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      Validation of Reliable Reference Genes for RT-qPCR Studies of Target Gene Expression in Colletotrichum camelliae During Spore Germination and Mycelial Growth and Interaction With Host Plants

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          Abstract

          The tea plant [ Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.

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          Most cited references28

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          How to do successful gene expression analysis using real-time PCR.

          Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting. Copyright 2009 Elsevier Inc. All rights reserved.
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            Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi.

            Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray. Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes. Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes. © 2012 The Authors New Phytologist © 2012 New Phytologist Trust.
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              Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100

              The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity. DOI: http://dx.doi.org/10.7554/eLife.07295.001
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                04 September 2019
                2019
                : 10
                : 2055
                Affiliations
                Laboratory of Molecular Plant Pathology, College of Plant Science, Jilin University , Changchun, China
                Author notes

                Edited by: Raffaella Balestrini, Institute for Sustainable Plant Protection, Italian National Research Council (IPSP-CNR), Italy

                Reviewed by: Taurai Tasara, University of Zurich, Switzerland; Arunachalam Ramaiah, University of California, Irvine, United States; Matteo Caser, University of Turin, Italy

                *Correspondence: Shouan Liu, shouan@ 123456jlu.edu.cn

                These authors have contributed equally to this work

                This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.02055
                6737088
                31551988
                4f4720e1-fee3-4d1d-8c24-330b6812fcea
                Copyright © 2019 He, An, A and Liu.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 29 April 2019
                : 20 August 2019
                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 41, Pages: 11, Words: 0
                Funding
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                reference genes,tea plant,colletotrichum camelliae,rt-qpcr,gene expression
                Microbiology & Virology
                reference genes, tea plant, colletotrichum camelliae, rt-qpcr, gene expression

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