OsCAF2 contains two CRM domains and is necessary for chloroplast development in rice

Abstract Background Chloroplasts play an important role in plant growth and development. The chloroplast genome contains approximately twenty group II introns that are spliced due to proteins encoded by nuclear genes. CAF2 is one of these splicing factors that has been shown to splice group IIB introns in maize and Arabidopsis thaliana. However, the research of the OsCAF2 gene in rice is very little, and the effects of OsCAF2 genes on chloroplasts development are not well characterized. Results In this study, oscaf2 mutants were obtained by editing the OsCAF2 gene in the Nipponbare variety of rice. Phenotypic analysis showed that mutations to OsCAF2 led to albino leaves at the seeding stage that eventually caused plant death, and oscaf2 mutant plants had fewer chloroplasts and damaged chloroplast structure. We speculated that OsCAF2 might participate in the splicing of group IIA and IIB introns, which differs from its orthologs in A. thaliana and maize. Through yeast two-hybrid experiments, we found that the C-terminal region of OsCAF2 interacted with OsCRS2 and formed an OsCAF2-OsCRS2 complex. In addition, the N-terminal region of OsCAF2 interacted with itself to form homodimers. Conclusion Taken together, this study improved our understanding of the OsCAF2 protein, and revealed additional information about the molecular mechanism of OsCAF2 in regulating of chloroplast development in rice.

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A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.

Blood ◽

10.1182/blood.v116.21.1552.1552 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1552-1552

Author(s):

Hirohiko Shibayama ◽

Yuri Saito ◽

Akira Tanimura ◽

Hirokazu Tanaka ◽

Itaru Matsumura ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Cyclin D1 ◽

P38 Mapk ◽

Conflicts Of Interest ◽

Biological Effects ◽

Terminal Region ◽

Yeast Two Hybrid ◽

A Cell ◽

Two Hybrid

Abstract Abstract 1552 [Background] Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated. [Methods & Results] In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells. [Conclusion] P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions. Disclosures: No relevant conflicts of interest to declare.

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IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites

Journal of General Virology ◽

10.1099/vir.0.81270-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3031-3038 ◽

Cited By ~ 13

Author(s):

WonKyung Kang ◽

Noriko Imai ◽

Yu Kawasaki ◽

Toshihiro Nagamine ◽

Shogo Matsumoto

Keyword(s):

Bombyx Mori ◽

Fluorescent Protein ◽

Coiled Coil ◽

Terminal Region ◽

Mutant Protein ◽

Yeast Two Hybrid ◽

Focus Formation ◽

Coiled Coil Domain ◽

Nuclear Foci ◽

Two Hybrid

The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1–110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1–78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

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Plastid caseinolytic protease OsClpR1 regulates chloroplast development and chloroplast RNA editing in rice

Rice ◽

10.1186/s12284-021-00489-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xi Liu ◽

Ziyi Xu ◽

Yanrong Yang ◽

Penghui Cao ◽

Hang Cheng ◽

...

Keyword(s):

Rna Editing ◽

Chloroplast Development ◽

Chlorophyll Biosynthesis ◽

Wild Type ◽

Yeast Two Hybrid ◽

Lethal Mutant ◽

Caseinolytic Protease ◽

Young Leaves ◽

Chloroplast Rna ◽

Two Hybrid

Abstract Background Plant plastidic caseinolytic protease (Clp) is a central part of the plastid protease network and consists of multiple subunits. The molecular functions of many Clps in plants, especially in crops, are not well known. Results In this study, we identified an albino lethal mutant al3 in rice, which produces albino leaves and dies at the seedling stage. Molecular cloning revealed that AL3 encodes a plastid caseinolytic protease, OsClpR1, homologous to Arabidopsis ClpR1 and is targeted to the chloroplast. Compared with the wild type, chloroplast structure in the al3 mutant was poorly developed. OsClpR1 was constitutively expressed in all rice tissues, especially in young leaves. The OsClpR1 mutation affected the transcript levels of chlorophyll biosynthesis and chloroplast development-related genes. The RNA editing efficiency of three chloroplast genes (rpl2, ndhB, ndhA) was remarkably reduced in al3. Using a yeast two-hybrid screen, we found that OsClpR1 interacted with OsClpP4, OsClpP5, OsClpP2, and OsClpS1. Conclusions Collectively, our results provide novel insights into the function of Clps in rice.

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Functional analysis of the cotton CLE polypeptide signaling gene family in plant growth and development

Scientific Reports ◽

10.1038/s41598-021-84312-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ke Wan ◽

Kening Lu ◽

Mengtao Gao ◽

Ting Zhao ◽

Yuxin He ◽

...

