S. meliloti transcriptome displays evidence for
genotype-by-genotype interactions
Given the large number of phenotypes showing S. melilotistrain-specific differential response to Trichoderma species, we
aimed to identify the transcriptome signatures (in terms of number and
type of differentially expressed genes, DEGs) of such
genotype-by-genotype interactions. The four S. meliloti strains
were then exposed to 1W spent media for 24h and their transcriptome was
evaluated by RNA sequencing.
The number of differentially expressed genes after treatment (here
termed as stimulon) identified is shown in Table 1 . The list of
DEGs can be found in Supplementary Datasets S2 and S3 . Overall,
with respect to the four S. meliloti strains, the stimulon due toT. gamsii spent medium elicited the highest number of genes
(average ca. 20.6%), while T. harzianum and T. tomentosumtreatments resulted in the lowest number of significant DEGs (from 6.0
to 10.0% of total genes) with limited differences between S.
meliloti strains. T. velutinum , on the contrary, showed the
ability to stimulate a high number of DEGs in 1021 and in the hybrid
strain (ca. 23.8-25%), but in BL225C and AK83 the stimulon dropped to
ca. 6.6-9.6%, indicating that a large part of the elicited
transcriptome is strictly dependent upon the combination between givenS. meliloti genotypes and fungal genotypes. It is worth noting
that the growth phenotypes under T. velutinum 1W spent medium
resulted in a quite different response among the 4 S. melilotistrains (Figure 1 ).
Since S. meliloti harbours an open pangenome with a core set of
genes shared by all strains and a dispensable set of genes present in a
fraction only of strains we can expect that transcriptomic signatures of
genotype-by-genotype interactions could be due to (mainly) the core set
or the dispensable set. To clarify if and how much of a S.
meliloti strain specific response is reflected in a transcriptional
rewiring of core genes, we performed a principal component analysis
(PCA) on the DEGs belonging to the core gene set (shared orthologs among
the four S. meliloti strains) (Supplementary Dataset
S4 ). Results (Figure 5 ) revealed that, for each strain, the
transcriptional responses of the core genome to the different fungal
spent media give rise to fungal-related grouping of samples, with the
first principal component separating T. tomentosum /T.
harzianum elicited transcriptomes from T. gamsii , the second
principal component differentiating T. tomentosum from T.
harzianum . This pattern resembles the separation based on spent media
metabolome composition (Figure 2 ) and partial S.
meliloti growth (Figure 1 ). Concerning up-regulated DEGs
(Figure 5a ), the transcriptional response in all the strains
treated with T. harzianum spent medium is very similar. This
clustering is even tighter in the down-regulated genes (Figure
5b ). Similarly, T. tomentosum treatment elicited a similar
response among the samples, both in up-regulated and down-regulated
genes. However, AK83 and BL225C treated with T. velutinum spent
medium were found in T. tomentosum clusters and, likewise, 1021
and the hybrid treated with T. velutinum were found clustering
among samples exposed to T. gamsii spent medium, indicating that
transcriptomic signatures of genotype-by-genotype interaction reside in
the shared set of genes (core genome) also. Figure 6 reinforces
the evidence that a large fraction of the transcriptome can be involved
in the genotype-by-genotype interaction, in particular concerning
strain-specific response to each fungal species. Indeed, most of DEGs
identified are unique to the response to single fungal species
(Figure 6a-d ) or at least can be shared among two fungal
species (Figure 6 e-h ), while only a very limited fraction of
DEGs is common to all fungal species. Interestingly, depending on the
strain, the number of unique up-regulated genes belonging to the
dispensable genome spanned from 40.4% to 69.9%, while for
down-regulated genes, the dispensable range varied from 51% to 85%,
suggesting the importance of the accessory genome in the strain-
specific response. The lists of unique genes are reported inSupplementary Dataset S5 .
These data highlight the relevance of transcriptome variation in
strain-specific molecular communication between soil fungi and rhizobia.
Such variations also underlie the presence of regulatory interactions in
the genome which are differentially affected by the presence of fungi.
In this context, the transcriptional response of the hybrid S.
meliloti strain (e.g. comparing the panels for 1021 and the hybrid
strain in Figure 5 ) is different from that of the parental 1021
strain, though they share the same core genome (being different in the
symbiotic megaplasmid pSymA only). We may consequently hypothesize the
presence of epistatic interactions between genes on pSymA and those
harboured by the chromosome and pSymB chromid related to the response to
the presence of Trichoderma , similar to what has been found for
the response to root exudates . This hypothesis broadens the relevance
of pSymA megaplasmid, which could be related not just to the symbiotic
interaction with the host plant (see for instance ), but also to the
molecular dialogue with soil and rhizospheric fungi.
To inspect the type of genes functions affected by fungal spent media,
we collapsed DEGs into Clusters of Orthologous Genes (COG) categories.
