Abstract
Soil microbiota can confer fitness advantages to plants and increase
crop resilience to drought and other abiotic stressors. However, there
is little evidence on the mechanisms correlating a microbial trait with
plant abiotic stress tolerance. Here, we report that Streptomyces
effectively alleviate drought and salinity stress by producing
spiroketal polyketide pteridic acid H (1) and its isomer F (2), both of
which promote root growth in Arabidopsis at a concentration of 1.3 nM
under abiotic stress. Transcriptomics profiles show increased
expression of multiple stress responsive genes in Arabidopsis seedlings
after pteridic acids treatment. We confirm in vivo a bifunctional
biosynthetic gene cluster for pteridic acids and antimicrobial
elaiophylin production. We propose it is mainly disseminated by
vertical transmission and is geographically distributed in various
environments. This discovery reveals a perspective for understanding
plant-Streptomyces interactions and provides a promising approach for
utilising beneficial Streptomyces and their secondary metabolites in
agriculture to mitigate the detrimental effects of climate change.
Subject terms: Natural products, Plant hormones, Applied microbiology
__________________________________________________________________
Soil microbiota can increase crop resilience to abiotic stressors. Here
the authors show that Streptomyces produce bioactive spiroketal
polyketides to enhance plant growth under drought and salt stress.
Introduction
According to the Food and Agriculture Organization of the United
Nations, climate change generates considerable uncertainty about future
water availability in many regions. Increased water scarcity under
climate change will present a major challenge for climate adaptation,
while sea-level rise will affect the salinity of surface and
groundwater in coastal areas. Stress caused by climate change has led
to increased agricultural losses and threatened global food
security^[42]1. Drought is considered the most damaging environmental
stress, which directly affects the entire growth period of plant seeds
from germination to final fruiting^[43]2. Drought stress can lead to
increased plant osmotic regulators, the inhibition of photosynthesis,
and the change of plant endogenous hormone content^[44]3–[45]5. Drought
stress also induces reactive oxygen species, such as superoxide
radicals, hydrogen peroxide, and hydroxyl radicals, leading to
oxidative stress^[46]6. Crop loss due to soil salinisation is another
increasing threat to agriculture worldwide, which is more severe in
agricultural land in coastal and arid regions^[47]7. Irrigation with
saline water, low precipitation, and high evapotranspiration are key
factors of the rapid salinisation of agricultural land^[48]8. These
abiotic stresses of drought and salinity have brought unprecedented
challenges to the development of crop farming. Compared to the heavy
use of chemical fertilisers, the use of plant growth-promoting bacteria
to improve plant growth under drought and salinity environments is more
sustainable and gaining more attention^[49]9,[50]10.
Soil microbial communities are critical to plant health and their
resistance to both biotic and abiotic stressors, such as pathogens,
drought, salinity, and heavy metal pollution^[51]11. A few studies have
demonstrated that many beneficial soil bacteria harbour plant
growth-promoting activities, e.g. by helping plants with disease
suppression^[52]12, nutrient acquisition^[53]13, phosphorus
uptake^[54]14 and nitrogen fixations^[55]15. Beneficial root microbiota
also regulate biosynthetic pathways in the plant itself, leading to
differential alterations in the plant metabolome in response to
stresses^[56]16. Streptomyces are Gram-positive filamentous bacteria,
widely distributed in soil and marine environments. While they have
long been considered the richest source of bioactive secondary
metabolites^[57]17, Streptomyces have recently drawn attention as a
class of plant growth-promoting bacteria that help plants respond to
adversity stress^[58]18. The growing evidence showed that Streptomyces
can promote plant growth or tolerance to stressors in direct or
indirect ways, by secreting plant growth regulator auxin
(indole-3-acetic acid, IAA) and siderophores, inducing systemic
resistance in plants, and regulating the rhizosphere microbiome via
producing antibacterial compounds or signalling
molecules^[59]19–[60]21. Notably, the commercial product Actinovate®
and Mycostop® are two Streptomyces-based formulations that have been
widely used to suppress a wide range of diseases in a variety of crop
groups as a biological fungicide/bactericide for the long term.
Recently, the enrichment of Streptomyces has also been shown to play a
subsequent role in the drought/salt tolerance of plants^[61]22. Despite
the widespread claims of efficacy of inoculation of plant
growth-promoting Streptomyces, the molecular basis of the
growth-promoting effects and the key role of secondary/specialised
metabolites in this process are largely unknown.
Here, we report that Streptomyces iranensis HM 35 has profound
beneficial effects on helping barley alleviate osmotic, drought and
salinity stress. The active components were identified as bioactive
spiroketal polyketides pteridic acids H (1) and its isomer F (2)
through large-scale fermentation and bioactivity-directed purification
followed by NMR, MS, and X-ray crystallography. The abiotic stress
mitigating effects of pteridic acids H and F have been confirmed on the
model plant Arabidopsis thaliana, where it effectively reversed both
drought and salinity stress as phytohormone-like small biomolecules at
concentrations as low as 0.5 ng mL^−1 (1.3 nM). RNA sequencing results
suggested that pteridic acids may assist plants in stress resistance
via activating photosynthesis and regulating multiple stress response
genes. Moreover, the Biosynthetic Gene Cluster (BGC) of pteridic acids
(pta) was identified and analysed in silico, and functionally confirmed
by in vivo CRISPR-based genome editing. We have furthermore conducted a
survey of 81 potential producers of pteridic acids, which are widely
distributed around the world. Phylogenetic and comparative genomic
analysis of pta-containing streptomycetes suggested that these strains
have evolutionary convergence in disseminating pta BGC through main
vertical transmission and occasional horizontal gene transfer. In
summary, we reveal a strategy of Streptomyces to secret plant growth
regulators that help plants cope with abiotic stresses, which is a
promising alternative solution for plant development and crop yields
under the current climate change-induced environmental stresses.
Results
Abiotic stress-mitigating activities exhibited by S. iranensis
The promotion of barley growth induced by S. iranensis was tested under
multiple abiotic stresses including osmotic, salinity and drought. The
osmotic stress experiment was simulated using soils supplemented with
20% (w/v) PEG-6000 by transiently reducing the water potential of the
plant. We found that S. iranensis played a significant role in
alleviating osmotic stress in barley seedlings. The treated seedlings
showed a significant increase in height, fresh weight, and dry weight
compared to the control group without any extra treatment
(Fig. [62]1a). The culture broth of S. iranensis also showed
considerable activity for the growth of barley in alleviating salinity
stress mediated by 100 mM NaCl (Fig. [63]1b), while S. iranensis was
not significantly enriched in the soil around the roots of barley
seedlings (Supplementary Fig. [64]1). Additionally, based on the
analysis of barley seedling phenotypes, the treatment with S. iranensis
resulted in a significant improvement in plant growth recovery from
drought stress (Fig. [65]1c). Surprisingly, S. iranensis promoted the
growth of barley seedlings even in non-stress growth condition and thus
indicate that S. iranensis may have potential for use as biostimulant
(Supplementary Fig. [66]2).
Fig. 1. The effects of S. iranensis on barley seedlings under abiotic stress
and the bioactive components produced by S. iranensis.
