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TaCAMTA4, a Calmodulin-Interacting Protein, Involved in Defense Response of Wheat to Puccinia triticina.

ABSTRACT: Leaf rust caused by Puccinia triticina is one of the main diseases affecting wheat (Triticum aestivum) production worldwide. Calmodulin (CaM) was found involved in the early stage of signal transduction pathway in response to P. triticina in wheat. To study the function and molecular mechanism of calmodulin (CaM) in signal transduction of wheat against P. triticina, we cloned a putative calmodulin-binding transcription activator (TaCAMTA4), and characterized its molecular structure and functions by using the CaM-encoding gene (TaCaM4-1) as a bait to screen the cDNA library from P. triticina infected wheat leaves. The open reading frame of TaCAMTA4 was 2505?bp encoding a protein of 834 aa, which contained all the four conserved domains of family (CG-1 domain, TIG domain, ANK repeats and CaM-binding domain). TaCaM4-1 bound to TaCAMTA4 by the C-terminal CaM-binding domain in Ca2+-dependent manner in the electrophoretic mobility shift assay (EMSA). Bimolecular fluorescence complementation (BiFC) analysis indicated that the interaction of TaCAMTA4 and TaCaM4-1 took place in the cytoplasm and nucleus of epidermal leaf cells in N. benthamiana. The expression level of TaCAMTA4 genes was down-regulated in incompatible combination after P. triticina infection. Furthermore, virus-induced gene silencing (VIGS)-based knockdown of TaCAMTA4 and disease assays verified that silencing of TaCAMTA4 resulted in enhanced resistance to P. triticina race 165. These results suggested that TaCAMTA4 function as negative regulator of defense response against P. triticina.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6345913 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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TaCAMTA4, a Calmodulin-Interacting Protein, Involved in Defense Response of Wheat to Puccinia triticina.

Wang Yuelin Y Wei Fengju F Zhou Hui H Liu Na N Niu Xiaonan X Yan Chao C Zhang Lifeng L Han Shengfang S Hou Chunyan C Wang Dongmei D

Scientific reports 20190124 1

Leaf rust caused by Puccinia triticina is one of the main diseases affecting wheat (Triticum aestivum) production worldwide. Calmodulin (CaM) was found involved in the early stage of signal transduction pathway in response to P. triticina in wheat. To study the function and molecular mechanism of calmodulin (CaM) in signal transduction of wheat against P. triticina, we cloned a putative calmodulin-binding transcription activator (TaCAMTA4), and characterized its molecular structure and functions ...[more]

PMID: 30679453

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Project description:Inoculation of wheat plants with Puccinia triticina (Pt) spores activates a wide range of host responses. Compatible Pt interaction with susceptible Thatcher plants supports all stages of the pathogen life cycle. Incompatible interaction with TcLr9 activates defense responses including oxidative burst and micronecrotic reactions associated with the pathogen's infection structures and leads to complete termination of pathogen development. These two contrasting host-pathogen interactions were a foundation for transcriptome analysis of incompatible wheat-Pt interaction.A suppression subtractive hybridization (SSH) library was constructed using cDNA from pathogen-inoculated susceptible Thatcher and resistant TcLr9 isogenic lines. cDNA represented steps of wheat-brown rust interactions: spore germination, haustorium mother cell (HMC) formation and micronecrotic reactions. All ESTs were clustered and validated by similarity search to wheat genome using BLASTn and sim4db tools. qRT-PCR was used to determine transcript levels of selected ESTs after inoculation in both lines.Out of 793 isolated cDNA clones, 183 were classified into 152 contigs. 89 cDNA clones and encoded proteins were functionally annotated and assigned to 5 Gene Ontology categories: catalytic activity 48 clones (54 %), binding 32 clones (36 %), transporter activity 6 clones (7 %), structural molecule activity 2 clones (2 %) and molecular transducer activity 1 clone (1 %). Detailed expression profiles of 8 selected clones were analyzed using the same plant-pathogen system. The strongest induction after pathogen infection and the biggest differences between resistant and susceptible interactions were detected for clones encoding wall-associated kinase (GenBank accession number JG969003), receptor with leucine-rich repeat domain (JG968955), putative serine/threonine protein kinase (JG968944), calcium-mediated signaling protein (JG968925) and 14-3-3 protein (JG968969).The SSH library represents transcripts regulated by pathogen infection during compatible and incompatible interactions of wheat with P. triticina. Annotation of selected clones confirms their putative roles in successive steps of plant-pathogen interactions. The transcripts can be categorized as defense-related due to their involvement in either basal defense or resistance through an R-gene mediated reaction. The possible involvement of selected clones in pathogen recognition and pathogen-induced signaling as well as resistance mechanisms such as cell wall enforcement, oxidative burst and micronecrotic reactions is discussed.

Project description:Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of Puccinia triticina (Pt) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six Pt pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the Pt pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of Pt9226 in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of Pseudomonas fluorescens EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of PtEF-1α was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the Pt pathotype THTT was inoculated on a transient expression of Pt9226 in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the Pt pathotype THTT and wheat.

Dataset Information

TaCAMTA4, a Calmodulin-Interacting Protein, Involved in Defense Response of Wheat to Puccinia triticina.

Publications

TaCAMTA4, a Calmodulin-Interacting Protein, Involved in Defense Response of Wheat to Puccinia triticina.

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