Abstract
Plants, arbuscular mycorrhizal (AM) fungi and soil bacteria coexist
stably in ecosystems. Resource availability can affect the mutualistic
relationship between plants and AM fungi, but it is unclear how it
affects the reciprocal cooperation between AM fungi and bacteria. Here,
we used an in vitro culture system containing a source of organic
phosphorus (P) to explore the early-stage reciprocal cooperation
between the AM fungus Rhizophagus irregularis and the
phosphate-solubilizing bacterium (PSB) Rahnella aquatilis under two
different levels of inorganic P. Under low inorganic P availability,
the reciprocal cooperation between the AM fungus and the PSB,
materialized by carbon (C)-P exchange, was strengthened. This was
evidenced at transcriptional level by the activation of multiple C
metabolism and P transport and regulation pathways of both partners.
Conversely, under high inorganic P availability, the exchange of P for
C with plants was slowed down. This was accompanied by the activation
of only P transport and regulation pathways and less C metabolism
pathway, weakening the reciprocal cooperation with the PSB. In
conclusion, the availability of inorganic P can mediate the reciprocal
cooperation between AM fungi and bacteria, which could also extend to
host plants since they are inseparable in ecosystems.
Subject terms: Microbiology, Physiology, Fungi
__________________________________________________________________
Reciprocal cooperation at the early stage between an arbuscular
mycorrhizal fungus and a phosphate-solubilizing bacterium is mediated
by external phosphorus availability.
Introduction
Most natural environments are home to an abundant diversity of
microbial species competing with each other for space and resources.
They generally display highly social behavior, using chemical signals
or shared low-cost resources as a means of communication, cooperation,
and competition within the complex network of the
ecosystem^[28]1,[29]2. Cooperation is the most effective way of
maintaining community stability and improving productivity among
conspecifics or heterospecifics. It refers to the interaction in which
one individual provides fitness benefits to other individuals and
obtains corresponding rewards in exchange^[30]3,[31]4.
Cooperation occurs in the world of plants and microbes, where plants
feed the complex and diverse soil microbiota through exudates, while
the soil microbiota extends the habitat range and metabolic capacity of
plants^[32]5,[33]6. An emblematic example of such cooperation is the
symbiosis established between arbuscular mycorrhizal (AM) fungi and
over two-thirds of plant species. These fungal symbionts increase the
access of host plants to minerals (especially phosphorus—P and
nitrogen—N), in exchange of carbon (C) resources that are vital for
their life cycle^[34]7,[35]8. At more complex levels, AM fungi engage
in reciprocal cooperation with soil bacteria, facilitated by their
extensive extraradical hyphae and highly branched hyphal networks.
These bacteria inhabit the hyphosphere and feed on hyphal exudates and,
in exchange, increase the accessibility of minerals for AM fungi and
improve their overall fitness^[36]9,[37]10.
The cooperation between plants, AM fungi, and soil bacteria has been
studied at genetic and evolutionary levels. Up to now, all the
sequenced AM fungi (e.g., Rhizophagus irregularis, Rhizophagus clarus,
and Gigaspora margarita) have genomes lacking key enzymes for de novo
fatty acid biosynthesis and organic substrates
degradation^[38]11–[39]13. Therefore, plant-derived fatty acids and
sugars support the abundant development of AM fungal extraradical
hyphae and the production of spores, enabling extensive exploration of
soil nutrients (e.g., P, N), with a beneficial return to the host
plants^[40]14,[41]15. However, AM fungi are unable to fully exploit
polymer organic matters in soil because they lack most genes encoding
carbohydrate-active enzyme (CAZyme)^[42]16. This has led AM fungi to
cooperate specifically with hyphospheric bacteria harboring a large
number of genes encoding CAZymes in their genome^[43]17,[44]18 rather
than cooperating randomly with soil bacteria^[45]19,[46]20.
Both plants and AM fungi can improve reciprocal cooperation by
rewarding their most compatible partners with increased resource
allocation, a process often regulated by host-derived C resources and
external nutrients availability^[47]21,[48]22. For example, high P
availability tends to limit root colonization by AM fungi and reduces
the transfer of C from plants to AM fungi^[49]23–[50]25. Conversely,
under low P conditions, plants can boost arbuscule development and
derive most benefits from AM fungi^[51]26. If the P demand of plants is
low, AM fungi can store the absorbed P within their hyphal network
until root demand escalates, thereby ensuring a greater C
return^[52]27,[53]28. When extraradical hyphae of AM fungi proliferate
in a P-limited environment, they tend to supply a greater proportion of
P to plants, as P transport can enable plants to donate more C for
fungal growth and metabolism^[54]28,[55]29. In this scenario, the
energetic cost of acquiring P from AM fungi may be increased,
necessitating the recruitment of soil bacteria to aid in enhancing P
accessibility and, in turn, offering C-rich metabolic compounds to
reward these bacteria^[56]30.
Among the many soil bacteria, phosphate solubilizing bacteria (PSB) are
presumably the most reliable associates of AM fungi. They efficiently
colonize the surface of extraradical hyphae and promote the
mineralization of organic P, which is omnipresent in the soil, into
inorganic P that is easily assimilated by AM fungi^[57]31,[58]32.
Recently, a study found that AM fungi can exploit C sources to rapidly
transport PSB to organic P patches via an extensive hyphal network,
thereby facilitating the establishment of cooperative
relationships^[59]31. AM fungi and bacteria have broader ecological and
agricultural significance, for example, accelerating nutrient cycling,
influencing soil C storage and promoting crop production and ecosystem
stability^[60]33–[61]35. However, it remains unclear how the
availability of external P affects the reciprocal cooperation and
synergistic mechanisms between AM fungi and PSB and how these two
organisms adapt their behavior in response to changes in P availability
in the environment.
In the present study, we used an in vitro culture system that comprises
a single AM fungus, R. irregularis, and a single PSB, Rahnella
aquatilis, to explore biological interactions in the hyphosphere and
provide insights into microbial-driven nutrient cycling and potential
plant production. The one-to-one model is particularly well-suited for
elucidating the potential mechanisms underlying reciprocal cooperation
between AM fungi and bacteria, as it allows the harvest of large
quantities of hyphae without any contamination. More specifically, we
conducted different experiments and transcriptome analysis to address
the hypothesis that the cooperation between R. irregularis and R.
aquatilis is mediated by the availability of inorganic P: (1) Under the
scenario of low inorganic P availabililty, R. irregularis enhances
metabolic activity in R. aquatilis and activate the P turnover pathway,
thereby accelerating the mineralization of organic P and improving P
availability for R. irregularis. (2) Under the scenario of high
inorganic P availabililty, R. irregularis directly absorbs P from the
environment and stores the excess P in search of more plant C return,
while weakening cooperation with the PSB. (3) Under low P availability,
the exchange of C and P between R. irregularis and R. aquatilis is
accelerated, while under high P availability, this process is slowed
down.
