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1. Distribution and the evolutionary history of G-protein components in plant and algal lineages

   https://doi.org/10.1093/plphys/kiac153  

   Mohanasundaram, Boominathan ; Dodds, Audrey ; Kukshal, Vandna ; Jez, Joseph M. ; Pandey, Sona ( April 2022 , Plant Physiology)

   Heterotrimeric G-protein complexes comprising Gα-, Gβ-, and Gγ-subunits and the regulator of G-protein signaling (RGS) are conserved across most eukaryotic lineages. Signaling pathways mediated by these proteins influence overall growth, development, and physiology. In plants, this protein complex has been characterized primarily from angiosperms with the exception of spreading-leaved earth moss (Physcomitrium patens) and Chara braunii (charophytic algae). Even within angiosperms, specific G-protein components are missing in certain species, whereas unique plant-specific variants—the extra-large Gα (XLGα) and the cysteine-rich Gγ proteins—also exist. The distribution and evolutionary history of G-proteins and their function in nonangiosperm lineages remain mostly unknown. We explored this using the wealth of available sequence data spanning algae to angiosperms representing extant species that diverged approximately 1,500 million years ago, using BLAST, synteny analysis, and custom-built Hidden Markov Model profile searches. We show that a minimal set of components forming the XLGαβγ trimer exists in the entire land plant lineage, but their presence is sporadic in algae. Additionally, individual components have distinct evolutionary histories. The XLGα exhibits many lineage-specific gene duplications, whereas Gα and RGS show several instances of gene loss. Similarly, Gβ remained constant in both number and structure, but Gγ diverged before the emergence ofmore »land plants and underwent changes in protein domains, which led to three distinct subtypes. These results highlight the evolutionary oddities and summarize the phyletic patterns of this conserved signaling pathway in plants. They also provide a framework to formulate pertinent questions on plant G-protein signaling within an evolutionary context.

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2. Deciphering the protein ubiquitylation system in plants

   https://doi.org/10.1093/jxb/erad354  

   Hua, Zhihua ; Murray, ed., James ( September 2023 , Journal of Experimental Botany)

   Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivalling several other major systems, such as transcription regulation and epigenetics, I describe the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarize the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I propose a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I present new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of ubiquitin E3 ligase genes.
3. Molecular Mechanisms of DNA Replication and Repair Machinery: Insights from Microscopic Simulations

   https://doi.org/10.1002/adts.201800191  

   Maffeo, Christopher ; Chou, Han‐Yi ; Aksimentiev, Aleksei ( February 2019 , Advanced Theory and Simulations)

   Reproduction, the hallmark of biological activity, requires making an accurate copy of the genetic material to allow the progeny to inherit parental traits. In all living cells, the process of DNA replication is carried out by a concerted action of multiple protein species forming a loose protein–nucleic acid complex, the replisome. Proofreading and error correction generally accompany replication but also occur independently, safeguarding genetic information through all phases of the cell cycle. Advances in biochemical characterization of intracellular processes, proteomics, and the advent of single‐molecule biophysics have brought about a treasure trove of information awaiting to be assembled into an accurate mechanistic model of the DNA replication process. This review describes recent efforts to model elements of DNA replication and repair processes using computer simulations, an approach that has gained immense popularity in many areas of molecular biophysics but has yet to become mainstream in the DNA metabolism community. It highlights the use of diverse computational methods to address specific problems of the fields and discusses unexplored possibilities that lie ahead for the computational approaches in these areas.
4. Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

   https://doi.org/10.1002/cpmc.130  

   Banta, Amy B. ; Ward, Ryan D. ; Tran, Jennifer S. ; Bacon, Emily E. ; Peters, Jason M. ( December 2020 , Current Protocols in Microbiology)

   Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors.

   Basic Protocol 1: sgRNA design

   Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors

   Basic Protocol 3: Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria

   Basic Protocol 4: ICEBs1transfer of Mobile‐CRISPRi to Bacillales

   Support Protocol 1: Quantification ofmore »CRISPRi repression using fluorescent reporters

   Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates

   Support Protocol 3: Transformation ofE. coliby electroporation

   Support Protocol 4: Transformation of CaCl₂‐competentE. coli

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5. A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in A rabidopsis

   https://doi.org/10.1111/tpj.14164  

   Zaman, Najia ; Seitz, Kati ; Kabir, Mohiuddin ; George‐Schreder, Lauren St. ; Shepstone, Ian ; Liu, Yidong ; Zhang, Shuqun ; Krysan, Patrick J. ( January 2019 , The Plant Journal)

   The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. SinceMAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitorMAPKactivity in living plant cells would be valuable. Here, we report the development of a genetically encodedMAPKactivity sensor for use inArabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, aMAPKsubstrate domain and a flexible linker. Usingin vitrotesting, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. TheFRETefficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor ofMAPKactivity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains inFRETefficiency. Control lines expressing a version ofSOMAin which the phosphosite was mutated to an alanine did not show any substantial changes inFRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the ArabidopsisMAPKgenesMPK3andMPK6. These experiments demonstrated thatMPK3/6 are necessary for the NaCl‐inducedFRETgain ofmore »the sensor, while otherMAPKs are probably contributing to the chitin and flg22‐induced increases inFRET. Taken together, our results suggest thatSOMAis able to dynamically reportMAPKactivity in living plant cells.

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