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Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum

Journal of Nanobiotechnology volume 22, Article number: 494 (2024) Cite this article

Abstract

Background

Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies.

Results

Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1.

Conclusions

Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.

Graphical Abstract

Introduction

Fungal diseases are a major menace to crop yield, responsible for annual losses ranging among 10-23%, along with an additional 10-20% post-harvest loss [1, 2]. These challenges are expected to intensify with the evolving climate [3]. Among the myriad of pathogens, the ascomycete Sclerotinia sclerotiorum (Lib.) de Bary is particularly notorious for its destructive and widespread impact. It can infect over 600 plant species, encompassing crucial crops like rapeseed and soybean [4]. Its unique dual feeding lifestyle, exhibiting characteristics of both biotroph and necrotroph, makes it a valuable subject for host-pathogen interaction studies [5]. Due to the considerable impact of S. sclerotiorum on crops yield and quality, it is imperative to conduct extensive research to identify the function of the genes involved [6]. Elucidating the roles of genes involved in pathogenicity and fungal development is fundamental for unearthing potential targets in disease management. This understanding relies heavily on the availability of an efficient genetic transformation system.

The primary techniques for genetic transformation in filamentous fungi include Agrobacterium tumefaciens (AT)- and polyethylene glycol (PEG)-mediated protoplast transformation, along with electroporation [7]. In the case of S. sclerotiorum, protoplast transformations mediated by PEG or AT have been successfully applied for gene transfer, gene knock-out, and insertional mutagenesis [8, 9]. Despite their utility, these methods have their drawbacks, including relatively low transformation rate, the need for precise osmotic balance, and the delicate and laborious handling and regeneration of protoplasts involved in the gene transfer process [7]. Biolistic transformation presents an alternative by delivering exogenous DNA into fungal spores or hyphae via microscopic particles [7]. However, the efficacy of this approach hinges on several critical factors, including the specificity of particle delivery apparatus, the type and density of target cells, the osmotic environment, and the methodology employed for particle coating [10, 11]. Shock wave-mediated transformation has emerged as an effective technique for some filamentous fungi [12]. It transiently modulates the permeability of fungal cell membranes, facilitating the entry of DNA with a high degree of reproducibility [13]. Nonetheless, the application of this method is impeded by the high costs of the shock wave sources and the specialized equipment required [13].

Nanomaterials have attracted significant interest for their role in delivering biological molecules into animal cells, leveraging their small size, large surface area, compatibility with biological systems, degradability, and low toxicity [14]. However, their use in plant cells faces the challenge posed by the robust cell wall, which acts as a natural barrier to nanoparticle penetration. Despite this, recent research has managed to overcome this hurdle, achieving successful delivery of DNA, RNA, and proteins into plant cells [15,16,17,18,19,20,21,22,23,24,25,26,27,28]. All the successful examples refuted the notion of the plant cell wall as an impenetrable barrier. The size, shape, compactness, stiffness of the nanomaterials contributes the ability to penetrate cell walls and internalize into plant cells [29, 30]. Similar to plant cells, the fungal pathogens’ cells are enveloped by a resilient cell wall. However, only two studies have reported the application of nanomaterials in successfully transferring exogenous DNA into fungal cell, such as yeast and Candida albicans [31, 32].

In our previous research, a kind of nanoparticle with fusiform morphology (FDAP-NP), synthesized with 2,6-diaminopyridine and Fe3+, possesses good permeability in solid tumors and can load drug doxorubicin to enhance thermal therapy and chemotherapy to tumor [33]. Base on its good permeability, we hypothesized that it may easily penetrate fungal cell. Therefore, this kind of fusiform nanoparticle (Fusiform-NP, FM) was resynthesized in this research with some modification and chosen as the carrier for the integration of exogenous DNA into the genome of S. sclerotiorum through either protoplast or mycelium transformation. We loaded a GFP expression construct onto the FM and successfully obtained the strains with green fluorescence via FM-mediated protoplast or mycelium transformation. Meanwhile, in order to verify the feasibility of FM-mediated mycelium transformation in gene function analysis of S. sclerotiorum, we transferred a gene-silencing vector to silence the oxaloacetate acetylhydrolase gene (Ss-oah1) [34]. The study highlights a much more convenient approach to the mycelium transformation and confirms the FM is non-toxic to S. sclerotiorum.

