Abstract
The emergence and rapid spread of multidrug-resistant Botrytis cinerea
strains pose a great challenge to the quality and safety of
agricultural products and the efficient use of pesticides. Previously
unidentified fungicides and targets are urgently needed to combat B.
cinerea–associated infections as alternative therapeutic options. In
this study, the promising compound Z24 demonstrated efficacy against
all tested plant pathogenic fungi. Thiamine thiazole synthase (Bcthi4)
was identified as a target protein of Z24 by drug affinity responsive
target stability (DARTS), cellular thermal shift assay (CETSA), and
surface plasmon resonance (SPR) assays. Molecular docking and enzyme
activity experiments have demonstrated that Z24 can affect the function
of Bcthi4. Last, mechanistic studies show that Z24 inhibits thiamine
biosynthesis by binding to Bcthi4 and induces up-regulation of
alternative splicing [alternative 5′ splice site (A5SS)] of the Bcthi4
gene. In conclusion, by targeting Bcthi4, Z24 has the potential to be
developed as a previously unidentified anti–B. cinerea candidate.
__________________________________________________________________
Thiazole synthase (Bcthi4) was identified as a target protein of Z24 by
drug affinity responsive target stability (DARTS).
INTRODUCTION
Botrytis cinerea is a necrotrophic plant pathogenic fungus with a broad
host range that causes large economic losses to crops ([44]1). Because
about 85% of fungicides now on the market target single enzymes, this
is particularly alarming. The emergence of resistance to azoles,
succinate dehydrogenase inhibitors (SDHIs), and quinone outside
inhibitors (strobilurins) poses a serious threat to agricultural
security ([45]2–[46]4). Therefore, the identification of potential
fungicides with previously unknown targets and low toxicity is urgently
needed to combat B. cinerea infections.
On earth, plants make up most of biomass. Plant-derived compounds have
been widely used in the treatment of various infections ([47]5, [48]6).
Neocryptolepine, an indoloquinoline alkaloid with a wide range of
pharmacological activities, was isolated from the traditional African
herb Cryptolepis sanguinolenta. Neocryptolepine and its derivatives
exhibit excellent antifungal activity against plant pathogenic fungi,
while compound Z24 exhibits the most effective inhibitory potency
against B. cinerea, as initially identified in our prior investigation
([49]7). However, there no studies have been done on its antifungal
properties and potential target, especially against B. cinerea, to
date.
Thiamine thiazole synthase (Thi4) is an intracellular membrane–bound
protein and serves as the potential target. It is a core protein
involved in the biological and metabolic processes of Fusarium solani.
Furthermore, the Thi4 protein exists in the thiamine thiazole
biosynthesis pathway, which is unique to pathogens and deficient in
humans ([50]8). Thiamine biosynthesis in fungi has been extensively
studied in Aspergillus species, and inhibiting thiamine biosynthesis by
targeting thiamine uptake has been proposed as an effective strategy
for antifungal development ([51]9). Thiamine, an important cofactor
required for the growth of fungi, is produced by the Thi6p-dependent
coupling of thiazole and pyrimidine ([52]10). The pyrimidine moiety is
synthesized from 5-aminoimidazole ribotide by
4-amino-2-methyl-5-phosphomethylpyrimidine phosphate synthase (THIC) in
prokaryotes, plants, and green algae, whereas in fungi, it is
synthesized from pyridoxal-5-phosphate and histidine by thiamine
biosynthesis protein 5/N-myristoyltransferase 1 (THI5/NMT1) ([53]11).
4-Methyl-5-(2-phosphooxyethyl)thiazole is produced in eubacteria via
ThiG from iminoglycine, pyruvate, glyceraldehyde-3-phosphate, and
cysteine. In archaea, fungi, plants, and green algae, it is produced
via THI1/THI4 using nicotinamide adenine dinucleotide (oxidized form),
glycine, and a sulfur atom from a cysteine residue in the active site
([54]12). The biosynthesis of the adenylated carboxythiazole (ADT)
precursor of thiamin is chemically complex and energetically expensive
([55]13). Plants, fungi, and some prokaryotes make ADT via the thiazole
synthase THI4, a single-turnover suicide enzyme ([56]14, [57]15), which
is also true for THI5/NMT1 ([58]16). Thiamine pyrophosphate (TPP) is a
cofactor required for the carboxylation and decarboxylation of various
metabolic intermediates in carbohydrate and amino acid metabolism. It
acts as a cofactor for key enzymes such as pyruvate dehydrogenase,
transketolase, and pyruvate decarboxylase and is central to the
activity of several enzymes, particularly those involved in the
metabolism of glucose ([59]13, [60]17). The TPP riboswitch is one of
the most common and abundant riboswitches in bacteria ([61]18). It is
also the main riboswitch found in eukaryotes, such as fungi ([62]19),
algae, and plants ([63]20, [64]21). The active form of thiamine, TPP,
is produced when thiamine is pyrophosphorylated by the enzyme thiamine
pyrophosphokinase ([65]22). Thiamine enters cells and is phosphorylated
to generate TPP, and the resulting coenzyme serves as a ligand for
riboswitch-mediated control of RNA splicing in fungi ([66]23). The
filamentous fungus Neurospora crassa has three riboswitches, two of
which are located in introns within the 5′ untranslated region of the
thiamin synthesis genes THI4 (NCU06110) and THI5 (also known as NMT1 or
NCU09345) ([67]24, [68]25). Eukaryotic riboswitches are often situated
within introns, where they function by regulating splicing. TPP
riboswitches regulate the expression of thiamin synthesis genes in
algae and marine phytoplankton ([69]26), plants ([70]27, [71]28),
filamentous fungi ([72]24, [73]25), and probably oomycetes ([74]29).
TPP riboswitches have been experimentally validated in a few species of
fungi where they are involved in splicing and regulate the expression
of TPP biosynthesis ([75]24, [76]30) and transporter ([77]25) genes.
However, the role of Bcthi4 in B. cinerea remains unclear to date.
Here, we found that Z24 is a potent fungicide candidate targeting
Bcthi4 and exhibited excellent antifungal activity against B. cinerea.
Our study first elucidated the mechanism of the neocryptolepine
derivative Z24 and identified a promising lead for the development of
anti–B. cinerea fungicides.
RESULTS
The candidate compound Z24 was evaluated for its activity in vitro
Neocryptolepine (Z1) and derivative Z24 were evaluated for their
antifungal activity against six different plant pathogenic fungi,
including B. cinerea Pers., Sclerotinia sclerotiorum, FusaHum
graminearum, Fusarium oxysporum, Rhizoctonia solani, and Phytophthora
capsici, taking pyrimethanil, boscalid, thiophanate-methyl,
carbendazim, and azoxystrobin as positive controls, and their half
maximal effective concentration (EC[50]) values against the tested
plant pathogenic fungi are listed in [78]Table 1. As indicated by the
EC[50] values, the findings demonstrated a notable enhancement in the
activity of the modified derivative Z24. Specifically, Z24 showed
notably lower EC[50] values, ranging from 0.52 to 4.93 μg/ml. In
comparison, the positive control drugs showed EC[50] values ranging
from 0.65 to 13.19 μg/ml. As a result, compound Z24 was chosen as a
representative for further investigation due to its superior antifungal
potency. As illustrated in [79]Fig. 1A, Z24 exhibited superior
inhibitory efficacy against S. sclerotiorum, B. cinerea Pers., F.
graminearum, and F. oxysporum at a concentration of 5 μg/ml. The
antifungal activity of Z24 against B. cinerea Pers. and B. cinerea
B05.10 was over 10-fold greater than that of Z1, as seen in [80]Fig. 1
(B and C). For B. cinerea Pers., the EC[50] values of compounds Z1 and
Z24 were 5.37 and 0.56 μg/ml, respectively ([81]Fig. 1, D and E). As
shown in [82]Table 1, compound Z24 was more effective than pyrimethanil
(EC[50] of 4.45 μg/ml), a commercially available fungicide, against B.
cinerea Pers. Additionally, compound Z24 exhibited noteworthy
inhibition of B. cinerea spore germination ([83]Fig. 1 F and I). The
germination rates of B. cinerea B05.10 spores were 36.69, 32.86, 30.56,
28.14, and 24.28%, following Z1 treatment, as shown in [84]Fig. 1H.
However, after Z24 treatment, the germination rate dropped to 19.93,
17.63, 10.87, 7.11, and 4.74% ([85]Fig. 1G). Meanwhile, B. cinerea
Pers. spores showed germination rates of 41.82, 37.89, 34.06, 29.20,
and 22.63%, following Z1 treatment ([86]Fig. 1J). When Z24 treatment
was applied, however, the germination rates decreased to 30.48, 21.29,
16.09, 9.95, and 3.54% ([87]Fig. 1K). To assess the anti–B. cinerea
mechanism of Z24, we preliminarily investigated the structural
integrity and oxidative state of B. cinerea. Examination of the
Z24-treated mycelium cell ultrastructure by transmission electron
microscopy (TEM) revealed thicker mycelium cell walls, organelle
degradation, reduced cytoplasmic vacuoles, disorganized cytoplasm, and
disrupted mitochondrial structures ([88]Fig. 1L). The intracellular
reactive oxygen species (ROS) was measured using a fluorescent probe,
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Observation under
a fluorescence microscope clearly confirmed that Z24 treatment
exhibited a strong ability to generate intracellular ROS in B. cinerea
compared to the control group ([89]Fig. 1M). To assess the toxicity of
Z24 (0.5 μg/ml) on B. cinerea cells in potato dextrose agar (PDA)
medium, we used the nuclear dye PI/Hoechst 33342 cell stain to stain
the treated cells. Adding the membrane-impermeable “propidium iodide
(PI) dye” and membrane-permeable “Hoechst 33342 dye” caused dying or
dead cells to turn bright red or bright blue, while live cells were
fluorescent baby blue ([90]Fig. 1N).
