1
Symbiosis with an endobacterium increases the fitness of a mycorrhizal fungus,
2
raising its bioenergetic potential
3
4
Alessandra Salvioli1, Stefano Ghignone2, Mara Novero1, Lorella Navazio3, Francesco
5
Venice1, Paolo Bagnaresi4, Paola Bonfante*1
6
7
Summary
8
The Supplementary Information includes Supplementary Materials containing
9
Methods and Results, eleven Supplementary Figures and twelve Supplementary
10
Tables.
11
Supplementary Materials and Methods
12
RNA extraction and sample preparation for sequencing
13
Samples belonging to Data Set 1 (see below) were subjected to a normalization
14
procedure that equalizes transcript concentrations to increase the sequencing
15
efficiency of rare transcripts. First, cDNA was obtained from 500 ng of total RNA for
16
each sample using the SMARTer PCR cDNA synthesis kit (Clontech, Mountain
17
View, CA, USA). Then the cDNA libraries were normalized using the Trimmer-2
18
cDNA normalization kit (Evrogen, Moskow, Russia) following the manufacturer's
19
instructions. The normalized cDNAs were purified using the QIAquick PCR
20
purification kit (Qiagen, Germany) and 1.5 μg of clean material was sent to the
21
sequencing company (Fasteris, Geneva, Switzerland), where independent indexed
22
libraries were prepared and run in a single Illumina Hiseq channel.
1
1
For Data Set 2 (not normalized, see below), 1.4 μg of total RNA was sent to the
2
sequencing facility for library construction and Illumina Hiseq sequencing.
3
4
Generation of Data Sets 1 and 2 and de novo transcriptome assembly
5
The normalized paired-end libraries were generated with the aim of enriching the
6
dataset for rare transcripts, whereas the single-end libraries were constructed to
7
investigate the differential gene expression in the presence and in the absence of the
8
endobacterium.
9
Data Set 1 was composed of four in vitro normalised paired-end libraries, obtained
10
from the B+ line of G. margarita, sampled in the following stages of the fungal life
11
cycle: quiescent spores (GOU-13), germinating spores (GOU-14), spores treated with
12
strigolactone (GOU-15), and extraradical mycelium (GOU-16).
13
The total number of reads in channel (PE) was 188'118'376 for a total 18'811'837'600
14
bases, with an average Q30 of 85.6%. A custom script, derived from Cutadapt (Martin
15
M., 2011) (courtesy P. Giannini & M. Malavasi) was used to remove any remaining
16
sequences from the primers used in library construction. Read quality was then
17
checked by FastQC script, and since quality scores dropped below 25,
18
TrimmomaticPE (Bolger et al., 2014) was used to trim poor quality ends from each
19
pair
20
SLIDINGWINDOW:4:25 MINLEN:25). After quality trimming, 76.00 to 79.13% of
21
both paired ends remained (Table S11).
22
Data Set 2 was composed of 14 single-end libraries, obtained from the B+ and B-
23
lines of G.margarita, sampled in the following stages of the fungal life cycle:
24
germinating spores (B+: GDR-25/26/27; B-: GDR-28/29/30), spores treated with
25
strigolactone (B+: GDR-31/32; B-: GDR-33/34), and mycorrhizal roots (B+: GDR-
(TrimmomaticPE
settings:
2
LEADING:10
TRAILING:10
1
35/36; B-: GDR-37/38). According to the ENCODE Consortium Standards,
2
Guidelines and Best Practices for RNA-Seq (v1.0), a minimum of two replicates were
3
collected for critical samples. (The total number of reads was 357,777,717, with an
4
average Q30 of 91.42%. No quality trimming was required for this dataset.
5
The de novo assembly of Data Set 1 and 2 libraries was performed on a 60 core and
6
256 GB RAM machine, running Ubuntu server 12.04 LTS, using Trinity
7
v.Trinityrnaseq_r20131110 (Grabherr et al., 2011). First trials indicated that the
8
available amount of memory was not sufficient to handle all the raw reads and,
9
following the Trinity manual, we performed in silico reads normalization for each of
10
the libraries from Data Set 1 and 2, to a max coverage of 30. Normalization results are
11
summarized in Table S11. Libraries from myc tips (GDR-35 to GDR-38) were not
12
subjected to normalization and were not used for the de novo assembly, since only a
13
fraction of reads were ascribable to the fungal transcriptome (13.4%, 17.7%, 16.6%,
14
7.6%), whereas the majority of reads were from the plant host (Lotus). The Data Set 1
15
and 2 read counts are reported in Table S11.