Materials and methods

Pathogenicity analysis of S. sclerotiorum

The S. sclerotiorum wild type strain 1980 (WT) and the transformants were routinely stored and cultured on potato dextrose agar media (PDA, 20% potato, 2% dextrose and 1.5% agar) (Macklin, Shanghai, China) at 22 °C [35]. Brassica napus (rapeseed) and Glycine max (soybean) were cultivated in the field according to the routine cultivation and management measures in Beibei, Chongqing, China. Nicotiana benthamiana (tobacco) was grown in the autoclaved medium (Pindstrup, Fabriksvej, Danmark) in climate chamber at the cycle of 28 ± 2 °C with 16 h light and 16 ± 2 °C with 8 h dark, under 70% relative humidity. To assess the pathogenicity of the transformed strains of S. sclerotiorum, detached leaves of rapeseed, soybean, and tobacco were inoculated with mycelia agar plugs (6 mm in diameter) at 22 ± 1 °C under approximate 95% relative humidity. The lesion size was calculated at 48 h post inoculation (hpi). Each experiment was performed in triplicate.

Preparation of FM-DNA complexes

The pEF1-GFP construct was generated by the insertion of the codon-optimized gene encoding Green Fluorescent Protein (GFP) into the pUC57 cloning vector [36], driven by the promoter of elongation factor 1 alpha (EF1α, SS1G_05520). The fragment of S. sclerotiorum Ss-oah1 was amplified using the primer pair Sioah-F and Sioah-R (Supplementary Table S1). The amplicons were then inserted into the corresponding multiple cloning sites of vector pCIT, constructing the gene-silencing vector pSioah1, following the protocol previously outlined [37]. The FM was synthesized according to the previous study with some modification [33]. Briefly, 24.0 mmol of FeCl3·6H2O was completely dissolved in 120 mL of double-distilled water followed by oxygen-removal with argon for 1 h, and then was mixed with 6.0 mmol of DAP (2,6-diaminopyrimidine) to polymerize FMs at 50 °C for 24 h. The synthesized product subsequently underwent purification via dialysis using a 3,500 Da molecular weight cutoff dialysis bag submerged in sterile distilled water at ambient temperature for 24 h. The purified FMs were thereafter preserved at 4 °C for the following use.

To confirm the indeed penetration of the nanoparticles through the cell wall of S. sclerotiorum, the FM was labelled with indocyanine Green (ICG), designated as FMICG (FMI). ICG emits red fluorescence at the wavelength of 785 nm, which can be detected using confocal microscopy (LSM800, ZEISS, Germany). For the preparation of FM-DNA or FMI-DNA complexes, plasmid was incubated with FMs or FMIs in a sterile centrifuge tube with a mass ratio of 1:400 and shaken overnight on an oscillatory table (OS-200, Hangzhou, China) at 50 rpm at room temperature. The mixture was then centrifuged (Micro-17, Thermo, Massachusetts, USA) at 5,000 rpm for 10 min to remove the unbound DNA. Diluted sample (10 µL) (FM or FM-DNA) was evenly dispersed on a hydrophilicity-treated copper grid and dried in a vacuum desiccator (Sigma-Aldrich (Shanghai), Shanghai, China) at room temperature. This process was repeated 5 to 6 times to amass a sufficient sample for morphology characterization using transmission electron microscope (TEM, HT7800, HITACHI, Japan) (HC-1, Accelerating voltage = 80,000; Magnification = 70,000; Spot number = 5; Emission = 10). The particle size distribution and charge on the surface were tested through dynamic light scattering (DLS) and zeta potential analysis, respectively [38]. The stability of FM-DNA complexes was periodically evaluated with DLS and polymer dispersity index (PDI), which were conducted bi-daily throughout the storage period [38].

