https://orcid.org/0000-0003-0741-9261
Figures
Abstract
Tomato is cultivated worldwide as a nutrient-rich vegetable crop. Tomato wilt disease caused by Fusarium oxysporum f.sp. Lycopersici (Fol) is one of the most serious fungal diseases posing threats to tomato production. Recently, the development of Spray-Induced Gene Silencing (SIGS) directs a novel plant disease management by generating an efficient and environmental friendly biocontrol agent. Here, we characterized that FolRDR1 (RNA-dependent RNA polymerase 1) mediated the pathogen invasion to the host plant tomato, and played as an essential regulator in pathogen development and pathogenicity. Our fluorescence tracing data further presented that effective uptakes of FolRDR1-dsRNAs were observed in both Fol and tomato tissues. Subsequently, exogenous application of FolRDR1-dsRNAs on pre-Fol-infected tomato leaves resulted in significant alleviation of tomato wilt disease symptoms. Particularly, FolRDR1-RNAi was highly specific without sequence off-target in related plants. Our results of pathogen gene-targeting RNAi have provided a new strategy for tomato wilt disease management by developing an environmentally-friendly biocontrol agent.
Author summary
Tomato wild disease caused by Fusarium oxysporum f. sp. lycopersici (Fol) threatens the global tomato production for both processing and the fresh-market system. An efficient and eco-friendly strategy to control wilt disease is urgent needed. SIGS is a novel discovered RNA silencing strategy for disease control, and effectively used for crops protection based on the uptake of external dsRNA by plant pathogens. By homologous comparison. We identified FolRDR1 in the existing gene annotation of Fol. Here, our data showed that exogenous application of FolRDR1-dsRNAs on pre-Fol-infected tomato leaves resulted in significant alleviation of tomato wilt disease symptoms. Particularly, FolRDR1-RNAi was highly specific without sequence off-target in related plants.
Citation: Ouyang S-Q, Ji H-M, Feng T, Luo S-J, Cheng L, Wang N (2023) Artificial trans-kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease. PLoS Pathog 19(6): e1011463. https://doi.org/10.1371/journal.ppat.1011463
Editor: Nian Wang, University of Florida Institute of Food and Agricultural Sciences, UNITED STATES
Received: December 18, 2022; Accepted: June 5, 2023; Published: June 20, 2023
Copyright: © 2023 Ouyang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw sequence data for this study are available in the National Genomics Data Center with accession no. CRA011174. https://bigd.big.ac.cn/gsa/browse/CRA011174.
Funding: This work was partially supported by a grant from the National Natural Science Foundation of China #31972351 and a grant from Zhejiang Natural Science Foundation #KYZ34423025 to S.Q.O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Tomato (Solanum lycopersicum L.) is one of the most important vegetable worldwide. Because tomato is susceptible to more than 200 pests and microbe pathogens, disease control for appropriate disease resistances is crucial to the commercial production [1]. Fusarium wilt of tomato, caused by Fusarium oxysporum f. sp. lycopersici (Fol), threatens the global tomato production for both processing and the fresh-market system. Fol penetrates tomato roots before colonizing the vascular tissue. Initial disease symptoms are visible at the early stage about one week with wilting of lower basal leaves, acropetally to the upper leaves until the entire plant die. In the absence of control strategies, such as fumigation and host resistance, Fusarium wilt disease can causes up to complete tomato crop loss [2]. Therefore, an efficient and eco-friendly strategy to control wilt disease is urgent needed.
Crops are always challenged by environmental stresses from different kinds of parasites, such as bacteria, fungi, viruses, oomycetes, insects, and parasitic plants throughout their life cycles. To maintain surveillance of progenies, plant hosts have evolved fine-tuned defense mechanisms [3,4,5]. In eukaryotic world, RNA interference (RNAi), triggered by small RNAs (sRNAs) such as small interfering RNAs (siRNAs) and miRNAs, is an evolutionarily conserved and sequence-specific mechanism that regulates target gene expression at either the transcriptional level (transcriptional gene silencing, TGS) or the posttranscriptional level (posttranscriptional gene silencing, PTGS) [6,7,8,9,10]. So far, different RNAi pathways including Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS) and Spray-Induced Gene Silencing (SIGS) are reported for the artificial silencing of genes [11]. SIGS is a novel discovered RNA silencing strategy for disease control, and effectively used for crops protection based on the uptake of external dsRNA by plant pathogens [12,13,14]. Therefore, SIGS is a valuable eco-friendly and advanced innovative strategy for plant disease control at pre-harvesting and post-harvesting stages but with limited off-target effects [15].
