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1. A temperature-sensitive FERONIA mutant allele that alters root hair growth

   https://doi.org/10.1093/plphys/kiaa051  

   Kim, Daewon; Yang, Jiyuan; Gu, Fangwei; Park, Sungjin; Combs, Jonathon; Adams, Alexander; Mayes, Heather B; Jeon, Su Jeong; Bahk, Jeong Dong; Nielsen, Erik (December 2020, Plant Physiology)

   null (Ed.)

   Abstract In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides. 

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2. VPS45 is required for both diffuse and tip growth of Arabidopsis thaliana cells

   https://doi.org/10.3389/fpls.2023.1120307  

   Mugume, Yosia; Roy, Rahul; Agbemafle, William; Shepard, Gabriella N.; Vue, Yee; Bassham, Diane C. (February 2023, Frontiers in Plant Science)

   IntroductionVPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously thatArabidopsis thalianaVPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on thetrans-Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth. ResultsAsvps45knockout mutation is lethal in Arabidopsis, we identified a mutant,vps45-3, with a point mutation in theVPS45gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However,vps45-3plants display severe growth defects with significantly reduced organ and cell size, similar tovps45RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised invps45-3. Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter invps45-3compared to WT. DiscussionWe thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth. 

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3. Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization

   https://doi.org/10.1128/mBio.00764-21  

   Zupan, John; Guo, Zisheng; Biddle, Trevor; Zambryski, Patricia (June 2021, mBio)

   Sloan Siegrist, M. (Ed.)

   ABSTRACT The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium -specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. IMPORTANCE Bacterial growth and division are extensively studied in model systems ( Escherichia coli , Bacillus subtilis , and Caulobacter crescentus ) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode—polar growth—is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae , in which many species, including Agrobacterium tumefaciens , exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth. 

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4. Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of β‐glucosidase function in Cellvibrio japonicus

   https://doi.org/10.1002/bit.27544  

   Hwang, Jeanice; Hari, Archana; Cheng, Raymond; Gardner, Jeffrey_G; Lobo, Daniel (September 2020, Biotechnology and Bioengineering)

   Abstract Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene‐deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacteriumCellvibrio japonicusgrown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics ofC. japonicususing either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild‐type strain as well as in β‐glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non‐diauxic growth and metabolite consumptions of the wild‐type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially‐useful strains. Importantly, the model is able to explain the role of the different β‐glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates. 

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5. Identification of structural and regulatory cell-shape determinants in Haloferax volcanii

   https://doi.org/10.1038/s41467-024-45196-0  

   Schiller, Heather; Hong, Yirui; Kouassi, Joshua; Rados, Theopi; Kwak, Jasmin; DiLucido, Anthony; Safer, Daniel; Marchfelder, Anita; Pfeiffer, Friedhelm; Bisson, Alexandre; et al (February 2024, Nature Communications)

   Abstract Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood.Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin’s cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains. 

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