FM-mediated transformation in S. sclerotiorum

The protoplast of S. sclerotiorum was prepared following the protocol described by Rollins [39]. Briefly, the harvested mycelia of S. sclerotiorum cultured on the PDA medium (90 mm in diameter) after 36 h incubation were treated with a solution comprising snailase (1 mg·mL− 1) (Solarbio, Beijing, China) and megalyase (10 mg·mL− 1) (Shenqi Biotechnology, Shanghai, China) at 30 °C for 4 h to digest the cell wall. The protoplasts were collected through filtration and washed with 0.8 M MgSO4 to remove any residual enzymes, followed by resuspension in a STC solution (QualityYard, Beijing, China). Two-hundred microliters of FM-DNA complexes were incubated with 100 µL S. sclerotiorum protoplasts (approximately 103 protoplasts·µL− 1) in a sterile centrifuge tube at room temperature for 2 h, followed by centrifugation at 4,000 rpm for 3 min to collect the transformed-protoplasts. The pelleted transformed-protoplasts were then washed twice with 0.01 M phosphate buffer solution (PBS, 8.00 g NaCl, 0.20 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1 L distilled water, pH = 7.40). Subsequently, the transformed protoplasts were resuspended on cell wall recovery medium (47.92 g sucrose, 0.10 g yeast extract, 0.10 g lactium, 2.40 g agar in 1 L distilled water). After cell wall reconstruction, the cells were transferred to the selective medium (PDA supplemented with 100 mg·L− 1 hygromycin (Sigma-Aldrich (Shanghai), Shanghai, China)) for the screening and proliferation of transformants. Parallel to this, the entire S. sclerotiorum mycelia cultured on the PDA medium (90 mm in diameter) after 2 days incubation, were directly incubated with 4 mL FM-DNA complexes (FM-DNA:0.01 M PBS = 1:3) at room temperature for 3 h. After incubation, the extra FM-DNA complexes were rinsed out with 0.01 M PBS for 3–5 times and then the mycelia with fluorescence signal were selected and transferred onto the selective medium. The process for selecting and cultivating transformants was consistent with the aforementioned methods.

The transformants obtained through protoplast or mycelium transformation were hyphal tipped and transferred onto the selective medium and screened and cultured up to the fourth generation (T4). GFP expression within the hyphae was monitored using confocal microscopy across T1, T2, T3, and T4 generations. Mycelial radial growth was quantified at 12, 24, and 36 hpi on the PDA medium. Mycelial morphology was examined at 3, 5, and 15 days post inoculation (dpi). The average number and dry weight of sclerotia per plate was counted at 15 dpi. The experiments were repeated three times.

Validation of the insertion of exogenous DNA into the genome of S. sclerotiorum

Purified genomic DNA was extracted from the mycelia of a putative transformant from T1 generation with cetyltrimethyl ammonium bromide (CTAB) following the standard procedure [40]. Subsequent polymerase chain reaction (PCR) analysis employing specific primers GFT-F/GFT-R (Supplementary Table S1) was performed to validate positive transformants. Furthermore, the precise genomic integration site of the exogenous DNA within S. sclerotiorum genome was determined through genome re-sequencing using the Illumina HiSeq platform employing the Sequencing by Synthesis (SBS) (Beijing Biomics Biotech Co. Ltd.). Raw data quality control assessments were performed using FastQC, and high-quality sequences were isolated following the removal of adaptor sequences and low-quality reads (including reads with a proportion of N greater than 10%; reads with a mass value of Q (Quality Score) ≤ 10 with an alkali base accounting for more than 50% of the entire Read). The clean reads of the transformant were assembled and aligned to the reference genome of S. sclerotiorum 1980 (GenBank: GCA_000146945.2) using the Adaptive Insertion Mapping with Sequencing (AIM-Seq) method [41]. Finally, insertion sites of the sequence were conclusively verified by PCR with specific primers TL_F1/ TR_R2 (Supplementary Table S1). PCR reaction program comprised of 94 °C for 3 min (1 cycle), then 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s (35 cycles), followed by 72 °C for 3 min.

The expression level of GFP and Ss-oah1 in S. sclerotiorum transformants were quantified using the CFX96™ Real-Time PCR system (Bio-Rad, Hercules, CA, USA). Total RNA was extracted from the mycelium using TRIzol reagent (TianGen, Dalian, China) and reversely transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, California, USA) following the corresponding instructions. The quantitative real-time PCR (qRT-PCR) was performed in triplicate for each gene, using SsTubulin (SS1G_04652) encoding tubulin protein as internal standard. The corresponding program was set as follows: predenaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 58–60 °C for 1 min, extension at 72 °C for 20 s. This cycle was repeated for 40 times. The primer sequences used for qRT-PCR are listed in Supplementary Table S1. Preprocessing of the raw data was undertaken using CFX Manager™ v 3.0 (Bio-Rad, California, USA) and the relative expression of target genes was determined with the 2−ΔΔCT method [42]. All experiments were conducted in triplicate.

