AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance

Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

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Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development

Development ◽

10.1242/dev.119.3.711 ◽

1993 ◽

Vol 119 (3) ◽

pp. 711-720 ◽

Cited By ~ 21

Author(s):

U. W. Rothenpieler ◽

G. R. Dressler

Keyword(s):

Kidney Development ◽

Morphological Changes ◽

Cell Formation ◽

Protein Accumulation ◽

Metanephric Mesenchyme ◽

Loss Of Function ◽

Organ Cultures ◽

Protein Levels ◽

Differentiated Cells ◽

Mesenchyme Cells

The conversion of mesenchyme to epithelium during the embryonic development of the mammalian kidney requires reciprocal inductive interactions between the ureter and the responding metanephric mesenchyme. The Pax-2 gene is activated in the mesenchyme in response to induction and is subsequently down-regulated in more differentiated cells derived from the mesenchyme. Pax-2 belongs to a family of genes, at least three of which encode morphogenetic regulatory transcription factors. In order to determine the role of Pax-2 during kidney development, we have generated a loss- of-function phenotype using antisense oligonucleotides in mouse kidney organ cultures. These oligonucleotides can specifically inhibit Pax-2 protein accumulation in kidney mesenchyme cells, where the intracellular concentrations are maximal. The kidney organ cultures were stained with uvomurulin and laminin antibodies as markers for epithelium formation. With significantly reduced Pax-2 protein levels, kidney mesenchyme cells fail to aggregate and do not undergo the sequential morphological changes characteristic of epithelial cell formation. The data demonstrate that Pax-2 function is required for the earliest phase of mesenchyme-to-epithelium conversion.

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The role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-20-0235-r ◽

2021 ◽

Author(s):

Jinxin Gao ◽

Jie Chen

Keyword(s):

Toxin Production ◽

Bimolecular Fluorescence Complementation ◽

Curvularia Lunata ◽

Yeast Two Hybrid ◽

Acetyl Xylan Esterase ◽

Btb Domain ◽

Intermediate Metabolites ◽

Regulative Mechanism ◽

Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular ﬂuorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

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FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

The Journal of Cell Biology ◽

10.1083/jcb.201805128 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3290-3306 ◽

Cited By ~ 1

Author(s):

Melody Atkins ◽

Laïla Gasmi ◽

Valérie Bercier ◽

Céline Revenu ◽

Filippo Del Bene ◽

...

Keyword(s):

Molecular Motor ◽

Motor Axon ◽

Regulatory Mechanisms ◽

Axon Pathfinding ◽

Loss Of Function ◽

Yeast Two Hybrid ◽

Neuronal Connectivity ◽

Transport Velocity ◽

Bidirectional Transport ◽

Two Hybrid

Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

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The chaperonin 60 protein SlCpn60α1 modulates photosynthesis and photorespiration in tomato

Journal of Experimental Botany ◽

10.1093/jxb/eraa418 ◽

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.

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Abscisic acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds

New Phytologist ◽

10.1111/nph.12785 ◽

2014 ◽

Vol 203 (1) ◽

pp. 81-93 ◽

Cited By ~ 46

Author(s):

Julio Maia ◽

Bas J. W. Dekkers ◽

Miranda J. Dolle ◽

Wilco Ligterink ◽

Henk W. M. Hilhorst

Keyword(s):

Abscisic Acid ◽

Desiccation Tolerance ◽

Aba Sensitivity

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Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNFα-mediated apoptosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806973115 ◽

2018 ◽

Vol 115 (26) ◽

pp. E5944-E5953 ◽

Cited By ~ 30

Author(s):

Palak Amin ◽

Marcus Florez ◽

Ayaz Najafov ◽

Heling Pan ◽

Jiefei Geng ◽

...

Keyword(s):

Complex I ◽

Tnf Receptor ◽

Loss Of Function ◽

Signaling Mechanism ◽

E3 Ligases ◽

Complex Ii ◽

Cell Death Pathways ◽

Distinct Cell ◽

Rapid Formation ◽

Als Patients

Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)—the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed “iuRIPK1.” iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson’s disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.

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The MATH-BTB BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases target PP2CA and other clade A PP2Cs for degradation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908677116 ◽

2019 ◽

Vol 116 (31) ◽

pp. 15725-15734 ◽

Cited By ~ 15

Author(s):

Jose Julian ◽

Alberto Coego ◽

Jorge Lozano-Juste ◽

Esther Lechner ◽

Qian Wu ◽

...