Results
The interaction between R. irregularis and R. aquatilis contributes to the
mineralization of organic P
We first quantified the concentrations of inorganic P remaining and
organic P consumed in the liquid medium of the hyphal compartment (HC),
24 h after introduction of R. aquatilis. The inorganic P concentrations
remaining in the P^Low and P^High RI^− RA^+ treatments were equivalent
to the initial concentrations of inorganic P (Fig. [62]1a). In
contrast, the concentrations of inorganic P in the P^Low or P^High RI^+
RA^− treatments were significantly lower as compared to the initial
concentrations as well as to the two other treatments (Fig. [63]1a). In
the RI^+ RA^+ treatment, whether under the P^Low or P^High
availability, the concentrations of inorganic P remaining in the liquid
medium of the HC was intermediate to the RI^+ RA^− and RI^− RA^+
treatments (Fig. [64]1a). Whatever the availability of inorganic P in
the HC, the consumption of organic P was significantly higher in the
RI^+ RA^+ treatment compared to the two other treatments (Fig. [65]1b).
In addition, the organic P in the P^Low RI^+ RA^+ treatment was almost
entirely consumed, an amount that was twice as much as that in the
P^High RI^+ RA^+ treatment (Fig. [66]1b).
Fig. 1. Inorganic P concentration, organic P consumption, ACP and ALP
production.
[67]Fig. 1
[68]Open in a new tab
Concentrations of a inorganic P remaining and b organic P consumed in
the MSR liquid medium of the HC under low (35 μM KH[2]PO[4]) and high
(700 μM KH[2]PO[4]) P availabilities. The horizontal dashed lines
represent the initial concentrations of inorganic and organic P after
volume increase due to the introduction of organic P and bacterial
suspension. c Acid and d alkaline phosphatase activity in the liquid
medium of the HC under low and high P availability. AM fungus-induced e
acid and f alkaline phosphatase production efficiency and g organic P
mineralization efficiency under low and high P availabilities. The
error bar represents the standard errors. For the same P availability,
different capital letters indicate a significant difference (p < 0.05
by Tukey’s HSD test, n = 4 biologically independent sample) between the
RI^− RA^+, RI^+ RA^− and RI^+ RA^+ treatments^. For the same RI^− RA^+,
RI^+ RA^− or RI^+ RA^+ treatment^, the asterisks indicate a significant
difference between low and high P availabilities (t-test, *p < 0.05;
***p < 0.001; n.s., p > 0.05, n = 4 biologically independent sample).
Abbreviations: RI^− RA^+ only R. aquatilis cells present in the HC,
RI^+ RA^−, only R. irregularis hyphae present in the HC, RI^+ RA^+ both
R. irregularis hyphae and R. aquatilis cells present in the HC.
We then measured the activity of acid phosphatase (ACP) and alkaline
phosphatase (ALP) in the P^Low and P^High treatments. Whatever the
concentration of inorganic P in the HC, a significant increase in ACP
(Fig. [69]1c) and ALP (Fig. [70]1d) activities was observed in the RI^+
RA^+ treatment compared with the RI^+ RA^− and RI^− RA^+ treatments,
while no difference was noticed between these two later treatments. In
addition, the ACP and ALP activities in the RI^+ RA^+ treatment were
significantly higher under P^Low availability compared to P^High
availability (Fig. [71]1c, d). R. aquatilis is known to secrete ACP and
ALP to mineralize phytate, a function that is not present in R.
irregularis. Therefore, we calculated the efficiency of AM
fungus-induced ACP and ALP production (EAP and ELP), and the efficiency
of AM fungus-induced organic P mineralization (EPM). EAP and ELP were
significantly higher at low P availability than at high P availability
(Fig. [72]1e, f), which resulted in higher EPM (Fig. [73]1g).
The exudates of R. irregularis stimulate the growth of R. aquatilis
Increased phosphatase activity can potentially result from an increase
in the population of R. aquatilis. In the absence of R. irregularis in
the HC (i.e., RI^− RA^+ treatment), no difference was observed in the
absolute abundance of R. aquatilis under P^Low and P^High
availabilities. Conversely, in the presence of R. irregularis (i.e.,
RI^+ RA^+ treatment), a significantly higher absolute abundance of R.
aquatilis was noticed at P^Low availability compared to P^High
availability (Fig. [74]2a). This was consistent with the results
obtained with the exudates of R. irregularis grown under the P^Low or
P^High availabilities (Fig. [75]2b). The OD[600] of R. aquatilis
increased rapidly from 2 to 24 h, followed by a slower rise from 26 to
48 h. Conversely, in the absence of exudates of R. irregularis and at
both P^Low and P^High availabilities, the OD[600] of R. aquatilis
increased slowly from 1 to 24 h, then remained unchanged from 26 to
48 h. The OD[600] of R. aquatilis in the 0.85% NaCl solution (i.e., the
control) remained unchanged over the 48 h of observation (Fig. [76]2b).
At each time-point, the OD[600] of R. aquatilis with the exudates of R.
irregularis in the P^Low or P^High treatments were significantly
greater than that in the other three treatments, with higher OD[600] of
R. aquatilis at P^Low availability compared to P^High availability
(Fig. [77]2b).
Fig. 2. Growth of R. aquatilis in the presence or absence of R. irregularis
and its hyphal exudates.
[78]Fig. 2
[79]Open in a new tab
a Absolute abundance of R. aquatilis 24 h after inoculation in the
medium of HC under low (35 μM KH[2]PO[4]) or high (700 μM KH[2]PO[4])
inorganic P availability. The asterisks indicate a significant
difference between the RI^+ RA^+ and RI^− RA^+ treatments under the
same P availability, and between the RI^+ RA^+ treatments under the
different P availabilities (t-test, ***p < 0.001, n = 4 biologically
independent sample). b Effect of R. irregularis exudates collected from
the HC under low or high inorganic P availability on the growth of R.
aquatilis. The data represent optical density (OD) of the culture from
2 to 48 h minus the initial OD of the bacterial liquid cultures. The
error bar represents the standard errors. Abbreviations: RI^− RA^+ only
R. aquatilis cells present in the HC, RI^+ RA^+ both R. irregularis
hyphae and R. aquatilis cells present in the HC, RI^− no R. irregularis
hyphae present in the HC, RI^+ only R. irregularis hyphae present in
the HC.
R. irregularis enhances the C metabolism and cell motility of R. aquatilis
C is an essential energy source for bacterial growth and motility. This
is confirmed by the significant growth of R. aquatilis in the presence
of R. irregularis exudates, whether grown in the P^Low or P^High
treatments, compared to the absence of AM fungal exudates. This
suggests that R. aquatilis has undergone an attenuation of C
limitation. Therefore, we conducted an in-depth analysis of R.
aquatilis gene expression and metabolic pathway change at the
transcriptional level, in the presence or absence of R. irregularis
grown under P^Low or P^High availabilities. A total of 1120 (565
upregulated and 555 downregulated) and 1446 (691 upregulated and 755
downregulated) genes were identified as differentially expressed
(DEGs) (adjusted P values < 0.05) in the P^Low and P^High RI^+ RA^+
treatment vs RI^+ RA^− treatment, respectively (Fig. [80]S1a, b). The
bubble chart depicted the top 20 functional categories that encompass
the highest number of DEGs. The number of DEGs within 14 and 10
functional categories constituted over 40% of the entire pathway at
P^Low and P^High availability, respectively (Fig. [81]S2a, b). Among
these functional categories, eleven exhibited enrichments at P^Low
availability (adjusted P values < 0.05). Specifically, seven were
associated with carbohydrate metabolism, one with amino acid
metabolism, two with cell motility, and another one with membrane
transport (Fig. [82]3a, Supplementary data [83]1). In contrast, only
six functional categories were found to be enriched (adjusted P
values < 0.05) at P^High availability. Four were associated with
carbohydrate metabolism, one with amino acid metabolism, and one with
cell motility. Furthermore, all these enriched functional categories
exhibited a tendency towards up-regulation (Fig. [84]3b, Supplementary
data [85]1).
