10.6092/issn.2531-7342/6647
L’obiettivo di questo lavoro è stato quello di applicare lo strumento della metaproteomica a suoli di tartufaia già approfonditamente caratterizzati tramite approcci di metagenomica. Per fare questo è stato impiegato un database creato ad hoc a partire dagli organismi fungini, batterici e dalle piante identificati negli studi di metagenomica. Attraverso l’identificazione delle proteine estratte dal suolo della tartufaia si è cercato di chiarire quali processi metabolici fossero ivi attivi e quali fossero specificatamente presenti nel pianello di Tuber melanosporum, cioè in un’area caratterizzata da scarsa vegetazione attorno alla sua pianta ospite. Gli studi di metagenomica avevano messo in evidenza una minore biodiversità dei funghi nel pianello e che T. melanosporum era il fungo dominante, mentre i funghi ectomicorrizici appartenenti al phylum dei Basidiomycota diminuivano, suggerendo competizione con il tartufo. All’interno del pianello risultavano anche scarsamente presenti batteri appartenenti sia al genere Pseudomonas che alla famiglia delle Flavobacteriaceae. Con l’applicazione della metaproteomica è stato possibile individuare quali processi biologici fossero maggiormente presenti nel pianello, o esclusivi. I processi legati a multipli tipi di stress erano principalmente rappresentati, e presenti soprattutto nelle piante erbacee. Questo studio ha permesso quindi di svelare il funzionamento di un ambiente così particolare come il pianello, dove il tartufo nero è il protagonista.
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Who is out there? What are they doing? Application of metagenomics and metaproteomics to reveal soil functioning
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46
A. Mello, E. Zampieri                               Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647




 Who is out there? What are they doing?
 Application of metagenomics and metaproteomics
 to reveal soil functioning
 ___________________________________________
                                   Antonietta Mello1, Elisa Zampieri2
 1
   Istituto per la Protezione Sostenibile delle Piante, SS di Torino, CNR, Viale P.A. Mattioli 25, I-10125
 Torino
 2
   Dipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA), Università di Torino, Largo Paolo
 Braccini 2, I-10095 Grugliasco (TO);
 CREA, Council for Agricultural Research and Economics, Rice Research Unit S.S. 11 to Torino, Km 2,5
 13100, Vercelli

 corresponding author: Antonietta Mello, antonietta.mello@ipsp.cnr.it

 Abstract

     In order to link microbial community composition to ecological processes happening in the brûlé, a
     metaproteomics analysis was applied to a brûlé previously characterized by metagenomics. The metagenomics
     data had showed a reduced fungal biodiversity, a dominance of Tuber melanosporum and a reduced presence
     both of ectomycorrhizal Basidiomycota and of bacteria belonged to Pseudomonas and Flavobacteriaceae inside
     the brûlé. By metaproteomics analysis, the identified proteins revealed which biological processes were more
     represented or only present in the brûlé, and, among them, processes related to multiple stresses were identified
     in herbaceous plants. This study demonstrates that combining the data of metagenomics and metaproteomics
     gives the opportunity to potentially reveal the functioning of any environment.


     Keywords: truffle-ground soil; Tuber melanosporum;fungi; bacteria; herbaceous plants


 Riassunto

     L’obiettivo di questo lavoro è stato quello di applicare lo strumento della metaproteomica a suoli di tartufaia
     già approfonditamente caratterizzati tramite approcci di metagenomica. Per fare questo è stato impiegato un
     database creato ad hoc a partire dagli organismi fungini, batterici e dalle piante identificati negli studi di
     metagenomica. Attraverso l’identificazione delle proteine estratte dal suolo della tartufaia si è cercato di
     chiarire quali processi metabolici fossero ivi attivi e quali fossero specificatamente presenti nel pianello di
     Tuber melanosporum, cioè in un’area caratterizzata da scarsa vegetazione attorno alla sua pianta ospite. Gli
     studi di metagenomica avevano messo in evidenza una minore biodiversità dei funghi nel pianello e che T.
     melanosporum era il fungo dominante, mentre i funghi ectomicorrizici appartenenti al phylum dei
     Basidiomycota diminuivano, suggerendo competizione con il tartufo. All’interno del pianello risultavano anche
     scarsamente presenti batteri appartenenti sia al genere Pseudomonas che alla famiglia delle Flavobacteriaceae.
     Con l’applicazione della metaproteomica è stato possibile individuare quali processi biologici fossero
     maggiormente presenti nel pianello, o esclusivi. I processi legati a multipli tipi di stress erano principalmente
                                                                                                                     1
A. Mello, E. Zampieri                              Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647



    rappresentati, e presenti soprattutto nelle piante erbacee. Questo studio ha permesso quindi di svelare il
    funzionamento di un ambiente così particolare come il pianello, dove il tartufo nero è il protagonista.