[67]Fig. 1
[68]Open in a new tab
a The box plots depict the plant height, fresh weight and dry weight of
barley seedlings growing under osmotic stress mediated by 20% (w/v)
PEG-6000 (mean ± SD, n = 18). b the box plots depict the plant height,
fresh weight and dry weight of barley seedlings growing under salinity
stress mediated by 100 mM NaCl (mean ± SD, n = 18). In a and b,
statistical significance was assessed by one-way ANOVA with post hoc
Dunnett’s multiple comparisons test. Asterisks indicate the level of
statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 and
****p < 0.0001. Abbreviation: Mock: control; Si: treatment of S.
iranensis culture broth; D: treatment of S. iranensis/ΔptaA culture
broth; M: treatment of blank medium (ISP2). c The box plots depict the
plant height, fresh weight and dry weight of barley seedlings growing
under drought stress (mean ± SD, n = 18). Different colours of box
plots indicate different growing conditions: green, 21 days water;
brown, 7 days treatment after 7 days water followed by 7 days drought;
red, 7 days water followed by 14 days drought. Statistical significance
was assessed by one-way ANOVA with Tukey test. Asterisks indicate the
level of statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001
and ****p < 0.0001. Abbreviation: Water: well water for 21 days; CK, 7
days treatment of water after 7 days water + 7 days drought; Si: 7 days
treatment of S. iranensis culture broth after 7 days water + 7 days
drought; D: 7 days treatment of S. iranensis/ΔptaA culture broth after
7 days water + 7 days drought; M: treatment of blank medium (ISP2);
Drought: 14 days drought after 7 days water. Statistical significance
was assessed by one-way ANOVA with Tukey test. d The metabolite profile
of the native S. iranensis growing in liquid ISP2 medium, the known
secondary metabolites were identified by HR-LC-MS and highlighted; e
the bioactive components pteridic acid H (1) and pteridic acid F (2)
isolated from S. iranensis. All box plots with centre lines showing the
medians, boxes indicating the interquartile range, and whiskers
indicating a range of minimum to maximum data beyond the box. Source
data are provided as a Source Data file.
Genomic and metabolomic profiles of S. iranensis
To reveal the potential bioactive components, we first annotated BGCs
responsible for the biosynthesis of secondary metabolites in S.
iranensis using antiSMASH 6.0^[69]23. Genome sequence analysis of S.
iranensis revealed the presence of 47 putative secondary metabolites
BGCs with a variety of biosynthetic categories (Supplementary
Table [70]1). The clusters 3, 6, 7, 8, 23, 31, 35 and 40 were annotated
to have greater than 80% similarity with BGCs responsible for the
biosynthesis of coelichelin, azalomycin, nigericin, elaiophylin,
desferrioxamin B, ectoine, rapamycin and hygrocin, respectively. Their
corresponding products were also detected and identified through
High-resolution Liquid Chromatography-tandem Mass Spectrometry
(HR-LC-MS/MS) as well as Global Natural Products Social (GNPS)
molecular networking (Fig. [71]1d, Supplementary Fig. [72]3)^[73]24.
However, a large number of metabolites from S. iranensis are still
unknown. Since none of the previously identified compounds have been
associated with mitigating abiotic stress in plants, we were prompted
to expand the fermentation process and identify the potential bioactive
compounds.
Characterisation of the bioactive compound pteridic acid
To uncover the bioactive components, fermentation of S. iranensis was
scaled up to 175 litres and the culture broth was subjected to
separation through open-column chromatography on Amberchrom CG161Me
resin, silica gel, and Sephadex LH-20. Bioactivity-guided fractionation
led to the isolation of bioactive compound 1 (15.0 mg) together with
its isomer compound 2 (4.0 mg) (Fig. [74]1e).
The bioactive component compound 1 was isolated as a white solid. Its
formula of C[21]H[34]O[6] was deduced by m/z 383.2439 [M + H]^+
(calculated for 383.2428, Δ 2.84 ppm). The ^1H NMR spectrum exhibited
signals for four olefinic protons (δ 7.33, 6.26, 6.13, 5.90)
corresponding to two conjugated double bonds, four oxygen-bearing
methines (δ 3.85, 3.69, 3.59, 3.43), five methyls, and other aliphatic
protons. The ^13C NMR spectrum indicated the presence of one carbonyl
group (δ 168.7) and one oxygen-bearing quaternary carbon (δ 103.2,
C-11). The COSY spectrum established two partial substructures, which
could be connected via a spiral function by analysing HSQC and HMBC
correlations (Supplementary Figs. [75]4–[76]11). Compound 1 was
crystallised in methanol solution, and the structure was determined via
X-ray crystallography (Supplementary Fig. [77]12). Therefore, compound
1 was identified as a new Streptomyces-derived natural product named
pteridic acid H.
Compound 2, a white solid, is an isomer of 1 deduced by MS with the
same molecular formula of C[21]H[34]O[6.] The ^1H NMR spectrum
exhibited signals for four olefinic protons (δ 7.16, 6.25, 6.07, 5.97)
corresponding to two conjugated double bonds, four oxygen-bearing
methines (δ 3.88, 3.66, 3.56, 3.32), five methyls, and other aliphatic
protons. The ^13C NMR spectrum indicated the presence of one carbonyl
group (δ 170.2) and one oxygen-bearing quaternary carbon (δ 103.2,
C-11). HSQC and HMBC correlations confirmed a spiroketal skeleton.
NOESY spectrum confirmed its relative configurations, where
correlations between H-21 and H-13 and H-15, H-7 and H-12a, and H6,
H-10, and Me-18 were observed. The key NOESY correlations between H-7
and H-12a revealed a different spiroketal structure than 1
(Supplementary Fig. [78]11, [79]13-[80]20). This can be reflected by
the relative up-field NMR data for C-12 (δ 33.8 vs δ 37.4 in 1).
Compound 2 was identified as pteridic acid F, previously isolated from
Streptomyces pseudoverticillus YN17707 and a marine-derived
Streptomyces sp. SCSGAA 0027^[81]25,[82]26.
Abiotic stress mitigation of pteridic acids in planta
Initially, we tested the effects of different concentrations of
pteridic acids H and F on Arabidopsis growth in the absence of abiotic
stress. A concentration of 0.5 ng ml^−1 of both pteridic acids H and F
was found to significantly promote the growth of Arabidopsis seedlings
(Supplementary Fig. [83]21). Under drought stress, pteridic acid H at a
concentration of 0.5 ng mL^−1 increased the root length and fresh
weight of Arabidopsis seedlings by 54.5% and 89%, respectively, and its
activity was significantly better than IAA and ABA at the same molar
concentration (Fig. [84]2a, c). The treatment of pteridic acid F also
showed great activity in alleviating drought stress, and the root
length and fresh weight were increased by 30.5% and 56.7%, respectively
(Fig. [85]2c). Pteridic acids H and F also showed significant activity
in alleviating NaCl-mediated salinity stress (Fig. [86]2b, d). Compared
to the non-treated groups, the treatment of 0.5 ng mL^−1 pteridic acids
H and F increased root length of Arabidopsis seedlings by 74.0% and
61.8%, as well as fresh weight by 126.2% and 110.9%, respectively
(Fig. [87]2d).
Fig. 2. The effect of pteridic acids H and F on Arabidopsis seedlings under
abiotic stress.
[88]Fig. 2
[89]Open in a new tab
a Phenotype of Arabidopsis seedlings growing under drought stress
mediated by 15% (w/v) PEG-6000 using other treatments (bars = 1 cm); b
Phenotype of Arabidopsis seedlings growing under salinity stress
mediated by 80 mM NaCl using other treatments (bars = 1 cm); c The box
plots depict the primary root length and fresh weight of Arabidopsis
seedlings growing on non-stress condition (mean ± SD, n = 16); d The
box plots depict the primary root length and fresh weight of
Arabidopsis seedlings growing on drought stress condition (mean ± SD,
n = 16). In c and d, statistical significance was assessed by one-way
ANOVA with Tukey test. Asterisks indicate the level of statistical
significance: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; e
differences of lateral root growth of Arabidopsis seedlings growing in
other conditions. Mock: control; PH: treatment of 0.5 ng mL^−1 pteridic
acid H; PF: treatment of 0.5 ng mL^−1 pteridic acid F; IAA: treatment
of 1.3 nM indole-3-acetic acid; ABA: treatment of 1.3 nM abscisic acid.
All box plots with centre lines showing the medians, boxes indicating
the interquartile range, and whiskers indicating a range of minimum to
maximum data beyond the box. Source data are provided as a Source Data
file.
To get a first understanding how pteridic acids help Arabidopsis
mitigate salinity stress, we used messenger RNA sequencing (mRNA-seq)
to profile the transcripts of Arabidopsis seedlings treated with
pteridic acid F or H under NaCl-mediated salt stress (Fig. [90]3a).