Table 1. Fungicidal activity of Z1/Z24.
Fungus EC[50] (μg/ml)
Z1 Z24 Positive control
Botrytis cinerea Pers. 5.37 0.56 4.45/Pyrimethanil
Sclerotinia sclerotiorum 17.65 2.80 1.94/Boscalid
Rhizoctonia solani 9.00 4.93 6.76/Thiophanate methyl
FusaHum graminearum 16.31 1.92 0.65/Carbendazim
Fusarium oxysporum 17.63 2.82 12.53/Azoxystrobin
Phytophthora capsici 37.15 0.52 13.19/Azoxystrobin
[91]Open in a new tab
Fig. 1. In vitro antifungal activity of Z24 against B. cinerea.
[92]Fig. 1.
[93]Open in a new tab
(A) Colony formation (n = 6) of phytopathogenic fungi after 3 to 7 days
of growth on agar plates supplemented with Z1 and Z24 (5 μg/ml). (B)
Compound Z1 moderately suppressed mycelial growth of B. cinerea Pers.
and B. cinerea B05.10 at concentrations ranging from 5 to 50 μg/ml. (C)
Compound Z24 effectively suppressed mycelial growth of B. cinerea Pers.
and B. cinerea B05.10 at concentrations of 0.5 to 5 μg/ml. (D and E)
Colony formation (n = 6) of B. cinerea Pers. after 4 days of growth on
agar plates, supplemented with increasing concentrations of compound Z1
and Z24. The green dotted line indicates EC[50] concentration. (F)
Representative images of the germination of B. cinerea B05.10 spores
after treatment with Z1 and Z24. Scale bars, 100 μm. (G and H) Effects
of (G) Z24 (n = 6 to 8) and (H) Z1 (n = 5 to 7) on the germination of
B. cinerea B05.10 spores. (I) Representative images of the germination
of B. cinerea Pers. spores after treatment with Z1 and Z24. Scale bars,
100 μm. (J and K) Effects of (J) Z1 (n = 6 to 10) and (K) Z24 (n = 7 to
8) on the germination of B. cinerea Pers. spores. (L) Transmission
electron microscopy (TEM) images reveal the ultrastructure of B.
cinerea Pers. after treatment with Z24 (1 μg/ml). Scale bars, 1.0 μm.
(M) Intracellular ROS levels [2′,7′-dichlorodihydrofluorescein
diacetate (DCFH-DA)] in B. cinerea Pers. spores after treatment with
Z24 (20 μg/ml) were measured using DCFH-DA. Scale bars, 100 μm. (N)
Nuclear dye propidium iodide (PI)/Hoechst 33342 was used to stain the
mycelial cells treated with Z24 (0.5 μg/ml). Scale bars, 50 μm.
The direct targets of Z24 against B. cinerea were identified and validated
In previous proteomics-based studies, we conducted a preliminary
investigation into the mechanism of action of neocryptolepine on R.
solani and found that it has a substantial effect on the thiamine
metabolism process of R. solani ([94]31). Therefore, neocryptolepine
was selected as the lead molecule for the subsequent optimization
phase, and the candidate compound Z24 was synthesized. The efficacy of
Z24 against a range of plant pathogenic fungi suggests that it may have
a mechanism of action distinct from that of the first-line fungicide
used to treat B. cinerea infection. Consequently, identifying the
direct targets of Z24 and further elucidating its unique mechanism of
action is of substantial importance. To investigate the molecular
mechanisms by which Z24 exerts antifungal bioactivity, drug affinity
responsive target stability (DARTS) technology and mass spectrometry
analysis were performed ([95]Fig. 2A). Coomassie brilliant blue
([96]Fig. 2B) and silver staining ([97]Fig. 2C) revealed the presence
of distinct protein bands (B1 and B2) in the 25- to 35-kDa range.
Additionally, DARTS experiments showed that compound Z1 produced the
same protein bands in the same location as compound Z24 (fig. S1).
Protease treatment resulted in decreased protein content in the
dimethylsulfoxide (DMSO) group and increased protein content in the Z24
group as concentration increased. A total of 367 proteins were
identified as potential targets of Z24 (tables S1 and S2). These
results suggest that Z24 may target multiple proteins in B. cinerea.
Among the candidate proteins, thiazole synthase (fig. S2), which is
involved in the thiamine biosynthesis pathway and has the highest
abundance, was selected for further investigation. The intensity-based
absolute quantification of the Bcthi4 protein was 37.11%, while that of
the Bcnmt1 protein was 8.08% ([98]Fig. 2D). Thiamine thiazole synthase,
also known as Bcthi4, is the key enzyme in the thiamine biosynthesis
pathway responsible for synthesizing the thiazole component. Bcnmt1 is
involved in pyrimidine production, and the thiazole and pyrimidine
components are coupled to form thiamine ([99]12, [100]16).
Additionally, high-abundance proteins BCIN_16g03380 and BCIN_03g01010
were identified, suggesting that Z24 may exert antifungal activity in
B. cinerea through a range of possible targets ([101]Fig. 2E).
Fig. 2. Z24 targets the Bcthi4 protein, which contributes to inhibit the
growth of B. cinerea cells in vitro and in vivo.
[102]Fig. 2.
[103]Open in a new tab
(A) DARTS identifies a molecular target of Z24. (B) B. cinerea Pers.
cell lysates were incubated with Z24 in vitro, followed by protease
digestion and Coomassie bright blue (CBB) stain. (C) B. cinerea Pers.
cell lysates were incubated with Z24 in vitro, followed by protease
digestion and silver staining (n = 3 exp.). The red and black arrows
indicate the locations of the different bands, B1 and B2. (D and E)
Information on the top 10 proteins with the highest relative abundance
in the B1 and B2 band. iBAQ, intensity-based absolute quantification.
(F and G) Predicted binding mode of Z24 with Bcthi4. (H) Expression and
purification of the Bcthi4^WT protein in a prokaryotic expression
system. (I) Surface plasmon resonance (SPR) sensorgrams obtained from
Bcthi4^WT-coated chips at different concentrations of Z24. (J)
Dissociation constant (K[d]) value of Z24 binding to the recombinant
Bcthi4^WT protein. (K) Expression and purification of the
Bcthi4^MT(Met304/Thr232/Thr231) mutant protein in a prokaryotic
expression system. (L) Detection of the binding of
Bcthi4^MT(Met304/Thr232/Thr231) to Z24 by SPR analysis. (M) K[d] value
of Z24 binding to the recombinant mutated
Bcthi4^MT(Met304/Thr232/Thr231) protein. “NA” indicates that no binding
activity was detected. K[a], acid constant. (N and O) Bcthi4 activity
in B. cinerea Pers. and B. cinerea B05.10 after treatment with Z24 at
0.05 and 0.1 μg/ml, respectively. (P) Fold change in Bcthi4 activity in
B. cinerea pers. and B. cinerea B05. (Q and R) The Bcthi4 rabbit
polyclonal antibodies, G2206 and G2207, were successfully prepared. (S)
Z24-enriched Bcthi4 protein was verified by DARTS (n = 3 exp.). (T)
Thermal stability of Bcthi4 protein with or without Z24 treatment
(n = 3 exp.).
Next, we explored the possible binding mode between Z24 and Bcthi4.
Because of a lack of Bcthi4 crystal structure, we used molecular
docking to predict the binding site of Z24. The three-dimensional
structure of Bcthi4 predicted by AlphaFold 3
([104]https://golgi.sandbox.google.com/) was chosen as the receptor
structure. The docking result showed that the binding energy between
Z24 and the Bcthi4 protein was −8.185 kcal/mol, showing a strong
binding activity ([105]Fig. 2F). The expression and purification of the
Bcthi4^WT protein was showed in [106]Fig. 2H and fig. S4. As indicated
by surface plasmon resonance (SPR) analysis, Z24 can dose-dependently
bind to immobilized Bcthi4^WT protein ([107]Fig. 2I) with a high
affinity [dissociation constant (K[d]) of 9.425 × 10^−5] ([108]Fig.
2J). The phenyl group forms hydrophobic interactions with the residues
Met^304 and Thr^232. The carbon-hydrogen bond formed by residue Thr^231
is located between these two residues and has the shortest interatomic
distance, which may influence the molecular binding and interactions
([109]Fig. 2G). To verify the predicted binding mode, we generated the
mutants of Bcthi4: Met^304, Thr^232, and Thr^231 ([110]Fig. 2K and fig.
S5), and retested the binding ability of Z24 by SPR. The results showed
that the mutant completely abolished the binding of Z24 to Bcthi4
([111]Fig. 2, L and M). Bcthi4 plays an essential role in the thiamine
biosynthesis pathway as a thiazole synthase ([112]12). Therefore, we
further investigated whether the combination of Bcthi4 and Z24 inhibits
Bcthi4 activity. Compared with the control group, Z24 significantly
inhibited the activity of Bcthi4 in B. cinerea Pers. ([113]Fig. 2N) and
B. cinerea B05.10 ([114]Fig. 2O) strains when the concentrations
reached 0.05 and 0.1 μg/ml. As depicted in [115]Fig. 2P, the inhibitory
effect of Z24 on Bcthi4 in B. cinerea B05.10 was stronger than that on
Bcthi4 in B. cinerea Pers. To further confirm the binding of Z24 to
Bcthi4, we successfully produced a rabbit polyclonal antibody against
the Bcthi4 protein ([116]Fig. 2, Q and R), which exhibited high
sensitivity and specificity (fig. S3). Subsequently, the binding of Z24
to Bcthi4 was further investigated using DARTS assay and cellular
thermal shift assay (CETSA). Consistent with the above results, Z24
significantly increased Bcthi4 accumulation at concentrations ranging
from 250 to 500 μg/ml (compared to the DMSO solvent control) ([117]Fig.