Generating Ss-oah1- silenced transformants via FM-mediated mycelium transformation

To investigate the efficacy of FM-mediated mycelium transformation in S. sclerotiorum, Ss-oah1 gene-silencing plasmid, pSioah1, was introduced into S. sclerotiorum via mycelium transformation. The Ss-oah1-silenced transformants were cultured on PDA media supplemented with 50 mg·L− 1 bromophenol (Macklin, Shanghai, China), which is utilized as an indicator monitoring the qualitative assessment of oxalic acid (OA) production, as the medium shifts to violet (at pH > 4.6) or yellow (at pH < 3.0), depending on the acid produced by S. sclerotiorum strains. Quantitative determination of OA production was subsequently performed using high performance liquid chromatography (HPLC) [43]. Specifically, three mycelium plugs (9 mm in diameter) of each transformant were inoculated into 50 mL of potato dextrose broth (PDB, 20% potato, 2% dextrose) (Macklin, Shanghai, China) and incubated at 22 °C with constant agitation at 150 rpm for 3 d. OA concentrations was designated as milligrams of OA per milliliter PDB solution. Three replicates were performed for each transformant.

Statistical analysis

The data from different biological treatments were subjected to one-way analysis of variance (ANOVA) and statistically analyzed using Prism 8 (GraphPad Software, Santiago, USA). The results were presented as the mean value ± standard deviation (SD). Significant difference was calculated using Student’s t-test (**P < 0.001).

Results

Characteristics of FM and FM-DNA complex

The FM-DNA complexes were assembled by loading plasmid DNA onto the surface of FM (Fig. 1). The characteristics of FM and FM-DNA complexes were investigated by TEM, DLS and zeta potential analysis. As shown in Fig. 2A, FMs and different FM-DNA complexes displayed a regular, elongated fusiform and uniform morphology. Specifically, the diameter of FM-GFP (FMG, average longitudinal length 70 nm and latitudinal length 30 nm) was marginally larger than those of FM (average longitudinal length 59 nm and latitudinal length 23 nm). DLS and PDI analyses indicated that the average hydrodynamic diameter of FM (average 24.9 nm) was less than that of different FM-DNA complexes (FMI, 46.7 nm; FMG, 41.2 nm; FMICG-GFP [FMIG], 46.5 nm) (Fig. 2B), which may be attributable to the addition of ICG or DNA to the surface of the nanoparticles. Similar PDI among the FM and the FM-DNA complexes suggested that the loading DNA or ICG did not significantly affect the disperity uniformity. As shown in Fig. 2C, zeta potential analysis showed positive values for different FM-DNA complexes (FMI, + 12.6 mV; FMG, + 23.0 mV; FMIG, + 8.9 mV), but these were lower than that of free nanomaterial particle (average about + 44 mV). This implies that the loading of negative DNA or ICG partially neutralized the positive charge on the surface of the nanomaterials. The stability of FMG was assessed by measuring DLS and PDI every two days in one-week storage at room temperature. The dynamics of DLS and PDI revealed no significant fluctuation in diameter and PDI throughout the 7-day period, suggesting robust binding between the DNA and FMs, which conferred substantial stability to the FM-DNA complexes (Fig. 2D).

Fig. 1
figure 1

Schematic illustration of FM-mediated transformation strategy in S. sclerotiorum

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Fig. 2
figure 2

Characteristics of FM and FM-DNA complexes. A: TEM images of FM, FMI, FMG and FMIG complexes. FM, free nanoparticle; FMI, ICG tagged-nanoparticle; FMG, nanoparticle loaded with the plasmid pEF1-GFP; FMIG, ICG tagged-nanoparticle loaded with the plasmid pEF1-GFP. Scale bars, 100 μm. B: Hydrodynamic diameter of FM and FM-DNA complexes. DLS, dynamic light scattering; PDI, polymer dispersity index. C: zeta potential of FM and FM-DNA complexes. Error bars represent SD from three replicates. D: Stability of FMG complexes. Error bars represent SD from three replicates

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Delivering exogenous DNA into S. sclerotiorum protoplasts via FM

