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1. A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

   https://doi.org/10.3389/fpls.2021.707378  

   Diwan, Danish ; Liu, Xiaoyu ; Andrews, Caroline F. ; Pajerowska-Mukhtar, Karolina M. ( July 2021 , Frontiers in Plant Science)

   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 activitymore »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.« less
2. Regulation of biotic interactions and responses to abiotic stresses by MAP kinase pathways in plant pathogenic fungi

   https://doi.org/10.1007/s44154-021-00004-3  

   Zhang, Xue ; Wang, Zeyi ; Jiang, Cong ; Xu, Jin-Rong ( December 2021 , Stress Biology)

   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 ismore »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).« less
3. Small spaces, big impacts: contributions of micro-environmental variation to population persistence under climate change

   https://doi.org/10.1093/aobpla/plaa005  

   Denney, Derek A ; Jameel, M Inam ; Bemmels, Jordan B ; Rochford, Mia E ; Anderson, Jill T ( April 2020 , AoB PLANTS)

   Abdelaziz, Mohamed (Ed.)

   Abstract Individuals within natural populations can experience very different abiotic and biotic conditions across small spatial scales owing to microtopography and other micro-environmental gradients. Ecological and evolutionary studies often ignore the effects of micro-environment on plant population and community dynamics. Here, we explore the extent to which fine-grained variation in abiotic and biotic conditions contributes to within-population variation in trait expression and genetic diversity in natural plant populations. Furthermore, we consider whether benign microhabitats could buffer local populations of some plant species from abiotic stresses imposed by rapid anthropogenic climate change. If microrefugia sustain local populations and communities in the short term, other eco-evolutionary processes, such as gene flow and adaptation, could enhance population stability in the longer term. We caution, however, that local populations may still decline in size as they contract into rare microhabitats and microrefugia. We encourage future research that explicitly examines the role of the micro-environment in maintaining genetic variation within local populations, favouring the evolution of phenotypic plasticity at local scales and enhancing population persistence under global change.
4. The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

   https://doi.org/10.1371/journal.pbio.3001569  

   Yoon, Jimin ; Nekongo, Emmanuel E. ; Patrick, Jessica E. ; Hui, Tiffani ; Phillips, Angela M. ; Ponomarenko, Anna I. ; Hendel, Samuel J. ; Sebastian, Rebecca M. ; Zhang, Yu Meng ; Butty, Vincent L. ; et al ( February 2022 , PLOS Biology)

   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 largestmore »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.« less
5. Diversifying evolution of the ubiquitin-26s proteasome system in Brassicaceae and Poaceae

   https://doi.org/10.3390/ijms20133226  

   Hua, Zhihua ; Yu, Peifeng ( June 2019 , International journal of molecular sciences)

   Genome amplification and sequence divergence provides raw materials to allow organismal adaptation. This is exemplified by the large expansion of the ubiquitin-26S proteasome system (UPS) in land plants, which primarily rely on intracellular signaling and biochemical metabolism to combat biotic and abiotic stresses. While a handful of functional genomic studies have demonstrated the adaptive role of the UPS in plant growth and development, many UPS members remain unknown. In this work, we applied a comparative genomic study to address the functional divergence of the UPS at a systematic level. We first used a closing-target-trimming annotation approach to identify most, if not all, UPS members in six species from each of two evolutionarily distant plant families, Brassicaceae and Poaceae. To reduce age-related errors, the two groups of species were selected based on their similar chronological order of speciation. Through size comparison, chronological expansion inference, evolutionary selection analyses, duplication mechanism prediction, and functional domain enrichment assays, we discovered significant diversities within the UPS, particularly between members from its three largest ubiquitin ligase gene families, the F-box (FBX), the Really Interesting New Gene (RING), and the Bric-a-Brac/Tramtrack/Broad Complex (BTB) families, between Brassicaceae and Poaceae. Uncovering independent Arabidopsis and Oryza genus–specific subclades of themore »26S proteasome subunits from a comprehensive phylogenetic analysis further supported a diversifying evolutionary model of the UPS in these two genera, confirming its role in plant adaptation.« less