CRA Open Workshop Applied Biology and Microbiology in the ...sito.entecra.it/portale/public/documenti/bonfante.pdf · Housekeeping genes: GAPDH Ubiquitin Actin Tubulin PP2A cRNA RNA

Paola BonfanteDepartment of Life Science and Systems Biology

Università di Torino, IPP-CNR, Italy

CRA Open Workshop

Applied Biology and Microbiology in the agricultura l industry: Research and Innovation

Plant –Microbe Interactions: Mycorrhizas

Symbiontfungi

Symbiontfungi

PlantsPlants

MycorrhizaeMycorrhizae

Plant Microbiota: Not only Prokaryotes

Fungi play a crucial role assymbiont and pathogenic microbes

AMs and Nodules: the symbioses 'that help feed the world'

(Marx, Science2004)

AM fungi

Plants

P, Nand other nutrients

How? Which services? Which mechanisms?

C

Plant Biology 2013: feeding the global world population

Bonfante and Anca, Ann. Rev. Microbiology 2009

The biodiversity of mycorrhizal fungi

Symbiont fungi:genomics and functional genomics

Plants: Cellular and Molecularresponses to AM fungido you speak plantish or fungish?

Focus onArbuscularMycorrhizas:

Mycorrhizas: research and innovation directives

AM fungi are one of the most widespread component ofthe plant microbiota

Ascomycota

Basidiomycota

Schüßler et al 2001, Kruger et al., 2012

Multigenomic organisms vs homogeneous mycelia

Obligate promiscuous biotrophs, multinucleated, asexual microbes

Glomeromycota, amonophylethic group

AM fungi: how to investigate their diversity

Lumini et al. 2010Disclosing arbuscular mycorrhizal fungal biodiversity in soil through a land-use gradientusing a pyrosequencing approachEnvironmental Microbiology 12 (8): 2165-2179

A.Orgiazzi et al 2012Unravelling Soil Fungal Communities from Different Mediterranean Land-Use BackgroundsPlos One, 2012

Barcoding approachesreveal AM fungi dynamics in diverse environments

NGS approaches454, Illumina

Fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological servicesas decomposers, plant mutualists and pathogens. The Mediterranean area is a biodiversity hotspot that isincreasingly threatened by intense land use. Therefore, to achieve a balance between conservation and humandevelopment, a better understanding of the impact of land use on the underlying fungal communities is needed.

Rice Microbiome RISINNOVA (Lupotto /Valè)

Microbial Biodiversity-

Rhizosphere functioning

Wetland versus upland 454-TitaniumRoots versus soil 16-18sRNA, ITSProkayotes versus Fungi

Aim: to dissect the microbiome associated with soil and roots of Oryza sativa

P. Abbruscato, P. PiffanelliE. Lumini, S.Ghignone

Prokaryotic Communities (Bacteria/Archaea)UNIFRAC WEIGHTED ANALYSIS UNIFRAC WEIGHTED ANALYSIS

MANAGEMENTMANAGEMENT

LOWLANDLOWLAND

UPLANDUPLAND

ARCHEAARCHEA

EUBATTERIEUBATTERI

Most significant differences are associated to the compartment (Root vs Soil)In soil no significant differences due to the management systems, even among rice maturation stages.

In root some differences are observed upon low/upland management along the different stages.

Upland- Soil

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

L5A1.1s L5A1.2s L5A1.3s L5A2.1s L5A2.2s L5A2.3s L5A3.1s L5A3.2s L5A3.3s

Unidentified

Unidentified Zygomycota

Zygomycota; Zygomycetes; Endogonales

Zygomycota; Incertaesedis; Zoopagales

Zygomycota; Incertaesedis; Mucorales

Zygomycota; Incertaesedis; Mortierellales

Zygomycota; Incertaesedis; Kickxellales

Zygomycota; Incertaesedis; Harpellales

Zygomycota; Incertaesedis; Entomophthorales

Neocallimastigomycota; Neocallimastigomycetes;Neocallimastigales

Incertae sedis

Glomeromycota; Glomeromycetes; Glomerales

Glomeromycota; Glomeromycetes;Diversisporales

Glomeromycota; Glomeromycetes;Archaeosporales

Unidentified Chytridiomycota

Chytridiomycota; Monoblepharidomycetes;unidentified

Chytridiomycota; Monoblepharidomycetes;Monoblepharidales

Chytridiomycota; Chytridiomycetes;Spizellomycetales

Chytridiomycota; Chytridiomycetes;Rhizophydiales

Chytridiomycota; Chytridiomycetes;Rhizophlyctidales

Chytridiomycota; Chytridiomycetes;Lobulomycetales

Chytridiomycota; Chytridiomycetes; Incertaesedis

Chytridiomycota; Chytridiomycetes; Chytridiales

Blastocladiomycota; Blastocladiomycetes;Blastocladiales

Unidentified Basidiomycota

Lowland - Soil

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

L6S1.1s L6S1.2s L6S1.3s L6S2.1s L6S2.2s L6S2.3s L6S3.1s L6S3.2s L6S3.3s

Unidentified










Incertae sedis















Upland - Root

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

L5A1.1r L5A1.2r L5A1.3r L5A2.1r L5A2.2r L5A2.3r L5A3.1r L5A3.2r L5A3.3r

Unidentified










Incertae sedis















Lowland - Root

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

L6S1.1r L6S1.2r L6S1.3r L6S2.1r L6S2.2r L6S2.3r L6S3.1r L6S3.2r L6S3.3r

Unidentified










Incertae sedis















Eukaryotic Communities (Fungi)