Principal Component Analysis on this dataset (Figure 7 ) showed
for the up-regulated genes, a relevant contribution of COG category K
(transcription). This observation reinforces the hypothesis about the
differential modulation of epistatic interaction in S. melilotigenomes triggered by fungal spent media, which may involve various
regulons. Among down-regulated genes, an important contribution to
variance was found for COG categories G (carbohydrate metabolism and
transport) and J (translation), suggesting that fungal spent media may
also have differential nutritional effects over S. melilotimetabolism. However, we should point out that the highest contribution
to transcriptional variance was for genes not found in COG or with
unknown function (COG category S), indicating that probably much of the
genes relevant for the modulation of interaction taking place in the
rhizosphere microbiota has still to be disclosed.
However, concerning specific functional genes with known relevance in
plant-rhizobium interaction and which can explain part of the
synergistic phenotypes observed (Figure 3 ), among the most
highly expressed genes in S. meliloti 1021 and in thecis -hybrid strain after exposure to T . gamsii ,T . harzianum and T . velutinum , were
components of the flagellar apparatus (flgA , C , D ,F , G , H , I , L and fliE ,G , I , K , L , M , N ),
(Supplementary Dataset S2 ). It is worth noticing that the
orthologs of these genes were not induced in BL225C or AK83. To
reinforce the presence of epistasis and novel roles for pSymA
megaplasmid, while for the cis -hybrid strain the same spent media
strongly reduced root adhesion (Figure 1 ), in line with the
expectation of an active flagellar apparatus,, the same was not true for
1021, which formed an abundant biofilm on the roots when treated withT. velutinum spent medium. However, regarding the general
differences among S. meliloti strains, we must consider that the
transcriptome was evaluated after 24h incubation, while the root biofilm
after 48h and in the presence of the plant root system. Indeed, the
motility-to-biofilm transition often includes an early phase with active
flagellar apparatus, followed by an inhibition of flagellar gene
transcription . We may speculate that the kinetics of this transition
can be differentially modulated by Trichoderma species among the
tested S. meliloti strains, however direct observations of
biofilm formation under co-inoculation are needed to sustain such
hypothesis.
No evidence was found for changes in the expression of galactoglucan
(EPS-II) and succinoglucan (EPS-I) biosynthesis genes, but exoDgene was overexpressed in all the strains. This gene has been
demonstrated to be needed for the invasion and development of alfalfa
nodules .
Regarding genes involved in nodulation and nitrogen fixation,
unexpectedly nodA expression was found induced in BL225C and 1021
in the presence of T. gamsii and T. velutinum ,
respectively, while nodD3 expression was induced in 1021 in the
presence of T. tomentosum and T. velutinum . The gene was
also induced in the hybrid strain when treated with T. velutinumand in AK83 in presence of T. gamsii . NodD3 does not require
specific plant compounds to activate nod gene expression , but
its expression is activated by a LysR family transcriptional regulator,syrM . This latter gene was indeed found overexpressed in the same
conditions. This gene also activates syrA , which when
overexpressed, causes an increase in EPS-I production. Strikingly,nifN and nifH expression was induced in AK83 in the
presence of T. velutinum spent medium, questioning the
possibility of activation of part of the nitrogenase complex in
nonsymbiotic conditions. Concerning other genes which could be relevant
for rhizobium phenotypes connected with symbiosis, among the
up-regulated genes under spent media treatment those encoding for
proteins involved in conjugal transfer (tra genes) were
retrieved, suggesting that presence of the fungus may promote the
mobilization of the pSymA megaplasmid. However, the rctA gene,
the repressor of the conjugation machinery was found differentially
expressed in 1021 and Bl225C strains under T. gamsii spent medium
treatment (Log2 Fold change ~ 2), suggesting repression
of the megaplasmid conjugation. Interestingly, the same gene was not
differentially expressed in the cis -hybrid strain, indicating the
presence of epistatic interaction among genes on the megaplasmid and
those residing on the chromosome and the pSymB chromid.
o quantitatively estimates the number of DEGs whose expression changes
among strains and treatments are explained by the rhizobial strain they
belong to, by the fungal species and by the interaction between strain
and species, a nested LRT model was constructed. Overall results are
reported in Table 2 and the list of genes inSupplementary Dataset S6. A total of 327 genes were shared as
DEGs among stimulons, which allowed to train a model for a total of 981
strain, fungus, and strain x fungus combinations. Within these genes, a
large fraction (ca. 2/3 of total DEGs) showed a significant effect of
strain, fungus, or strain x fungus, only 34.1% of their variability in
expression not being supported by the models. For 146 genes changes in
relative expression levels were modeled with respect to the rhizobial
strain, for 270 to Trichoderma (giving a total of 416
combinations for both rhizobium and Trichoderma ), and for 230
genes to rhizobium x fungus interaction. Within this latter list of
genes, several are related to redox balance (as sox gst ,
and nuo genes), suggesting that part of the genotype-species
specific response could be related to a general differential response to
stressful conditions.