2S). Notably, with the addition of Z24, the stability of Bcthi4 was
considerably improved, suggesting the possible formation of a
Z24/Bcthi4 complex. Moreover, as shown in [118]Fig. 2T, Z24
significantly increased Bcthi4 accumulation at temperatures ranging
from 50° to 80°C (compared to the DMSO solvent control), indicating a
direct interaction with Bcthi4 via thermal stability. Together, these
results suggest that Bcthi4 has the potential to be an attractive
antifungal target, and screening for Bcthi4 inhibitors could be an
effective strategy for developing previously unidentified fungicides.
Transcriptomic analysis of B. cinerea under Z24 treatment
To investigate the mechanism of action of Z24 in B. cinerea at the
genetic level, we performed transcriptomic sequencing analysis. As
shown in [119]Fig. 3A, 273 differentially expressed genes (DEGs) were
identified in B. cinerea after treatment with Z24, with 1907
up-regulated and 2366 down-regulated. The top 10 enriched Kyoto
Encyclopedia of Genes and Genomes (KEGG) database pathways were
up-regulated, including ribosome (42 DEGs), aminoacyl-tRNA biosynthesis
(30 DEGs), amino acid biosynthesis (56 DEGs), cysteine and methionine
metabolism (27 DEGs), and citric acid cycle (16 DEGs). The DEGs were
predominantly associated with ribosome function, aminoacyl-tRNA
synthesis, and amino acid biosynthesis ([120]Fig. 3B). Thiamine is an
essential coenzyme in cell metabolism, playing a crucial role in
various biochemical processes, including sugar metabolism and energy
production. When thiamine synthesis is inhibited, cells may enhance
protein synthesize by up-regulating ribosomal gene expression to
compensate for the metabolic stress caused by thiamine deficiency.
Moreover, in fungi, a significant increase in the expression of
aminoacyl-tRNA synthetase genes may occur as a biological response to
thiamine deficiency, aimed at improving protein synthesis efficiency
and thus supporting cell function and growth. In fungi, the TPP
riboswitch regulates gene expression through alternative splicing (AS)
of the pre-mRNA ([121]32). THI4 riboswitches, found in gene introns,
may contribute to AS, as demonstrated in N. crassa ([122]24).
Meanwhile, the role of AS in eukaryotic gene regulation is becoming
increasingly clear ([123]33, [124]34). AS is a crucial
posttranscriptional regulatory mechanism that enhances transcriptome
diversity and protein complexity. However, few studies have focused on
AS in fungi. Further analysis of AS data revealed a total of 70,420
splicing sites in the RNA sequencing (RNA-seq) data, with 47,107 being
previously unknown splicing sites and 23,313 being known splicing
sites. A total of 4290 variable splicing events were identified, with
889 known (annotated) and 3401 unannotated variable splicing events,
which account for 79.28% of all observed AS events. Common types of
splicing events included alternative 3′ splice site (A3SS), A5SS, and
intron recognition (IntronR), as shown in [125]Fig. 3C. The types and
quantities of up-regulated and down-regulated AS events are presented
in [126]Fig. 3D. For up-regulated events, these include A3SS (11), A3SS
and exon skipping (A5SS&ES) (1), A5SS (14), ES (2), and IntronR (71),
while, for down-regulated events, the types and numbers are A3SS (18),
A5SS (18), A5SS&ES (1), IntronR (158), and cassette exon (2). KEGG
pathway enrichment analysis of differentially variable splicing genes
revealed that the thiamine metabolic pathway showed the most notable
enrichment. Three differentially variable splicing genes were enriched
in this pathway, including the target gene Bcthi4, as well as Bcthi6
and Bcrpb8 ([127]Fig. 3E). As shown in [128]Fig. 3F, the thiamine
metabolic pathway was also notably enriched in overlap analysis of
differentially variable splicing genes and DEGs. Notably, a
significantly up-regulated A5SS splicing event was detected in the
Bcthi4 riboswitch of B. cinerea, suggesting that Z24 treatment induced
selective splicing of the Bcthi4 precursor mRNA in B. cinerea and
increased its splicing ([129]Fig. 3G). This indicates that, similar to
plants, fungi may use AS to increase proteome complexity and respond to
transcriptional changes induced by stress, thereby reducing the
metabolic cost of processing all AS transcripts ([130]35).
Fig. 3. DEGs and RASG analysis.
[131]Fig. 3.
[132]Open in a new tab
(A) Volcano plot analysis of differential gene expression, including
1907 up-regulated genes and 2366 down-regulated genes. FDR, false
discovery rate. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG)
enrichment analysis of up-regulated differentially expressed genes
(DEGs). (C) Overview of all detected AS events. (D) Types and numbers
of AS events detected in up-regulated and down-regulated AS genes. (E)
KEGG enrichment analysis of variable AS genes (RASGs). (F) KEGG
enrichment analysis of the overlap between RASGs and DEGs. (G)
Differential variable splicing events of the Bcthi4 gene.
High Bcthi4 expression is correlated with B. cinerea survival under Z24
stress
When exposed to external stress, plant pathogenic fungi exhibit a
certain degree of tolerance and can withstand damage by activating
their defense mechanisms. These processes may involve modifications to
metabolic pathways, the synthesis of protective chemicals, and other
adaptive responses. For example, the transcription of drug target genes
(FgCYP51s) was significantly induced in the wild-type Fusarium
graminearum after treatment with the azole compound tebuconazole, but
not with iprodione or fludioxonil, which act via different mechanisms
([133]36). Therefore, we further investigated the effects of Z24 on the
genes and metabolism of B. cinerea. Phenotypic characterization
revealed that Z24 and the tebuconazole treatment groups showed
increased mycelial growth on PDA plates on days 6 and 8, respectively
([134]Fig. 4, A to C). We hypothesized that, during this process, the
target genes and related metabolites of B. cinerea affected by Z24
would also change notably. Expectedly, genes involved in thiamine
metabolism are tightly regulated by thiamine levels ([135]37). A study
showed that the green alga Chlamydomonas reinhardtii uses TPP
riboswitches to down-regulate the expression of THIC and THI4 when
exposed to exogenous thiamine ([136]38).
Fig. 4. Available thiamine promotes the growth of B. cinerea under Z24 stress
by up-regulating genes and metabolites.
[137]Fig. 4.
[138]Open in a new tab
(A and B) Colony diameter measurement of B. cinerea Pers. and B.
cinerea B05.10 following treatment with Z24 (2.5 and 5 μg/ml). (C)
Colony diameter of B. cinerea Pers. after treatment with tebuconazole
(2.5 μg/ml). d, days. (D and E) Reverse transcription quantitative
polymerase chain reaction (RT-qPCR) analysis of Bcthi4 gene expression
in B. cinerea Pers. and B. cinerea B05.10 after Z24 treatment. (F to H)
Measurement of thiamine content in (F) B. cinerea Pers. and (G) B.
cinerea B05.10, as well as the (H) fold change after Z24 treatment. (I
to K) Measurement of TPP content in (I) B. cinerea Pers. and (J) B.
cinerea B05.10, along with the (K) fold change after Z24 treatment. (L)
Z24 specifically inhibits the growth of B. cinerea by targeting Bcthi4
in the thiamine biosynthesis pathway. HET-P,
4-methyl-5-(2-phosphooxyethyl)thiazole; NAD, nitric acid dihydrate.
To investigate whether thiamine biosynthesis is inhibited by compound
Z24, we examined the transcription of the Bcthi4 gene, which encodes a
key enzyme in thiamine biosynthesis. Reverse transcription quantitative
polymerase chain reaction (RT-qPCR) assays showed that the expression
of Bcthi4 was significantly up-regulated in B. cinerea Pers. ([139]Fig.
4D) and B. cinerea B05.10 ([140]Fig. 4E) under Z24 stress. Notably, as
a typical suicide enzyme, the transcriptional level of Bcthi4 was
markedly up-regulated following Z24 treatment, likely as a compensatory
response to thiamine deficiency in the cytoplasm. These findings
indicate that Z24 inhibits the thiamine biosynthesis pathway in B.
cinerea. The utilization of energy during fungal growth and metabolism,
as well as the synthesis of various substances required for cell
growth, is highly efficient, especially for thiamine biosynthesis,
which is an energy-intensive pathway. This may also explain the
presence of feedback regulatory mechanisms in species with complete
biosynthesis pathways ([141]38). To further investigate the effect of
Z24 on the thiamine pathway mediated by Bcthi4, we compared thiamine
and TPP content related to this pathway in B. cinerea Pers. and B.
cinerea B05.10 strains. As shown in [142]Fig. 4F, thiamine content in
B. cinerea Pers. mycelial cells increased by 1.4, 1.5, and 1.9 times in
a concentration-dependent manner under Z24 stress ([143]Fig. 4H). In B.
cinerea B05.10 mycelial cells, the thiamine content increased by 1.4,
1.6, and 2.7 times ([144]Fig. 4H) in a concentration-dependent manner
([145]Fig. 4G). Thiazole synthase is a key enzyme in the thiamine
biosynthesis pathway, and changes in its expression level may affect
thiamine synthesis. These results suggest that, under conditions of
limited thiamine synthesis, fungi may up-regulate the expression of
enzymes such as thiazole synthase to increase the production of
thiamine precursors, thereby partially compensating for the thiamine
deficiency. TPP, the active form of thiamine, was also quantified. The
experimental results revealed that the TPP content in B. cinerea Pers.