Compared to the control (CK, treatment with an equal amount of water
under NaCl-mediated salt stress), we observed significant differences
in the gene expression patterns upon treatment with pteridic acid (PH
or PF) by Pearson correlation analysis (Supplementary Fig. [91]22). The
Differentially Expressed Genes (DEGs) analysis revealed 3575 DEGs (1405
upregulated and 2170 downregulated), and 3727 DEGs (1555 upregulated
and 2172 downregulated) in the pteridic acid H treatment versus control
(PH vs. CK) and pteridic acid F treatment versus control (PF vs. CK),
respectively (Supplementary Data [92]1). Meanwhile, 1226 up-regulated
genes and 1860 down-regulated genes were shared between PH and PF
treatments. Several abiotic stress-related genes were significantly
upregulated after pteridic acids treatments (Fig. [93]3b), including
SLAC1 HOMOLOGUE 1 (SLAH1, AT1G62280)^[94]27, PIN-FORMED 6 (PIN6,
AT1G77110)^[95]28, FERRIC REDUCTION OXIDASE 6 (FRO6, AT5G49730)^[96]29
and TOO MANY MOUTHS (TMM, AT1G80080)^[97]30. Compared with the control,
ALPHA CARBONIC ANHYDRASE 8 (ATACA8, AT5G56330)^[98]31, and IAA-LEUCINE
RESISTANT 2 (ILR2, AT3G18485)^[99]32 were uniquely upregulated in the
PH treatment samples, while ATP-BINDING CASSETTE G17 (ABCG17,
AT3G55100)^[100]33, DIACYLGLYCEROL KINASE 4 (DGK4, AT5G57690)^[101]34
and MAP KINASE KINASE 7 (MKK7, AT1G18350)^[102]35 were upregulated in
the PF treatment samples.
Fig. 3. Effect of exogenous pteridic acids on transcription in Arabidopsis
seedlings under salt stress.
[103]Fig. 3
[104]Open in a new tab
a The phenotype of Arabidopsis seedling samples in different treatments
under salinity stress. CK: control; PF: pteridic acid F treatment; PH:
pteridic acid H treatment. b The volcano plots of DEGs identified by
mRNA-seq in Arabidopsis seedlings treated by pteridic acid H and F
under salinity stress. c heat-maps of DEGs enriched in photosynthesis,
response to stimulus, auxin-activated signalling pathway, abiotic
stress defense and TFs.
Next, we performed the Gene Ontology (GO) enrichment analysis of DEGs
focusing primarily on the biological processes (Supplementary
Data [105]2). Among all upregulated genes, several highly enriched GO
terms such as “Photosynthesis (GO:0015979)”, “Plastid organization
(GO:0009657)”, “Chloroplast organization (GO:0009658)” and “Response to
light stimulus (GO:0009416)” suggested pteridic acids could enhance
photosynthesis under abiotic stress. For these DEGs with
log[2]FoldChange ≥ 2, we observed upregulation of 12 genes belonging to
the “auxin-activated signalling pathway (GO:0009734)” (Fig. [106]3c),
suggesting that pteridic acids also trigger auxin-activated signalling
transduction. Intriguingly, we observed the downregulations of genes
related to “response to stimulus (GO:0050896)”, “response to chemical
(GO:0042221)” and “response to stress (GO:0006950)”, which was
speculated to be an interference between different signalling pathway
or a negative feedback mechanism of plants to maintain homoeostasis
(Fig. [107]3c)^[108]36. Moreover, similar conclusions were reached by
the most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway, pointing to the “Photosynthesis (ath00195)” and “Plant hormone
signal transduction (ath04075)” pathways in the Arabidopsis seedlings.
In addition, the pathway of “Motor proteins (ath04814)”, “Ribosome
(ath03010)”, “Glucosinolate biosynthesis (ath00966)”, “Porphyrin
metabolism (ath00860)” and “Flavonoid biosynthesis (ath00941)” were
also activated, which had been previously reported that these are
closely associated with plant abiotic stress
resistance^[109]37,[110]38. On the contrary, pteridic acids
downregulated several genes enriched in “Phenylpropanoid biosynthesis
(ath00940)”, “Cyanoamino acid metabolism (ath00460)”, “Glutathione
metabolism (ath00480)”, “MAPK signalling pathway-plant (ath04016)” and
“Starch and sucrose metabolism (ath00500)” pathways.
Transcription Factors (TFs) play an important function in coping with
abiotic stress tolerance. Several TFs have been proved to participate
in plant salt stress responses, such as AP2/ERF, bZIP and
MYB^[111]39–[112]41. Within these DEGs, we identified various TFs,
comprising AP2/ERF (42 unigenes), MYB (35 unigenes), WRKY (27
unigenes), bHLH (31 unigenes) and bZIP families (6 unigenes)
(Supplementary Data [113]3). A handful of plant stress
resistance-related TFs were observed, such as MYB25, MYB111, WRKY46,
PRE1 and SCRM2^[114]42–[115]46. TFs associated with resistance to
abiotic stress, such as MYB60 and ERF120, exhibited discernible
upregulation in the PH treatment samples compared to the
control^[116]47,[117]48. Conversely, MYB29, MYB113 and PRE6 showed
unique upregulation in the PF vs. CK group^[118]49,[119]50.
A previous study suggested that pteridic acids A and B might have a
plant growth-promoting effect like IAA and could stimulate the
formation of adventitious roots in kidney beans^[120]25. However, we
observed that pteridic acids H and F displayed different IAA-induced
phenotypes. Pteridic acids did not exhibit the function to
significantly promote lateral root growth of Arabidopsis seedlings like
IAA (Fig. [121]2e)^[122]51. They were also not capable of promoting the
formation of adventitious roots in kidney beans, as shown in
Supplementary Fig. [123]23. Except for drought and salinity stress, we
also tested the CuSO[4]-mediated heavy metal stress alleviation
activity of pteridic acids on mung beans. The results showed that the
1 ng mL^−1 pteridic acid H was as effective as ABA in helping mung
beans to relieve heavy metal stress (Supplementary Fig. [124]24). In
conclusion, pteridic acids H and F are widely applicable potent plant
growth regulators produced by Streptomyces to assist plants in coping
with different abiotic stress.
Biosynthesis of pteridic acids
The retro-biosynthesis analysis indicated that pteridic acids could
derive from a modular type I polyketide synthase. A putative pta BGC
was identified in the whole genome sequence of S. iranensis, which
shows 87% antiSMASH similarity to the BGC of elaiophylin (BGC0000053 in
MiBiG database)^[125]52. The pta BGC spans approximately 56 kb and
encodes 20 individual biosynthetic genes responsible for the
biosynthesis of core polyketide backbones, precursor, glycosylated
substituents, transporters and regulators (Supplementary Table [126]2).
The five consecutive Type I polyketide synthase (PKS) encoding genes
within the pta BGC consist of one loading module and seven extender
modules, which are sequentially extended to form a linear polyketide
chain by ketosynthase (KS) domain, acyltransferase (AT) domain, acyl
carrier protein (ACP), with additional ketoreductase (KR), dehydratase
(DH), and enoyl reductase (ER) domains. The substrate specificity
predictions for individual AT domains fit well with the structure of
pteridic acids (Supplementary Table [127]3). The last “Asn” residue is
absent in the conserved Lys-Ser-Tyr-Asn tetrad of the KR domain in
module 3 (PtaB), which is predicted to be inactive (Supplementary
Fig. [128]25). This is consistent with the nonreduced carbonyl group on
the α-carbon in module 3. The first DH domain in module 1 is inactive
since it does not have a conserved active motif LxxHxxGxxxxP
(Supplementary Fig. [129]26). Following the thioesterase-mediated
release of the polyketide chain, the 6,6-spiroketal core structure is
likely formed by spontaneous spiroketalisation of the carbonyl group on
C11 and the two hydroxyl groups on C17 and C25. Following a loss of
H[2]O, two differentially oriented spirocyclic rings were formed to
yield pteridic acids F and H (Fig. [130]4a). Remarkably, pteridic acid
H showed molecular instability under extreme conditions. In the water
solution with high temperature (65°C) or acidity (pH = 3), pteridic
acid H is transformed into pteridic acid F (Supplementary
Fig. [131]27). A similar spontaneous transformation from (S) to (R)
chirality at the centre of the spiroketal ring was also observed in
6,6-spiroketal avermectin^[132]53.