Keyword(s):

Gene Family ◽

Growth And Development ◽

Peptide Hormone ◽

Luciferase Assay ◽

Yeast Two Hybrid ◽

Short Peptide ◽

C Terminus ◽

Development Regulation ◽

Subcellular Localization Pattern ◽

Two Hybrid

AbstractThe CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)–RELATED (CLE) gene family encodes a large number of polypeptide signaling molecules involved in the regulation of shoot apical meristem division and root and vascular bundle development in a variety of plants. CLE family genes encode important short peptide hormones; however, the functions of these signaling polypeptides in cotton remain largely unknown. In the current work, we studied the effects of the CLE family genes on growth and development in cotton. Based on the presence of a conserved CLE motif of 13 amino acids, 93 genes were characterized as GhCLE gene family members, and these were subcategorized into 7 groups. A preliminary analysis of the cotton CLE gene family indicated that the activity of its members tends to be conserved in terms of both the 13-residue conserved domain at the C-terminus and their subcellular localization pattern. Among the 14 tested genes, the ectopic overexpression of GhCLE5::GFP partially mimicked the phenotype of the clv3 mutant in Arabidopsis. GhCLE5 could affect the endogenous CLV3 in binding to the receptor complex, comprised of CLV1, CLV2, and CRN, in the yeast two-hybrid assay and split-luciferase assay. Silencing GhCLE5 in cotton caused a short seedling phenotype. Therefore, we concluded that the cotton GhCLE gene family is functionally conserved in apical shoot development regulation. These results indicate that CLE also plays roles in cotton development as a short peptide hormone.

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An Exon Skipping in CRS1 Is Associated with Perturbed Chloroplast Development in Maize

International Journal of Molecular Sciences ◽

10.3390/ijms221910668 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10668

Author(s):

Mao Wang ◽

Kaiwen Li ◽

Yang Li ◽

Lingyu Mi ◽

Zhubing Hu ◽

...

Keyword(s):

Growth And Development ◽

Chloroplast Development ◽

Higher Plants ◽

Exon Skipping ◽

Mutant Gene ◽

Single Mutation ◽

Plant Growth And Development ◽

Transmission Electron ◽

Phenotype Analysis ◽

Morphological Defects

Chloroplasts of higher plants are semi-autonomous organelles that perform photosynthesis and produce hormones and metabolites. They play crucial roles in plant growth and development. Although many seedling-lethal nuclear genes or regulators required for chloroplast development have been characterized, the understanding of chloroplast development is still limited. Using a genetic screen, we isolated a mutant named ell1, with etiolated leaves and a seedling-lethal phenotype. Analysis by BN-PAGE and transmission electron microscopy revealed drastic morphological defects of chloroplasts in ell1 mutants. Genetic mapping of the mutant gene revealed a single mutation (G-to-A) at the 5′ splice site of intron 5 in CRS1, resulting in an exon skipping in CRS1, indicating that this mutation in CRS1 is responsible for the observed phenotype, which was further confirmed by genetic analysis. The incorrectly spliced CRS1 failed to mediate the splicing of atpF intron. Moreover, the quantitative analysis suggested that ZmCRS1 may participate in chloroplast transcription to regulate the development of chloroplast. Taken together, these findings improve our understanding of the ZmCRS1 protein and shed new light on the regulation of chloroplast development in maize.

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YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Cellular & Molecular Biology Letters ◽

10.2478/s11658-011-0017-9 ◽

2011 ◽

Vol 16 (3) ◽

Cited By ~ 1

Author(s):

Akin Sevinc ◽

Marta Witek ◽

Leslie Fung

Keyword(s):

Membrane Skeleton ◽

Terminal Region ◽

Yeast Cells ◽

Titration Calorimetry ◽

Single Mutation ◽

Wild Type ◽

Yeast Two Hybrid ◽

Terminal Fragment ◽

Kd Values ◽

Two Hybrid

AbstractYeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the Kd values for V22F were similar to those for the wild-type (about 7 nM), whereas the Kd values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.

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Phosphorylation of BIK1 is critical for interaction of downstream signaling components

10.21203/rs.3.rs-213662/v1 ◽

2021 ◽

Author(s):

Jae-Han Choi ◽

Eun-Seok Oh ◽

Man-Ho Oh

Keyword(s):