([146]Fig. 4I) and B. cinerea B05.10 ([147]Fig. 4J) mycelial cells
significantly increased with increasing Z24 concentrations ([148]Fig.
4K). A previous study showed that the expression of the Bcthi6 gene is
negatively regulated by intracellular TPP, suggesting that fungi can
regulate thiamine biosynthesis through a feedback mechanism ([149]39).
According to the transcriptome data, the expression of the Bcthi6 gene
in B. cinerea was also significantly up-regulated after Z24 treatment,
further indicating that the thiamine biosynthesis process was
inhibited.
Collectively, the thiamine biosynthetic pathway is tightly regulated to
ensure that TPP production meets cellular demands. This is typically
achieved by controlling the expression of one or more enzyme genes. For
instance, exogenous thiamine notably inhibits the transcription of
thiamine-related genes in bacteria and fungi ([150]40). In contrast,
under thiamine deficiency, the expression of the thiamine synthase gene
is markedly up-regulated. Together, the data indicate that compound Z24
specifically inhibits the thiamine biosynthesis pathway ([151]Fig. 4L).
Pathway validation of Z24-induced disturbance in thiamine metabolism
To further verify the mechanism of action of Z24, exogenous thiamine
(250 and 500 μg/ml) was supplemented to support B. cinerea growth and
assess the inhibitory effects of Z24 and pyrimethanil, both alone and
in combination. Both B. cinerea Pers. and B. cinerea B05.10 strains
exhibited severe growth defects after pyrimethanil treatment. Further
validation with exogenous thiamine supplementation (500 μg/ml) showed
that both B. cinerea Pers. and B. cinerea B05.10 strains displayed
similar inhibitory activity, regardless of the presence or absence of
thiamine ([152]Fig. 5, A and B). Pyrimethanil is known to inhibit the
production of infection-causing enzymes, rather than disrupting the
thiamine metabolic pathway, to prevent fungal infections and kill
fungi. In contrast, B. cinerea Pers. and B. cinerea B05.10 strains
exhibited significantly reduced growth inhibition when grown in the
presence of thiamine after Z24 treatment ([153]Fig. 5, C and D). We
also investigated whether thiamine could enhance B. cinerea growth in
PDA medium. Thiamine did not significantly enhance fungal growth, and
high concentrations of thiamine (500 μg/ml) actually inhibited B.
cinerea growth ([154]Fig. 5, E and F). Similarly, both B. cinerea Pers.
and B. cinerea B05.10 strains showed significantly reduced
concentration-dependent growth inhibition when grown in the presence of
thiamine after treatment with Z24 (0.1 μg/ml) in liquid PDA medium
([155]Fig. 5, G to I). Meanwhile, thiamine also exhibited significant
inhibitory activity against B. cinerea in liquid PDA medium ([156]Fig.
5, J and K). Last, B. cinerea showed significantly reduced growth
inhibition when cultivated with thiamine (500 μg/ml) after treatment
with Z24 (10 μg/ml) on grapes ([157]Fig. 5L). Together, these results
strongly suggest that thiamine is crucial for B. cinerea growth, and
Z24 likely interferes with the B. cinerea thiamine biosynthesis
pathway.
Fig. 5. Metabolic pathway blockade identifies fungicide Z24.
[158]Fig. 5.
[159]Open in a new tab
(A and B) The effect of exogenous thiamine (500 μg/ml) on the growth of
B. cinerea Pers. and B. cinerea B05.10 after treatment with
pyrimethanil (4 μg/ml) on PDA plates. (C and D) The growth inhibitory
activity of B. cinerea Pers. and B. cinerea B05.10 in the presence of
thiamine (500 μg/ml) after treatment with Z24 (0.25 μg/ml) on PDA
plates. (E and F) The inhibitory activity of thiamine (at 50, 100, 250,
500 μg/ml) against B. cinerea Pers. and B. cinerea B05.10 on PDA
plates. (G and H) The effect of exogenous thiamine (at 250 and 500
μg/ml) on the growth of B. cinerea Pers. after treatment with Z24 in
liquid PDA medium. (I) The growth inhibitory activity of B. cinerea
B05.10 in the presence of thiamine (250 and 500 μg/ml) after treatment
with Z24 (0.1 μg/ml) in liquid PDA medium. (J and K) Inhibitory
activity of thiamine (at 100, 250, and 500 μg/ml) against B. cinerea
Pers. and B. cinerea B05.10 in liquid PDA medium. (L) The growth
inhibitory activity of B. cinerea B05.10 in the presence of thiamine
(500 μg/ml) after treatment with Z24 (10 μg/ml) on grapes.
Metabolite analysis of thiamine metabolism disruption by Z24
Metabolite analysis was conducted to gain a comprehensive understanding
of the antifungal mechanism of Z24 and to verify its impact on the
thiamine metabolism pathway. TPP, the active form of thiamine, is an
essential cofactor in glycolysis, the tricarboxylic acid cycle, the
pentose phosphate pathway, and the synthesis of branched-chain amino
acids. It plays a critical role in the carboxylation and
decarboxylation of various metabolic intermediates ([160]41, [161]42).
To analyze the metabolites of B. cinerea after treatment with Z24, we
performed liquid chromatography–tandem mass spectrometry. As
illustrated in fig. S6A, metabolites identified in the positive ion
model and annotated to biological processes include amino acids,
carbohydrates, coenzymes, vitamins, and others. Figure S6B shows the
primarily enriched pathways, including transmembrane transport,
translation, amino acid metabolism, coenzyme and vitamin metabolism,
carbohydrate metabolism, lipid metabolism, and others. Principal
components analysis (PCA) revealed a clear distinction between the
control and Z24 treatment groups, with the contribution rates of PC1
and PC2 being 57.87 and 21.55%, respectively ([162]Fig. 6A). As a
result, 331 differential metabolites were identified in positive ion
mode, including 184 up-regulated and 147 down-regulated metabolites
([163]Fig. 6B). KEGG pathway analysis revealed that Z24 primarily
affected amino acid biosynthesis, including alanine, aspartate, and
glutamate metabolism; tryptophan metabolism; phenylalanine metabolism;
niacin and niacinamide metabolism; histidine metabolism; arginine and
proline metabolism; aminoacyl-tRNA biosynthesis; and adenosine
5′-triphosphate (ATP)–binding cassette (ABC) transporter in B. cinerea
([164]Fig. 6C). The abundance of l-histidinol, argininosucinic acid,
and l-arginine was significantly up-regulated, while d-aspartate and
l-asparagine were significantly down-regulated in the amino acid
metabolism and aminoacyl-tRNA biosynthesis pathways ([165]Fig. 6, D to
G).
Fig. 6. The relative content of up-regulated and down-regulated metabolites
shows a substantial correlation with amino acid metabolism in B. cinerea.
[166]Fig. 6.
[167]Open in a new tab
(A) Principal components analysis (PCA) score plots in electrospray
ionization (ESI)^+ mode. (B) Volcano plot analysis of differential
metabolomics in ESI^+ mode. FC, fold change. (C) Pathway enrichment
analysis of differential metabolomics in ESI^+ mode. (D to G) The fold
change of metabolites in (D) alanine, aspartate, and glutamate
metabolism; (E) biosynthesis of amino acids; (F) arginine biosynthesis;
and (G) aminoacyl-tRNA biosynthesis in ESI^+ mode. (H) PCA score plots
in ESI^− mode. (I) Volcano plot analysis of differential metabolites in
ESI^− mode. (J) Pathway enrichment analysis of differential metabolites
in ESI^− mode. (K to N) The fold change of metabolites in (K) alanine,
aspartate and glutamate metabolism; (L) biosynthesis of amino acids;
(M) C5-branched dibasic acid metabolism; and (N) lipoic acid metabolism
in ESI^− mode. The q value is obtained by correcting the P value using
the FDR. BHcorrect, Benjamini-Hochberg correction.
Carbohydrates and amino acids, which are also involved in biological
processes, were classified as metabolites in the negative ion mode, as
shown in fig. S7A. The enrichment pathways included lipid metabolism,
coenzyme and vitamin metabolism, carbohydrates metabolism, and amino
acids metabolism (fig. S7B). PCA revealed that PC1 contributed 63.73%
and PC2 contributed 21.04%, as shown in [168]Fig. 6H. A total of 247
different metabolites were identified in the Z24 group in negative ion
mode, with 119 up-regulated and 128 down-regulated, compared to those
in the control group ([169]Fig. 6I). KEGG pathways analysis revealed
that Z24 primarily affected amino acid biosynthesis; alanine,
aspartate, glutamate metabolism; and ABC transporter in B. cinerea
([170]Fig. 6J). The abundance of l-histidinol, argininosucinic acid,
and N-acetyl-l-aspartic acid was significantly up-regulated, while
2-oxobutyric acid, trans-aconitic acid, lipoic acid, and
2-keto-glutaramic acid were significantly down-regulated in amino acid
metabolism, C5-branched dibasic acid metabolism, and lipoic acid
metabolism ([171]Fig. 6, K to N). TPP-dependent enzymes, such as
branched-chain α-ketoacid dehydrogenase (BCKDC) and acetolactate
synthase (AHAS), play a crucial role in promoting cell growth and
preventing metabolic stress ([172]43). AHAS and BCKDC are responsible
for the synthesis and degradation of branched-chain amino acids,
respectively ([173]44). These findings support the notion that Z24
notably affects TPP-mediated microbial metabolism activities and play a
key role in the metabolic profile of B. cinerea, primarily interfering
with amino acid metabolism in fungi.