The temporal and spatial distribution of FM and FMG complexes within S. sclerotiorum were tracked using confocal fluorescent microscopy at 488 nm wavelength. After 2 h incubation of FMI with S. sclerotiorum protoplasts, 75.0% of protoplasts exhibited red fluorescence, signifying the successful permeation of FMI into the cells (Fig. 3A). Simultaneously, transformation of pEF1-GFP plasmid into S. sclerotiorum protoplasts was attempted utilizing both FM and FMI as vectors. The results indicated that 75.6% of the protoplasts displayed green fluorescence following transformation with the FMG complexes, whereas 68.6% of the protoplasts exhibited dual fluorescence (green and red) after transformation with the FMIG complexes (Fig. 3A). However, S. sclerotiorum transformed solely with FM or the pEF1-GFP plasmid did not exhibit green fluorescence (Fig. 3A). Protoplasts transformed with FMG were cultured, yielding mycelial growth exhibiting green fluorescence (T1 generation) (Fig. 3B). Selective culture of intensely fluorescent mycelia continued over successive generations (T2, T3, and T4) via hyphal-tipping culture. The green fluorescence was constantly observed in T2, T3, and T4 generations upon re-culturing (Fig. 3B). Molecular validation of GFP integration within the S. sclerotiorum genome was conducted by PCR analysis, employing the genomic DNA of T1, T2, T3, and T4 transgenic S. sclerotiorum as templates. The presence of PCR amplification products matching the expected length of 250 base pairs was consistent with the observed fluorescence, affirming the success of genetic transformation (Fig. 3C). Furthermore, the GFP expression level in T1, T2, T3, and T4 transgenic S. sclerotiorum validated by qRT-PCR further confirmed the internalization of the GFP in S. sclerotiorum genome (Fig. 3D). Collectively, these results endorse the efficacy of FM in mediating the transfer and stable integration of exogenous DNA into S. sclerotiorum via protoplast incubation.

Fig. 3
figure 3

DNA delivery into protoplasts with FM transformation. A: FM internalization into S. sclerotiorum protoplasts and tracking of fluorescence in protoplasts via confocal microscopy. Scale bars, 10 μm. B: Tracking of green fluorescence in the regenerated S. sclerotiorum hyphae and the hyphae of re-cultured generations. S. sclerotiorum protoplasts were re-walled to generate T1 generation, and subsequently re-cultured to generate T2, T3 and T4 generations. WT, wild type strain. Scale bars, 100 μm. C: Amplifying GFP from transgenic S. sclerotiorum. M, DNA ladder; T1-T4, T1 to T4 generation of transformant. D: qRT-PCR analysis of transgenic S. sclerotiorum. Sstubulin was used as the internal standard. Error bars represent SD from three replicates

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Delivering exogenous DNA into S. sclerotiorum mycelia by FM

The successful transformation in protoplast of S. sclerotiorum mediated by FM does not offset the relative pitfalls including time-consuming, and complicated manipulation. To streamline this procedure, we explored direct incubation methods. S. sclerotiorum mycelia (cultivated for 2 d) were directly incubated with the FMI for 3 h, after which the fluorescence in mycelia was assessed. As anticipated, red fluorescence was detected among transformed S. sclerotiorum mycelia (Fig. 4A), indicating the FMI could successfully penetrate the cell wall and the plasmalemma of S. sclerotiorum mycelia without any external assistance. Likewise, green fluorescence was observed in the S. sclerotiorum mycelia incubated with FMG and FMIG (Fig. 4A). Additionally, we observed that the exogenous GFP was stably expressed across T1-T4 generations derived from the FMG-mediated mycelium transformant via hyphal-tipping culture (Fig. 4B, C).

Genome re-sequencing was employed in the strain from the T1 generation to ascertain the precise locational integration of the pEF1-GFP construct within the S. sclerotiorum genome. A total of 2,652,632,200 bases comprising 8,838,774 pair-end clean reads were obtained (SRA: PRJNA1126013). The GC content of clean reads was approximately 49.56%, with 92.01% clean reads with Q value ≥ to 30. Junction reads, which include sequences from both the S. sclerotiorum reference genome and the inserted exogenous DNA sequence, were utilized for clustering. The results showed that two fragments of pEF1-GFP construct were integrated within the genome of S. sclerotiorum (Fig. 4D). Specifically, the insert fragment 1 (6557 bp) covering the entire expression cassette of GFP and hygromycin resistance gene (hygromycinphosphtolransferase, hph) was inserted into Chromosome NW_001820820.1 (977055.977062) with six-base pairs’ deletion in S. sclerotiorum genome (Fig. 4D). The insert fragment 2 (2475 bp) covering part of the expression cassette of GFP and hph was inserted into Chromosome NW_001820835.1 (681,961) (Fig. 4D). The green fluorescence and the GFP expression were detected among four generations, indicating that the insert fragment 1 was functional. Subsequently, we further validated the insertion site 1 via PCR with two pairs of primers specifically designed to amplify the genomic regions flanking the integration locus. Sequencing analyses confirmed the stable integration of pEF1-GFP in the genomes of T2, T3, and T4 generations (Supplementary Figs. 1, 2). qRT-PCR analysis was further performed among the WT, T1, T2, T3, and T4 generations, detecting high expression of GFP gene in transgenic S. sclerotiorum strains (Fig. 4E). Collectively, these findings underscore the capability of FM to directly and efficaciously mediate the transformation and stable genomic integration of exogenous genes in S. sclerotiorum via mycelia transformation.