Fig. 3. Functional enrichment and pathway map of C metabolism gene expression
in R. aquatilis.
[86]Fig. 3
[87]Open in a new tab
Enrichment of functional categories (P < 0.05) among differentially
expressed genes in R. aquatilis in the RI^+ RA^+ treatment vs RI^− RA^+
treatment under a low (35 μM KH[2]PO[4]) and b high (700 μM KH[2]PO[4])
P availabilities. Each section of the circle plot represents an
enrichment pathway. Inner bar plot indicates the log[10] of the
adjusted P-value for each enrichment pathway and the color shows the
z-score value, indicating global upregulation (the value > 0) of genes
within each pathway. Detailed log[2] fold-change (log[2]FC) value of
each gene within each pathway is plotted as a dot plot in the outer
circle (upregulated in red and downregulated in blue). c
Transcriptional responses of key genes in R. aquatilis pathways
including sugar transport and metabolism, TCA cycle, chemotaxis,
two-component system, and flagellar synthesis and regulation under low
and high P availabilities. The heat map shows the log[2] of transcripts
per kilobase million (TPM) calculated based on the initial reads count.
The dashed lines represent simplified pathways. The asterisks are used
to indicate a significant difference in gene expression level between
(2) P^Low RI^+ RA^+ and (1) P^Low RI^− RA^+ treatments; between (4)
P^High RI^+ RA^+ and (3) P^High RI^− RA^+ treatments; and between (2)
P^Low RI^+ RA^+ and (4) P^High RI^+ RA^+ treatments (DESeq2 test,
*p < 0.05; **p < 0.01; ***p < 0.001, n = 4 biologically independent
sample). Abbreviations: RI^- RA^+ only R. aquatilis cells present in
the HC, RI^+ RA^− only R. irregularis hyphae present in the HC, RI^+
RA^+ both R. irregularis hyphae and R. aquatilis cells present in the
HC.
Based on these results, we generated a pathway heatmap depicting gene
expression for sugar transport, C metabolism, and bacterial motility,
aiming for more precise insights into key metabolic processes of R.
aquatilis. The expression of several sugar transporter genes,
specifically those encoding for trehalose (treB), glucose (crr),
maltose/glucose (malX), and fructose (fruB) transport, was
significantly upregulated in the P^Low and P^High RI^+ RA^+ treatments
(Fig. [88]3c, Supplementary data [89]2). The sugar assimilated by R.
aquatilis was first metabolized via the glycolysis pathway, and then,
following several conversion steps, produced acetyl-CoA, which
subsequently entered the tricarboxylic acid (TCA) cycle to produce
energy. Among them, the expression of multiple genes associated with
the glycolysis pathway, such as glucokinase (glk), fructokinase,
6-phosphofructokinase, and fructose-bisphosphate aldolase (FBA), were
significantly upregulated in the P^Low and P^High RI^+/RA^+ treatments.
R. irregularis specifically stimulated genes encoding key enzymes in
the TCA cycle at P^Low availability (Fig. [90]3c, Supplementary
data [91]2). This includes bifunctional aconitate hydratase
2/2-methylisocitrate dehydratase (ancB), E1 and E2 components of
2-oxoglutarate dehydrogenase (OGDH and DLST), and alpha subunit of
succinyl-CoA synthetase. In addition, some genes involved in sugar
metabolism and TCA cycle, such as trehalose 6-phosphate phosphatase
(otsB), isocitrate dehydrogenase (IDH1) and succinate dehydrogenase
flavoprotein subunit (frdA), whose expression was stimulated by R.
irregularis, showed higher expression levels at P^Low availability
compared to P^High availability. The chemotactic two-component system
and flagellar assembly were pivotal in governing the motility of R.
aquatilis. The expression of numerous genes within these pathways, such
as cheA, cheB, cheV, cheY, flhD, flgN, motA, and motB, was
significantly upregulated in the P^Low or P^High RI^+/RA^+ treatments.
Particularly, many genes of these pathways exhibited higher expression
levels at P^Low availability compared to P^High availability
(Fig. [92]3c, Supplementary data [93]2).
The presence of R. irregularis enhances the P turnover function in R.
aquatilis
To explore whether R. aquatilis has an important contribution to
phosphatase activity in liquid medium, we analyzed at the
transcriptional level the genes associated with P turnover and
calculated their variance-stabilized count abundance (Fig. [94]4a,
Supplementary data [95]3). The abundance of six genes inducing P
mineralization, eight involved in P transportation, and four
responsible for P regulation were significantly increased in the P^Low
RI^+ RA^+ treatment vs RI^+ RA^− treatment. In contrast, only five
genes involved in P mineralization, four in P transport, and two in P
regulation showed a significantly increased abundance in the P^High
RI^+ RA^+ treatment vs RI^+ RA^- treatment. Furthermore, R. aquatilis
reduced the variance stabilized count abundance of most P
solubilization genes at both P^Low and P^High availability
(Fig. [96]4a). The regulation genes (i.e., phoB, phoP, phoQ, and phoR)
of the two-component system were responsible for sensing nutrient
changes in the environment and the stimulation of AM fungi, thereby
activating or inhibiting the expression of other P turnover functional
genes. The variance stabilized count abundance of P regulation genes
was significantly positively correlated with genes involved in P
mineralization (R^2 = 0.57, P = 0.0008) and in P transport (R^2 = 0.40,
P = 0.0090), but negatively correlated with genes involved in P
solubilization (R^2 = 0.41, P = 0.0072) (Fig. [97]4b).
Fig. 4. Expression levels of genes involved in P cycling function in R.
aquatilis.
[98]Fig. 4
[99]Open in a new tab
a Heat map showing variance stabilized counts of P functional genes in
R. aquatilis: P solubilization, mineralization, transport, and
regulation pathways. The eight lines above represent
variance-stabilized counts of related genes in the RI^− RA^+ and RI^+
RA^+ treatments under low (35 μM KH[2]PO[4]) P availability. The eight
lines below represent variance-stabilized counts of related genes in
the RI^− RA^+ and RI^+ RA^+ treatments under high (700 μM KH[2]PO[4]) P
availability. The red circles and blue circles indicate that
variance-stabilized counts were significantly increased and decreased
in the RI^+ RA^+ treatment compared with the RI^− RA^+ treatment. b
Spearman correlation between P regulation genes and P solubilization
genes, mineralization genes, and transport genes based on the
calculation of variance stabilized counts. c Transcriptional responses
of genes in R. aquatilis P solubilization, mineralization, transport,
and regulation pathways under low and high P availability. The genes
with higher expression level in the P^Low RI^+ RA^+ treatment than in
the P^High RI^+ RA^+ treatment were selected and log[2] of transcripts
per kilobase million (TPM) were calculated based on the initial reads
count. The asterisks indicate a significant difference in gene
expression level between P^Low RI^+ RA^+ and P^Low RI^− RA^+
treatments; between P^High RI^+ RA^+ and P^High RI^− RA^+ treatments
and between P^Low RI^+ RA^+ and P^High RI^+ RA^+ treatments (DESeq2
test, *p < 0.05; **p < 0.01; ***p < 0.001; n.s., p > 0.05, n = 4
biologically independent sample). Abbreviations: RI^− RA^+ only R.
aquatilis cells present in the HC, RI^+ RA^− only R. irregularis hyphae
present in the HC, RI^+ RA^+ both R. irregularis hyphae and R.
aquatilis cells present in the HC.