Representative compound Z24 demonstrates favorable safety
The effects of compounds Z1, Z24, and pyrimethanil on cell viability
were assessed using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay in normal human intestinal epithelial cells (HIECs), normal human
hepatocytes (HL-7702), and gastric epithelial cells (GES-1). Cell
viability was measured after exposure to different concentrations of
these compounds. As shown in [174]Fig. 7 (A to C), compound Z24
exhibited lower cytotoxicity, with cell viability ranging from 71 to
95%, compared to Z1 (36.5 to 80.5%) and pyrimethanil (84.75 to 94%).
Compound Z24 demonstrated the lowest cytotoxicity, making it a
promising candidate for further research.
Fig. 7. In vitro and in vivo toxicity evaluation of compound Z24.
[175]Fig. 7.
[176]Open in a new tab
(A to C) Inhibitory activity of Z24 on (A) HIECs, (B) HL-7702 cells,
and (C) GES-1 cells. HIECs, HL-7702 cells, and GES-1 cells were treated
with Z24, Z1, and pyrimethanil for 48 hours, and cell viability was
assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. (D to H) Effects of Z24 (2000 mg/kg) on
pathological changes in the (D) heart, (E) liver, (F) spleen, (G) lung,
and (H) kidney tissues of Wistar rats. (I to M) Relative body weights
of the (I) heart, (J) liver, (K) spleen, (L) lung, and (M) kidney in
the control and Z24 (2000 mg/kg) groups. (N) Body weight changes in
Wistar rats after Z24 administration. (O to R) Changes in blood
biochemical markers: (O) GPT, (P) GOT, (Q) AKP/ALP, and (R) urea
nitrogen in rats from the control and Z24 groups. (S and T)
Representative images and quantitative statistical data for the Z24 and
Triton X-100 hemolysis rates (n = 3).
Compound Z24 was also evaluated for acute oral toxicity in Wistar rats
following the Organization for Economic Co-operation and Development
(OECD) Guideline 423. The medium lethal dose of Z24 was found to be
greater than 2000 mg/kg after 14 days of close observation, indicating
that Z24 has an acceptable safety profile when administered orally.
Histological examination of tissue sections from the heart, liver,
spleen, lungs, and kidneys revealed no signs of inflammation, and the
organ architecture appeared normal. Gross necropsy of the female rat
revealed no lesions or abnormalities in the organs. Microscopical
analysis of cardiac sections showed no changes in the cardiomyocyte
architecture. There were no indications of cardiac myopathy, myofiber
degeneration, necrosis, vacuolation, or mononuclear cell infiltration,
leading to the conclusion that Z24 therapy is safe for the rat heart
([177]Fig. 7D). Z24 also appears to be safe for normal liver function,
as histological examination of rat hepatic sections treated with Z24
revealed no signs of periportal hepatocellular vacuolation, lipid
accumulation, localized inflammation, focal tension,
hepato-diaphragmatic nodules, or inflammatory cell infiltration
([178]Fig. 7E). Additionally, the presence of immune cells in the
lymphoid tissue and blood vessels within the connective tissue
trabeculae extending from the hilus into the lymph parenchyma indicated
that Z24 had no effect on the rat spleen ([179]Fig. 7F). The absence of
invasive lymphocytes, plasma cells, and histocytes in the subpleural
regions of rat lung sections treated with Z24 indicates the absence of
inflammation ([180]Fig. 7G). Rats treated with Z24 displayed delicate
and thin glomerular capillary loops, with a normal number of mesangial
cells and endothelium, suggesting normal glomerular filtration
([181]Fig. 7H). The heart, kidney, spleen, and lung remained relatively
unchanged, with the exception of the liver ([182]Fig. 7, I to M).
Furthermore, no significant body weight loss was observed after the
various treatments, as shown in [183]Fig. 7N.
Subsequently, the biochemical indicators of the liver and kidney
functions were assessed using blood samples taken from the rats
mentioned above. As shown in [184]Fig. 7 (O to R), compound Z24 did not
induce any significant changes in glutamic pyruvic transaminase (GPT),
glutamic oxalacetic transaminase (GOT), alkaline phosphatase (AKP/ALP),
or urea nitrogen levels, suggesting that Z24 has no noticeable negative
impact on liver or kidney function. [185]Figure 7 (S and T) presents
the results from the in vitro hemolysis tests. No visible hemolysis was
observed after co-incubating compound Z24 with sheep erythrocyte
suspensions. Z24 exhibited no hemolysis at a concentration of 64 μg/ml
(114 times the EC[50]), indicating that the compound Z24 has negligible
hemolytic activity on human red blood cells.
Compound Z24 exhibits promising in vivo efficacy against B. cinerea
Compound Z24 was evaluated for its in vivo antifungal activity using
tomato and cucumber pot models. Several opportunistic pathogenic fungi
produce a large number of airborne spores. Therefore, we sprayed
various concentrations of Z24 onto tomatoes, incubated the tomatoes for
72 hours after inoculating them with B. cinerea Pers. spores, and
quantified the disease symptoms. In the control group, B. cinerea Pers.
spores formed colonies on the tomatoes after 72 hours, causing gray
mold, a characteristic symptom of tomato gray mold disease. Symptom
development was inhibited when tomatoes were pretreated with Z24, and
the protective effect was concentration dependent ([186]Fig. 8A). Z24
almost completely suppressed infection by B. cinerea Pers. spores and
provided full protection against gray mold disease at a concentration
of 100 μg/ml. Therefore, we sprayed an equal concentration (100 μg/ml)
of Z24 and pyrimethanil onto cucumber leaves, incubated the treated
cucumber leaves for 7 days after applying B. cinerea Pers. spores, and
quantified the disease symptoms. In the control experiments, B. cinerea
Pers. spores formed colonies on cucumber leaves after 7 days, causing
gray mold on the leaves. Symptom development was inhibited when
cucumber leaves were pretreated with Z24 and pyrimethanil. In contrast,
Z24 inhibited symptom development in cucumber leaves at 100 μg/ml as
effectively as pyrimethanil ([187]Fig. 8B).
Fig. 8. Phytotoxicity and plant protection or therapy by Z24 in tomato and
cucumber.
[188]Fig. 8.
[189]Open in a new tab
(A) Gray mold disease symptoms in tomatoes after treatment with various
concentrations of Z24 (n = 5). (B) Gray mold disease symptoms on
cucumber leaves; data are presented as means ± SD, n = 7 to 10
biologically independent samples. (C and D) Protective effects of Z24
and pyrimethanil on cucumber leaves after infection with B. cinerea
Pers. plugs. (E and F) Therapeutic effects of Z24 and pyrimethanil on
cucumber leaves after infection with B. cinerea Pers. plugs. h, hours.
(G and H) Inhibitory activity of Z24 and carbendazim (a positive
control against S. sclerotiorum strain) on sclerotia formation (n = 6).
(I) Phytotoxicity of Z24 on radish seed (n = 12). (J) Effect of Z24 on
the germination rate of radish seeds. (K) Effect of Z24 on the bud
length of radish seeds. Note that control contains 0.25% DMSO.
**P < 0.001, ***P = 0.0001, and ****P < 0.0001. n.s., not significant.
Furthermore, the mycelium formed by B. cinerea spore germination
contacts the plant epidermis, penetrates the cuticle, and damages the
plant tissues by releasing numerous degrading enzymes and toxins. We
further evaluated the protective and therapeutic effects of Z24 on
mycelia-infected cucumber leaves. Infection with B. cinerea Pers. plugs
led to the development of gray mold lesions after 48 and 72 hours of
incubation. Symptom development was significantly inhibited when
cucumber leaves were pretreated with Z24 (50, 100, and 200 μg/ml) and
pyrimethanil, and this protective effect was concentration dependent.
In contrast, Z24 exhibited a more effective protective effect than the
positive control, pyrimethanil ([190]Fig. 8, C and D). As shown in
[191]Fig. 8 (E and F), Z24 also demonstrated a more potent therapeutic
effect than pyrimethanil. Additionally, Z24 completely inhibited
sclerotia formation in the tested S. sclerotiorum strain at a dose of 5
μg/ml ([192]Fig. 8G), whereas carbendazim, a positive control,
effectively inhibited sclerotium formation at 0.25 μg/ml ([193]Fig.
8H). Subsequently, Raphanus raphanistrum seeds were grown in Murashige
and Skoog agar medium treated with Z24 (0 to 200 μg/ml) and
pyrimethanil to assess their phytotoxicity on seed germination and bud
tube growth ([194]Fig. 8I). As shown in [195]Fig. 8J, Z24 had no effect
on seed germination at concentrations of 0 to 200 μg/ml. However, at
concentrations of 10 to 200 μg/ml, it significantly delayed R.
raphanistrum tube growth ([196]Fig. 8K). Pyrimethanil did not affect
seed germination at concentrations of 0 to 100 μg/ml ([197]Fig. 8J) but
significantly slowed R. raphanistrum tube growth at 100 to 200 μg/ml
([198]Fig. 8K). Collectively, compound Z24, with its safety and
efficiency, exhibits antifungal activity in crops and is a promising
fungicide for agriculture use.