Fig. 4. Biosynthesis mechanism of pteridic acids and CRISPR base editing
application in S. iranensis.
[133]Fig. 4
[134]Open in a new tab
a the proposed biosynthetic pathway of pteridic acids H (1) and F (2);
b the proposed biosynthesis mechanism of elaiophylin (3), and its
macrodiolide formation is catalysed by thioesterase (TE) domain. c
Sanger sequencing and HR-LC-MS output of CRISPR base editing
application of STOP codon introduction targeting the ptaA of S.
iranensis. The 20-nt protospacer sequence is highlighted in light
green, whereas the 3-nt PAM sequence is shown in yellow. The codons and
corresponding amino acids are indicated, and the black double-headed
arrow represents the position of the editing window; Extracted Ion
Chromatography (EIC) for 1 and 2 (m/z 383.2428 [M + H]^+) and 3 (m/z
1047.5863 [M+Na]^+) in the wild type S. iranensis (Control) and the
mutant S. iranensis/ΔptaA; d Sanger sequencing and HR-LC-MS output of
CRISPR base editing application of site-directed mutagenesis targeting
the TE domain of pta BGC. EIC for 1 and 2 (m/z 383.2428 [M + H]^+) and
3 (m/z 1047.5863 [M+Na]^+) in the wild type S. iranensis (Control) and
the mutant S. iranensis/M2089I + E2090K + D2091N.
CRISPR base editing in S. iranensis
To validate the in silico prediction, we utilised the efficient base
editing tool CRISPR-cBEST to experimentally confirm the pta
BGC^[135]54. As a non-model Streptomyces strain, S. iranensis is hard
to genetically manipulate through intergeneric conjugation^[136]55.
Therefore, the conjugation process was systematically optimised in this
study (Supplementary Fig. [137]28). The core polyketide synthase ptaA
was targeted and inactivated by converting a TGG (Trp) codon at
position 916 into the stop codon TAA using CRISPR base editing. The
editing event was confirmed by PCR amplification and Sanger sequencing
of the editing site (Fig. [138]4c). As expected, the production of both
pteridic acids and elaiophylin was abolished in S. iranensis/ΔptaA
(Fig. [139]4c). Plant experiments showed that the treatment of S.
iranensis/ΔptaA fermentation suspension led to the abolishment of the
abiotic stress mitigating effects (Fig. [140]1a–c). To further confirm
the pta gene cluster, a bacterial artificial chromosome (BAC) library
of S. iranensis was constructed. BAC-based cross-complementation of
ptaA in S. iranensis/ΔptaA restored the production of pteridic acids
and elaiophylin (Supplementary Fig. [141]29).
Interestingly, based on isotope-labelled precursor feeding and partial
cosmid sequencing-based bioinformatics prediction, this BGC has long
been inferred to be responsible for the biosynthesis of the
antibacterial elaiophylin^[142]56,[143]57. In 2015, Zhou et al.
reported that the thioesterase in the last module catalysed the
formation of symmetrical macrodiolide using two units of linear
elaiophylin monomeric seco acid (Fig. [144]4b)^[145]58. To confirm
whether the biosynthesis of pteridic acids is also
thioesterase-dependent, site-specific mutations of residues Met-Glu-Asp
to Ile-Lys-Asn were introduced into the active sites of the TE domain
in vivo (Supplementary Fig. [146]30)^[147]59. HR-LC-MS analysis showed
that the mutant strain (M2089I + E2090K + D2091N) no longer produced
pteridic acids and elaiophylin (Fig. [148]4d). Hence, we provide
additional evidence via in vivo inactivation and site-directed
mutagenesis. Co-production of the plant growth-regulating pteridic
acids and the antimicrobial elaiophylin through a shared BGC is
intriguing and points to possible joint efforts in helping plants cope
with both biotic and abiotic stress.
Geographical distribution of pteridic acid producers
We surveyed available gene cluster family (GCF) data for all bacteria
in the BiG-FAM database^[149]60. We found that the pta BGC (GCF_02696)
is strictly restricted to the Streptomyces genus. In addition, a total
of 55 BGCs with high similarity to the pta BGC were detected by
BiG-SCAPE^[150]61, among a total of 9386 type I polyketides BGCs in
1965 Streptomyces from the NCBI assembly database. Through literature
supplementation and data dereplication of other reported producers
without sequence information, at least 81 Streptomyces are known to
produce pteridic acids/elaiophylin or have specific pta BGC up to date
(Supplementary Table [151]4). Based on the known sampling information,
the pta-containing Streptomyces display a variety of geographic
distribution and biological origins (Fig. [152]5a). We selected two
available Streptomyces strains (Streptomyces violaceusniger Tu 4113 and
Streptomyces rapamycinicus NRRL 5491) to test the potential plant
growth-promoting activity of these potential pteridic acid producers.
The HR-LC-MS analysis of both culture broths revealed that they shared
similar metabolite profiles, and both produced pteridic acids H and F
(Supplementary Fig. [153]31). Treatment with both culture broths on
barley seedlings also exhibited significant plant growth-promoting
activities under osmotic, salinity and drought stress (Supplementary
Fig. [154]32). This evidence suggests that this class of Streptomyces
and its specific secondary metabolite pteridic acids have unique
ecological significance involved in plant abiotic stress resistance.
Fig. 5. Geographical distribution and phylogenetic analysis of pta-containing
streptomycetes possessing pta BGC.
[155]Fig. 5
[156]Open in a new tab
a A total of 61 streptomycetes were displayed on the map and
distinguished by different colours. For detailed strain information,
see (Supplementary Table [157]4). This figure was partly generated
using Servier Medical Art ([158]http://smart.servier.com/), licensed
under a Creative Common Attribution 3.0 Generic License; b the
phylogenetic tree of 16S rRNA nucleotides sequences of
pta-containing streptomycetes and other streptomycetes; c the heatmap
depicts similarity differences of core biosynthetic genes of pta BGC
between S. iranensis and other 14 pta-containing streptomycetes.
Phylogeny and evolution of pta BGC
To explore the evolutionary clues of pteridic acid producers, 16S rRNA
genes were initially used to assess the relatedness of the collected 34
potential producers of pteridic acids with other streptomycetes that do
not contain pta BGC (Fig. [159]5b). The results revealed that, except
for Streptomyces albus DSM 41398 and Streptomyces sp. GMR22, and other
pta-containing streptomycetes cluster together and are distinct with
divergent lineages. To further confirm this hypothesis, two
high-resolution Streptomyces housekeeping genes, tryptophan synthase
subunit beta (trpB) and RNA polymerase subunit beta (rpoB) were
employed to analyse the phylogeny relationship among these
strains (Supplementary Fig. [160]33)^[161]62. Consequently, only S.
albus DSM 41398 was classified in a distinct phylogenetic lineage among
the Streptomyces strains containing pta BGC. The strict congruence
among the clades of the housekeeping genes indicated dominant vertical
transmission and potential horizontal gene transfer of the pta BGC in
Streptomyces.