The RNAi silencing process is initiated with long dsRNAs being cleaved into small fragments of sRNAs (21–25 nts) by the Dicer (DCL), followed by loading into the RNA Induced Silencing Complex (RISC) which contains an essential member such as Argonaute (AGO) protein [16]. The mechanism is fulfilled by amplifying sRNA molecules through RNA-dependent RNA polymerase (RDRs) through the amplification of double-stranded RNA (dsRNA), which are further cleaved and processed by DCLs and trigger the next round of RNAi [17]. A crucial role of RDRs is to interact with RNAi machinery and provide defense against pathogens. RDRs are evolutionarily distributed into four subclasses (RDR1, 2, 3, and 6) in plants [18]. Similarly, RDRs are also widely distributed in three major groups of fungi Ascomycetes, Basidiomycetes and Zygomycetes [18]. However, no RDR-like gene has been reported in F. oxysporum so far. In this study, we tried to find the genes, such as DCL, RDR, AGO and other gene families, related to the production and function of sRNA that might exist in the genome of Fol by homologous comparison. However, with the existing gene annotation of Fol, only FolRDR1 was identified.
Literatures have provided proof-of-concept that RNAi-based plant protection is an effective strategy for controlling crop fungal diseases. Here, we explored the potential and the mechanism of an RNAi-based crop protection strategy using direct applications of FolRDR1-dsRNA to inhibit F. oxysporum. We found that FolRDR1 mediated the pathogen development and pathogenicity. Both Fol and host plant efficiently took up FolRDR1-dsRNA from the environment. Finally, exogenous application of FolRDR1-dsRNAs significantly alleviated the progress of tomato wilt disease symptoms. In summary, our data established that SIGS based on FolRDR1-dsRNA-RNAi contributed to the resistance to tomato Fusarium wilt disease.
Results
FolRDR1 is required for the vegetative growth and asexual reproduction in Fol
By analysis of sequence homology, we found that FolRDR1 (Sequence ID: XM_018384343.1) was highly conservative in F. oxysporum including F. oxysporum f. sp. Lycopersici, F. oxysporum f. sp. Cepae, F. oxysporum f. sp. Cubense, F. oxysporium f. sp. Cucumerinum, F. oxysporum f. sp. Pisi, F. oxysporum f. sp. Raphani and F. oxysporum f. sp. Conglutinans (S1 Fig). These results indicated that FolRDR1 may possess essential biological functions in F. oxysporum. To confirm this hypothesis, we generated two FolRDR1-knockout (FolRDR1-KO) strains FolRDR1-KO-36# and 126# by approach of homologous recombination (S2 Fig).
To assess the potential regulated gene by impaired FolRDR1, the miRNA levels were evaluated by sRNA-seq using the KO-strains FolRDR1-KO-36 (named as FolRDR1-1 in library), FolRDR1-KO-126 (named as FolRDR1-2 in library) and wild type strain (named as Fol-WT in library). The DEGs of miRNAs were listed in S2 Table. Well correlation was showed between FolRDR1-KO-36 and FolRDR1-KO-126 (S3A Fig). Compared with WT strain, the abundances of miRNAs declined significantly in both FolRDR1-KO strains (S3B and S3C Fig). We further analyzed the biological functions of predicted targets of miRNAs (Listed in S3 Table), and the results showed that knockouting of FolRDR1 mainly affected the metabolic pathway in both KO strains (S4 Fig). With above results, we concluded that the levels of miRNA were correlated with FolRDR1 in Fol.