To evaluate the potential cytotoxicity of nanomaterials on S. sclerotiorum, the physiological characteristics were compared between WT and T4 generation. The mycelia of the transgenic strain showed no significant difference from that of the WT strain at 3, 5, and 15 dai (Fig. 5A). The colony area (cm2) of the transgenic strains was measured at 12 h, 24 h and 36 h. No significant difference was detected in hyphal growth rate between the transgenic S. sclerotiorum strains and WT (P > 0.05) (Fig. 5B). At 25 dai, no significant variance was detected in the average number of sclerotia (P = 0.4676) and sclerotia dry weight per plate (P = 0.4414) between the transgenic S. sclerotiorum and the WT strain (Fig. 5C). Moreover, there were no significant differences in the size of the lesions on leaves of rapeseed (P = 0.1875) inoculated with the transgenic S. sclerotiorum and WT strain, as well as that in leaves of soybean (P = 0.6887) and tobacco (P = 0.4925) (Fig. 5D, E). These results indicated that the FM exerted no discernible impact on the hyphal growth, sclerotia development, and pathogenicity of S. sclerotiorum.

Fig. 4
figure 4

DNA delivery into S. sclerotiorum mycelium with FM transformation platform. A: FM internalization into S. sclerotiorum mycelium and tracking of fluorescence in mycelia via confocal microscopy. Scale bars, 100 μm. B: Tracking of green fluorescence in the re-cultured S. sclerotiorum generations. WT, wild type strain. T1-T4, transgenic hyphae. Scale bars, 100 μm. C: Amplifying of GFP from transgenic S. sclerotiorum. bp, base pair; M, DNA ladder. D: Genome re-sequencing revealed the integration sites in the transgenic S. sclerotiorum genome. hph: Hygromycinphosphtolransferase. E: qRT-PCR analysis of transgenic S. sclerotiorum. Sstubulin was used as the internal standard. Error bars represent SD from three replicates

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Fig. 5
figure 5

Characteristics of the T4 transgenic S. sclerotiorum. A: Colony morphology and sclerotia formation of wild type and T4 transgenic S. sclerotiorum on PDA medium. WT, wild type strain. B: Colony area (cm2) of wild type and T4 transgenic S. sclerotiorum. Error bars represent SD from three replicates. ‘ns’ means no significant difference. C: Number and mass of sclerotia per petri plate with 90 mm diameter. Error bars represent SD from three replicates. D: Pathogenicity of wild type and transgenic S. sclerotiorum on leaves of rapeseed, soybean, and tobacco. Lesion size was recorded at 48 hpi. Scale bars, 30 mm. E: Evaluation of lesion size (cm2) produced by transgenic S. sclerotiorum and WT strains on leaves of rapeseed, soybean and tobacco. Error bars represent SD from three replicates. ‘ns’ means no significant difference between WT and T4 transgenic strains

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OA has been demonstrated experimentally to be a critical virulence factor for S. sclerotiorum colonization and Ss-oah1 is pivotal for OA accumulation [34]. Consequently, S. sclerotiorum Ss-oah1 was then selected as a target for gene silence analysis to ascertain the viability of FM-mediated-mycelium transformation system. The FM-Sioah1, by loading the Ss-oah1 gene RNAi construct (pSioah1) onto the surface of FM (Supplementary Fig. 3), was directly transformed with S. sclerotiorum mycelium and re-cultured on hygromycin-PDA medium up to T4 generation. Five hygromycin-resistant transformants were obtained and three genetically modified strains (designated as Sioah1-4, Sioah1-6, and Sioah1-11) with remarkable reduction in Ss-oah1 expression were selected based on the qRT-PCR analysis (Fig. 6A). Compared to WT, the expression of Ss-oah1 was reduced to 0.27 in Sioah1-4, 0.31 in Sioah1-6, and 0.41 in Sioah1-11, respectively.