DISCUSSION
Globally, the prevalence of B. cinerea infection remains notable. Given
the increasing prevalence of drug-resistant B. cinerea strains, it is
crucial to develop previously unidentified therapeutic approaches that
overcome the limitations of existing treatments. Using antifungal
agents with previously unknown mechanisms of action against B. cinerea
is essential to prevent cross-resistance with current fungicides.
In this study, a chemical modification was successfully developed to
synthesize previously unidentified antifungal agents, and a candidate
Z24, specifically targeting B. cinerea, was synthesized ([199]7).
Because of its efficacy against plant pathogenic fungi such as B.
cinerea, S. sclerotiorum, F. graminearum, R. solani, P. capsica, and F.
oxysporum, along with its favorable safety profile and unique mechanism
of action observed in this study, Z24 may offer a potential alternative
to current pesticide to address the growing issue of pesticide
resistance. According to the China Pesticide Information Network,
fungicides such as dicarboximides, anilinopyrimidines, phenylpyrroles,
methyl-benzimidazole carbamates, quinone outside inhibitors, and SDHIs
have been registered to combat gray mold. However, the environmental
pressure caused by the widespread use of fungicides has driven the
emergence of resistance to all major fungicides ([200]45), and
resistance to several fungicides has been frequently observed in B.
cinerea across different nations ([201]46, [202]47). Meanwhile,
broad-spectrum agricultural fungicides, such as difenoconazole,
propiconazole, and tebuconazole, not only are chemically similar to
first-line medical triazoles (itraconazole, posaconazole, and
voriconazole) but also are also being used in increasing quantities
worldwide ([203]48). These findings serve as a “smoking gun,” linking
the use of agricultural fungicides to clinical resistance ([204]49).
Mechanistically, antifungal resistance is typically caused by
modifications that either directly or indirectly affect the drug-target
interaction. Therefore, it is urgent to identify key pathogenic or
growth regulatory factors in B. cinerea as previously unknown
therapeutic targets and to develop previously unidentified fungicides
for its management.
DARTS is a proteomics approach that has been widely adopted and applied
in various biological research domains due to its unique
characteristics. It facilitates the identification of potential
treatment targets by assessing the active proteome in plant pathogenic
fungi-relevant tissues or cells. These validated targets could be
critical for understanding the key participants in disease pathways and
developing targeted therapies. Several verifications were conducted to
ensure the reliability of Z24-based target exploration. Additionally,
molecular docking predicted the identical interactions between Z24 and
the specific binding sites of Bcthi4, further confirming the validity
of the target exploration results. Additionally, Z24 dose-dependently
inhibited the activity of the thiazole synthase Bcthi4 in two strains
of B. cinerea. As a result, Z24 could be considered a suitable tool for
DARTS-based direct target fishing. The verification results strongly
supported the mechanisms proposed in this study, as well as those
observed in morphological analysis and transcriptome investigation
(including variable splicing).
Bcthi4, the validated target of compound Z24, and Bcnmt1, an
indispensable synthetase involved in thiazole and pyrimidine synthesis
in thiamine biosynthesis pathway, are essential for fungal viability,
making them promising targets for antifungal therapy. The relative
abundance of thiazole synthase Bcthi4 in the identified list was
37.11%, while pyrimidine synthase Bcnmt1 accounted for 8.08%. Bcthi4
and Bcnmt1 catalyze the two metabolic branches of thiazole and
pyrimidine synthesis in the fungal TPP biosynthesis pathway, which are
regulated by ribose switches ([205]10, [206]50). Now, limited studies
have been conducted on inhibitors targeting the B. cinerea Bcthi4 and
Bcnmt1 systems. Compound Z24 was predicted and found to act on both
systems, potentially inhibiting the entire TPP biosynthesis pathway. As
a result, fungi would experience a TPP deficit because of the
suppression of Bcthi4. To confirm that Z24 induces a TPP deficit in B.
cinerea by targeting Bcthi4 and Bcnmt1, thiamine and TPP levels were
measured in both the untreated control group and the Z24-treated group.
As shown in [207]Fig. 4 (F to K), a significant increase in thiamine
and TPP levels was observed in the Z24-treated group during Z24-induced
stress growth, further confirming the effect of Z24 on thiazole
synthase and pyrimidine synthase in the TPP biosynthesis. As a result,
TPP deficiency disrupts other TPP-related metabolic processes. The
absence of TPP, particularly in synthase-related proteins (Bcthi4 and
Bcnmt1), could impair the coenzyme function of B. cinerea in various
metabolic pathways. This effect was observed in B. cinerea after
treatment with Z24.
Thiamine is an essential cofactor in all organisms, from microorganisms
to mammals, because its active form, TPP, is required by several
enzymes involved in key cellular metabolic pathways, including amino
acid and central carbon metabolism, branched-chain amino acid
metabolism, and lipid metabolism ([208]12, [209]17, [210]51–[211]54).
It also plays a critical role in numerous cellular processes in the
cytoplasm, mitochondria, and peroxidase ([212]55). Thiamine enters
cells, is phosphorylated to form TPP, and the resulting coenzyme acts
as a ligand for riboswitch-mediated regulation of RNA splicing in fungi
([213]23). Numerous studies have shown that TPP directly bind to
aptamers, inducing conformational changes that interfere with the
expression of thiamine biosynthesis genes ([214]56–[215]59). In
eukaryotes, only TPP riboswitches have been found ([216]60, [217]61),
which regulate gene expression through AS ([218]24, [219]25). However,
riboswitches have not been found in mammals ([220]62). A previous study
on Aspergillus oryzae thiA mRNA, which carries a TPP aptamer, found
that thiamine (vitamin B1) supplementation in medium inhibits gene
expression and that deletion of riboswitch aptamer regions disrupts
thiamine responsiveness ([221]30). In another study, the roles of three
N. crassa TPP aptamers were investigated. NMT1 and CyPBP37 (a homolog
of THI4, hereafter referred to as THI4) are two of these genes
recognized as involved in thiamine metabolism. The third gene,
NCU01977.1, encodes a protein with no known function ([222]63–[223]65).
The findings show that thiamine induces AS of NMT1 and THI4 precursor
mRNAs in N. crassa, as well as increasing precursor mRNA splicing of
NCU01977.1. Thiamine has no impact on the splicing of RNA lacking the
TPP riboswitch ([224]24). Although the THI4 riboswitch mechanism has
not been fully studied, it has been shown that an excess of TPP in the
medium results in the synthesis of longer, nonfunctional mRNA
([225]24). Both Bcthi4 and Bcnmt1, which are involved in thiazole and
pyrimidine synthesis in thiamine biosynthesis, exhibited up-regulated
AS events and A5SS patterns. According to these findings, compound Z24
induced AS of Bcthi4 and Bcnmt1 precursor mRNAs, enhancing their
splicing. Further investigation revealed that the Bcthi4 gene produced
two distinct AS transcripts. Under Z24 stress, the transcription level
of the Bcthi4 gene in B. cinerea increased in a concentration-dependent
manner, ranging from 1.4 to 11.2 times. These results demonstrated that
AS plays an increasingly important role in the regulation of eukaryotic
thiamine biosynthesis genes ([226]33, [227]34). When extracellular
thiamine levels are low, the ribosome binding site becomes available,
TPP is not bound, and translation continues, leading to increased
transcription of downstream genes. Compound Z24 regulates thiamine
metabolism in B. cinerea by altering the binding of TPP to the Bcthi4
riboswitch. In response to intracellular thiamine deficiency, B.
cinerea up-regulates the expression of thiamine synthesis genes through
a feedback mechanism. The high metabolic cost of thiamine production
may explain the presence of a feedback control mechanism in organisms
with a complete biosynthesis pathway. The TPP riboswitch is the primary
riboswitch in fungi, often involved in the feedback regulation of
metabolite production genes. After treatment with Z24, the expression
of Bcthi4 in B. cinerea cells increased rapidly, leading to elevated
intracellular thiamine levels and improved cell survival. In summary,
Z24 inhibits thiamine biosynthesis by targeting Bcthi4, altering the
binding of TPP to the Bcthi4 riboswitch, which disrupts thiamine
metabolism in B. cinerea.
To sum up, neocryptolepine and its derivatives were designed and
synthesized in our previous study ([228]7). A novel derivative, Z24,
was successfully synthesized and demonstrated high efficacy against all
tested plant pathogenic fungi, with an EC[50] range of 0.52 to 4.93
μg/ml, while exhibiting a favorable safety profile both in vitro and in
vivo. Compound Z24 also showed more potent therapeutic and protect
effects than pyrimethanil in vivo. Using DARTS technique, Bcthi4,
involved in thiamine biosynthesis, was identified as a direct target of
Z24, and its binding activity was further confirmed by DARTS, CETSA,
and SPR. By inhibiting Bcthi4, Z24 disrupts the thiamine biosynthesis
pathway in B. cinerea, thereby protecting plant cells from fungal
infection. Given its previously unknown mechanism of action, the risk
of cross-resistance between Z24 and existing fungicides is relatively
low. To the best of our knowledge, compound Z24 is the first molecule
discovered to inhibit the catalytic activity of Bcthi4 in fungi,
representing a potential previously unidentified class of fungicides.
In conclusion, compound Z24, with its safety profile and previously
unknown mechanism of action, demonstrates anti–B. cinerea activity
through targeting Bcthi4, making it a promising chemical entity for
fungicide development.
MATERIALS AND METHODS
Chemicals, reagents, fungal strains, cells, and experimental animals
Compounds Z1 and Z24 were synthesized in our previous study ([229]7).
Pyrimethanil (purity ≥ 98%; CAS no. 53112-28-0), carbendazim (purity ≥
98%; CAS no. 10605-21-7), and tebuconazole (purity ≥ 99%; CAS no.