16
All the normalised single-end Data Set 2 libraries were merged together with the
17
paired-end Data Set 1 left ends.
18
Trinity was run with the following characterizing options, suited to assemble a gene-
19
dense compact genome, such as a fungal genomes, and to minimize the number of
20
isoforms per transcript: Trinity.pl --seqType fq --CPU 30 --JM 150G --
21
min_contig_length
22
group_pairs_distance 300 –extended_lock.
23
Transcript abundance estimation (computing expression values) was performed using
24
RSEM, using the original Data Set 2 reads (not in silico normalized), which were
25
mapped against the Trinity transcripts. RSEM was executed to estimate expression
350
--jaccard_clip
3
--min_kmer_cov
2
--CuffFly
--
1
values based on the resulting alignments. All the libraries from Data Set 2 were used
2
in this step, including the four libraries (GDR-35 to GDR-38) from mycorrhizal roots
3
not used for the de novo assembly. Identification and Analysis of Differentially
4
Expressed Trinity Genes and Transcripts were performed as described below.
5
Further downstream analyses were performed on nucleotide sequences of transcripts
6
or on likely coding regions, extracted from Trinity transcripts using TransDecoder,
7
including PFAM domain searches as ORF retention criteria. Sequence similarities
8
were captured with BLAST suite programs, using an e-value of 1E-5, querying all
9
Trinity transcripts or peptide predictions against the refseq_protein and R. irregularis
10
protein databases. Protein domains were identified using HMMER and TransDecoder.
11
SignalP was used to predict signal peptides and tmHMM was used to predict
12
transmembrane regions. RNAMMER was used to identify rRNA transcripts. All
13
downstream analyses were loaded into a Trinotate SQLite Database. Whole Trinity
14
transcript annotation was performed with Blast2Go.
15
Since the smallest contig size was set to 350 bp to reduce the number of isoforms per
16
component, the current assembly is not well suited to investigate the effector-like and
17
secreted protein repertoire of genes encoded by the fungus. Only one transcript
18
(comp7520_c0_seq1) annotated as secreted protein was in fact found in the Best
19
Reciprocal Hit (BRH) dataset with Rhizophagus (transcript ID:336770). Nevertheless,
20
the list of the 1,340 transcripts predicted in G. margarita as putatively secreted
21
proteins by SignalP is reported in Table S12.
22
GOslim representations (based on S. pombe GOslim, Figure S10) were produced to
23
summarize G. margarita biological processes and pinpoint highly represented GO
24
groups.
25
4
1
GO enrichment analyses
2
GO enrichment analyses were conducted with the goseq bioconductor package
3
version 1.14.0, while KEGG pathway pictures, KO (KEGG Orthology) mappings
4
were
5
http://www.genome.jp/kegg/kaas/) using as query
6
transcripts against the KEGG GENES database. All the details are provided in the
7
Supplemental material.
8
The Goseq package is specifically designed to limit length bias, which may affect
9
RNA-seq data (Secco et al., 2012). Gene lengths were obtained from the Trinity
first
obtained
from
KAAS
(KEGG
Automatic
Annotation
Server;
trinity-assembled Gigaspora
10
assembler output file (i.e. genes_feature_lengths file).
11
GO mappings for each gene were obtained by first running BLASTX against the
12
RefSeq_protein database (December 2013) using as query trinity-assembled isoforms
13
(86,183 isoforms) with an Expect value of 1e-05. The resulting XML file was then
14
loaded in Blast2GO (Gotz et al., 2008) and annotated with the following settings: E-
15
value filter: 1.0E-6; Annot_cutoff: 55; GO _weight: 5; HSP-Hit_Cov_cutoff: 0). GO
16
terms associated with isoforms were exported from Blast2GO and collapsed over the
17
corresponding genes (4,554 unique genes with at least one GO). An FDR cutoff of 0.1
18
was used as goseq parameter for GO enrichments.
19
20
KEGG maps
21
In order to obtain KEGG pathway pictures, KO (KEGG Orthology) mappings were
22
first
23
http://www.genome.jp/kegg/kaas/) using as query trinity-assembled Gigaspora
24
transcripts against the KEGG GENES database. The selected fungal database
obtained
from
KAAS
(KEGG
5
Automatic
Annotation
Server;
1
organisms were: uma, spo, tml, pcs, ang, ani, bfu, ncr, mgr, fgr, sce, yli, ecu, mgl,
2
mpr, ppl, cne, ure, cpw, aor, afm, ssl, ago, kla, ppa, vpo, cgr, and dha.
