The Mildew Resistance Locus O 4 Interacts with CaM/CML and Is Involved in Root Gravity Response

The plant-specific mildew resistance locus O (MLO) proteins, which contain seven transmembrane domains and a conserved calmodulin-binding domain, play important roles in many plant developmental processes. However, their mechanisms that regulate plant development remain unclear. Here, we report the functional characterization of the MLO4 protein in Arabidopsis roots. The MLO4 was identified as interacting with CML12 in a screening for the interaction between the proteins from Arabidopsis MLO and calmodulin/calmodulin-like (CaM/CML) families using yeast two hybrid (Y2H) assays. Then, the interaction between MLO4 and CML12 was further verified by Luciferase Complementation Imaging (LCI) and Bimolecular Fluorescence Complementation (BiFC) assays. Genetic analysis showed that the mlo4, cml12, and mlo4 cml12 mutants displayed similar defects in root gravity response. These results imply that the MLO4 might play an important role in root gravity response through interaction with CML12. Moreover, our results also demonstrated that the interaction between the MLO and CaM/CML families might be conservative.

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CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium

Horticulture Research ◽

10.1038/s41438-021-00534-x ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Jingjing Xin ◽

Ye Liu ◽

Huiyun Li ◽

Sumei Chen ◽

Jiafu Jiang ◽

...

Keyword(s):

Signaling Pathways ◽

Alternaria Alternata ◽

Defense Responses ◽

Chrysanthemum Morifolium ◽

Bimolecular Fluorescence Complementation ◽

Resistance Locus ◽

Plant Defense Responses ◽

Yeast Two Hybrid ◽

Two Hybrid ◽

Insight Into

AbstractThe Mildew Resistance Locus O (MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemum MLO genes. We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection. Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A. alternata infection. Genes in the abscisic acid (ABA) and Ca2+ signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants. We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca2+ signaling pathways, resulting in reduced susceptibility of chrysanthemum to A. alternata infection. In addition, a candidate gene, CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus. CmKIC overexpression increased the susceptibility of chrysanthemum to A. alternata, whereas CmKIC silencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together in C. morifolium to support the growth of A. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

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Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures

Nature Protocols ◽

10.1038/nprot.2006.32 ◽

2006 ◽

Vol 1 (3) ◽

pp. 1066-1091 ◽

Cited By ~ 36

Author(s):

Marc B T Bickle ◽

Eric Dusserre ◽

Olivier Moncorgé ◽

Hélène Bottin ◽

Pierre Colas

Keyword(s):

Yeast Two Hybrid ◽

Hybrid Procedures ◽

Peptide Aptamers ◽

Two Hybrid

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Use of the Yeast Two-Hybrid System for the Characterization of Integrin Signal Transduction Pathways: Identification of the Integrin-Linked Kinase, p59ILK

Signaling through Cell Abhesion Molecules ◽

10.1201/9780429129384-3 ◽

2019 ◽

pp. 33-48

Author(s):

Gregory E. Hannigan ◽

Shoukat Dedhar

Keyword(s):

Signal Transduction ◽

Hybrid System ◽

Signal Transduction Pathways ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Integrin Linked Kinase ◽

Two Hybrid

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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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A novel yeast two-hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein1

FEBS Letters ◽

10.1016/j.febslet.2006.01.013 ◽

2006 ◽

Vol 580 (3) ◽

pp. 904-911 ◽

Cited By ~ 50

Author(s):

Miguel A. Rodriguez Milla ◽

Yuichi Uno ◽

Ing-Feng Chang ◽

Jared Townsend ◽

Eileen A. Maher ◽

...

Keyword(s):

Zinc Finger ◽

Hybrid Approach ◽

Yeast Two Hybrid ◽

Two Hybrid

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Characterization of Interactions between the Neurofilament Triplet Proteins by the Yeast Two-hybrid System

Journal of Biological Chemistry ◽

10.1074/jbc.271.24.14041 ◽

1996 ◽

Vol 271 (24) ◽

pp. 14041-14044 ◽

Cited By ~ 28

Author(s):

Conrad L. Leung ◽

Ronald K. H. Liem

Keyword(s):

Hybrid System ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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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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Yeast Two-Hybrid System for Studying Protein-Protein Interactions--Stage 1: Construction and Characterization of a Bait Protein

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot5429 ◽

2010 ◽

Vol 2010 (5) ◽

pp. pdb.prot5429-pdb.prot5429 ◽

Cited By ~ 1

Author(s):

I. Serebriiskii

Keyword(s):

Hybrid System ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Bait Protein ◽

Yeast Two Hybrid System ◽

Stage 1 ◽

Two Hybrid

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Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.200107126 ◽

2001 ◽

Vol 155 (2) ◽

pp. 239-250 ◽

Cited By ~ 110

Author(s):

Maren Heese ◽

Xavier Gansel ◽

Liliane Sticher ◽

Peter Wick ◽

Markus Grebe ◽

...

Keyword(s):

Cell Division ◽

Membrane Fusion ◽

Functional Characterization ◽

Cell Plate ◽

Yeast Two Hybrid ◽

Division Plane ◽

Plant Cytokinesis ◽

Cell Plate Formation ◽

Plate Formation ◽

Two Hybrid

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

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