Keyword(s):

Feedback Mechanism ◽

Dependent Manner ◽

Aba Signaling ◽

E3 Ligases ◽

Negative Feedback Mechanism ◽

Protein Levels ◽

Aba Sensitivity ◽

Regulate Protein ◽

Protein Components ◽

Ring E3

Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas bpm3 bpm5 plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.

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CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane

Plant and Cell Physiology ◽

10.1093/pcp/pcaa091 ◽

2020 ◽

Vol 61 (9) ◽

pp. 1669-1682

Author(s):

Lina Yao ◽

Changqing Ding ◽

Xinyuan Hao ◽

Jianming Zeng ◽

Yajun Yang ◽

...

Keyword(s):

Plasma Membrane ◽

Freezing Tolerance ◽

Sugar Transport ◽

Bimolecular Fluorescence Complementation ◽

Freezing Stress ◽

Sugar Uptake ◽

Wild Type ◽

Alternatively Spliced ◽

Germination And Growth ◽

Two Hybrid

Abstract Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.

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suz12 inactivation in p53- and nf1-deficient zebrafish accelerates the onset of malignant peripheral nerve sheath tumors and expands the spectrum of tumor types

Disease Models & Mechanisms ◽

10.1242/dmm.042341 ◽

2020 ◽

Vol 13 (8) ◽

pp. dmm042341 ◽

Cited By ~ 1

Author(s):

Felix Oppel ◽

Dong H. Ki ◽

Mark W. Zimmerman ◽

Kenneth N. Ross ◽

Ting Tao ◽

...

Keyword(s):

Peripheral Nerve ◽

Chromatin Modification ◽

Control Fish ◽

Loss Of Function ◽

Zebrafish Model ◽

Reading Frame ◽

Nerve Sheath Tumors ◽

Peripheral Nerve Sheath Tumors ◽

Nerve Sheath ◽

Tumor Types

ABSTRACTPolycomb repressive complex 2 (PRC2) is an epigenetic regulator of gene expression that possesses histone methyltransferase activity. PRC2 trimethylates lysine 27 of histone H3 proteins (H3K27me3) as a chromatin modification associated with repressed transcription of genes frequently involved in cell proliferation or self-renewal. Loss-of-function mutations in the PRC2 core subunit SUZ12 have been identified in a variety of tumors, including malignant peripheral nerve sheath tumors (MPNSTs). To determine the consequences of SUZ12 loss in the pathogenesis of MPNST and other cancers, we used CRISPR-Cas9 to disrupt the open reading frame of each of two orthologous suz12 genes in zebrafish: suz12a and suz12b. We generated these knockout alleles in the germline of our previously described p53 (also known as tp53)- and nf1-deficient zebrafish model of MPNSTs. Loss of suz12 significantly accelerated the onset and increased the penetrance of MPNSTs compared to that in control zebrafish. Moreover, in suz12-deficient zebrafish, we detected additional types of tumors besides MPNSTs, including leukemia with histological characteristics of lymphoid malignancies, soft tissue sarcoma and pancreatic adenocarcinoma, which were not detected in p53/nf1-deficient control fish, and are also contained in the human spectrum of SUZ12-deficient malignancies identified in the AACR Genie database. The suz12-knockout tumors displayed reduced or abolished H3K27me3 epigenetic marks and upregulation of gene sets reported to be targeted by PRC2. Thus, these zebrafish lines with inactivation of suz12 in combination with loss of p53/nf1 provide a model of human MPNSTs and multiple other tumor types, which will be useful for mechanistic studies of molecular pathogenesis and targeted therapy with small molecule inhibitors.

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Identification of Beet necrotic yellow vein virus P25 Pathogenicity Factor–Interacting Sugar Beet Proteins That Represent Putative Virus Targets or Components of Plant Resistance

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-8-0999 ◽

2009 ◽

Vol 22 (8) ◽

pp. 999-1010 ◽

Cited By ~ 15

Author(s):

Heike Thiel ◽

Mark Varrelmann

Keyword(s):

Sugar Beet ◽

Resistance Mechanism ◽

Yellow Vein ◽

Bimolecular Fluorescence Complementation ◽

Plant Proteins ◽

Yeast Two Hybrid ◽

Response To Stress ◽

Bimolecular Fluorescence Complementation Assay ◽

Two Hybrid ◽

Important Disease

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

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