Growth And Development ◽

Kinase Activity ◽

Higher Plants ◽

Site Directed Mutagenesis ◽

Bimolecular Fluorescence Complementation ◽

Yeast Two Hybrid ◽

Downstream Signaling ◽

Signaling Components ◽

Two Hybrid

Abstract Botrytis-induced Kinase 1 (BIK1) is a receptor-like cytoplasmic kinase (RLCK) involved in the defense, growth, and development of higher plants. It interacts with various receptor-like kinases (RLKs) such as Brassinosteroid Insensitive 1 (BRI1), Flagellin Sensitive 2 (FLS2), and Perception of the Arabidopsis Danger Signal Peptide 1 (PEPR1), but little is known about signaling downstream of BIK1. Interestingly, Arabidopsis thaliana BIK1 (AtBIK1) displays strong autophosphorylation kinase activity on tyrosine and threonine residues, whereas Brassica rapa BIK1 (BrBIK1) does not exhibit autophosphorylation kinase activity in vitro. Herein, we demonstrated that four proteins (RGP2, PATL2, PP7, and SULTR4.1) interact with BrBIK1 but not AtBIK1 in a yeast two-hybrid (Y2H) system. We subsequently employed bimolecular fluorescence complementation (BiFC) to confirm interactions between BIK1 and candidates in Nicotiana benthamiana, and found that only BrBIK1 bound the three proteins tested. We selected three phosphosites, T90, T362, and T368, based on amino acid sequence alignment between AtBIK1 and BrBIK1, and performed site-directed mutagenesis (SDM) on AtBIK1 and BrBIK. S90T, P362T, and A368T mutations in BrBIK1 restored autophosphorylation kinase activity on threonine residues comparable with AtBIK1. However, T90A, T362P, and T368A mutations in AtBIK1 did not alter autophosphorylation kinase activity on threonine residues compared with wild-type AtBIK1. Interestingly, BiFC results showed that BIK1 mutations restored kinase activity but not binding to RGP2, PATL2, or PP7 proteins. Our results suggest that phospho-BIK1 might be involved in plant innate immunity, while non-phospho BIK1 may regulate plant growth and development through interactions with RGP2, PATL2, and PP7.

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Characterization of Burkholderia glumae Putative Virulence Factor 11 (PVF11) via Yeast Two-Hybrid Interaction and Phenotypic Analysis

The Plant Pathology Journal ◽

10.5423/ppj.nt.02.2019.0026 ◽

2019 ◽

Vol 35 (3) ◽

pp. 280-286

Author(s):

Juyun Kim ◽

Namgyu Kim ◽

Mohamed Mannaa ◽

Hyun-Hee Lee ◽

Jong-Seong Jeon ◽

...

Keyword(s):

Virulence Factor ◽

Putative Virulence Factor ◽

Yeast Two Hybrid ◽

Phenotypic Analysis ◽

Burkholderia Glumae ◽

Two Hybrid

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Synaptic protein UNC-13 interacts with an F-box protein that may target it for degradation by proteasomes.

Acta Biochimica Polonica ◽

10.18388/abp.2006_3372 ◽

2006 ◽

Vol 53 (1) ◽

pp. 145-148 ◽

Cited By ~ 3

Author(s):

Cristina Polinsky ◽

Chanelle Houston ◽

Jaynine Vado ◽

Azizahmed Shaikh ◽

Rebecca E Kohn

Keyword(s):

Neurotransmitter Release ◽

Coiled Coil ◽

Proteasomal Degradation ◽

Terminal Region ◽

Splicing Factor ◽

Synaptic Protein ◽

Yeast Two Hybrid ◽

Amino Terminal ◽

F Box Protein ◽

Two Hybrid

UNC-13 protein participates in regulating neurotransmitter release. In Drosophila melanogaster, proteasomal degradation controls UNC-13 levels at synapses. Function of the amino-terminal region of a 207 kDa form of Caenorhabditis elegans UNC-13 is unknown. Yeast two-hybrid and secondary yeast assays identified an F-box protein that interacts with this amino-terminal region. As F-box proteins bind proteins targeted for proteasomal degradation, this protein may participate in degrading a subset of UNC-13 proteins, suggesting that different forms of UNC-13 are regulated differently. Yeast assays also identified an exonuclease, a predicted splicing factor, and a protein with coiled-coil domains, indicating that UNC-13 may affect RNA function.

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Genome-Wide Identification and Molecular Characterization of the Growth-Regulating Factors-Interacting Factor Gene Family in Tomato

Genes ◽

10.3390/genes11121435 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1435

Author(s):

Guo Ai ◽

Dedi Zhang ◽

Rong Huang ◽

Shiqi Zhang ◽

Wangfang Li ◽

...

Keyword(s):

Growth And Development ◽

Regulatory Elements ◽

Tomato Genome ◽

Yeast Two Hybrid ◽

Promoter Regions ◽

Cis Acting ◽

Functional Studies ◽

Genome Wide ◽

Two Hybrid

Growth-regulating factors-interacting factor (GIF) proteins play crucial roles in the regulation of plant growth and development. However, the molecular mechanism of GIF proteins in tomato is poorly understood. Here, four SlGIF genes (named SlGRF1a, SlGIF1b, SlGIF2, and SlGIF3) were identified from the tomato genome and clustered into two major clades by phylogenetic analysis. The gene structure and motif pattern analyses showed similar exon/intron patterns and motif organizations in all the SlGIFs. We identified 33 cis-acting regulatory elements (CAREs) in the promoter regions of the SlGIFs. The expression profiling revealed the four GIFs are expressed in various tissues and stages of fruit development and induced by phytohormones (IAA and GA). The subcellular localization assays showed all four GIFs were located in nucleus. The yeast two-hybrid assay indicated various growth-regulating factors (SlGRFs) proteins interacted with the four SlGIF proteins. However, SlGRF4 was a common interactor with the SlGIF proteins. Moreover, a higher co-expression relationship was shown between three SlGIF genes and five SlGRF genes. The protein association network analysis found a chromodomain helicase DNA-binding protein (CHD) and an actin-like protein to be associated with the four SlGIF proteins. Overall, these results will improve our understanding of the potential functions of GIF genes and act as a base for further functional studies on GIFs in tomato growth and development.

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