    Parole chiave: suolo di tartufaia; Tuber melanosporum; funghi; batteri; piante erbacee

Metagenomics is the genomic analysis of the microorganisms contained in environmental samples such as
ocean, soil and gut (Venter et al., 2004, Vogel et al., 2009, Feltman, 2013). It is based on direct extraction of
DNA of microorganisms from any environment, in this way avoiding their cultivation through the classical
microbiology methods. The transition from classical microbiology to modern metagenomics studies has
required the development of new technologies, such as the high-throughput sequencing technologies and the
development of new computational methods to collect and extract information from complex datasets
(Escobar-Zepeda et al., 2015). The species richness and the evenness are the two attributes that describe a
microbial community although the use of diversity indexes is a better way to quantify and compare microbial
diversity among samples. According to Escobar-Zepeda et al. (2015), the microbial diversity can be studied
using two different approaches: (1) Amplicon sequencing or (2) Shotgun metagenomics. In the first
approach, taxonomical informative markers such as 16S rRNA gene for prokaryotes and intergenic
transcribed spacers (ITS) or the large ribosomal subunit (LSU) gene for eukaryotes are amplified from DNA
extracted from communities. Since the amplicon sequence analysis relies on just one gene, rather than on all
the genes from the community, is more correctly defined “metaprofiling” instead of metagenomics. In the
second approach, large fragments or even complete genomes from a community can be reconstructed
through the characterization of several coding and non-coding sequences that can be used as phylogenetic
markers. Metaprofiling has been widely used to answer the predominant question in microbial ecology:
“Who is out there? “
A study-case among the various studied environmental samples is represented by the ”brûlé”, which is an
area of the truffle-grounds characterized by scarce vegetation around the host plant of the black truffle Tuber
melanosporum. Analyses of ITS and 16S of the rRNA genes directly amplified from the soil of a brûlé, in a
French truffle-ground, have revealed its bacterial and fungal communities composition (Napoli et al., 2010;
Mello et al., 2011; Mello et al., 2013; Mello et al., 2015). Clear differences were shown between the fungal
communities present inside and outside the brûlé, together with lower fungal biodiversity inside the brûlé
where T. melanosporum was the dominant fungus whereas the other ectomycorrhizal fungi decreased in
abundance, indicating that T. melanosporum competes with them (Napoli et al., 2010; Mello et al., 2011).
The patchy herbaceous plants in the brûlé were extensively colonized by arbuscular mycorrhizal fungi, as
were the plants outside the brûlé. Examination of this fungal diversity in the soil showed reduced species
richness inside the brûlé, compared with that outside the brûlé. Furthermore, members of Diversispora,
Acaulospora, and Archaeospora were only found in the brûlé, in roots or soil, revealing clear differences
between inside and outside the brûlé (Mello et al., 2015). Also bacterial communities living in the brûlé were
investigated. According to Mello et al. (2013), Firmicutes (e.g., Bacillus), several genera of Actinobacteria,
and a few Cyanobacteria were more abundant inside the brûlé, whereas Pseudomonas and several genera
within the family Flavobacteriaceae were more abundant outside the brûlé. Taking together, these
metagenomics studies clearly revealed the fungal and bacterial community composition inside and outside
the brûlé (Table 1), answering to the question: Who is there? But in microbial ecology it is necessary
answering also to a second question: What are they doing?
In order to link microbial community composition to ecological processes happening in the brûlé, Zampieri
et al. (2016) applied a metaproteomics analysis to the brûlé previously characterized by metagenomics.