Fig. 6
figure 6

Ss-oah1 silencing mediated by FM via mycelium transformation. A: Relative expression levels of Ss-oah1 in different Ss-oah1-silenced isolates and wild type strain were determined by qRT-PCR. Sstubulin was used as the internal standard. WT, wild type strain. B: Pathogenicity of Ss-oah1-silenced strains (Sioah1-4, Sioah1-6, and Sioah1-11) on rapeseed leaves. Lesions were recorded at 48 hpi. Scale bars, 30 mm. C: Evaluation of lesion size (cm2) produced by Ss-oah1-silenced strains and wild type strains on leaves of rapeseed. D: Medium acidification is indicated by color change from violet to yellow. E: OA accumulation in PDB. Error bars in (A), (C) and (E) represent SD from three replicates. ** in (C) and (E) means significant difference between WT and Ss-oah1 gene-silenced strains

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The pathogenicity assay revealed a pronounced reduction in virulence among the Ss-oah1-silenced strains relative to WT (Fig. 6B, C; P < 0.0001). The WT strain produced an average lesion size of approximate 6.04 cm2 on the rapeseed leaves at 48 hpi, whereas the Ss-oah1-silenced strains Sioah1-4, Sioah1-6, and Sioah1-11 produced significantly smaller lesion sizes of 2.24 cm², 2.43 cm², and 2.32 cm², respectively (Fig. 6C). This represented a decrease in lesion size of roughly 63% for the silenced strains compared to WT. Further assessments were carried out to evaluate the OA-producing capacity of the Ss-oah1-silenced strains qualitatively and quantitatively. On PDA medium supplemented with bromophenol blue, a colorimetric pH indicator, the WT strain induced color shift from violet to yellow at 2 dai, indicative of OA production. However, the medium inoculated with the transgenic strains exhibited slightly yellow (Fig. 6D), suggesting a significantly diminished production of OA. Quantitative evaluation of OA-producing reduction was confirmed by HPLC analysis, significant decreased OA content among transgenic strains compared to the WT was detected (P < 0.001). Specifically, the WT strain accumulated approximate 559.72 µg OA in 1 mL PDB solution, whereas Sioah1-4, Sioah1-6, and Sioah1-11 strains produced markedly lower concentrations of OA, measuring at 226.82 µg·mL− 1, 196.47 µg·mL− 1, and 200.38 µg·mL− 1, respectively (Fig. 6E). These findings indicate that the silencing of Ss-oah1 effectively reduced the OA-production capacity of the Ss-oah1-silencied strains, coincided with the Ss-oah1 expression level (Fig. 6A). These evidences further reinforce the utility of the FM-mediated genetic transformation system for the elucidation of gene function in S. sclerotiorum.

Discussion

Genetic engineering in S. sclerotiorum plays a pivotal role in rapidly and directly identifying the pathogenesis genes, thereby facilitating the development of targets for effective disease management. Nonetheless, the primary challenges in genetic engineering of filamentous fungi include low transformation efficiency and intricate and labor-intensive procedural requirements [7]. The pursuit of an efficient genetic transformation system remains an urgent aim and a fundamental prerequisite. This study presents a novel approach employing FM that efficiently deliver exogenous DNAs into the S. sclerotiorum genome via direct incubation with the mycelium. This method circumvents the traditional requirement for protoplast preparation and the subsequent recovery process, presenting an innovative strategy for genetic transformation in S. sclerotiorum and potentially other filamentous fungi. The FM emerges as a promising tool to surmount the existing obstacles in genetic engineering, thereby facilitating more efficient gene identification processes.

The application of nanomaterials in genetic transformation in plants demonstrate the remarkable advantages of nanomaterials, such as reduced recipient-dependence, high transformation efficiency, excellent biocompatibility, and effective protection of exogenous nucleic acids from nuclease degradation [18, 20, 21]. In current study, we pioneered the transformation of GFP into S. sclerotiorum cells using FM. While exhibiting high efficiency, AT- and PEG-mediated transformation still required a labor-intensive protoplast preparation and recovery procedure [8, 44]. In Aspergillus niger, the direct use of mycelia or conidia as recipients for gene transformation has eliminates the need for extensive recovery and screening steps [45]. Leveraging this reference, we successfully employ FM to transform GFP and Ss-oah1-silencing plasmid into S. sclerotiorum mycelia, as confirmed by qRT-PCR, re-genome sequencing, and PCR confirmation. Consequently, this FM-mediated mycelium transformation platform emerges as a simpler, time-saving, and promising strategy for genetic transformation in S. sclerotiorum presenting potential advancements in genetic manipulation for more fungi.