40.2%

28.6%

12.4%

7.8%5.5%

3.7%

0.2%

0.3%

0.2%

Most significant differences are associated to the compartment (Root vs Soil)In soil no significant differences due to the management systems, even among rice maturation stages.In root the most important differences are observed upon low/upland management especially because one important group of the eukaryotic community, the Arbuscular Mycorrhizal Fungi is not present in submerged condition

GLOBAL DIVERSITYGLOBAL DIVERSITY

Soil

Root

General Conclusions General Conclusions

The analysis of sequence datasets from rice rhizosphere revealedstatistically supported differences in microbial microbiome between the two agricultural management systems (i.e. aerobic versus anaerobic growth conditions).

The most important differences detected are associated to rice root compartment rather than soil compartment. In bulk soil the wholemicrobiota (Fungi, AMF, Bacteria, Archaea) is less prone to fluctuations.

NGS analysis allows us to determine the main forces driving the structure, relationships and the composition of different microbial communities associated with rice.

Perspectives and Innovation

To develop microbiota -soil cards which allow to better define agronomical procedures(cultivar selection, fertilizer amounts; microbial dynamics/environment)

Development of platforms for microbiota -soil databases at international scale to offer provisional models (climate change; CO2 rise; nutrient flushes …)

Bonfante and Anca,

Ann. Rev. Microbiology2009

The biodiversity of mycorrhizal fungiand of their associated bacteria

Symbiont fungi:genomics and functional genomics

Plants: Cellular and Molecularresponses to AM fungido you speak plantish or fungish?

Focus onArbuscularMycorrhizas:

Mycorrhizas: research and innovation directives

Medicago Carrot

The functional key for AM success: A flow of nutrients

Bonfante e Genre 2010, Nature communications

Genomics, transcriptomics and metabolomics reveal that the interaction between fungal symbionts and plants is characterized by a balanced nutritional exchange

2. Lessons from the genomic approach

The genome of an arbuscular mycorrhizal fungus

provides insights into the oldest plant symbiosis

A long and hard way

Tisserant et al submittedPNAS

Tisserant et al 2013 submitted

The genome of an arbuscular mycorrhizal fungusprovides insights into the oldest plant symbiosis

Rhizophagus irregularis strain DAOM-197198

Size of the genome assembly 153 Mbcoding space 98% complete on the basis of conserved core eukaryoticsingle-copy genes

28,232 protein coding genes predictedGenome is rich in A and T bases (A + T content 72%)Transposable elements (TEs) make up 11% of the assemblyBut much higher TE abundance (36%), including several retrotransposons. retrotransposons are long (9 to 25 kb), highly repetitive and nested: explanation for the observed fragmentation

of the assembly?

Lack of an efficient elimination mechanism?

Genome polymorphism: are AM fungi heterokaryotic a nd harbour genetically different nuclei?Neither segmental duplication nor distinct haplotypic contigs were detected suggesting that the assembled data is NOT composed of multiple genomes

Tisserant et al 2013 submitted

The genome of an arbuscular mycorrhizal fungusprovides insights into the oldest plant symbiosis

The Rhizophagus gene repertoire.

Hallmarks: -a lack of genes encoding plant cell wall degrading enzymes-a lack of genes involved in toxin and thiamine synthesis.-a battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues

Perspectives and Innovation

Very limited information on AM genomes

One sequenced genome provides a too narrow view

Why AMF are obligate biotrophs ?

If we overcome this difficulty we can grow them Producing efficient inocula

Target : AMF for different crops, environments, agronomic procedures, drought stressesCan we decrease the fertilization cost?

Lessons from tomatoes…..