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A. Mello, E. Zampieri                                Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647


 Tab.1. Metagenomics data concerning the bacterial and the fungal communities present in the truffle-ground analysed
 by Napoli et al. (2010); Mello et al. (2011); Mello et al. (2013); Mello et al. (2015).
 Tab.1 Dati di metagenomica riguardanti le comunità batteriche e fungine presenti nella tartufaia analizzata da Napoli et
 al. (2010); Mello et al. (2011); Mello et al. (2013); Mello et al. (2015).

                                    Bacteria                                  Fungi

                     Actinomyces                              Acaulospora sp.
                     Arthrobacter                             Acremonium sp.
                     Bacillus                                 Amanita sp.
                     Chryseobacterium                         Aporospora terricola
                     Flavobacterium                           Archaespora trappei
                     Frankia                                  Arthrographis sp.
                     Geobacillus                              Ascobolus sp.
                     Geodermatophilus                         Aspergillus insuetus
                     Massilia                                 Aspergillus ustus
                     Microbacterium                           Beauveria felina
                     Micromonospora                           Bionectria ochroleuca
                     Mycobacterium                            Cladophora sp.
                     Myxococcus                               Chaetomium globosum
                     Nitrospira                               Claroideoglomus sp.
                     Pedobacter                               Coniosporium sp.
                     Prochlorococcus                          Cordyceps bassiana
                     Pseudomocardia                           Cryptococcus aerius
                     Pseudomonas                              Curvularia inaequalis
                     Riemerella                               Cylindrocarpon sp.
                     Rubrobacter                              Didymella bryoniae
                     Saccharothrix                            Diversispora sp.
                     Streptosporangium                        Epicoccum sp.
                                                              Funneliformis caledonius
                                                              Funneliformis mosseae
                                                              Fusarium oxysporum
                                                              Fusarium solani
                                                              Genea
                                                              Geoglossum
                                                              Geosmithia sp.
                                                              Gibberella moniliformis
                                                              Glomus indicum
                                                              Glomus sp.
                                                              Helminthosporium solani
                                                              Hymenogaster sp.
                                                              Hypocrea koningii
                                                              Inocybe sp.
                                                              Leptosphaerulina sp.
                                                              Lewia infectoria

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A. Mello, E. Zampieri                           Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647



                                                       Lophiostoma sp.
                                                       Microdochium sp.
                                                       Mortierella alpina
                                                       Myrothecium cinctum
                                                       Naucoria (Alnicola) sp.
                                                       Nectria sp.
                                                       Neonectria radicicola
                                                       Paraglomus majewskii
                                                       Penicillium canescens
                                                       Periconia macrospinosa
                                                       Peziza sp.
                                                       Phaeosphaeria sp.
                                                       Phoma sp.
                                                       Pluteus sp.
                                                       Podospora sp.
                                                       Preussia sp.
                                                       Psathyrella
                                                       Pulvinula constellatio
                                                       Pyrenochaeta sp.
                                                       Rhizophagus irregularis
                                                       Rhizophydium sp.
                                                       Sclerocystis sp.
                                                       Scleroderma sp.
                                                       Scutellospora sp.
                                                       Septoglomus sp.
                                                       Spizellomyces sp.
                                                       Tetracladium sp.
                                                       Tomentella sp.
                                                       Trichoderma sp.
                                                       Tricholoma sp.
                                                       Tuber magnatum
                                                       Tuber melanosporum
                                                       Tuber rufum
                                                       Tuber sp. (T. borchii complex)
                                                       Vermispora sp.
                                                       Verrucaria sp.
                                                       Verticillium catenulatum
                                                       Verticillium dahliae
                                                       Xerocomus rubellus


Metaproteomics has been recently applied to different environments, i.e., soil, sediments, marine, and
freshwater systems, proving to be a powerful tool to describe metabolic processes active in these
environments (Siggins et al., 2012). It is the study of all the proteins expressed by the organisms within an
ecosystem at a specific time (Bastida et al., 2014). It has the potentiality to provide a link between
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A. Mello, E. Zampieri                                   Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647