A total of 15 pta-containing streptomycetes with complete genome
sequence information were selected to conduct the comparative genomics
investigation. The genetic diversity in these streptomycetes was
initially revealed using genome sequence similarity analysis. Except
for S. albus DSM 41398 and Streptomyces sp. NA02950, we observed a high
degree of similarity in the aligned region, as indicated by both the
average nucleotide identity (ANI) and the alignment percentage (AP)
among these strains (Supplementary Fig. [162]34). Genome synteny
analysis revealed that partial genome rearrangements happened among
strains even with high sequence similarities (Supplementary
Fig. [163]35). Notably, the pta BGC in S. albus DSM 41398 (pta-alb) is
located at the end of the chromosome, a high variable region in
Streptomyces, suggesting its existence by accepting heterologous
biosynthetic gene fragments. The nucleotide sequence alignment of pta
BGC results showed that pta-alb is relatively complete, and the
similarity of core genes is proportional to evolutionary relatedness
(Fig. [164]5c). The metabolite profile of S. albus DSM 41398 also
confirmed the integrity of the pta-alb by detecting the production of
elaiophylin and pteridic acid H (Supplementary Fig. [165]36). To
further assess the biosynthesis diversity in remaining genetically
related strains, we performed the similarity analysis of these
BGCs (Supplementary Figs. [166]37 and [167]38). The connections between
their secondary metabolites, BGCs, revealed that these vertically
inherited Streptomyces strains also harbour striking similarities.
Combining phylogenetic and comparative genomics analysis, we expect
that S. albus DSM 41398 is evolutionarily the most distinct member from
other pta-containing streptomycetes and obtained the pta BGC via
horizontal gene transfer. However, most pta-containing streptomycetes
have vertically inherited pta and other BGCs from their ancient
ancestors that may be ecologically important and rarely studied.
Discussion
Drought and salinisation of soil are increasing globally, driving a
reduction in crop yields that threatens food security. Plant
growth-promoting bacteria is a class of beneficial microorganisms that
positively interact with the plant to confer environmental
stresses^[168]63. Although some Streptomyces species have been reported
to have plant growth-promoting activity, the molecules mediating such
positive effects are poorly understood. Deciphering the molecular
mechanism is key to understanding the complex plant microbiota
interaction. In this study, we present an example of S. iranensis
secreting a family of secondary metabolites, pteridic acids, to assist
plants to cope with abiotic stresses like osmotic, salinity and
drought. Pteridic acids H and F were chemically isolated, structurally
characterised and functional validated as plant-beneficial molecules.
Plants respond to harsh environments by changing their physiological
processes for better survival^[169]64. Salt and drought stress signal
transduction consists of ionic and osmotic homoeostasis signalling
pathways, detoxification (i.e., damage control and repair) response
pathways, and pathways for growth regulation^[170]65,[171]66. Based on
mRNA-seq analysis, we identified a total of 3086 DEGs, some of which
are associated with diverse abiotic stress defenses in plants. For
example, we observed that the upregulation of SLAH1 is crucial for
alleviating the toxicity of salt by root-to-shoot Cl^− transport in
Arabidopsis^[172]27. PIN6 was previously identified as the key salt
tolerance-related gene in the roots of the mangrove Avicennia
officinalis^[173]28. AtACA8 is a plasma membrane localised Ca^2+ pump
and plays a role in sucrose signalling, ion homeostasis and root
development during early seedling germination^[174]31. MKK7 positively
regulates plant salt tolerance and promotes primary root growth in
Arabidopsis seedlings^[175]35. The differentiation and development of
Guard cells (GCs), which are in the epidermis of leaves and stems that
regulate stomatal development, are regulated by TMM^[176]30. Under
abiotic stress conditions, the upregulation of ABCG17 expression
facilitated the translocation of ABA from the shoot to the root,
consequently stimulating lateral root growth^[177]33. Additionally, our
study revealed an enhancement in the lateral root growth of Arabidopsis
seedlings upon exposure to pteridic acid F, with the expression of the
associated ABCG17 gene observed exclusively in the PF vs. CK group.
Salt stress negatively affects photosynthesis in plants, prompting them
to regulate the photosynthetic process, either through intrinsic
mechanisms or in response to external stimuli, to enhance their salt
tolerance^[178]67. Based on GO and KEGG pathway enrichment analysis,
photosynthesis and its related physiological events of Arabidopsis
seedlings treated with pteridic acids were immensely upregulated.
Interestingly, previous studies also showed that small molecules, such
as exogenously applied melatonin, can assist plants in resisting salt
stress by improving photosynthesis in different plants^[179]68,[180]69.
Some studies have confirmed that IAA is involved in response to salt
stress in plants and a link between IAA signalling and salt stress has
been established^[181]70,[182]71. Herein, we speculated that pteridic
acids might also promote plant growth under salt stress via activating
auxin signal transduction to promote plant growth under salt stress.
B-ARR, as a positive regulator that regulated downstream activity in
the cytokinin signalling pathway, was also uniquely upregulated in PF
vs. CK group.
Among the various TFs, MYBs participate in various biological processes
in plants such as growth, reproduction, secondary metabolism and stress
responses^[183]72. For example, upregulated MYB25 has been shown to
reduce sensitivities toward osmotic and salt stress in Arabidopsis and
upregulated MYB111 is a positive regulator of salt stress in
Arabidopsis by binding directly to the cis-acting element in the
promoter region of genes encoding flavonoid synthesis
enzymes^[184]42,[185]43. MYB60, which was upregulated in the PH vs. CK
group, was previously demonstrated to play a dual role in abiotic
stress responses in Arabidopsis through its involvement in stomatal
regulation and root growth for increased water uptake^[186]47. MYB29,
which was uniquely upregulated in the PF vs. CK group, has been
demonstrated to be an important factor in promoting Arabidopsis lateral
root growth under salinity stress^[187]49. MYB113 has also been
reported to promote anthocyanin biosynthesis in Arabidopsis and pear
for defense against abiotic and biotic stresses^[188]50. Previous
studies have shown that overexpression of WRKY46 enhanced root
development during salt stress in Arabidopsis through modulation of ABA
signalling^[189]44. We found that WRKY46 was upregulated in pteridic
acid-treated samples, which may also serve as a positive regulator in
the ABA signalling pathway to confer abiotic stress resistance to
plants, although we didn’t observe other ABA-related DEGs. The two
upregulated bHLH family TFs PRE1 and SCRM2 have also been
experimentally proved to promote plant growth and resist abiotic
stress^[190]45,[191]46.
Horizontal gene transfer is an integral driver of BGC evolution,
revealing the independent processes of species phylogeny and BGCs
distribution^[192]73. However, vertical inheritance also influences
BGCs evolutionary dynamics, evident from BGCs conservation among
closely related strains^[193]74. We found that the pta BGC in
Streptomyces are widely dispersed geographically and mainly inherited
through vertical gene transmission. Some of these strains have also
been described to have remarkable biocontrol capabilities. For example,
Streptomyces sp. AgN23 activates Arabidopsis defence responses to
fungal pathogen infection by secreting plant elicitors^[194]75,
Streptomyces rhizosphaericus 0250 and Streptomyces sp. 5–10 displayed
significant biocontrol potential to fusarium wilt of bitter
gourd^[195]76,[196]77. The family of pta-containing Streptomyces was
also previously described as a specific phylogenetic lineage with the
highest BGC abundance and largest genome size across
diverse streptomycetes^[197]78. Although the biosynthetic diversity of
these Streptomyces strains is likely due to horizontal transfer events
that occurred relatively recently in their evolutionary history instead
of genetic diversification through a vertical transfer of BGCs. The
multiple Type I PKSs presenting among these strains are highly
conserved based on genetic similarity network analysis. There is
currently some evidence supporting potential complex cross-BGC
regulation in this class of Streptomyces strains. Jiang et al.
demonstrated that a TetR family transcriptional regulator, GdmRIII,
controls the biosynthesis of geldanamycin and elaiophylin meanwhile, in
Streptomyces autolyticus CGMCC 0516^[198]79. Recently, He et al. found
that the rapamycin BGC-situated LAL family regulator RapH co-ordinately
regulated the biosynthesis of both rapamycin and elaiophylin in S.
rapamycinicus NRRL 5491^[199]80. Although these reports correspond to
cross-regulation between evolutionarily conserved BGCs, more details of
these communications need to be investigated.
In conclusion, pteridic acids are secondary metabolites produced by
streptomycetes enhancing plant resistance to abiotic stress.