3
The resulting KO mappings were collapsed to genes and combined with the whole
4
expression data estimated by DESeq2. The Bioconductor package pathview version
5
1.2.3 (Luo & Brouwer, 2013) was used to generate relevant KEGG pathway pictures.
6
7
Phosphate measurements in roots and shoots from mycorrhizal clover plants
8
About 5 g of frozen roots and shoots from clover plants mycorrhized with B+ or B- G.
9
margarita were dried at 70°C for 48 hours, and digested in 2 ml 6 M HNO3 at 90°C
10
for one hour. The digestion product was diluted to 6 ml with milliQ water and filtered.
11
The P contents were determined using Inductively Coupled Plasma Atomic Emission
12
Spectrometry (ICP-AES) performed using a Liberty 100 Varian apparatus equipped
13
with a VGroove nebulizer and a Czerny–Turner monochromator (Department of
14
Mineralogical and Petrological Science, University of Turin).
15
16
Supplementary Results
17
The de novo assembly
18
We found that most of the transcripts described as key features of the R. irregularis
19
genome (Liu et al., 2013; Tisserant et al., 2013) were expressed by G. margarita as
20
well. In addition to the features illustrated in the main text, the following traits are
21
noteworthy.
22
For primary metabolism, the enzymes involved in glycolysis, gluconeogenesis, and
23
TCA cycle were all expressed at high levels; galactose metabolism was also highly
6
1
represented. The genes for the metabolism of purines, pyrimidines, and amino acids
2
were all well represented in the transcriptome of G. margarita at the asymbiotic
3
phase. By contrast, some of the central enzymes involved in fatty acid (FA)
4
biosynthesis seemed not to be expressed, but FA degradation was highly represented.
5
The genes involved in cell cycle and ribosome biogenesis were well represented in the
6
global transcriptome, as were the genes for ubiquitin-mediated proteolysis,
7
proteaseome, and the sec-dependent pathway. Also the BTB/POZ domain, a common
8
structural domain, was over-represented in both lines. The BTB/POZ domain is a
9
versatile protein-protein interaction motif that participates in a wide range of cellular
10
functions, including transcriptional regulation, cytoskeletal dynamics, ion channel
11
assembly and gating, and targeting proteins for ubiquitination (Stogios et al., 2005).
12
We found that other features were shared with mycorrhizal fungi whose genomes had
13
been sequenced. The expansion of the tyrosine kinase-encoding gene family involved
14
in signaling pathways was also a key feature of the ectomycorrhizal L. bicolor
15
genome (Martin & Selosse, 2008). The Sel1 repeat domain is a subclass of the
16
tetratricopeptide repeat region (TPR), a structural motif present in a wide range of
17
proteins, which are also overrepresented in G. margarita transcriptome. The TPR
18
domain mediates protein-protein interactions and the assembly of multiprotein
19
complexes (Blatch & Lassle, 1999). Proteins containing TPRs are involved in a
20
variety of biological processes, such as cell cycle regulation, transcriptional control,
21
mitochondrial and peroxisomal protein transport, and protein folding (D'Andrea &
22
Regan, 2003). Sel1-like repeat proteins have been shown to be involved in signal
23
transduction (Mittl & Schneider-Brachert, 2007). In fungi, bimA, a member of the
24
tetratricopeptide repeat family of proteins, has been found to be essential for the
25
completion of mitosis in A. nidulans (Odonnell et al., 1991); the TPR domain of a
7
1
serine/threonine phosphatase of A. oryzae was necessary for the full activity of the
2
enzyme (Feng et al., 2007). Other evidences indicate that the TPR domain may be
3
involved in virulence of fungal pathogens; C. neoformans with a mutation in the TPR-
4
containing gene CCN1 failed to cause systemic infection in mice and regained the
5
virulence when complemented with the wild-type CCN1 gene (Chung et al., 2003).
6
Candida albicans Tcc1p, a TPR domain containing protein, interacts with Tup1p to
7
regulate the morphological transition and virulence (Kaneko et al., 2006).
