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      A Transcriptomic and Proteomic Analysis of the Diaphorina citri Salivary Glands Reveals Genes Responding to Candidatus Liberibacter asiaticus

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          Abstract

          The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the principal vector of the Candidatus Liberibacter asiaticus (CLas) bacterium that causes Huanglongbing (HLB) disease. The D. citri salivary glands (SG) is an important barrier to the transmission of CLas. Despite its importance, the transcriptome and proteome of SG defense against CLas are unstudied in D. citri. In the present study, we generated a comparative transcriptome dataset of the SG in infected and uninfected D. citri using an Illumina RNA-Seq technology. We obtained 407 differentially expressed genes (DEGs), including 159 upregulated DEGs and 248 downregulated DEGs. Functional categories showed that many DEGs were associated with the ribosome, the insecticide resistance, the immune response and the digestion in comparison with CLas-infected SG and CLas-free SG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases confirmed that metabolism and immunity were important functions in the SG. Among the DEGs, 68 genes (35 upregulated and 33 downregulated) encoding putative-secreted proteins were obtained with a signal peptide, suggesting that these genes may play important roles in CLas infection. A total of 673 SG proteins were identified in uninfected D. citri by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, and 30 DEGs (15 upregulated and 15 downregulated) were found using the local tBLASTP programs. Among the 30 DEGs, many DEGs mainly involved in the metabolism and cellular processes pathways. This study provides basic transcriptome and proteome information for the SG in D. citri, and helps illuminate the molecular interactions between CLas and D. citri.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          Kenneth Livak, Thomas D. Schmittgen (2001)
          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

            Michael Love, Wolfgang Huber, Simon Anders (2014)
            In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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              TopHat: discovering splice junctions with RNA-Seq

              Cole Trapnell, Lior Pachter, Steven Salzberg (2011)
              Motivation: A new protocol for sequencing the messenger RNA in a cell, known as RNA-Seq, generates millions of short sequence fragments in a single run. These fragments, or ‘reads’, can be used to measure levels of gene expression and to identify novel splice variants of genes. However, current software for aligning RNA-Seq data to a genome relies on known splice junctions and cannot identify novel ones. TopHat is an efficient read-mapping algorithm designed to align reads from an RNA-Seq experiment to a reference genome without relying on known splice sites. Results: We mapped the RNA-Seq reads from a recent mammalian RNA-Seq experiment and recovered more than 72% of the splice junctions reported by the annotation-based software from that study, along with nearly 20 000 previously unreported junctions. The TopHat pipeline is much faster than previous systems, mapping nearly 2.2 million reads per CPU hour, which is sufficient to process an entire RNA-Seq experiment in less than a day on a standard desktop computer. We describe several challenges unique to ab initio splice site discovery from RNA-Seq reads that will require further algorithm development. Availability: TopHat is free, open-source software available from http://tophat.cbcb.umd.edu Contact: cole@cs.umd.edu Supplementary information: Supplementary data are available at Bioinformatics online.
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                Author and article information

                Contributors
                Journal
                Journal ID (nlm-ta): Front Physiol
                Journal ID (iso-abbrev): Front Physiol
                Journal ID (publisher-id): Front. Physiol.
                Title: Frontiers in Physiology
                Publisher: Frontiers Media S.A.
                ISSN (Electronic): 1664-042X
                Publication date (Electronic): 25 September 2020
                Publication date Collection: 2020
                Volume: 11
                Electronic Location Identifier: 582505
                Affiliations
                [1] 1Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University , Chongqing, China
                [2] 2International Joint Laboratory of China-Belgium on Sustainable Crop Pest Control, Academy of Agricultural Sciences, Southwest University , Chongqing, China
                [3] 3Guangxi Key Laboratory of Citrus Biology, Guangxi Citrus Research Institute , Gulin, China
                Author notes

                Edited by: Ya-Nan Zhang, Huaibei Normal University, China

                Reviewed by: Lu Xu, Jiangsu Academy of Agricultural Sciences (JAAS), China; Hao Guo, Chinese Academy of Sciences (CAS), China; Peng He, Guizhou University, China

                †These authors have contributed equally to this work

                This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

                Article
                DOI: 10.3389/fphys.2020.582505
                PMC ID: 7546269
                SO-VID: c9c1919b-f891-480a-94e8-e11ff03f89fd
                Copyright © Copyright © 2020 Liu, Jiang, Liu, Yang, Fan, Xiong, Jing, Lou, Dou and Wang.
                License:

                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
                Date received : 12 July 2020
                Date accepted : 02 September 2020
                Page count
                Figures: 5, Tables: 3, Equations: 0, References: 38, Pages: 11, Words: 0
                Categories
                Subject: Physiology
                Subject: Original Research

                ScienceOpen disciplines: Anatomy & Physiology
                Keywords: diaphorina citri,candidatus liberibacter asiaticus,salivary glands,comparative transcriptome,shotgun lc–ms/ms
                Data availability:
                ScienceOpen disciplines: Anatomy & Physiology
                Keywords: diaphorina citri, candidatus liberibacter asiaticus, salivary glands, comparative transcriptome, shotgun lc–ms/ms

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