The EAP, ELP, and EPM were significantly greater at P^Low availability
compared to P^High availability, potentially attributable to the
increased expression of related genes regulating or encoding
phosphatase proteins. The expression of eleven genes involved in P
mineralization, transport and regulation were significantly higher in
the P^Low RI^+ RA^+ treatment (Fig. [100]4c). Among them, four genes
encode phosphatase proteins and four regulate phosphatase synthesis and
transport, namely inositol phosphatase gene phy, acid phosphatase gene
phoA, alkaline phosphatase genes phoN1 and phoN2, P regulon response
regulator phoB, P sensor histidine kinase phoQ, P regulon sensor
histidine kinase phoR, and transcriptional regulator phoU
(Supplementary data [101]3).
Low inorganic P availability stimulates R. irregularis to absorb and
transport P to the host plant while enhancing its C and energy metabolism
The sustained growth of R. aquatilis by R. irregularis at P^Low
availability, can be attributed to an increase in C metabolism,
deriving from altered P needs of R. irregularis. Therefore, we
conducted a thorough analysis of R. irregularis gene expression
patterns at the transcriptional level. In the presence of R. aquatilis,
R. irregularis exhibited 449 (373 upregulated and 76 downregulated) and
347 (255 upregulated and 92 downregulated) DEGs (adjusted P
values < 0.05) in the P^Low and P^High RI^+ RA^+ treatment vs RI^− RA^+
treatment, respectively (Fig. [102]S3a, b). Seven functional categories
were found to be enriched (adjusted P values < 0.05) at P^Low
availability. Specifically, two were associated with carbohydrate
metabolism, three with lipid metabolism, two were involved in amino
acid metabolism, and another one was involved in transport and
catabolism. Conversely, only one functional category associated with
amino acid metabolism was enriched (adjusted P values < 0.05) at P^High
availability (Fig. [103]S4a, b, Supplementary data [104]4).
AM fungi can change P transfer to host plants based on inorganic P
availability in the environment. The expression of several P-absorbing
protein genes (i.e., pho84 and pho89) located on the membrane of
extraradical hyphae and P transporter genes (i.e., vtc1 and vtc4)
located on the membrane of vacuoles were significantly upregulated in
the P^Low or P^High RI^+ RA^+ treatment, and pho84, pho89, and vtc1
genes had higher expression levels at P^Low availability compared to
P^High availability (Fig. [105]5a, Supplementary data [106]5). Once
entering the vacuole, P was converted into Poly-P and subsequently
transferred with arginine to intraradical hyphae, where it underwent
further hydrolysis and transport to the host. The expression of
multiple genes, such as argG, argH, argI, ypq2, ppn, and pho91,
associated with arginine synthesis, Poly-P degradation and the
subsequent transport of P was only induced in the P^Low RI^+ RA^+
treatment. In addition, at P^Low availability, the positive regulator
pho4 in the PHO regulatory system was stimulated, while the negative
regulator pho85 was inhibited (Fig. [107]5a, Supplementary
data [108]5).
Fig. 5. Pathway map of C and P metabolism gene expression in R. irregularis.
[109]Fig. 5
[110]Open in a new tab
Transcriptional responses of genes in R. irregularis a P transport and
regulation and arginine biosynthesis and b fatty acid elongation and
degradation, energy metabolism, TCA cycle, gluconeogenesis and
glycolysis pathways, under low (35 μM KH[2]PO[4]) and high (700 μM
KH[2]PO[4]) P availabilities. The heat map shows the log[2] of
transcripts per kilobase million (TPM) calculated based on the initial
reads count. The dashed lines represent simplified pathways. The
asterisks indicate a significant difference in gene expression level
between (2) P^Low RI^+ RA^+ and (1) P^Low RI^+ RA^− treatments; between
(4) P^High RI^+ RA^+ and (3) P^High RI^+ RA^− treatments; and between
(2) P^Low RI^+ RA^+ and (4) P^High RI^+ RA^+ treatments (DESeq2 test,
*p < 0.05; **p < 0.01; ***p < 0.001, n = 4 biologically independent
sample). Abbreviations: RI^− RA^+ only R. aquatilis cells present in
the HC, RI^+ RA^− only R. irregularis hyphae present in the HC, RI^+
RA^+ both R. irregularis hyphae and R. aquatilis cells present in the
HC.
AM fungi transfer P to the host plant in return for fatty acids, which
may result in enhanced C and energy metabolism, thereby strengthening
the reciprocal cooperation between AM fungi and bacteria. The
expression of multiple genes involved in R. irregularis fatty acid
elongation and degradation, TCA cycle and gluconeogenesis/glycolysis
was significantly upregulated in the P^Low RI^+ RA^+ treatment, mainly
including fatty acid elongase 2, long chain-3-oxoacyl-CoA reductase
(HSD17), long chain-3-hydroxyacyl-CoA dehydratase, acetyl-CoA
C-acetyltransferase (ACAT), citrate synthase, OGDH, enolase,
phosphoenolpyruvate carboxykinase (pckA), FBA,
fructose-1,6-bisphosphatase I (FBP), glk. In contrast, the expression
of only a few genes, such as HSD17, ACAT, and IDH1, was stimulated in
the P^High RI^+ RA^+ treatment (Fig. [111]5b, Supplementary
data [112]5). Furthermore, several genes, specifically ATPeF0B,
ATPeF1G, and ATPeV0D, which are crucial in energy metabolism, had their
expression exclusively induced in the P^Low RI^+ RA^+ treatment
(Fig. [113]5b, Supplementary data [114]5).
Low inorganic P accelerates C-P reciprocity between R. irregularis and R.
aquatilis
To further explore the cooperation strategies employed by R.
irregularis and R. aquatilis at varying P availability, we calculated
the P uptake ratio (PUR) of R. irregularis and the C acquisition ratio
(CAR) of R. aquatilis to characterize the C-P exchange rate. The
results showed that under P^Low availability, R. irregularis had a PUR
4.78 times higher than that under P^High availability (Fig. [115]6a).
Under P^Low availability, R. aquatilis also acquired C more efficiently
(i.e., 1.91 times higher) compared to P^High availability
(Fig. [116]6b).
Fig. 6. R. aquatilis C acquisition ratio and R. irregularis P uptake ratio.
[117]Fig. 6
[118]Open in a new tab
a R. aquatilis CAR and b R. irregularis PUR under low (35 μM
KH[2]PO[4]) and high (700 μM KH[2]PO[4]) P availabilities. The
asterisks indicate a significant difference between low and high P
availabilities (t-test, ***p < 0.001, n = 4 biologically independent
sample).