8
9
Quantitative analysis of differentially expressed genes: the presence of the
10
endobacterium is more relevant during the presymbiotic phase
11
Due to the complexity of the experimental design (two fungal lines investigated at
12
three stages, Figure S1) as a first step we performed a quantitative analysis of the
13
differentially expressed genes (DEGs) to understand whether the main driver of gene
14
expression in G. margarita depended on the life cycle stage or on the
15
presence/absence of the endobacterium. When the whole number of DEGs was
16
considered (B+ and B- lines considered together, purple arrows in Figure S1) as
17
shown in Venn diagrams (Figure S12), we concluded that the major transcriptional
18
changes take place during the transition from the asymbiotic to the symbiotic stage
19
(1559 versus 8420 transcripts, respectively). This quantitative analysis shows that the
20
association of G. margarita with its host plant leads to an important change in the
21
fungal transcriptomic profile in agreement with the patterns already shown by other
22
symbiotic fungi (i.e. Laccaria, Tuber, Rhizophagus) irrespective of their taxonomic
23
position and of the mycorrhizal typology they support: Laccaria and Tuber are
24
ectomycorrhizal fungi belonging to the Basidiomycota and Ascomycota, respectively
8
1
(Martin & Selosse, 2008; Martin et al., 2010) while Rhizophagus is an arbuscular
2
mycorrhizal fungus (Tisserant et al., 2013). Gigaspora seems therefore to follow the
3
trend typical of mycorrhizal fungi as well as of other plant-interacting fungi, such as
4
the endophyte Pirimorphospora indica (Zuccaro et al., 2011).
5
By contrast, when the data sets were investigated by comparing the fungus with and
6
without the endobacterium, Venn diagrams (Figure S4), showed that the major
7
changes in the transcriptome landscape related to the endobacterium presence were
8
recorded during the pre-symbiotic phase, i.e. in the germinating stage (G) and
9
strigolactone-treated condition (SL). As commented in the main text, the most DEGs
10
were identified in the G condition, followed by SL (9609 and 3427 transcripts,
11
respectively), while very few transcripts were differentially expressed during the
12
symbiotic phase. The low number of fungal transcripts may have two explanations:
13
the fungal transcripts may have been diluted by the massive number of plant
14
transcripts, or, more likely, the low number is related to a limited impact of the
15
endobacterium during the mycorrhizal phase. As demonstrated, the presence or the
16
absence of the endobacterium does not have any impact on the success of the
17
mycorrhization (Figure S5).
18
19
Gene expression and differential expression analysis between B+ and B- lines
20
In addition to the genes commented in the main text, other relevant differentially
21
expressed genes were also detected:
22
Phosphate transporter: a sequence sharing a very high similarity with P transporters
23
from filamentous fungi and yeasts, was downregulated in the B+ line
24
(comp27005_c0). It shares the highest similarity with Pho87, a P transporter
25
containing an SPX domain involved in Pi signaling and homeostasis (Secco et al.,
9
1
2012). According with what we observed in our dataset, in Saccharomyces, the P
2
transporters Pho87 and Pho84 were reported to behave in opposite ways, being active
3
at high and low Pi levels, respectively (Secco et al., 2012). Interestingly, a similar
4
trend was obtained for sulphur: three sulfate transporters of G. margarita were down
5
regulated in the presence of the endobacterium, but one was consistently upregulated,
6
also leading to a higher S content in the plant (not shown).
7
Starch and sucrose metabolism, and fatty acid beta oxidation appeared to be up-
8
regulated in the G condition, while components of the spliceosome were rather up-
9
regulated in both G and SL. Some genes involved in N-glycan biosynthesis were
10
slightly repressed in SL, as well as many enzymes of the shikimate pathway.
11
Confirming the GO analysis, nitrogen metabolism was downregulated by the
12
endobacterium especially in the G condition, while sulfur metabolism appeared to be
13
up-regulated exclusively in SL.
14
A transcript encoding a protein kinase C (K0: 2677), involved in the calcium-
15
mediated signalling pathway was downregulated in SL. Along the same line, the
16
phosphatidylinositol signaling system (KO: 04070) was downregulated.
17
Some genes encoding to two-component systems (KO: 02020) seem to be influenced
18
by the endobacterial presence. In many bacteria the expression of phosphate-regulated
19
genes is modulated by the two-component system PhoR–PhoP (Sola-Landa et al.,
20
2003). In our transcriptome, a putative alkaline phosphatase phoA was downregulated
21
in SL, and a phosphate sensing membrane transporter phoP was upregulated in the
22
presence of the endobacterium.