We further checked the growth rate of wild type (WT) Fol and FolRDR1-KO strains cultured on PDA plates, respectively. Statistic data showed that no significant difference was observed between Fol and FolRDR1-KO strains (S5 Fig). Further, no significant difference in colony morphology was observed either under salt, alkali and osmotic pressure stress at different concentrations (S6 Fig). The above results indicated that the absence of FolRDR1 did not change the response to abiotic stresses in Fol. However, we found that the colony edge of both FolRDR1-KO strains were more loose than Fol. Furthermore, the mycelia of FolRDR1-KO strains presented abnormal growth such as mycelia ablation and increased sclerotia (Fig 1A). To filamentous fungi, sporulation ability is an important physiological index to measure the pathogenicity. Knocking out FolRDR1 also leaded to lower sporulation but larger size of conidia compare to WT strain (Fig 1B–1D). Intriguingly, the growth and penetrability of Fol on PDA plate covered with cellophane were nearly unchanged, contrarily, both FolRDR1-KO strains showed dramatic decreased penetrability (Fig 1E). There results indicated that FolRDR1 was essential to the vegetative growth and conidiogenesis in Fol as well as penetrability.
A the mycelia of FolRDR1-KO strains presented abnormal growth such as mycelia ablation and increased sclerotia (indicated by red arrow). All three strains were cultured on PDA plates, and images were taken at fourth days. B-D Knocking out FolRDR1 leaded to lower sporulation but larger size of conidia compare to WT strain. * indicates significant difference when compared to WT at P < 0.05, chi-square test, Error bars indicate the Standard Deviation of three replicates. 40 x scale bars, 50 μm, 100 x scale bars, 20 μm. E Knocking out FolRDR1 resulted in dramatic decreased penetrability in Fol. All three strains were cultured on the center of PDA plates covered with half cellophane, and images were taken at fourth days. Front, images were taken from the front of plate. Back, images were taken from the back of plate. Three biological replicates were used in each experiment.
FolRDR1 is required for pathogenicity in Fol
To investigate the response of FolRDR1upto Fol infection in tomato roots, two-week susceptible cultivar Moneymaker seedlings were infected by Fol. Fusarium wilt symptoms were developed at early stage (7 day post infection, 7dpi) (Fig 2A). Total RNA was extracted from infected tomato roots which included both tomato and pathogen RNA. The transcript level of FolRDR1was further valuated by Northern blot, and the results indicated that FolRDR1was constantly induced during the pathogen infection (Fig 2B).
A The wilt disease symptoms of susceptible cultivar Moneymaker infected with Fol. B The transcript level of FolRDR1 was induced under the infection of Fol. 10 μg of total RNA was resolved by electrophoresis using urea polyacrylamide gel electrophoresis (PAGE) and transferred to anylon N+ membrane. [γ-32P]ATP-labelled specific nucleotide probe sequences were used for hybridization. Sly-18s rRNA was used as a loading control. C FolRDR1 was required for pathogenicity in Fol. The FolRDR1-KO strains and control WT Fol were used to inoculate tomato seedlings. Wilt disease symptoms were photographed 2 weeks after inoculation. D Cotton blue staining results reflect the abundance of Fol in the stem of tomato plants. More intense cotton blue staining correlates with higher abundance of Fol. E The outgrowth of fungi from tomato stems of plants inoculated with the indicated strains on PDA, and images were taken at 1.5 and 2 days, respectively. Front, images were taken from the front of plants. Top, images were taken from the top of plants. Three biological replicates were used in each experiment.
To further evaluate the pathogenicity of FolRDR1, tomato seedlings were inoculated with Fol and two FolRDR1-KO strains, respectively. We observed impaired infection of Moneymaker supported by alleviated Fusarium wilt symptoms, less presence of the fungus within the plant stem and fungal mycelium regeneration compared to Fol-treated Moneymaker, while no Fusarium wilt symptoms were observed in resistant cultivar Motelle under infection with all three individual strains (Fig 2C–2E). Based on above results, we concluded that FolRDR1 was a critical pathogenic factor in Fol.