Discussion
Plants, AM fungi, and bacteria cooperate intimately by metabolic
exchanges or cross-feeding to ensure the stable development of all the
players^[119]36. While it is well established that resource
availability affects the cooperative relationship between plants and AM
fungi^[120]28,[121]29,[122]37, the way in which such cooperation is
regulated between AM fungi and bacteria in the hyphosphere has not yet
been fully elucidated. Here, we studied at the ecological and molecular
levels how the availability of inorganic P in the environment impacts
the mechanisms of cooperation at the early stage between an AM fungus
and a PSB. The results confirmed our hypothesis that low inorganic P
availability (35 μM KH[2]PO[4]) stimulates the reciprocal cooperation
between the AM fungus and the bacterium, accompanied by an increased
acquisition of C by the bacterium and an enhanced uptake of P by the AM
fungus. Conversely, under high inorganic P availability (700 μM
KH[2]PO[4]), the AM fungus directly absorbs and stores excess P,
slowing down the exchange of C with plants and weakening the
cooperation with the PSB. These findings contribute to elucidating the
mechanisms underlying cross-kingdom interactions facilitated by
resource availability. It may establish a foundation for
comprehensively understanding microbial-mediated soil nutrient cycling,
enhancing plant productivity, and optimizing P fertilizer application
strategies to safeguard soil health.
Low inorganic P availability can strengthen cooperation between the AM fungus
and the PSB, based on C and P exchange
In previous studies^[123]30–[124]32,[125]38, the existence of
reciprocal cooperation between the AM fungus R. irregularis and the PSB
R. aquatilis, was demonstrated based on the exchange of C and P. This
cooperation may result from the functional complementarity of the
metabolism of the two partners as well as the scarcity of resources
available in the soil. In the absence of exploitable C sources,
bacteria typically enter a dormant state, a temporary adaptation that
slows down metabolism to reduce energy expenditure and sustains
viability during a prolonged period of C limitation^[126]39,[127]40. In
contrast, a dense network of hyphae is known to release significant
amounts of C-containing compounds, which are mainly composed of low
molecular weight sugars, amino acids, and carboxylates (see
Table [128]S1). Sugars, in particular glucose, fructose, and trehalose,
have been shown to promote the growth of soil bacteria, so that they
emerge from dormancy and develop rapidly^[129]9. This is consistent
with our findings showing that the expression of genes encoding
glucose, fructose, and trehalose transporters on the cell membrane of
R. aquatilis was significantly upregulated in the presence of R.
irregularis. In addition, the growth of the bacterial population was
increased in the presence of either the AM fungus or only its hyphal
exudates: the increase was 21% and 75% respectively (Fig. [130]2).
Cooperation requires both parties to engage in an exchange of resources
aimed at achieving greater benefits at minimum cost^[131]3,[132]41. The
AM fungus provides the PSB with easily accessible sources of C and, in
exchange, the PSB provides the AM fungus with more important sources of
inorganic P^[133]30,[134]42. This is further supported by the increase
in phosphatases secreted by R. aquatilis in the presence of R.
irregularis, which accelerates the mineralization of organic P, thus
increasing the availability of inorganic P for the AM fungus
(Fig. [135]1). This scenario is, however, dependent on the availability
of P in the environment. Under a high inorganic P scenario, we observed
that R. irregularis was able to directly assimilate inorganic P from
the medium via P transporters (e.g., Pho80, Pho84, and Pho89) that
belong to the phosphate transporter 1 family, located on the plasma
membranes of extraradical hyphae^[136]43–[137]46. This process may
consume less energy and costs than cooperating with the PSB for the
acquisition of inorganic P from organic sources. Conversely, under low
inorganic P scenario, the AM fungus was more likely to cooperate with
the PSB to increase the metabolic exchange of C and P, improving its
overall ability to adapt to the environment (Fig. [138]1 and
Fig. [139]2). At the transcriptional level, we confirmed that, as well
as acting as a source of C to stimulate PSB growth and activate its
glycolysis, TCA cycle and other C metabolic pathways, hyphal exudates
may also contain signaling molecules and chemotactic substances that
induce flagellum synthesis and cell motility in the bacterium, further
promoting the formation of reciprocal cooperation (Fig. [140]3). The
two-component signaling system is a ubiquitous and conserved regulatory
component in bacteria that can specifically recognize and sense
external environmental signals and induce specific metabolic responses
by activating or inhibiting gene expression in downstream
pathways^[141]47. Higher gene expression levels of C metabolism and
cell motility mediated by sensor and response regulatory receptor, as
well as more enriched metabolic pathways, were observed in R. aquatilis
under low versus high inorganic P availability (Fig. [142]3).
Bacteria are the main drivers of the P cycle in the soil, facilitating
the solubilization of non-soluble P and the mineralization of organic
P, and participating in the transport and regulation of
P^[143]48–[144]50, while AM fungi are mainly responsible for P uptake,
storage, and transport, owing to their limited saprophytic
capabilities^[145]16,[146]51. In the present study, we identified
potential regulatory trade-offs of R. aquatilis in solubilizing
non-soluble P and mineralizing organic P. Specifically, in the presence
of organic P as the available P source in addition to inorganic P, R.
aquatilis stimulated the organic P mineralization pathway at the same
time suppressing the non-soluble P solubilization pathway via the P
regulatory pathway (Fig. [147]4) as earlier observed^[148]47. This
suggests that the PSB can accurately identify the form of P in the
environment and make strategic adjustments to effectively cooperate
with AM fungi. The exchange of C and P leads to the reciprocal
cooperation between AM fungi and bacteria, which is a common mode of
cooperation, widely documented in the
literature^[149]20,[150]52–[151]54. In the hyphosphere, bacteria from
multiple orders, such as Myxococcales, Betaproteobacteriales,
Fibrobacterales, Cytophagales, and Chloroflexales, interact closely
with AM fungi and form the hyphobiome^[152]52,[153]53. These bacteria
thrive in the hyphosphere, dominate the soil bacterial community, and
secrete phosphatases that enhances the availability of soil P to AM
fungi^[154]20,[155]53.
Inorganic P availability alters the P delivery and C acquisition of the AM
fungus with the host to enhance or weaken cooperation with the PSB
Upon uptake by the extraradical hyphae of AM fungi, inorganic P is
first polymerized into Poly-P, then accumulated in tubular vacuoles or
transported with arginine to the intraradical hyphae, where it is
transferred from fungal cell to plant cell^[156]55. AM fungi are able
to regulate P homeostasis and control P transfer to the host via the
PHO regulatory pathway^[157]56,[158]57, depending on the nutritional
status of the host and the availability of inorganic P in the
environment, to maximize the return of C^[159]27,[160]28. In the
present study, we found that high inorganic P availability allowed R.
irregularis to directly absorb large amounts of P from the environment
(Fig. [161]1). The AM fungus may accumulate the inorganic P rather than
transport it to the host, as the expression of core regulatory factor
Pho4, the hydrolase genes involved in the degradation of Poly-P, and
multiple genes responsible for arginine synthesis were not increased in
the presence or absence of R. aquatilis (Fig. [162]5). On the contrary,
low inorganic P availability enhanced reciprocal cooperation between
the AM fungus and the PSB, allowing R. irregularis to efficiently
acquire P from the environment and transfer it to the host when
resources were scarce. This process was accompanied by increased gene
expression of AM fungal C and energy metabolism, suggesting that the AM
fungus may obtain more C returns (Fig. [163]5).
