Name: Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine (Video) | JoVE Journal
Uploaded: 2017-12-22T12:30:00.000+00:00
Duration: 10 min 40 s
Description: Learn a comprehensive workflow for genome-wide identification and expression meta-analysis of the ATL E3 ubiquitin ligase gene family in grapevine. This method enables complete gene family characterization for plant genetics research.

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The work was supported by the University of Verona within the frame of Joint Project 2014 (Characterization of the ATL gene family in grapevine and of its involvement in resistance to Plasmopara viticola).

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</ol>

The authors have nothing to disclose.

In the genomic era, many gene families have been deeply characterized in several plant species. This information is preliminary to functional studies and provide a frame to investigate further the role of different members in a family. In this context, there is also a need for a nomenclature system allowing to uniquely identify each member in a family, avoiding the redundancy and confusions that may arise when names are assigned independently to different genes by different research groups.
Af...

During the last decade, much research has been carried out in grapevine genomics. Grapevine is a recognized economically relevant crop, which has become a model for research on fruit development and on the responses of woody plants to biotic and abiotic stresses. In this context, the release of the Vitis vinifera cv. PN40024 genome in 20071 and its updated version in 20112 led to a rapid accumulation of &#34;Omics&#34;-scale data and to a burst of high-throughput studies. Based on the published sequence data, the comprehensive analysis of a given gene family (generally composed of proteins sharing conserved motifs, structural and/or functional similarities and evolutionary relationships), can now be performed to uncover its molecular functions, evolution, and gene expression profiles. These analyses can contribute to understanding how gene families control physiological processes at a genome-wide level.
Many aspects of the plant life cycle are regulated by ubiquitin-mediated degradation of key proteins, which require a fine-tuned turnover to ensure regular cellular processes. Important components of the ubiquitin-mediated degradation process are the E3 ubiquitin ligases, which are responsible for system flexibility, thanks to the recruitment of specific targets3. Accordingly, these enzymes represent a huge gene family, with around 1,400 E3 ligase-encoding genes predicted in Arabidopsis thaliana genome4, each E3 ubiquitin ligase acting for the ubiquitination of specific target proteins. Despite the importance of substrate-specific ubiquitination in cellular regulation in plants, little is known about how the ubiquitination pathway is regulated and target proteins have been identified only in a few cases. The deciphering of such specificity and regulation mechanisms relies first on the identification and characterization of the different components of the system, in particular the E3 ligases. Among ubiquitin ligases, the ATL subfamily is characterized by 91 members identified in A. thaliana displaying a RING-H2 finger domain5,6, some of them playing a role in defense and hormone responses7.
The first crucial step to define the members of a new gene family is the precise definition of the family features, such as consensus motifs, key domains, and protein sequence characteristics. Indeed, the reliable retrieval of all gene family members based on BLAST analysis requires some mandatory sequence characteristics, in particular protein domains responsible for protein function/activity, serving as protein signature. This can be facilitated by previous characterization of the same gene family in other plant species or achieved by analyzing different genes putatively belonging to the same family in different plant species, to isolate common sequences. The family members can then be individually named following common rules settled by international consortia for a given plant species. In grapevine, for instance, such procedure is subjected to the recommendations of the Super-Nomenclature Committee for Grape Gene Annotation (sNCGGa), establishing the construction of a phylogenetic tree including V. vinifera and A. thaliana gene family members to allow gene annotation based on nucleotide sequences8.
Chromosome localization of family members and gene duplication survey allow highlighting the presence of whole-genome or tandem duplicated genes. Such information appears useful to unravel putative gene functions, since it might show functional redundancy or reveal different situations, i.e., non-functionalization, neo-functionalization, or sub-functionalization9. Both neo- and sub-functionalization are important events that create genetic novelty, providing new cellular components for plant adaptation to changing environments10. In particular, duplications of ancestral genes and production of new genes were very frequent during the evolution of the grapevine genome and newly formed genes originating from proximal and tandem duplications in grapevine were more likely to produce new functions11.
Another key factor in deciphering gene family function is the transcriptomic profile. The availability of public databases giving access to a huge amount of transcriptomic data can be thus exploited to assign putative functions to gene family members using large-scale in silico expression analyses. Indeed, the peculiar expression of some genes in specific plant organs or in response to certain stresses can give some hints regarding the putative roles of the corresponding proteins in defined conditions, and give support to hypotheses about possible sub-functionalization of duplicated genes to respond to different challenges. For that purpose, it is important to consider several datasets: these can be already available gene expression matrixes, such as the genome-wide transcriptomic atlas of grapevine organs and developmental stages12, or can be built ad hoc by retrieving transcriptomic datasets for the particular plant species subjected to defined stresses. Moreover, a simple approach using two matrices, one with pairwise similarity data and the other one with pairwise co-expression coefficients can be applied to evaluate the relationships between sequence similarity and expression patterns within a gene family.
The aim of this work is to provide a global approach, defining gene structure, conserved protein motifs, chromosomal location, gene duplications, and expression patterns, as well the prediction of protein localization and phosphorylation sites, to attain an exhaustive characterization of a gene family in plants. Such a comprehensive approach is applied here to the characterization of the ATL E3 ubiquitin ligase family in grapevine. According to the emerging role of ATL subfamily members in regulating key cellular processes7, this work can well assist the identification of strong candidates for functional studies, and eventually unravel the molecular mechanisms governing the adaptation of this important crop to its environment.