 phylogenetic microbial community data and functional ones (Bastida and Jemlich, 2016; Keblinger et al.,
 2016; Wang et al., 2016), but it is negatively influenced by different factors (e.g., soil heterogeneity, high
 microbial diversity, incomplete metagenomic information, etc.) (Keblinger et al., 2016 and references inside;
 Wang et al., 2016). The extraction of proteins from soils is a challenge because of organic compounds, such
 as complex carbohydrates, lipids and phenolic compounds, humic acids, and inorganic compounds, such as
 silt and clay minerals (Keblinger et al., 2016). Another obstacle in metaproteomics analysis could be the
 protein identification, which depends on the features (design, capacity, quality) of the database chosen for
 the analysis (Wang et al., 2016). To overcome these difficulties, Zampieri and colleagues (2016) used three
 different protein extractions methods to increase the yield of proteins from the studied brûlé and they built a
 homemade database based on the metagenomics data previously obtained from the same truffle-ground. In
 this study, the authors managed to extract 638 proteins, and in particular, 411 and 309 proteins were specific
 for inside and outside the brûlé, respectively. The organismal classification demonstrated that bacterial
 proteins were more abundant than those of Eukaryota, both inside and outside the brûlé, and surprisingly that
 proteins of Viridiplantae were more inside than outside (Zampieri et al., 2016) (Fig. 1).




 Fig. 1. Organismal classification of the identified proteins inside and outside the brûlé at superkingdom level (a) and at
 fungal and viridiplanta level (b) on the base of the metaproteomics data of Zampieri et al. (2016).
 Fig. 1. Classificazione degli organismi delle proteine identificate dentro e fuori il pianello a livello di superregno (a) e a
 funghi e di piante (b) sulla base dei dati di metproteomica di Zampieri et al. (2016).




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A. Mello, E. Zampieri                            Italian Journal of Mycology vol. 46 (2017) ISSN2531-7342
DOI: 10.6092/issn.2531 – 7342/6647



The identified proteins were characterized on the basis of Gene Ontology that allows to define the biological
pathways in which they are involved, as well as their specific molecular functions. Moreover, the authors
identified over-represented biological processes and molecular functions by using the Fisher’s Exact Test: in
particular, 57 processes were over-represented inside the brûlé and, among them, 14 were only specific
inside, and related to stress, glycolysis and the Krebs cycle, sulphur metabolism, etc. Interestingly, the
processes related to response to some kinds of stress (osmotic and salt), stimuli (abiotic and temperature),
inorganic substances and metal ions (cadmium) were present only inside the brûlé, demonstrating how the
two environments were different from a metabolic point of view, and the stress proteins were identified in
plants, fungi and bacteria, suggesting that the organisms living in the brûlé face stress experience (Zampieri
et al., 2016). Moving to T. melanosporum proteins, the search was done against the T. melanosporum
genome      database     from    MycorWeb         (version    of    February     2015;     12826     sequences
(http://mycor.nancy.inra.fr/IMGC/TuberGenome/download.php?select= fast). Overall, 265 T. melanosporum
proteins were identified, whose 148 were specific inside the brûlé and 92 outside. Although the majority of
T. melanosporum truffles are usually collected inside the brûlé, the presence of black truffle proteins outside
the brûlé was expected, in agreement with the metagenomics data, namely nucleotidic sequences of T.
melanosporum. T. melanosporum proteins as heat shock proteins and co-chaperonine, laccase and tyrosinase
were identified only inside the brûlé, but the differences between inside and outside were not significant on
the base of the Fisher Exact Test (Zampieri et al., 2016). This finding may suggest that the truffle proteins
are not involved in the phenomenon of brûlé, i.e., the decrease of herbaceous plants, which are on the
contrary affected by volatile organic compounds, delivered by T. melanosporum (Splivallo et al., 2007,
2011; Streiblová et al., 2012). Truffle volatiles could be then responsible of the stress and defence response
in the organisms living inside the brûlé, principally the herbaceous plants where chaperonine proteins,
superoxide dismutase, heat shock proteins, late embryogenesis abundant protein were over-represented
(Zampieri et al., 2016). Even if the biodiversity is reduced where the vegetation is scanty, the brûlé is an
active environment.
This study demonstrates that combining the data of metagenomics and metaproteomics we have nowadays
the opportunity to potentially reveal the functioning of any environment. The year of the soil has been just
celebrated and new frontiers are open to disclose not only “Who there is” but also “What they are doing”.

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