107534-96-3) were purchased from Bide Pharmatech Ltd., Shanghai, China.
Triton X-100 (20%; CAS no. 9002-93-1) and thiamine (purity ≥ 99%; CAS
no. 59-43-8) were obtained from Yuanye Biotechnology Co. Ltd.,
Shanghai, China. F. graminearum Sehw, B. cinerea Pers., R. solani Kuhn,
S. sclerotiorum (Lib.) de Bary, and F. oxysporum f. sp. vasinfectum
(Atk.) Snyder & Hansen were provided by the Institute of Plant
Protection, Gansu Academy of Agricultural Science. The standard strain
B. cinerea B05.10 and P. capsici Leonian were provided by Nanjing
Agricultural University and Sichuan Agricultural University,
respectively.
HIECs, HL-7702 cells, and GES-1 cells were stored in our laboratory.
All specific pathogen–free animals were purchased from the Veterinary
Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China).
In vivo acute toxicity studies were conducted under the approval and
supervision of the Institutional Animal Care and Use Committee of
Lanzhou University [approval no. SCXK (甘) 2023-0003].
Antifungal susceptibility tests in vitro
The fungitoxicity of Z24 against B. cinerea Pers. and B. cinerea B05.10
were assessed by measuring spore germination and mycelial growth. The
effect of Z24 on B. cinerea spore germination was determined using
previously reported methods ([230]66). Z24 was dissolved in DMSO to
prepare a stock solution of 100 μg/ml. This stock solution was then
added to liquid PDA medium containing conidial suspension (1.5 × 10^6
spores/ml) to obtain final concentrations of 0, 2, 4, 6, 8, 10, 12, 16,
and 20 μg/ml. Spores were incubated at 26°C for 10 hours to assess the
spore germination rate. Three replicates were performed for each
experiment.
The inhibitory activity of Z24 against phytopathogenic fungi was
determined using previously described methods ([231]67). Briefly, PDA
medium containing various concentrations of Z24 was prepared and poured
into sterilized petri dishes for testing. As a blank control, 0.25%
(v/v) DMSO was added to the PDA medium. A 5-mm fungal plug was
inoculated at the center of each plate, which was then incubated in the
dark at 26° ± 1°C (28°C for molds). Three replicates were performed for
each parallel experiment. The rate of mycelial growth inhibition was
calculated using the following formula
[MATH: Mycelial growth inhibition
ratio=[(dc−dt)(dc−5 mm)]×100%
:MATH]
where dc and dt represent the mean diameters in the control and
treatment groups, respectively.
Anti–B. cinerea efficacy in vivo
Protective effect evaluation (B. cinerea Pers. spores): Compound Z24
was tested for its protective effect on inoculated tomatoes using
previously reported methods ([232]33). The fruits were first
surface-sterilized for 5 min with 2% (v/v) sodium hypochlorite, then
washed with 75% ethanol, and rinsed with sterile distilled water. Each
fruit was punched (3 mm deep by 3 mm wide) at the equatorial section
and inoculated with 10 μl of B. cinerea Pers. conidial suspension
(1.5 × 10^6 spores/ml) after 24 hours of treatment with Z24 (0, 25, 50,
and 100 μg/ml), respectively. The fruits were cultured at 26° ± 1°C for
3 days. Five replicates were performed for each treatment group. For
the cucumber leaves, they were washed twice with sterile distilled
water and dried naturally. Then, Z24 (100 μg/ml) and pyrimethanil (20
ml per pot) were evenly sprayed onto the surface of each cucumber leaf,
and sterile water containing the same amount of DMSO was used as a
blank control. After 24 hours of spraying Z24, 10 μl of B. cinerea
Pers. conidial suspension (1.5 × 10^7 spores/ml) was inoculated. The
pots were incubated at 26° ± 1°C for 7 days. For each parallel
experiment, eight to nine biological replicates were performed.
Pyrimethanil was used as a positive control. The lesion area was
determined using ImageJ software.
Protective effect evaluation (B. cinerea Pers. mycelia): To assess
Z24’s ability to protect cucumber leaves from B. cinerea Pers.
mycelium, each pot of cucumber leaves was sprayed with 20 ml of various
Z24 concentrations, mixed with 0.1% (v/v) Tween 80. After 24 hours of
spraying, freshly cultured B. cinerea Pers. plugs were inoculated and
cultured for 48 and 72 hours under 12-hour light, 26° ± 1°C, 90%
relative humidity. The diameters of the lesions on cucumber leaves were
measured in millimeters.
Therapeutic effect evaluation (B. cinerea Pers. mycelia): After 24
hours of inoculation with B. cinerea Pers. plugs, each pot was sprayed
with 20 ml of Z24 solution, and other experimental conditions were the
same as described above.
Sclerotia formation assay
For the sclerotia formation inhibition assay ([233]68), PDA plates
treated with different concentrations of Z24 were inoculated with
5-mm S. sclerotiorum plugs. The plates were then covered with tin foil
and incubated at 26° ± 1°C for 15 days. Last, the wet weight of formed
sclerotium was measured.
Transmission electron microscopy
TEM observation was performed following the method described in our
previous study ([234]66). B. cinerea mycelia treated with Z24 (1 μg/ml)
were incubated at 25° ± 1°C for 4 days. The mycelia were then fixed
with 2.5% glutaraldehyde at 4°C for 4 hours, rinsed three times with
0.01 M phosphate-buffered saline (PBS) (pH 7.2), and dehydrated through
a graded ethanol series (25, 50, 70, 95, and 100% three times),
followed by dehydration in acetone. The dehydrated mycelia were
subsequently embedded in Epon 812 and polymerized in Spurr’s resin for
48 hours at 60°C. Last, the samples were stained with 2.5% lead citrate
and 2% uranyl acetate solutions before being examined under a TEM
(JEM-1010 TEM; NEC, Japan).
Fluorescence staining
Intracellular ROS level was determined using the fluorescent probe
DCFH-DA (Solarbio, Beijing, China) according to the manufacturer’s
instructions. B. cinerea Pers. spores were treated with Z24 (20 μg/ml)
for 24 hours, collected by centrifugation at 4°C, and washed three
times with 0.01 M PBS buffer (pH 7.2). After centrifuge at 6000 rpm for
30 min at 4°C, the supernatant was discarded. The spores were then
incubated in a 10 μM solution of DCFH-DA at 37°C for 30 min in the
dark. The dye solution was removed, and the spores were resuspended in
PBS buffer. The spore suspension was photographed and examined under a
fluorescence microscope (Zeiss Axioskop 40, Germany). For Hoechst 33342
and PI staining, B. cinerea Pers. mycelium treated with Z24 (0.5 μg/ml)
was incubated with 5 μl of Hoechst 33342 (1 mg/ml) and 5 μl of PI (5
mg/ml) for 15 min at 37°C. After thoroughly removing the stains, the
mycelium was observed and imaged under a confocal laser microscope.
DARTS (SDS-PAGE analysis)
Total protein was extracted using a Fungal Protein Extraction Kit
(Kanglang, Shanghai, China), and the protein was quantified to 5 mg/ml.
Cell lysates were aliquoted into equivalent volumes containing 500 μg
of protein and incubated for 60 min at 25°C with or without Z24.
Pronase E from Streptomyces griseus (Solarbio, Beijing, China) was
added to all samples at a pronase E:substrate mass ratio of 1:500 and
incubated at 37°C for 30 min. To identify differential protein bands,
SDS–polyacrylamide gel electrophoresis (PAGE) was performed, followed
by Coomassie bright blue and silver staining.
Mass spectrometry analysis
Gel bands were excised and prepared for mass spectrometry analysis
using trypsin digestion, as described in Supplementary Text. Mass
spectrometry was performed using Thermo’s Q Exactive Plus liquid
chromatography–mass spectrometry system. The mass spectrometry data
generated by the Q Exactive Plus were analyzed using MaxQuant
(V1.6.2.10) and the MaxLFQ database search algorithm. The Proteome
Reference Database of standard strain B. cinerea B05.10 was used for
the retrieval.
Molecular modeling analysis
The 3D structure of Bcthi4 was predicted using the AphaFold3 server
([235]https://golgi.sandbox.google.com/) ([236]69). The SDF format of
the two-dimensional (2D) structure of Z24 (ligand) was obtained from
the PubChem database ([237]https://pubchem.ncbi.nlm.nih.gov/). The 3D
structure of the receptor was processed by dehydrogenating and
dewetting using AutoDock tools. Molecular docking analysis was
performed using AutoDock vina, and docking interactions between the
receptor and Z24 were visualized using PyMOL software.
Rabbit polyclonal antibody preparation
Primers were designed on the basis of the B. cinerea B05.10 Bcthi4 gene
sequence (Gene ID: 5430120) from GenBank. The recombinant plasmid
Bcthi4-pET-B2M was constructed by amplifying the Bcthi4 gene via PCR
and inserting it into the pET-B2M vector. The Bcthi4-pET-B2M plasmid
was then transferred into Escherichia coli BL21 (DE3) cells, and
high-purity recombinant Bcthi4 protein was obtained after
isopropyl-β-d-thiogalactopyranoside (IPTG)–induced culture. Following
animal immunization, indirect enzyme-linked immunosorbent assay (ELISA)
and Western blotting assays confirmed that the rabbit polyclonal
antibody against the Bcthi4 gene exhibited high sensitivity and
specificity, making it suitable for further Western blot applications.
The specific binding of Z24 to Bcthi4 was subsequently detected by
Western blot analysis.