Environmental dsRNA-FolRDR1 is effectively taken up by Fol
To validate whether SIGS of FolRDR1 control Fusarium wilt disease, we generated FolRDR1-dsRNA2 (1–804 bp) and FolRDR1-dsRNA1 (787–1523 bp) expressing constructs under double T7 promoter. Full length GFP-dsRNA was constructed as a control (S7 Fig). These constructs were transformed into RNase III deficient E. coli HT115 (DE3) strain to express dsRNA in vitro under optimum condition: 0.8 mmol/L IPTG, 280 rpm/min, 37°C and 8-hour induced expression (S8 Fig).
To trace external FolRDR1-dsRNA, we synthesized fluorescein-labeled FolRDR1-dsRNA using Fluorescein-12-UTP in vitro. Fluorescein-12-UTP and water treatment were used as negative controls. WT conidia were cultured with fluorescein-labeled FolRDR1-dsRNA for 24 hours on PDA plate followed by detecting fluorescence signals. Fluorescence signals of FolRDR1-dsRNAs and GFP-dsRNA were observed in conidia (Fig 3A, left). To further confirm whether the dsRNAs enter Fol cells, the Fol mycelium were cultured with the fluorescein-labelled FolRDR1-dsRNAs and GFP-dsRNA respectively in liquid culture for 48 hours. Subsequently, conidia were collected for protoplast preparation. Similarly, fluorescence signals of FolRDR1-dsRNAs and GFP-dsRNA were observed clearly in protoplast (Fig 3A, middle). Further, we also observed the fluorescence signals in 48-hour hyphae (Fig 3A, right). Above results indicated that Fol cells took up dsRNAs from the environment, and this action is not likely to be selective.
A Conidia were cultured in Vogel’s minimal medium at a concentration of 105 spores/mL with 150 ng/mL dsRNA for 24 hours (Left, scale bars, 10 μm), and conidia protoplast were made subsequently (Middle, scale bars, 10 μm). Hyphae were collected after 48 hours (Right, scale bars, 1 mm). Samples were treated with micrococcal nuclease (MNase) 30 min before images were taken using the confocal microscopy laser scanner (CMLS). B Two vertical glass slides (1 cm x 2 cm) were inserted into the PDA medium about 2 cm far away from the inoculation site of Fol to minimize the dissociative fluorescein-labeled dsRNA in the plate. Front, images were taken from the front of plate. Back, images were taken from the back of plate. C After 3 days of mycelium growth and expansion, the fluorescence signals in the mycelia climbing the glass slides were checked using CMLS. Scale bars, 1 mm. Three biological replicates were used in each experiment. D The corresponding dsRNA fragments in the marginal mycelia were further detected by RT-PCR. E FolRDR1-dsRNA-derived siRNAs were enriched in both FolRDR1-dsRNA treated fungal mycelia resulting in repressed the transcript levels of FolRDR1. Total ten 30-nt DNA fragments (detailed in S5 Table), which uniformly distributed in the predicted regions in the CDS of FolRDR1, were synthesized and mixed as a pool followed by labeling with [γ-32P]ATP as probes for to detect the enrichment of FolRDR1-dsRNA-derived siRNAs (Up panel). [γ-32P]ATP-labeled specific nucleotide probe sequences of FolRDR1 were used for hybridization to detect the transcript levels of FolRDR1 (Bottom panel). Sly-18s rRNA was used as a loading control in both Northern blots, respectively.
To determine whether dsRNA can be effectively transported in Fol mycelium, we applied fluorescein-labeled dsRNA on the center of PDA plate inoculated with Fol. Two glass slides (1 cm x 2 cm) were inserted into the medium about 2 cm far away from the inoculation site to minimize the dissociative dsRNA in the plate (Fig 3B). After 3 days of mycelium expansion, we detected the fluorescence signals in the fungal mycelia climbing on the glass slides (Fig 3C). The corresponding dsRNA fragments in the fungal mycelia were further detected (Fig 3D).