23
24
The fungal-bacterial symbiosis provides protection from ROS induced by oxidative
25
stress or strigolactone treatment
10
1
According to the behavior of other plant-and animal interacting fungi for
2
Magnaporthe and Beauveria, spores were treated with variable concentrations of
3
hydrogen peroxide (from 100 mM to 0.25mM) to check their germination responses
4
after seven days. Spores (both cured and WT) treated with hydrogen peroxide at 100
5
mM, 10 mM, 2 mM, 1 mM, 0.75 mM and 0.5 mM did not germinate and in addition
6
at 100 mM and 10 mM, the spore walls lost their natural coloration and became
7
completely white. Spores start to germinate when treated with hydrogen peroxide 0.3
8
mM. This concentration of 0.3 mM was therefore selected for the transcriptomic
9
experiments (see main text).
10
The phenotypic impact of SL on the two lines of G. margarita was checked again,
11
revealing that the presence of the bacterium had some very subtle effects: the hyphae
12
showed a curved/waving phenotype quite different from hyphae developing in water
13
or from those originating from spores that do not contain the endobacterium.
14
15
16
17
18
19
20
21
22
11
1
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1
Titles and legends to Supplementary figures:
2
Figure S1: Schematic representation of the experimental set up followed in the
3
present work. Two fungal lines (G. margarita with and without endobacteria, B+ and
4
B- , respectively) were investigated at three stages: germinating spores, strigolactone
5
treated spores, and symbiotic mycelium.
6
Figure S2: BLAST Top Hits species sharing similarities with the G. margarita whole
7
transcriptome assembly.
8
Figure S3: Cladogram based on the comparison of the whole proteome of G.
9
margarita with fungal genomes, constructed using CVTree v3. Proteomes were
10
obtained from the CVtree built-in databases with the exception of the dark red
11
genomes, which were provided by ENSEMBL and NCBI.
12
Figure S4: Venn diagram illustrating the number of differentially expressed
13
sequences (DEGs) retrieved from the comparison of G. margarita with endobacteria,
14
B+, versus the cured line of the same fungus (without endobacteria, B-). The three
15
experimental conditions considered were: germinating spores (G), germinating,
16
strigolactone treated spores (SL), and symbiotic stage (Myc).
17
Figure S5: Mycorrhizal colonization intensity evaluated according to the method of
18
Trouvelot et al., 1986. F= frequency of mycorrhization of root fragments, M=
19
intensity of root cortex colonization, a= average presence of arbuscules within the
20
infected areas and A= arbuscule abundance in the entire root system. Different letters
21
indicate significantly different values according to the Kruskal-Wallis test (p<0,05).
22
Figure S6: KEGG-based depiction of metabolic pathways differentially regulated in
23
G. margarita line B+ versus B- a) pentose phosphate pathway. b) selenocompound
24
metabolism. Functions supported by up-regulated, down-regulated or not-regulated
14
1
transcripts are shown in red, green and grey, respectively.
2
Figure S7: Mitochondria diameter in B+ and B- spores. Data are mean ± SD of 89
3
mitochondria belonging to 8 B+ spores and 76 mitochondria belonging to 7 cured
4
spores. Statistically supported differences are indicated with different letters
5
according to a Kruskal-Wallis non parametric test at p<0.05.
6
Figure S8: Relative quantification of gene expression obtained for a set of
7
mitochondrial and ROS-related genes. For each target gene the differential expression
8
has been calculated within the B+ and B- line using as the reference sample the
9
corresponding germinating spore condition (threshold line at Fold change=1).
10
Statistical significant data (Kruskal-Wallis non parametric test, p<0.05) are marked
11
with an asterisk.
12
Figure S9: Relative quantification of gene expression obtained for a set of ROS
13
related
14
gigasporarum. For each gene the fold change has been calculated comparing the
15
expression levels obtained in the H2O2- and strigolactone- treated fungal spores with
16
the basal expression recorded in the germinating spore condition (threshold line at
17
Fold change=1). Statistical significant data (Kruskal-Wallis non parametric test,
18
p<0.05) are marked with an asterisk.
19
Figure S10: GO Biological process (BP) distribution of G. margarita transcripts. For
20
clarity, only GO terms represented by at least 1,000 sequences are shown.
21
Figure S11: Clustering and heatmap of samples. DESeq2-normalized expression data
22
for all samples were rlog-transformed and clustering was performed with the
23
heatmap.2 function in the ‘gplots’ 2.14.2 Bioconductor package.
24
Figure S12: Venn representation of all the fungal genes differentially expressed
genes
belonging
to
the
endobacterium
15
Candidatus
Glomeribacter
1
during the presymbiotic phase (germinating spores and germinating spores after GR24
2
treatment) versus the symbiotic phase. The fungal DEGs from the line with the
3
endobacterium and the cured line were grouped together to reveal the impact of the
4
fungal life phase on the gene expression.
16