The interaction between the nanomaterials and the exogenous nucleic acids is governed by electrostatic attraction, which not only facilitates transformation but also shields the nuclei acids from nuclease degradation [46, 47]. In the present research, the cationic charge on the surface of the FM was partially neutralized by the anionic charge of exogenous DNA, thereby promoting the tight absorbability of the plasmid DNA to form FM-DNA complexes. We speculated that this may enable the internalization of the DNA into the fungal cells and protect the exogenous DNA from nucleolytic activity within the S. sclerotiorum cells. Successful penetration through the fungal cell walls and delivery of FM-DNA complexes into the nucleus is another critical aspect in successful application of this FM-mediated transformation platform. Here, the unique morphology of the FM-DNA complexes likely plays a role in facilitating this process. Future research should aim at elucidating potential structure alterations or pore-forming mechanisms on the cell wall, plasma membrane, or nuclear membrane of the recipient cell.

The successful integration of GFP mediated by FM in S. sclerotiorum was confirmed by genome-sequencing and validated by PCR analysis. However, the occurrence of random deletions or insertions in the transformant was observed. We speculated that it might be due to the morphological attributes or other characteristics of the FM, which needs further verification. Therefore, effectively controlling the random damage and decreasing the random insertion in our transformation system needs to be further explored to get more effective transformation and avoid the tedious screening for the desired transformants.

Despite the numerous advantages of nanomaterial, the non-cytotoxicity remains a crucial concern when it is employed for genetic transformation [48, 49]. While recognizing the utility, it is imperative to evaluate the potential risks and adverse effects associated with nanotechnology applications [50]. In the present study, the GFP-transformed S. sclerotiorum strains mediated by the FM grew normally and maintained the pathogenic properties, indicating negligible toxicity from FM. This biological compatibility holds promise for its widespread application in the genetic transformation of filamentous fungi. Despite the apparent biocompatibility of FM, the investigation in the removal or degradation within recipient cell is a worthwhile pursuit.

Considering the diversity of fungal species and the complexity of their cell wall structures, species-specific transformation protocols must be tailored for individual strains. In some instances, the development of entirely novel methods may be imperative to facilitate efficient transformation [51]. Therefore, further exploration and empirical validation are indispensable to ascertain the general applicability of the present transformation system in other fungi. A more profound understanding of the characteristics of the nanoparticles will accelerate its deployment in filamentous genetic engineering.

Conclusion

In the present study, we have successfully demonstrated the use of FM as a novel carrier for the transgenic transformation in S. sclerotiorum. The FM-mediated mycelium transformation circumvents the traditional requirement for protoplast preparation and the subsequent recovery process and exhibits excellent repeatability, efficiency, and high-throughput. This innovative approach represents a significant stride toward achieving tissue-independent delivery of genetic material into the S. sclerotiorum cells. By capitalizing on the distinctive properties of nanoparticles, the present system successfully overcomes the inherent challenge posed by the fungal cell wall, presenting a promising tool for advancing genetic manipulation in notable phytopathogenic fungus.

Data availability

We have deposited the sequencing data in the NCBI database, and it has been assigned the Sequence Read Archive (SRA) accession number PRJNA1126013.

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Funding

The authors thank the financial support from the National Key Research and Development Program of China (2023YFF1000700), the National Natural Science Foundation of China (32372077, 32072021), the Fundamental Research Funds for the Central Universities (SWU120075), and the Natural Science Foundation of Chongqing (CSTB2023NSCQ-MSX0355, CSTB2024NSCQ-MSX0293).

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Author notes

  1. Yijuan Ding and Nan Yang contributed to this work equally.

Authors and Affiliations

  1. Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Chongqing and Southwest University, College of Agronomy and Biotechnology, Southwest University, Beibei, Chongqing, 400715, China

    Yijuan Ding, Nan Yang, Kusum Rana, Yangui Chen, Wei Qian & Huafang Wan

  2. Academy of Agricultural Sciences, Southwest University, Beibei, Chongqing, 400715, China

    Yijuan Ding, Nan Yang, Kusum Rana, Yangui Chen, Wei Qian & Huafang Wan

  3. School of Materials and Energy, Southwest University, Beibei, Chongqing, 400715, China

    Yi Lu, Jiming Xu & Zhigang Xu

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Contributions

H. W., W. Q. and Y. D. conceived and designed the research. Y. D., N. Y., Y. L. and J. X. performed experiments. Y. D., H. W., W. Q., K. R., Y. C., and Z. X. Analyzed the data and wrote the manuscript. Z. X. provided suggestions and technical support. All authors contributed to this study, read, and approved the final manuscript.

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Ding, Y., Yang, N., Lu, Y. et al. Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum. J Nanobiotechnol 22, 494 (2024). https://doi.org/10.1186/s12951-024-02736-6

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