In ecosystems, plants, AM fungi and their associated bacteria can drive
top-down C flows as well as bottom-up nutrient flows^[164]36. The
accelerated P transfer and enhanced C metabolism of R. irregularis
under low inorganic P availability are likely to furnish more C
resources for R. aquatilis and induce this PSB to accelerate the
mineralization of organic P (Figs. [165]2, [166]4, and [167]5). This
ultimately improves the PUR of the AM fungus and the CAR of the PSB,
forming a cross-kingdom closed loop of C and P exchange (Fig. [168]6).
Unlike fatty acids delivered by plants to AM fungi, sugars are
generally thought to be passively exuded by AM fungi and acquired by
PSB (Table [169]S1), which may represent the surplus of nutrients for
AM fungi^[170]58,[171]59. However, in the present study, we found that
the continuous stimulation of PSB growth by hyphal exudates under low
inorganic P availability is attributed to the transfer of P from AM
fungi to the host, thereby enhancing C transfer from plant to fungus
and increasing energy metabolism in the AM fungus (Figs. [172]2,
[173]5). This finding suggests that the exudates released by the hyphae
may not be a purely passive process, but a conscious effort by AM fungi
and presumably the host to quest reciprocal cooperation with PSB, which
support the reciprocity theory that both partners need to proactively
provide resources^[174]60,[175]61.
What are the significances of cooperation between AM fungi and PSBs for
agricultural management?
Increasing the yield of food systems is highly reliant on mineral
fertilizers, whose production is energy-intensive, contributes
massively to global greenhouse gas emissions, and, in the case of P, is
dependent on finite resources^[176]62. In farmland soil, many crops
have low P fertilizer use efficiency, resulting in low recovery of
applied fertilizer^[177]63. Nowadays, there is a growing interest to
reduce our agricultural footprint and reliance on chemical fertilizers
and pesticides through the use of microbial inoculants^[178]64. AM
fungi and their associated bacteria, among which the PSBs, are an
important component of agricultural systems. They modify soil
structure, mineralize organic matter, and improve the uptake and
transfer of nutrients and water to the plants, thereby increasing crop
productivity^[179]36,[180]65. They represent potential tools in
increasing the sustainability of our food production systems. However,
the results are often lagging behind the expectations for many reasons
attributed to the microorganisms, as well as the environmental
conditions and agricultural practices. For instance, under intensive
agricultural systems, a reduction in the diversity of these fungi and
bacteria and an inhibition of their ecological functions have been
observed and attributed partly to the wide application and accumulation
of P fertilizers^[181]66,[182]67. Consequently, exploiting beneficial
soil microbial communities to increase food production while minimizing
the use of mineral P fertilizer and preserving non-renewable soil
resources is an essential objective of sustainable agricultural
development. There is thus a crucial need to better understand how AM
fungi and their associated PSBs cooperate under conditions of low to
high P concentration. For instance, a recent study on maize has
reported that in the presence of a well-established AM fungal
community, the application of P fertilizer can be reduced by 30 kg per
hectare^[183]68. Maize can fully exploit the potential of AM fungi to
obtain P under limited soil P supply conditions^[184]25. In the present
study, we demonstrated that cooperation between the PSB R. aquatilis
and the AM fungus R. irregularis accelerated the mineralization of
organic P when inorganic P availability was low, a process that may
give an indication of the reuse of organic P in the soil. Therefore,
optimizing the application of mineral fertilizers to increase crop
yields based on the mutualistic cooperation strategies between PSBs and
AM fungi, as well as AM fungi and plants, may represent a promising
strategy for achieving sustainable agricultural
development^[185]34,[186]69.
Conclusion
Under low inorganic P availability, the reciprocal cooperation between
the two partners, materialized by C-P exchange, was strengthened,
accompanied by the activation of multiple C metabolism and P transport
and regulation pathways. Conversely, under high inorganic P
availability, the exchange process and metabolism involved were less
solicited, and only glycolysis and phosphatase synthesis pathways of
the PSB as well as P transport and regulatory pathway of the AM fungus
were activated (Fig. [187]7). Therefore, P availability serves as a
mediator for the reciprocal cooperation between AM fungi and soil
bacteria. However, the existing understanding is confined solely to
reciprocal cooperation between two individual species, and the extent
to which plant C allocation in roots can facilitate mutualistic
interactions between multiple AM fungi and soil bacteria remains an
unresolved question.
Fig. 7. Schematic representation of reciprocal cooperation based on C and P
exchange between R. irregularis and R. aquatilis.
[188]Fig. 7
[189]Open in a new tab
Schematic representation of reciprocal cooperation based on C and P
exchange between the arbuscular mycorrhizal fungus R. irregularis and
the phosphate solubilizing bacterium R. aquatilis under under a low
(35 μM KH[2]PO[4]) and b high (700 μM KH[2]PO[4]) P availabilities. A
series of pathways for C metabolism and transport (green arrows) as
well as P metabolism and transport (purple arrow) in the AM fungus and
the bacterium are described. Pathways with enrichment and upregulated
are highlighted in red. Pase, phosphatase; TCA cycle, tricarboxylic
acid cycle.
Although root organ cultures (ROC) is a well-established and widely
recognized model system, with nutritional, metabolic, and resource
transfer characteristics close to those of whole plant
systems^[190]19,[191]21,[192]27, it presents some constraints to fully
understand the dynamics of nutrient exchange that occur within
ecosystems. In the invisible environment of the soil, multiple fungi
and bacteria coexist in micro-ecological niches created by plants,
maintaining a microbial network of interactions and
interdependencies^[193]70. The role of individual species in the
community and the cooperative mechanism between species need to be
verified using more realistic potted and field systems. Consequently,
future studies should focus on extending the influence of external
resources on the strategies and mechanisms of cooperation between two
individual species to the whole plant-AM fungi-bacteria community,
which is necessary if researchers are to explore the complex ecological
and evolutionary implications of plant-microbe social relationships.
Materials and methods
Biological material
The AM fungus used was R. irregularis (Błaszk., Wubet, Renker & Buscot)
C. Walker & A. Schüßler as “irregulare” MUCL 43194. It was isolated
from the roots of Fraxinus americana L. (American ash) in Pont Rouge,
Québec, Canada^[194]71 and maintained in bi-compartmented Petri plates
on ROC of Daucus carota L. clone DC2 on the Modified Strullu-Romand
(MSR) medium^[195]72. The PSB used was R. aquatilis HX2. It was
isolated from the rhizosphere soil of a vineyard located in Beijing,
China^[196]73. The plant Medicago truncatula L. (barrel clover) Gaertn.
cv. Jemalong A17 was used for experiments.