<table><tbody><tr><td>Personal computer</td><td></td><td></td><td></td></tr><tr><td>Basic Local Alignment Search Tool (BLAST)</td><td></td><td></td><td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td></tr><tr><td>Molecular Evolutionary Genetics Analysis (MEGA)</td><td></td><td></td><td>http://www.megasoftware.net/</td></tr><tr><td>Motif-based sequence analysis tools (MEME)</td><td></td><td></td><td>http://meme-suite.org/</td></tr><tr><td>Geneious</td><td>Biomatters Limited</td><td></td><td>http://www.geneious.com/</td></tr><tr><td>ProtParam Tool</td><td></td><td></td><td>http://web.expasy.org/protparam/</td></tr><tr><td>ngLOC</td><td></td><td></td><td>http://genome.unmc.edu/ngLOC/index.html</td></tr><tr><td>TargetP v1.1 Server</td><td></td><td></td><td>http://www.cbs.dtu.dk/services/TargetP/</td></tr><tr><td>Protein Prowler</td><td></td><td></td><td>http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-2/</td></tr><tr><td>MUsite</td><td></td><td></td><td>http://musite.sourceforge.net/</td></tr><tr><td>Pfam</td><td></td><td></td><td>http://pfam.xfam.org/</td></tr><tr><td>TMHMM Server v. 2.0</td><td></td><td></td><td>http://www.cbs.dtu.dk/services/TMHMM/</td></tr><tr><td>ProtScale</td><td></td><td></td><td>http://web.expasy.org/protscale/</td></tr><tr><td>Grape Genome Database (CRIBI)</td><td></td><td></td><td>http://genomes.cribi.unipd.it/grape/</td></tr><tr><td>PhenoGram</td><td></td><td></td><td>http://visualization.ritchielab.psu.edu/phenograms/plot</td></tr><tr><td>MCScanX</td><td></td><td></td><td>http://chibba.pgml.uga.edu/mcscan2/</td></tr><tr><td>Interactive Tree Of Life (iTOL)</td><td></td><td></td><td>http://itol.embl.de/</td></tr><tr><td>UniProt</td><td></td><td></td><td>http://www.uniprot.org/</td></tr><tr><td>Phylogeny.fr</td><td></td><td></td><td>http://www.phylogeny.fr/index.cgi</td></tr><tr><td>MUSCLE</td><td></td><td></td><td>http://www.ebi.ac.uk/Tools/msa/muscle/</td></tr><tr><td>Gblocks Server</td><td></td><td></td><td>http://molevol.cmima.csic.es/castresana/Gblocks_server.html</td></tr><tr><td>&lt;em&gt;Vitis vinifera&lt;/em&gt; cv. Corvina gene expression Atlas datamatrix</td><td></td><td></td><td>https://www.researchgate.net/publication/273383414_54sample_&lt;br /&gt;datamatrix_geneIDs_Fasoli2012</td></tr><tr><td>Multi Experiment Viewer (MeV)</td><td></td><td></td><td>http://mev.tm4.org/#/welcome</td></tr><tr><td>Sequence Read Archive (SRA)</td><td></td><td></td><td>https://www.ncbi.nlm.nih.gov/sra</td></tr><tr><td>R</td><td></td><td></td><td>https://www.r-project.org/</td></tr><tr><td>EMBOSS Needle (EMBL-EBI)</td><td></td><td></td><td>http://www.ebi.ac.uk/Tools/psa/emboss_needle/</td></tr></tbody></table>

comprehensive workflow for the genome-wide identification and expression meta-analysis of the atl e3 ubiquitin ligase gene family in grapevine

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

The VIT_05s0077g01970 gene, identified as the most similar to A. thaliana ATL2 (At3g16720) through a BLASTp search, was used as probe to survey the ATL family members in the grapevine genome (V. vinifera cv Pinot Noir PN40024). The PSI-BLAST analysis converged after a few cycles revealing a list of putative genes belonging to the grapevine ATL gene family (Figure 1A). The presence of the canonical RING-H2 domain for each candidate was evalua...

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

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Biology

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Learn a comprehensive workflow for genome-wide identification and expression meta-analysis of the ATL E3 ubiquitin ligase gene family in grapevine. This method enables complete gene family characterization for plant genetics research.

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine (Video) | JoVE Journal

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

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