. Furthermore, some infection processes or stress responses are co-regulated by MAPK pathways with cAMP or Ca 2+ /CaM signaling. Overall, functions of individual MAP kinase pathways in pathogenesis and stress responses have been well characterized in a number of fungal pathogens, showing the conserved genetic elements with diverged functions, likely by rewiring transcriptional regulatory networks. In the near future, applications of genomics and proteomics approaches will likely lead to better understanding of crosstalk among the MAPKs and with other signaling pathways as well as roles of MAPKs in defense against other microbes (biotic interactions).more » « less
-
more » « less
Summary Relative to homozygous diploids, the presence of multiple homologs or homeologs in polyploids affords greater tolerance to mutations that can impact genome evolution. In this study, we describe sequence and structural variation in the genomes of six accessions of cultivated potato (
Solanum tuberosum L.), a vegetatively propagated autotetraploid and their impact on the transcriptome. Sequence diversity was high with a mean single nucleotide polymorphisms (SNP ) rate of approximately 1 per 50 bases suggestive of high levels of allelic diversity. Additive gene expression was observed in leaves (3605 genes) and tubers (6156 genes) that contrasted the preferential allele expression of between 2180 and 3502 and 3367 and 5270 genes in the leaf and tuber transcriptome, respectively. Preferential allele expression was significantly associated with evolutionarily conserved genes suggesting selection of specific alleles of genes responsible for biological processes common to angiosperms during the breeding selection process. Copy number variation was rampant with between 16 098 and 18 921 genes in each cultivar exhibiting duplication or deletion. Copy number variable genes tended to be evolutionarily recent, lowly expressed, and enriched in genes that show increased expression in response to biotic and abiotic stress treatments suggestive of a role in adaptation. Gene copy number impacts on gene expression were detected with 528 genes having correlations between copy number and gene expression. Collectively, these data suggest that in addition to allelic variation of coding sequence, the heterogenous nature of the tetraploid potato genome contributes to a highly dynamic transcriptome impacted by allele preferential and copy number‐dependent expression effects.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41598-020-76114-1