Transcriptomics profile revealed a higher expression of a diverse set
of genes, e.g., in photosynthesis and abiotic stress response genes
after pteridic acids treatment. This is a useful illustration of the
bacterial metabolite-mediated alteration of plants in response to
environmental stress. It will open avenues for utilising Streptomyces
to rewild plant microbiomes and improve plant abiotic stress resistance
to tackle climate change^[200]81.
Methods
Strains, plasmids, and cultivation
All strains and plasmids used in this study are listed in
(Supplementary Table [201]5). All Streptomyces strains were obtained
from the German Collection of Microorganisms and Cell Cultures GmbH
(DSMZ, Germany). All Escherichia coli strains were grown in
liquid/solid LB medium (5.0 g L^–1 yeast extract, 10.0 g L^–1 peptone,
10.0 g L^–1 NaCl) at 37 °C. All Streptomyces strains were grown on SFM
medium (20.0 g L^–1 mannitol, 20.0 g L^–1 soya flour, 20.0 g L^–1
agar), and the SFM medium with the addition of 120 mM calcium chloride
solution was used for the step of conjugation at 28 °C. The ISP2 medium
(4.0 g L^–1 yeast extract, 10.0 g L^–1 malt extract, 4.0 g L^–1
dextrose, and 1.0 L distilled water) was used for liquid fermentation
of all Streptomyces strains used in plant assay and metabolomics
analysis. Appropriate antibiotics were supplemented with the following
working concentrations: apramycin (50 µg mL^–1), chloramphenicol
(25 µg mL^–1), and kanamycin (50 µg mL^–1). All chemicals utilised in
this study were from Sigma-Aldrich, USA.
Metabolomics analyses
High performance liquid chromatography was carried out on the Agilent
Infinity 1290 UHPLC system (Agilent Technologies, USA). The 250 ×
2.1 mm i.d., 2.7 μm, Poroshell 120 Phenyl Hexyl column (Agilent
Technologies, USA) was used for separation. The 2-μL samples were
eluted at a flow rate of 0.35 mL min^−1 using a linear gradient from
10% acetonitrile in Milli-Q water buffered with 20 mM formic acid
increasing to 100% in 15 min. Each starting condition was held for
3 min before the next run. Mass spectrometry detection was performed on
an Agilent 6545 QTOF MS equipped with Agilent Dual Jet Stream
electrospray ion source (ESI) with a drying gas temperature of 160 °C,
a gas flow of 13 L min^−1, sheath gas temperature of 300 °C, and flow
of 16 L min^−1. The capillary voltage was set to 4000 V and the nozzle
voltage to 500 V in positive mode. MS spectra were recorded as centroid
data at an m/z of 100–1700, and auto MS/HRMS fragmentation was
performed at three collision energies (10, 20, and 40 eV) on the three
most intense precursor peaks per programme. Data were analysed with
MassHunter software (Agilent Technologies, USA) and compared with known
compounds and crude extract spectral libraries stored in the GNPS
platform^[202]45. The precursor and fragment ion mass tolerance were
set as 0.1 Da and 0.02 Da, respectively. In addition, the minpairs cos
was set as 0.65, and the minimum matched fragment ions were set as 6.0.
The metabolites profile of wild-type S. iranensis was visualised by
MS-Dial 4.9.2^[203]82.
Large-scale fermentation and isolation
S. iranensis was cultivated in medium 2 (3.0 g L^–1 CaCl[2]·2H[2]O,
1.0 g L^–1 citric acid/Fe III, 0.2 g L^–1 MnSO[4]·H[2]O, 0.1 g L^–1
ZnCl[2], 0.025 g L^–1 CuSO[4]·5H[2]O, 0.02 g L^–1
Na[2]B[4]O[7]·10H[2]O, 0.01 g L^–1 Na[2]MoO[4]·2H[2]O, and 20.0 g L^–1
oatmeal in 1.0 L distilled water), at 175 L filling volume in a 300 L
fermentation vessel (Sartorius, Germany). The fermentation was carried
out for 6 days with aeration of 25-50 L min^–1, stirring at 200 rpm
with a temperature of 28 °C and at a pH range of 5.4-6.4. The
fermentation broth was separated, filtered, and loaded onto an
Amberchrom CG161Me resin LC column (200 × 20 cm, 6 L). Elution with a
linear gradient of H[2]O-MeOH (from 30% to 100% v/v, flow rate
0.5 L min^−1, in 58 min) afforded seven fractions (A–G). Fraction G was
firstly fractionated by silica gel chromatography with a
CH[2]Cl[2]/CH[3]OH gradient to yield 16 fractions, F01-F16. F07 was
separated by a Sephadex LH-20 (MeOH) column and twelve sub-fractions
F07a-l were obtained. From F07e, 1 (15.0 mg) and 2 (4.0 mg) were
obtained by repeated HPLC RP-C[18] (CH[3]CN/H[2]O as gradient).
Pteridic acid H (1): white solid;
[MATH:
[α]
D20 :MATH]
81 (0.32 mg mL^−1, CH[3]OH), ^1H NMR (800 MHz, MeOD): 7.33 (dd,
15.4 Hz, 11.1 Hz, 1H), 6.26 (dd, 15.2 Hz, 10.8 Hz, 1H), 6.13 (dd,
15.3 Hz, 8.8 Hz, 1H), 5.90 (d, 15.4 Hz, 1H), 3.85 (dd, 10.2 Hz, 2.2 Hz,
1H), 3.69 (overlapping, 1H), 3.59 (m, 1H), 3.43 (m, 1H), 2.48 (m, 1H),
2.29 (dd, 14.9 Hz, 6.1 Hz, 1H), 2.01 (m, 1H), 1.64 (dd, 14.9 Hz,
1.9 Hz, 1H), 1.52 (m, 1H), 1.21 (d, 6.1 Hz, 3H), 1.49 (m), 1.01 (d,
6.8 Hz, 3H), 0.96 (d, 6.8 Hz, 3H), 0.93 (t, 7.3 Hz, 3H), 0.91 (d,
7.0 Hz, 3H); ^13C NMR (200 MHz, MeOD): 168.7, 151.0, 146.7, 129,6,
120.6, 103.2, 75.5, 72.8, 70.3, 70.1, 51.3, 42.2, 40.4, 37.5, 37.4,
24.8, 20.9, 16.0, 12.1, 10.1, 5.0; UV/vis (CH[3]CN/H[2]O) λ[max]
262 nm; IR (ATR) v[max] 2967, 2934, 2879, 1712, 1642, 1600, 1458, 1410,
1383, 1300, 1266, 1223, 1187, 1142, 1109, 1058, 1002, 973 cm^−1;
(+)-HR-ESI-MS (m/z) [M + H]^+ calcd for C[21]H[35]O[6], 383.2428;
found, 383.2439. ^1H NMR and ^13C NMR see Supplementary Tables [204]6
and [205]7.
Pteridic acid F (2): white solid;
[MATH:
[α]
D20 :MATH]
−18 (10 mg mL^−1, CH[3]OH), ^1H NMR (800 MHz, MeOD): 5.97 (d, 15.1 Hz,
1H), 7.16 (dd, 15.1 Hz, 10.9 Hz, 1H), 6.25 (dd, 15.1 Hz, 10.9 Hz, 1H),
6.07 (dd, 15.1 Hz, 8.6 Hz, 1H), 3.88 (m, 1H), 3.66 (td, 10.8 Hz,
4.3 Hz, 1H), 3.56 (dd, 11.5 Hz, 4.7 Hz, 1H), 3.32 (m, 1H), 2.49 (m,
1H), 2.19 (dd, 13.1 Hz, 4.3 Hz, 1H), 2.02 (m, 1H), 1.69 (m, 1H), 1.32
(dd, 13.2 Hz, 11.2 Hz, 1H), 1.14 (d, 6.2 Hz, 3H), 1.02 (m, 1H), 1.02
(d, 6.8 Hz, 3H), 0.95 (d, 6.9 Hz, 3H), 0.98 (d, 6.8 Hz, 3H), 1.60 (m,
1H), 1.44 (m, 1H), 0.82 (t, 7.6 Hz, 3H); ^13C NMR (200 MHz, MeOD):
170.2, 148.1, 148.1, 129,6, 122.9, 103.2, 78.0, 74.7, 66.3, 66.3, 52.2,
42.1, 40.5, 38.0, 33.8, 20.4, 15.9, 12.7, 5.3; UV/vis (CH[3]CN/H[2]O)
λ[max] 264 nm; IR (ATR) v[max] 2968, 2931, 2877, 1692, 1643, 1618,
1458, 1410, 1380, 1299, 1270, 1188, 1138, 1106, 1059, 1002, 973,
850 cm^−1; (+)-HR-ESI-MS (m/z): [M + H]^+ calcd for C[21]H[35]O[6],
383.2428; found, 383.2433. ^1H NMR and ^13C NMR see Supplementary
Tables [206]6 and [207]7.