In vitro culture system design
The AM fungus was grown with ROC of D. carota or M. truncatula in
bi-compartmented Petri plates, which is the most reliable method for
generating substantial quantities of extraradical hyphae without any
microbial contaminant. Briefly, in one compartment (i.e., the root
compartment—RC), a carrot or barrel clover root was associated with the
AM fungus, while in the other compartment (i.e., the hyphal
compartment—HC) only the extraradical hyphae of the AM fungus were
allowed to proliferate (Fig. [197]S5a, b). 25 mL of sterilized (121 °C
for 15 min) MSR medium solidified with 3 g L^−1 Phytagel (Gelzan^TM CM,
G3251, CP Kelco, USA) was added in the RC. To facilitate the passage of
extraradical hyphae from the RC to the HC, 4 mL of the same medium
lacking sucrose, inorganic P, EDTA, calcium, and vitamins was added to
create a slope from the top of the HC to the bottom of the HC next to
the barrier. In addition, 10 mL of liquid MSR medium with the same
composition as the slope (except for the absence of gelling agent) was
added to the HC. A control treatment was also included under identical
conditions, except that a 0.5 cm wide strip of MSR medium was excised
from the RC next to the barrier to prevent the passage of hyphae into
the HC.
Experiment 1: impact of inorganic P availability on the mechanism of
cooperation between R. irregularis and R. aquatilis
The experiment was conducted in an in vitro bi-compartmented culture
system on MSR medium. Briefly, ROCs of M. truncatula were associated
with R. irregularis MUCL 43194 in the RC, while only the hyphae were
allowed to develop on a slope in the HC. After hyphae had crossed the
partition wall separating the RC from the HC and densely covered the
slope, 10 mL of liquid MSR medium with the same composition as the
slope (except for the absence of gelling agent) was added to the HC.
After another two weeks, the HC was covered with numerous actively
growing extraradical hyphae. At this point, the medium was removed and
replaced with 10 mL of fresh liquid MSR medium containing two different
concentrations of P. In half of the experimental systems, 35 μM
KH[2]PO[4] was added, representing a low P concentration in soil
solution and corresponding to the low inorganic P availability
treatment, while in the other half, 700 μM KH[2]PO[4] was added,
representing a high P concentration in soil solution and corresponding
to the high inorganic P availability treatment^[198]21. Prior to this
change, the HC of the experimental systems were gently rinsed three
times with sterilized (121 °C for 15 min) deionized water. The inoculum
of R. aquatilis was prepared as follows: The bacterium was cultured in
liquid Luria–Bertani (LB) medium with shaking at 180 rpm at 28 °C until
optical density (OD)[600] reached 0.4–0.6 (logarithmic phase). Then it
was centrifuged at 5878 g for 10 min. The supernatant was removed, and
the pellet was re-suspended and washed three times with inorganic
P-free liquid MSR medium. The supernatant was subsequently adjusted to
OD[600] = 0.6 and stored at 4 °C before use.
One week later, the remaining liquid medium in the HC was collected and
mixed homogeneously with 3 mL of R. aquatilis supernatant
(concentration of ~10^8 CFUs mL^−1) and 1 mL of 280 μM phytate
(Na-phytate, Sigma-Aldrich). The solution was then carefully returned
to the HC of each experimental system using a micropipette. 24 h later,
R. irregularis hyphae and R. aquatilis cells, as well as the remaining
liquid medium, were collected. To satisfy the sequencing requirements
for the AM fungus and PSB, each set of three experimental systems was
pooled into one. To collect R. aquatilis cells, the culture medium was
carefully agitated with a micropipette, and a 9 mL aliquot of medium
containing cells was removed using a micropipette and immediately
stored in liquid nitrogen for R. aquatilis absolute abundance and
transcriptome analysis. Likewise, R. irregularis hyphae were carefully
picked out with sterilized forceps, placed into a 2 mL tube, and
immediately frozen in liquid nitrogen for transcriptome analysis. The
remaining liquid medium was filtered through an Acrodisc Syringe Filter
(0.2 μm Supor Membrane, Pall Corporation, Cornwall, UK) to eliminate
bacterial cells, and stored at −20 °C for P concentration and
phosphatase activity analysis. In total 6 treatments were considered,
namely, the experimental systems with (1) only R. irregularis hyphae
developing in the HC under high and low inorganic P availability (i.e.,
the P^High RI^+ RA^− and P^Low RI^+ RA^− treatments), (2) only R.
aquatilis cells in the HC under high and low inorganic P availability
(i.e., the P^High RI^− RA^+ and P^Low RI^− RA^+ treatments), and (3)
both R. irregularis hyphae and R. aquatilis cells in the HC under high
and low inorganic P availability (i.e., the P^High RI^+ RA^+ and P^Low
RI^+ RA^+ treatments) (Fig. [199]S5a, b). Each treatment was replicated
four times, resulting in a total of 72 experimental systems.
RNA isolation, library preparation, and sequencing
The total RNA was extracted from the frozen hyphae of R. irregularis
and cells of R. aquatilis using the RNeasy Plant Mini Kit and RNeasy
Mini Kit (QIAGEN GmbH, Hilden, Germany), respectively. The
concentration and integrity of the isolated RNA were determined using
NanoDrop-ND 1000 UV–Vis Spectrophotometer, after which the samples were
shipped to Novogene Co., Ltd (Cambridge, UK) for library preparation
and sequencing.
For the AM fungus, Poly-T oligo-attached magnetic beads were used to
isolate mRNA from the total RNA. The process involved synthesizing two
strands of cDNA using random hexamer primers dUTPs. In contrast, for
the PSB, rRNA was first removed from the total RNA, and then the
remaining RNA was subjected to ethanol precipitation. During synthesis
of the second cDNA strand, dTTPs were replaced by dUTPs in the reaction
buffer.
The directional library was prepared following end repair, A-tailing,
adapter ligation, size selection, USER enzyme digestion, amplification,
and purification. It was then assessed using Qubit and real-time q-PCR
for quantification and a bioanalyzer for size distribution analysis.
The quantified libraries were subsequently pooled and sequenced on
Illumina platforms, in accordance with their effective concentrations
and the desired data output.
Bioinformatics analysis of R. irregularis and R. aquatilis at the
transcriptional level
The raw data (raw reads) were first processed by an internal Perl
script. Clean data (clean reads) were obtained by removing reads
containing adapter, reads containing ploy-N and low-quality reads. All
subsequent analyses were based on these high-quality clean data. For
the AM fungus, Hisat2 v2.0.5 was used to build the reference genome
index and align the paired clean reads to the reference genome^[200]74.
For PSB, building the reference genome index and aligning the clean
reads to the reference genome were done using Bowtie2^[201]75.
FeatureCounts v1.5.0-p3 was used to calculate the number of aligned
reads for each gene. Subsequently, the transcripts per kilobase million
(TPM) for each gene was calculated, considering the gene length and the
count of reads mapped to that gene^[202]76. As a quantitative metric of
gene expression, TPM is currently the most used method for comparing
gene expression levels across different groups^[203]77.