DARTS assay and CETSA (Western blot)
For the DARTS assay, 100 μl of lysates (5 mg/ml) from B. cinerea strain
were incubated with DMSO or Z24 (100, 250, and 500 μg/ml). Pronase E
was added to all samples at a pronase E:substrate mass ratio of 1:500,
and the mixture was incubated at 37°C for 30 min. The reaction was
terminated by adding 5 μl of protease inhibitor and incubating on ice
for 5 min. For the CETSA, 100 μl of lysates (5 mg/ml) from B. cinerea
strain were incubated with DMSO or Z24 (500 μg/ml) at temperatures
ranging from 50° to 80°C for 5 min. The samples were then centrifuged
at 10,000g for 15 min at 4°C to separate the supernatant and pellet.
The supernatant (32 μl) was mixed with 8 μl of 5× loading buffer and
heated in a metal bath for 10 min. The samples were then separated on a
12% SDS-PAGE gel for immunoblotting analysis of Bcthi4. After exposure,
the grayscale values of the Western blot bands were analyzed using
ImageJ software.
Expression and purification of the Bcthi4 protein
The pET-SUMO plasmids expressing His-tagged Bcthi4 were transformed
into E. coli BL21 Star (DE3) cells. Protein expression was induced with
0.5 mM IPTG, and the culture was incubated at 18°C for 18 hours with
shaking at 200 rpm. After incubation, the bacterial cells were lysed by
sonication, and the collected supernatant was loaded onto a
Ni–nitrilotriacetic acid Superflow affinity column. The His-tagged
Bcthi4 fusion protein was then eluted with 300 mM imidazole. The eluted
protein was analyzed by SDS-PAGE. The primers used for protein
expression are listed in table S3.
SPR analysis
Binding between Z24 and Bcthi4 was assessed using SPR with a Biacore
T200 instrument (Cytiva, USA). Z24 was prepared at concentrations of 0,
6.25, 12.5, 25, 50, and 100 μM in running buffer (0.05% polysorbate 20
and 5% DMSO). Bcthi4 was immobilized as the ligand on a CM5 sensor chip
(Cytiva, USA), and Z24 was used as the analyte.
Thiamin thiazole synthase activity
A 5-mm plug from the edge of 3-day-old colonies (B. cinerea Pers. and
B. cinerea B05.10) was inoculated into liquid PDA medium supplemented
with Z24 (0.05 and 0.10 μg/ml) and then incubated at 26° ± 1°C and 170
rpm for 3 days under darkness/light conditions. As a blank control,
0.25% (v/v) DMSO was used. The activity of Bcthi4 was measured using a
thiazole synthase (Thi4) kit (Shanghai XinYu Bio-Technology Co. Ltd.)
according to the manufacturer’s protocol.
RNA-seq and AS analysis
Total RNA from B. cinerea was extracted using the TRIzol method,
following the manufacturer’s protocol. The RNA samples were then sent
to Wuhan Ruixing Biotechnology Co. Ltd. (Wuhan, China) for RNA-seq
analysis. Differential expression was defined as a fold change ≥ 2
or ≤ 0.5 and a P value (probability value) < 0.05. HISAT2 software was
used to evaluate the splice junctions of each sample. AS events were
considered differential if the P value ≤ 0.05 compared to the DMSO
group. The signaling pathways were analyzed using the KEGG
([238]https://www.kegg.jp/).
Reverse transcription quantitative polymerase chain reaction
Total RNA was extracted with a Fungal RNA Kit (Feiyang, Guangzhou,
China). RNA reverse transcription was performed following the
instructions of the FastKing gDNA Dispelling RT SupperMix Reverse
Transcription Kit (Tiangen, Beijing, China), with TransScript One-Step
gDNA Removal. RT-qPCR was then performed according to the instructions
of the SuperReal PreMix Plus (SYBR Green) Fluorescence Quantitation Kit
(Tiangen, Beijing, China) in an Eco 48 real-time fluorescent
quantitative PCR system. Glyceraldehyde-3-phosphate dehydrogenase was
used as the internal control. Primer sequences specific to each gene
are listed in table S4. Relative transcription abundance was calculated
using the 2^−ΔΔCT method.
Thiamine and TPP content assay
A 5-mm plug from the edge of 3-day-old colonies (B. cinerea Pers. and
B. cinerea B05.10) was inoculated into liquid PDA medium supplemented
with Z24 (0, 0.05, 0.10, and 0.20 μg/ml) and incubated at 26° ± 1°C
with shaking at 170 rpm. A 0.25% (v/v) DMSO solution was used as a
blank control. The same amount of mycelium was harvested for metabolite
content determination. The contents of thiamine and TPP were measured
using a VB1 ELISA kit (Shanghai Enzyme Linked Biotechnology Co. Ltd.)
and a Fungi TPP ELISA kit (Shanghai Enzyme Linked Biotechnology Co.
Ltd.) following the manufacturer’s protocol.
Metabolome analysis
Metabolomics analysis was performed by Shenzhen BGI Co. Ltd. with three
biological replicates for each treatment group. Briefly, a 5-mm plug
from the edge of 3-day-old colonies of B. cinerea Pers. was inoculated
into liquid PDA medium treated with Z24 (0 and 0.1 μg/ml) and incubated
at 26 ± 1°C with shaking at 170 rpm for 3 days in darkness/light. A
0.25% (v/v) DMSO solution was used as a blank control. Waters 2D Ultra
Performance Liquid Chromatography (UPLC; Waters, USA) in tandem with a
Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific,
USA) was used for the separation and detection of metabolites.
Multivariate statistical analysis [PCA and Partial Least Squares
Discriminant Analysis (PLS-DA)] and univariate analysis (fold
change/Student’s t test) were combined to screen for differential
metabolites between groups.
In vitro cytotoxicity assay (MTT assay)
The cytotoxicity of Z1 and Z24 to HIECs, HL-7702 cells, and GES-1 cells
was evaluated in vitro. HIECs, HL-7702 cells, and GES-1 cells were
cultured at 37°C in RPMI medium (HyClone, USA) and Dulbecco’s modified
Eagle’s medium high-glucose medium (HyClone, USA) containing 1%
penicillin/streptomycin and 10% fetal bovine serum with 5% CO[2]. The
cell inhibition rate was determined colorimetrically using MTT. In
brief, 100 μl of exponentially growing HIECs, HL-7702 cells, and GES-1
cells were plated at a density of 6000 cells per well in a 96-well
plate and cultured for 24 hours in an incubator at 37°C with 5% CO[2].
The cells were then treated with various concentrations of Z1 and Z24
(2.5, 5, 10, and 25 μM) in complete medium for 48 hours, with
pyrimethanil as a positive control. After treatment, each well received
10 μl of MTT solution (5 mg/ml) and was incubated in the dark for 4
hours. A microplate reader was used to measure the absorbance at 490
nm. Four replicate wells were used for each condition.
In vivo acute toxicity test
The acute oral toxicity test was conducted using six nulliparous,
nonpregnant female rats in compliance with OECD Guideline 423
([239]70). Given that females are more sensitive than males in toxicity
tests, the use of female rats was chosen to provide a more conservative
estimate of the toxic effects ([240]71). Z24, DMSO, and more than 95%
pure corn oil were used to prepare the Z24 suspension. First, Z24
suspension was administered to three randomly selected female rats
(n = 3) at a dose of 2000 mg/kg. The animals were monitored for 2
weeks, and any instances of death were recorded. After the observation
period, blood and tissues from the heart, liver, spleen, lung, and
kidney were collected following a fasting period. These samples were
then used for histological assessment (hematoxylin and eosin staining).
The liver and kidney functions were evaluated by measuring GOT, GPT,
ALP, and urea nitrogen levels.
Hemolysis assay
The hemolysis assay was performed following the National Cancer
Institute procedure ([241]72). Briefly, erythrocytes were isolated from
sheep blood by centrifugation at 1500 rpm for 15 min. The collected
erythrocytes were diluted in a 0.9% sodium chloride solution to achieve
a final concentration of 4%. The compound Z24 to be tested was added to
the erythrocyte suspension, with PBS and Triton X-100 used as the
negative and positive controls, respectively. The mixture was incubated
at 37°C for 1 hour at 60 rpm in a rotatory shaker, followed by
centrifugation at 1000g for 3 min. Then, 100 μl of the supernatant was
transferred to a 96-well plate, and a multifunctional microplate reader
was used to measure the absorbance at 450 nm
[MATH: Hemolysis ratio (%)=[(As−An)(Ap−An)]×100%
:MATH]
where
[MATH: As :MATH]
,
[MATH: Ap :MATH]
, and
[MATH: An :MATH]
represent the absorbance of the compound Z24, Triton X-100, and PBS,
respectively.
Phytotoxicity assay
The phytotoxicity of Z24 was assessed using an in vitro seed
germination experiment on Murashige and Skoog agar plates, as
previously described ([242]66). R. raphanistrum seeds were sterilized
by immersing them in a 1% sodium hypochlorite solution for 20 min after
soaking overnight in sterile distilled water. After three rinses with
sterilized water, the seeds were placed on Murashige and Skoog agar
plates with Z24 concentrations ranging from 10 to 200 μg/ml. Rhizome
length and seed germination rate were assessed after 3 days of
incubation at 26° ± 1°C.
Statistical analysis
SPSS 26.0 was used to statistically analyze the experimental data,
which are presented as means ± SD. Data were further analyzed using
GraphPad Prism 9.5 software (San Diego, CA, USA). One-way analysis of
variance (ANOVA) followed by Duncan’s multi range test was performed to
compare the statistical differences between groups. Significant
differences were considered when P < 0.05.
Acknowledgments