Structure identification
NMR spectra were recorded on an 800 MHz Bruker Avance III spectrometer
equipped with a TCI CryoProbe using standard pulse sequences. NMR data
were processed using MestReNova 11.0. UHPLC-HRMS was performed on an
Agilent Infinity 1290 UHPLC system equipped with a diode array
detector. UV-Vis spectra were recorded from 190 to 640 nm. Specific
rotations were acquired using Perkin-Elmer 241 polarimeter. IR data
were acquired on Bruker Alpha FTIR spectrometer using OPUS version 7.2.
TLC analysis was performed on silica gel plates (Sil G/UV[254],
0.20 mm, Macherey-Nagel). The Biotage Isolera One Flash Chromatography
system was used for flash chromatography and performed on silica gel 60
(Merck, 0.04–0.063 mm, 230–400 mesh ASTM). Sephadex LH-20 was from
Pharmacia.
Crystal structure determination
X-ray data collection of 1 was performed on an Agilent Supernova
Diffractometer using CuKα radiation. Data were processed and scaled
using the CrysAlisPro software (Agilent Technologies, USA). The
structure was solved using SHELXS and refined using SHELXL. Hydrogen
atoms were included in ideal positions using riding coordinates. The
absolute configuration was determined based on the Flack parameter.
Crystal Data for 1: C[21]H[34]O[6], M = 382.50, monoclinic,
a = 8.4619(1) Å, b = 15.6161(2) Å, c = 8.4994(1) Å, α = 90.00°,
β = 107.768(1)°, γ = 90.00°, V = 1069.55(2)Å^3, T = 120.(2) K, space
group P21, Z = 2, μ(Cu Kα) = 0.698 mm^–1, 17514 reflections collected,
4275 independent reflections (R[int] = 0.0226, R[sigma] = 0.0155). The
final R[1] values were 0.0249 (I > 2σ(I)). The final wR[2] values were
0.0648 (I > 2σ(I)). The final R[1] values were 0.0252 (all data). The
final wR[2] values were 0.0651 (all data). The goodness of fit on F^2
was 1.057. Flack parameter = 0.13(10).
Genetic manipulation
All primers used were synthesised by IDT (Integrated DNA Technologies,
USA) and listed in (Supplementary Table [208]8). Plasmids and genomic
DNA purification, PCR and cloning were conducted according to standard
procedures using manufacturer protocols. PCR was performed using OneTaq
Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs,
USA). DNA assembly was done by using NEBuilder HiFi DNA Assembly Master
Mix (New England Biolabs, USA). DNA digestion was performed with
FastDigest restriction enzymes (Thermo Fisher Scientific, USA).
NucleoSpin Gel and PCR Clean-up Kits (Macherey-Nagel, Germany) were
used for DNA clean-up from PCR products and agarose gel extracts. One
Shot Mach1 T1 Phage-Resistant Chemically Competent E. coli (Thermo
Fisher Scientific, USA) was used for cloning. NucleoSpin Plasmid
EasyPure Kit (Macherey-Nagel, Germany) was used for plasmid
preparation. Sanger sequencing was carried out using a Mix2Seq Kit
(Eurofins Scientific, Luxembourg). All DNA manipulation experiments
were conducted according to standard procedures using manufacturer
protocols.
Gene inactivation and site-directed mutagenesis
To use the pCRISPR-cBEST for base editing applications, an oligo was
designed as Del-ptaA by the online tool CRISPy-web, and the
pCRISPR-cBEST plasmid was linearised by NcoI. Mixing the linearised
pCRISPR-cBEST plasmid and Del-ptaA with the NEBuilder HiFi DNA Assembly
Master Mix (New England Biolabs, USA). The linearised pCRISPR-cBEST
plasmid was then bridged by Del-ptaA, ending with the desired
pCRISPR-cBEST/ΔptaA. Chemically competent E. coli were transformed with
the recombinant plasmid and confirmed via PCR amplification (programme:
94 °C for 30 s, followed by 30 cycles consisting of 94 °C for 15 s;
54 °C for 15 s; 68 °C for 40 s, and 68 °C for 2 min) and Sanger
sequencing. The experimental procedure for site-directed mutagenesis
for the TE domain is the same as described above. The E.
coli-Streptomyces conjugation experiment was conducted according to the
modified protocol in this study, and the mutant Streptomyces strains
were also confirmed by PCR and Sanger sequencing (Eurofins, France).
Construction of BAC and genetical complementation
The BAC library of S. iranensis was constructed using pESAC13-A from
Bio S&T (Montreal, Canada). Based on a high-throughput screening method
(unpublished), we selected two BACs 1J23 and 6M10 that cross-cover the
ptaA gene (Supplementary Fig. [209]28). The selected BAC clones were
further confirmed using four sets of primers, including
ID-1J23-right-F/R, ID-1J23-left-F/R, ID-6M10-right-F/R, and
ID-6M10-left-F/R (Supplementary Table [210]8). Subsequently, we
introduced these two BAC clones 1J23 and 6M10 into S. iranensis/ΔptaA
(remove the pCRISPR-cBEST/ΔptaA to obtain antibiotics resistance free
strain) separately by conjugation. Exconjugants of mutants were further
validated by apramycin resistance screening and PCR.
Enrichment evaluation of S. iranensis in rhizosphere soil
The S. iranensis (with the apramycin resistance gene) spore suspension
was well mixed with fully sterilised soil and was transferred to a
250 mL flask. The sterilised germinated barley seed was placed in the
centre position of soil in flask and grown at 24 ± 2 °C, 8 h dark/16 h
light in the growth chamber for 7 days. Samples were collected from
soil within 0.1 cm and 3 cm distance from the barley root, with a
specification of 0.1 g soil per sample. Then, these samples were
transferred to sterilised 1.5 mL Eppendorf tubes and mixed with 500 μL
sterilised H[2]O. 200 μL of each sample was spread evenly over the
solid MS medium with the addition of 50 μg mL^−1 apramycin and grown at
28°C for 7 days. The number of Streptomyces colonies grown on each
plate were counted and statistically analysed.