The differential expression analysis was done comparing P^Low RI^+ RA^+
versus P^High RI^+ RA^+ treatments, P^Low RI^+ RA^+ versus P^Low RI^+
RA^− treatments, P^High RI^+ RA^+ versus P^High RI^+ RA^− treatments,
P^Low RI^+ RA^+ versus P^Low RI^− RA^+ treatments, P^High RI^+ RA^+
versus P^High RI^− RA^+ treatments, using the DESeq2 R package. DESeq2
proposes statistical methods designed to identify DEGs in digital gene
expression datasets, using a model based on the negative binomial
distribution. The P-values obtained were subsequently adjusted using
the Benjamini and Hochberg method to control the false discovery
rate^[204]78. The genes identified by DESeq2 with an adjusted P-value
of less than 0.05 were considered to be differentially expressed.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis of DEGs was conducted using the clusterProfiler R package.
Pathways from KEGG with a corrected P-value of less than 0.05 were
considered to be enriched by the DEGs^[205]79. The KEGG pathway for the
non-model species R. irregularis was annotated by referencing the model
fungus Saccharomyces cerevisiae (budding yeast).
DNA isolation and real-time q-PCR analysis of the absolute abundance of R.
aquatilis
DNA was extracted from the frozen cells of R. aquatilis using the
QlAprep Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany). Each sample
was analyzed in triplicate via real-time q-PCR using a Roche
LightCycler^® 96 System with R. aquatilis specific primers (atpD-F,
5’-GATGTGTCCGTTCGCTAA-3’ and atpD-R, 5’-GATAACCTACCGCTGATGG-3’). Each
q-PCR reaction was conducted in a total volume of 20 μL, comprising
2 μL of DNA, 10 μL of Master mix, 0.5 μL of each primer, and 7 μL of
ddH[2]O. The following thermal cycling conditions were applied: 95 °C
for 300 s, 40 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for
45 s. The melting curve, baseline range, and cycle threshold (Ct)
values were monitored and calculated. The number of copies was
calculated using the Ct values, which are directly proportional to the
biomass of R. aquatilis in the samples. This was done through the
application of standard curves, which was established in triplicate
using five serial 10-fold dilutions. The number of copies was then
determined by identifying the initial copy number, factoring in the
plasmid concentration and the aggregate number of base pairs
(encompassing both vector and primer sequences). Following this, the
DNA isolation efficiency was calculated by referencing the Ct values
from the standard curves and normalizing the relative abundance of PSB
to account for the DNA isolation efficiency.
Quantification of organic and inorganic P concentrations and ACP and ALP
activities in the HC
Inorganic P concentration in the liquid medium of the HC was quantified
using the molybdate-blue method^[206]80. Total P concentration was
assessed through inductively coupled plasma atomic emission
spectroscopy. Organic P (i.e., Na-phytate) concentration was calculated
by subtracting the inorganic P concentration from the total P
concentration. ACP and ALP activities in liquid medium (pKatal mL^−1)
were measured according to the method of Neumann (2006)^[207]81.
Evaluation of phosphatase production efficiency, organic P mineralization
efficiency, C acquisition ratio by R. aquatilis, and P uptake ratio by R.
irregularis
EAP, ELP and EPM were calculated by Eqs. [208]1 and [209]2,
respectively:
[MATH: EAPELP=ZRI+RA+
ZRI−RA+ :MATH]
1
[MATH:
EPM=C<
/mi>RI+RA
+CRI−RA+
:MATH]
2
(Z [RI+ RA+], enzyme activity detected in the RI^+ RA^+ treatment; Z
[RI− RA+], enzyme activity detected in the RI^− RA^+ treatment; C [RI+
RA+], the consumption of organic P in the RI^+ RA^+ treatment; C [RI−
RA+], the consumption of organic P in the RI^− RA^+ treatment)
The R. aquatilis CAR and R. irregulars PUR were calculated by
Eqs. [210]3 and [211]4, respectively:
[MATH: PUR=
CRI+
RA+−C
RI−RA+CRI+RA− :MATH]
3
[MATH:
CAR=X<
/mi>RI+RA
+−XR<
/mi>I−RA+XRI−<
/mo>RA+ :MATH]
4
(C [RI+ RA+], the reduction of total P in the RI^+ RA^+ treatment; C
[RI− RA+], the reduction of total P in the RI^− RA^+ treatment; C [RI+
RA−], the reduction of total P in the RI^+ RA^− treatment; X [RI+ RA+],
bacterial absolute abundance in the RI^+ RA^+ treatment; X [RI− RA+],
bacterial absolute abundance in the RI^− RA^+ treatment)
Experiment 2: effects of AM fungal exudates, produced in the presence of low
and high availability of P, on the growth of R. aquatilis
Bi-compartmented experimental systems were prepared with ROC of M.
truncatula associated with R. irregularis MUCL 43194 in the RC. After
eight weeks, extraradical hyphae developed profusely in the solid slope
and in the liquid MSR medium of the HC. At this point, the medium was
removed and replaced with 10 mL of fresh liquid MSR medium containing
35 μM KH[2]PO[4] or 700 μM KH[2]PO[4] as described in Experiment 1.
After one week, the hyphal exudates were collected. The inoculum of R.
aquatilis was prepared as described in Experiment 1 with slight
modifications: the LB medium containing R. aquatilis cells was
centrifuged to eliminate the supernatant, and R. aquatilis cells were
re-suspended threefold in a sterilized 0.85% NaCl solution. The
supernatant was then diluted to OD[600] = 0.1.
In a microwell plate, 500 μL of sterilized 0.85% NaCl solution was
introduced into the peripheral wells to mitigate potential edge
effects, and 450 μL of liquid medium collected from HC (i.e., P^High
RI^+, P^High RI^-, P^Low RI^+ and P^Low RI^− treatments) or 450 μL of
sterilized 0.85% NaCl solution (i.e., control treatment) were dispensed
into the central wells of the microwell plate. Then, 50 μL of the
prepared R. aquatilis suspension was introduced into the central wells
and uniformly mixed with the liquid medium. Each treatment was
replicated four times. The growth curve of PSB was monitored at 37 °C
over a 48-h period and the OD[600] measurements of the liquid cultures
were taken at 2-h intervals.
Statistics and reproducibility
Statistical analyses were conducted using SPSS v. 16.0 (SPSS Inc.,
Chicago, IL, USA). Tukey’s honest significant difference test
(P-value < 0.05) was used to evaluate the significant differences among
RI^+ RA^+, RI^− RA^+ and RI^+ RA^− treatments at the same P
availability. In addition, a t-test (P-value < 0.05) was conducted to
determine the significant differences between the low and high P
treatments.
To ensure the normal distribution of data, the TMPs of various genes
were subjected to a log transformation. The DESeq2 R package was used
to obtain variance-stabilized counts for genes associated with the P
function by transforming read counts. Heatmaps were generated using the
heatmap R package to depict the abundance of P cycle-related genes and
the gene expression levels of multiple metabolic pathways. The Spearman
method was used to assess the correlation between variance-stabilized
count-based P regulation genes and genes involved in P solubilization,
mineralization, and transport.
Reporting summary
Further information on research design is available in the [212]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[213]Supplementary material^ (905.7KB, pdf)
[214]Supplementary data 1-5^ (29.2KB, xlsx)
[215]Supplementary data 6^ (2.9MB, xlsx)
[216]42003_2025_8501_MOESM4_ESM.pdf^ (41.8KB, pdf)
Description of additional supplementary files
[217]Reporting summary^ (2.9MB, pdf)
[218]Transparent peer review file^ (943.8KB, pdf)
Acknowledgements