The fruit phosphorus concentration

Non Myc-Low Fer Non Myc-High Fert Myc-Low Fert

CA

Parniske 2008

Reprogramming of plant cells

3. Genetics bases, Signalling and Accomodation processDissecting plant responses to AM fungi

In the rhizosphere Signalling

At the root surface Physical Contact

Inside the root Colonization- Functioning

Myc Factor/s

Akiyama et al., Nature June 2005Plant sesquiterpenes inducehyphal branching in AM fungi

Besserer et al, PLOS 2006Plant Physiol., 2008

Enod 11 activationLateral root formation

Signaling events in the rhizosphere: Dating in the darkBonfante and Requena, 2011

Maillet et al Nature, 2011

Lotus Affymetrix Microarray •Chip designed by the group of MichaelUdvardi (MPI Golm, Germany)

• based on the genomic sequences from the Lotus japonicus sequencing project (http://www.kazusa.or.jp) and the EST list at http://www.tigr.org

Lotus japonicus + Gigaspora margarita

No. of probe sets: L.j. >50,000

No. of transcripts: L.j. >50,000

No. of probe sets: M.loti ~ 11,000

No. of transcripts: M.loti ~ 11,000

Array format: 49

Feature size: 11µm

Oligo probe length: 25mer

Housekeeping genes:

GAPDH

Ubiquitin

Actin

Tubulin

PP2A

cRNA

RNA

Regulated genes

Mike Guether

95 protein turnover, cell wall, membrane dynamics

47 transporters24 TFs Guether et al., New Phytol 2009

Genome-wide reprogramming: new emerging functions

PUTATIVE ANNOTATION

N°DI GENI

Phosphate transporters 1

Peptide transporters 7

Ammonium transporters Guether et al 2009 1

Nitrate transporters 4

Amino acid transporters Guether et al 2011 3

Potassium transporters 1

Sulfate transporters work in progress

3

Aquaporins/water channels Giovannetti et al 2012 5

Sugar transporters 2

Zinc transporters 1

Other metal ion transporters 1

PUTATIVE TF

N°DI GENI

Myb like 1

Scarecrow 3

Other 20

LjPT4: FD=1.400

Omologo di MtPT4

LjMAMI:FD= 20.300

LjMYB expression is only revealed in AM roots and is Pi independent

The AM responsive LjMYB is a putative TF (Volpe et al 2012,Plant Journal)

Veronica VOLPE

LjMYB is related to P-starvation TF genes

LjMYB expression is co-regulated with LjPT4

LjMYB is localized in the nuclei of arbusculated cells in Lotus plants

pLjMYB:eGFP:LjMYB

Genes which are AM dependent may be important in the whole plant development

Effect on fruit production

and phenology

Phenotypicalapproach

Transcriptomic approach

Effect on global fruit gene

expression

Metabolicapproach

Effect on the amino acid

fruit content

Different ApproachesSalvioli et al 2012

Different cultivars

Micro-Tom Moneymaker

Ailsa Craig and mutants in ripening light signalling

Different fertilization conditions

3,2µM P 300µM P 3,8mM P

Systemic effects: another key for AM success ?

A. Salvioli I. Zouari

Do fruits from tomato plants respond to AM fungi?

3. Perspectives and Innovation

To better decipher the molecular dialogue to promotecolonization under in field conditions

To better describe the systemic effects of AM fungi on edible parts of the plant

To better understand whether we can improve thenutraceutical properties of fruits from mycorrhizal plants

AM fungi: Crucial components of the Plant Microbiome andDrivers of new functions

A commercial inoculum

50 days post germination

Beneficial Microbes as AMF are crucial for plant health

They need to control Plant Immunity System to colonize roots

Their genome indicates how they do not activate plant defense

They deeply change the transcriptomic plant profile

But…Not only a a nutrient-transport business

New Knowledge on signalling and functioning

To manipulate the symbiosis, applying fungal molecules required for starting the interaction

To identify genes which have an impact on general plant processes (development, root branching, transition to flowering, ripening)

And to move to the field!

LIPM - INRA/CNRSCastanet Tolosan (France)

David BarkerG.Becard

Noble Foundation, USAMichael Udwardi

Wageningen UniversityThe Netherlands

J.G.M. Pierre de Witt

Ton Bisseling, Sergey Ivanov

University of TubingenUwe Ludewig, Benjamin Neuhäuse,

Marek Dynowski

Cornell UniversityJ.Giovannoni

Hannover UniversitatHelge Kuster

MIUR, PRIN 2008, INTEGRAL, BIOBIT 2008-2012, ARaS, Risinnova

Department of Biology, University of MilanoAlex Costa

Many thanks to you! And to…

Department of Life Science and System Biology – IPP-CNR

Biodiversity: Erica Lumini; Stefano Ghignone, Alberto Orgiazzi V.Bianciotto IPP CNR

Genome Sequencing: Raffaella Balestrini, Luisa Lanfranco in the frame of international collaborations

F Martin, INRA

Plant Fungal Interactions: Andrea Genre, Marco Giovannetti, Veronica Volpe, Inés Zouari, Alessandra Salvioli, Matteo Chialva, Mara Novero, Mike Guether,

A.Faccio

Paola Bonfante

Cra- Fiorenzuola Paolo Bagnaresi

Bonfante and Anca, Ann. Rev. Microbiology 2009

Mycorrhizas: a tripartite association

Plants

Symbiont fungi

Endobacteria and associated bacteria

Documents

CRA Open Workshop Applied Biology and Microbiology in the ...sito.entecra.it/portale/public/documenti/bonfante.pdf · Housekeeping genes: GAPDH Ubiquitin Actin Tubulin PP2A cRNA RNA