scholarly journals Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus

2021 ◽  
Vol 7 (4) ◽  
pp. 252
Author(s):  
Rundong Liu ◽  
Wonyong Kim ◽  
Jaycee Augusto Paguirigan ◽  
Min-Hye Jeong ◽  
Jae-Seoun Hur
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Incubation Temperature ◽  
Single Copy ◽  
Insertion Mutant ◽  
Symbiotic Association ◽  
Slow Growth Rate ◽  
Cultivation Period ◽  
C Terminus ◽  
Genetic Mechanisms ◽  
Dna Insertion

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

scholarly journals The chaperonin 60 protein SlCpn60α1 modulates photosynthesis and photorespiration in tomato

2020 ◽  
Vol 71 (22) ◽  
pp. 7224-7240
Author(s):  
Jie Ye ◽  
Weifang Chen ◽  
Longwei Feng ◽  
Genzhong Liu ◽  
Ying Wang ◽  
...  
Keyword(s):  
Calvin Cycle ◽  
Photosynthetic Electron Transport ◽  
Single Copy ◽  
Chlorophyll Synthesis ◽  
Insertion Mutant ◽  
Loss Of Function ◽  
Virus Induced Gene Silencing ◽  
Dna Insertion ◽  
Dimensional Electrophoresis ◽  
Two Hybrid

Abstract Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement.

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

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
John Zupan ◽  
Zisheng Guo ◽  
Trevor Biddle ◽  
Patricia Zambryski
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Agrobacterium Tumefaciens ◽  
Polar Growth ◽  
Content Type ◽  
Growth Pole ◽  
Human Apolipoprotein ◽  
Apolipoprotein A ◽  
C Terminus ◽  
Organizing Center

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.

scholarly journals A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

2021 ◽  
Vol 118 (17) ◽  
pp. e2021293118
Author(s):  
Yonglun Zeng ◽  
Baiying Li ◽  
Changyang Ji ◽  
Lei Feng ◽  
Fangfang Niu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Higher Plants ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Secretory Proteins ◽  
Insertion Mutant ◽  
Intermediate Metabolites ◽  
Dna Insertion ◽  
Autophagy Pathway ◽  
Copii Vesicles

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

scholarly journals Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Chuanling Li ◽  
Jian-Xiu Shang ◽  
Chenlei Qiu ◽  
Baowen Zhang ◽  
Jinxue Wang ◽  
...  
Keyword(s):  
Chloroplast Development ◽  
Developmental Process ◽  
Higher Plants ◽  
Critical Role ◽  
Elongation Factor ◽  
Early Embryo ◽  
The Body ◽  
Insertion Mutant ◽  
Cell Stage ◽  
Dna Insertion

Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.

scholarly journals The C-terminus of CBFβ-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 638-642 ◽  
Author(s):  
Yasuhiko Kamikubo ◽  
R. Katherine Hyde ◽  
Ling Zhao ◽  
Lemlem Alemu ◽  
Cecilia Rivas ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Myeloid Leukemia ◽  
Single Copy ◽  
Full Length ◽  
Myeloproliferative Disease ◽  
Chromosome 16 ◽  
C Terminus ◽  
Acute Myeloid

Abstract The C-terminus of CBFβ-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFβ-SMMHC. To test the role of the fusion protein's C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFβ-SMMHC (CBFβ-SMMHCΔC95). Embryos with a single copy of CBFβ-SMMHCΔC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFβ-SMMHCΔC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFβ-SMMHC. Importantly, unlike mice expressing full-length CBFβ-SMMHC, none of the mice expressing CBFβ-SMMHCΔC95 developed leukemia, even after treatment with a mutagen, although some of the older mice developed a nontransplantable myeloproliferative disease. Our data indicate that the CBFβ-SMMHC's C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis.

Cloning, Heterologous Expression and Kinetic Analysis of Glycerol Kinase (TbGLK1) from Trypanosoma brucei

2000 ◽  
Vol 381 (11) ◽  
pp. 1071-1077 ◽  
Author(s):  
K. Steinborn ◽  
A. Szallies ◽  
D. Mecke ◽  
M. Duszenko
Keyword(s):  
Heterologous Expression ◽  
Kinetic Analysis ◽  
Trypanosoma Brucei ◽  
Blot Analysis ◽  
Spatial Separation ◽  
Single Copy ◽  
Glycerol Kinase ◽  
Mrna Levels ◽  
Atp Production ◽  
C Terminus

Abstract We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

scholarly journals UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus

2008 ◽  
Vol 191 (5) ◽  
pp. 1429-1438 ◽  
Author(s):  
Richard C. Centore ◽  
Michael C. Leeson ◽  
Steven J. Sandler
Keyword(s):  
Excision Repair ◽  
Single Copy ◽  
Multicopy Plasmid ◽  
Uv Treatment ◽  
E Coli ◽  
Uv Sensitivity ◽  
C Terminus ◽  
Lexa Repressor ◽  
Nucleotide Excision ◽  
Terminal Amino

ABSTRACT Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UVs), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UVs phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell.

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