Arabidopsis growth assays
A. thaliana ecotype Columbia (Col-0) was used to test the effects of
pteridic acids treatment under drought stress mediated by PEG-6000
(Duchefa Biochemie BV) and salinity stress mediated by NaCl (Duchefa
Biochemie BV). The modified Murashige & Skoog medium (2.2 g L^–1
Murashige & Skoog medium including B5 vitamins, 5.0 g L^–1 of sucrose,
250 mg L^–1 MES monohydrate, 7.0 g L^–1 agar, and adjusted pH to 5.7
with KOH) was used in this study. PEG-6000 (15% w/v) was dissolved in
water and filtered through 0.2-micron Sartorius Minisart™ Plus Syringe
Filters (Fisher Scientific). 50 mL of the filtered solution was
overlaid onto the surface of solidified Murashige & Skoog medium. The
plates were left for 24 h to diffuse the PEG into the Murashige & Skoog
medium. NaCl was added to the medium to a final concentration of 80 mM
for the salinity stress alleviation test. The pure compound pteridic
acids H and F (0.50 ng mL^−1), IAA (0.23 ng mL^−1), and ABA
(0.34 ng mL^−1) were mixed with different media to a final
concentration of approximately 1.3 nM and poured into the plates. Seeds
were surface sterilised by washing with 70% ethanol for 2 min, then in
sterilisation solution (10% bleach) for 1 min by inverting the tubes,
and finally washed five times with sterilised water. The seeds were
stratified for 2 days at 4 °C in the dark. Sterilised seeds were placed
in Petri dishes (approx. 100 seeds per Petri dish) on Murashige & Skoog
medium and grown for 3–4 days in the vertical position in a culture
chamber at 22 °C under standard long-day conditions (16/8 h light/dark
photoperiod). After three days of growth, seedlings with similar root
lengths (7-10 mm) were transferred to square plates containing
Murashige & Skoog medium (control) or Murashige & Skoog medium
supplemented with 15% (w/v) PEG-6000 or 80 mM NaCl. 16 seedlings were
used per replicate for each treatment. The initial position of the
plant root tip was marked with a marker. The plants were grown in the
vertical position under standard long day conditions (22 °C, 16/8 h
light/dark) for 8 days, and then each plate was scanned using Image
Scanner. Primary and lateral root lengths and the total plant weight
were then scored. The primary and lateral root length measurements were
performed by analysing pictures with the Image J software. Fresh weight
measurements were estimated using a precision balance. Whole 8-day-old
plants grown in the medium were removed using forceps, dried in tissue
paper, and then weighed using a precision balance.
Barley and mung bean growth assays
The pteridic acid streptomycetes producers were tested for their
effects on barley cultivars Guld grown in soil. S. iranensis HM 35, S.
rapamycinicus NRRL 5491, and S. violaceusniger Tu 4113 were cultivated
in ISP2 medium for 7 days at 28 °C. 500 µL culture broth (ca. 3 × 10^9
CFU/mL) was added to 100 g sand soil and well mixed. Treated soils were
infiltrated with Milli-Q water, 20% PEG-6000 solutions to simulate
osmotic stress, and 100 mM NaCl solutions to simulate salinity stress
in soil environments. To simulate drought stress during barley growth,
the plant was initially watered for 7 days, then subjected to 7 days of
drought stress, and finally allowed to recover with various treatments
for another 7 days. Barley seeds were rinsed in distilled water and
sterilised with 1% sodium hypochlorite for 15 min. They were then
washed with distilled water and germinated in distilled water at 24 °C
for 2 days. Barley seeds were planted in each plastic pot (5 cm × 5 cm
× 6 cm, six seedlings per pot) supplemented with different treated soil
and grown at 24 ± 2 °C, 8 h dark/16 h light in the growth chamber for 7
days. Plant height (cm) was measured as the aerial part of the plant,
and the fresh shoot weight (g) and fresh root weight (g) of each
seedling were measured separately. Then, the seedlings were dried in
the hot-air oven at 70 °C for 6 h to obtain the dry shoot and dry root
weights (g). For heavy metal stress experiment, mung beans were
pre-germinated and placed on top of the modified Murashige & Skoog
medium agar, supplemented with 10 mM CuSO[4], with 1.0 ng mL^−1 pure
substances. All mung beans were grown in the dark at 24 ± 2 °C for 4
days.
Kidney beans growth assays
The seeds of kidney beans (appr. 2 cm in length, from organic farming)
were firstly sterilised successively with ethanol (70% v/v) and sodium
hypochlorite (5% v/v), each for 2 min and then rinsed with sterile
Milli-Q water (three times). The sterilised seeds were cultivated on
modified Murashige & Skoog agar plates for 3–4 days. After germination,
the seedlings (with 1.5–2-cm-long roots) were soaked in 10 mL aliquots
of testing compounds (pteridic acids H and F, 1.0 ng mL^−1, dissolved
in sterile Milli-Q water) in ultra-clear polypropylene containers (ø
34 mm, vol. 20 mL) with polyethylene caps. The control group was
treated with 10 mL sterile Milli-Q water. For each treatment, three
repetitions (containers) were used, and each repetition included four
seedlings. After 24 h, the seeds were transferred into a cut square
petri dish, put on the top layer of sandy soil, and then incubated
vertically in a growth chamber (24/22 °C, day/night cycle of 16/8 h,
50%, 60%, 70%, 100% of circulated wind velocity for 12 h, 2 h, 2 h,
8 h) for 7 days. Solutions of pteridic acids H and F (2 mL,
1.0 ng mL^−1 for both) were added separately into corresponding
containers, with sterile Milli-Q water as control, and an extra 8 mL of
Milli-Q water was added to each petri dish every other day.
RNA extraction and mRNA-seq
Arabidopsis seeds were sterilised, and seven to ten seeds were cultured
in each well of a 6-well plate in 2 mL medium containing modified
Murashige & Skoog and 30 mM sucrose with 16 h light, at 24 °C in a
controlled environment room. After 7 days, seedlings were washed in
20 mL modified Murashige & Skoog liquid medium and then moved to 20 mL
of fresh liquid medium containing 80 mM NaCl and cultured in 50 ml
E-flasks with 1 ng mL^−1 of each pteridic acids H or F (or equal amount
of water as control). Whole-seedling plant samples were collected after
72 h when significant phenotypic variation occurred between groups.
Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Germany).
mRNA was purified from total RNA using poly-T oligo-attached magnetic
beads. After fragmentation, the first strand cDNA was synthesised using
random hexamer primers, followed by the second strand cDNA synthesis
using dTTP for the non-directional library construction. The reads were
generated using an Illumina NovaSeq 6000 (Novogene, UK) with a
paired-end 150 bp configuration. DESeq2 was used to estimate DEGs
between different treatments with the threshold of FDR-adjusted p
values ≤ 0.05 and |log2(FoldChange)|≥1 if there is no additional
statement^[211]83. The online software g:Profiler
([212]http://biit.cs.ut.ee/gprofiler/gost) was used for GO enrichment
and clusterProfiler R package was used to test the statistical
enrichment of DEGs in KEGG pathways^[213]84,[214]85.
Bioinformatics analyses
The identification and annotation of all BGCs of Streptomyces secondary
metabolites were carried out with antiSMASH 6.0^[215]26. The threshold
for similar BGCs was selected as greater than 45% sequence similarity.
The BiG-FAM database and BiG-SCAPE software were used to identify the
distribution of pta gene clusters in different bacteria and generate
the pta-gene cluster family similarity network^[216]60,[217]61. A
cut-off of 0.3 was used as a raw index similarity metric for the
BiG-SCAPE analysis. The alignment of 15 pta-containing Streptomyces
genome sequences was performed using the whole-genome alignment plugin
of the CLC Genomics Workbench version 22.0.2 (Qiagen). The minimum
initial seed length was 15 bp, and the minimum alignment block length
was 100 bp. The networks of Big-SCAPE and GNPS analysis were visualised
using Cytoscape 3.9. The phylogenetic analysis was conducted by the
online multiple sequence alignment tool MAFFT and visualised by iTOL
v5^[218]86,[219]87.
Statistical analysis
Statistical significance was assessed by one-way ANOVA with post hoc
Dunnett’s multiple comparisons test, one-way ANOVA with Tukey test or t
test (see each figure legend). All analyses were performed using
GraphPad Prism version 9. p values < 0.05 were considered significant.
Asterisks indicate the level of statistical significance: *p < 0.05,
**p < 0.01, ***p < 0.001, and ****p < 0.0001. For all relevant figures,
source data and exact p values are provided in the Source Data file.
Reporting summary
Further information on research design is available in the [220]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[221]Supplementary Information^ (5.3MB, pdf)
[222]Peer Review File^ (1.7MB, pdf)
[223]41467_2023_43177_MOESM3_ESM.pdf^ (349.2KB, pdf)
Description of Additional Supplementary Files
[224]Supplementary Data 1^ (2.5MB, xlsx)
[225]Supplementary Data 2^ (63.3KB, xlsx)
[226]Supplementary Data 3^ (27.7KB, xlsx)
[227]Reporting Summary^ (194.4KB, pdf)
Source data
[228]Source Data^ (4.2MB, xlsx)
Acknowledgements