To verify the production of FolRDR1-dsRNA-derived siRNAs, firstly, the efficient siRNAs generated in different regions of FolRDR1 were analyzed using siRNA-Finder (Si-Fi) (http://www.wheatgenome.info/) (S9 Fig) [19]. Total ten 30-nt DNA fragments (detailed in S5 Table), which uniformly distributed in the predicted regions, were synthesized and mixed as a pool followed by labeling with [γ-32P]ATP as probes for Northern blot. The data clearly shown that FolRDR1-dsRNA-derived siRNAs were enriched in both FolRDR1-dsRNA1 and FolRDR1-dsRNA2 treated fungal mycelia, respectively (Fig 3E, up panel). Furthermore, the transcript level of FolRDR1 in both FolRDR1-dsRNAs treated fungal mycelia were repressed significantly compared to Fol or water treatment samples (Fig 3E, bottom panel). These results indicated that external dsRNAs were effectively transported in Fol growing-mycelium and repressed the transcript level of FolRDR1.
Application of external FolRDR1-dsRNA destructs the biological functions of FolRDR1 in Fol
Previously, we have shown that FolRDR1 was essential to the vegetative growth and conidiogenesis in Fol. We further explore whether external FolRDR1-dsRNA impair the biological functions of FolRDR1 in Fol. We applied FolRDR1-dsRNA on the center of the plate colony and observed the colony growth at 5 dpi. The data showed that the growth rate of the mycelium were unchanged under the treatments of FolRDR1-dsRNAs and GFP-dsRNA (Fig 4A and 4B). To the conidiogenesis, however, both FolRDR1-dsRNA1 and FolRDR1-dsRNA2, but not GFP-dsRNA, significantly suppressed the production of conidia (Fig 4C). Moreover, the transcript level of FolRDR1 was repressed accordingly at optimal treatment with the concentration of 150 ng/mL and 24 hours in liquid PDA medium (Fig 4D and 4E). We also noticed that the RNAi-based silencing efficiency of two dsRNAs in interference processing was about 90% for FolRDR1-dsRNA1 and 81% for FolRDR1-dsRNA2, respectively (Fig 4D and 4E), which was partially addressed by the analysis using siRNA-Finder (Si-Fi) (S9 Fig). Above results indicated that FolRDR1-dsRNA1 generated efficient siRNAs more than FolRDR1-dsRNA2, and application of external FolRDR1-dsRNAs effectively destructed the biological functions of FolRDR1 in Fol.
A The growth rate of the mycelium were unchanged under the treatments of either FolRDR1-dsRNAs or GFP-dsRNA at a concentration of 150 ng/mL. Fol strain was cultured on PDA plates with the treatments of either FolRDR1-dsRNAs or GFP-dsRNA, respectively, and images were taken at 1 day and 5 days. Front, images were taken from the front of plate. Back, images were taken from the back of plate. B The growth rate of the mycelium was scaled at different time points. C The treatments of FolRDR1-dsRNAs suppressed the production of conidia. D The transcript levels of FolRDR1 in both FolRDR1-dsRNAs treated marginal mycelia were concentration dependent. E The transcript level of FolRDR1 in both FolRDR1-dsRNAs treated marginal mycelia were time dependent. Three biological replicates were used in each experiment. a presents no significant differences (p>0.05), c, d, e present significant differences (P<0.01).
Host plant takes up and transfers environmental FolRDR1-dsRNAs
To evaluate the residual period of the external dsRNA on the host leaves, FolRDR1-dsRNAs and GFP-dsRNA were daubed on the 2-week tomato seedling leaves. The treated leaves, stems and roots were collected at different time points for detecting dsRNA by RT-PCR. All three dsRNAs were detected until 7 dps (day post spray) which indicated that the environmental dsRNA could remain on leaves, stems and roots for at least 7 days without dsRNA selectivity (Fig 5A).
A FolRDR1-dsRNAs and GFP-dsRNA were sprayed on the 2-week tomato seedling leaves. The different tissues of treated plant including leaf, stem and root were collected at different time points for detecting dsRNA by RT-PCR. B Fluorescein-labeled dsRNAs were daubed on one side leaf followed by checking the fluoresce signals using CMLS after 24 hours. Scale bars, 1 mm. C The fluoresce signals on undaubed leaf were detected using CMLS 3 days after treatment described above. Scale bars, 1 mm. D The fluoresce signals in stem were detected using CMLS 3 days after treatment described above. Vascular bundles were pointed by white arrows. Scale bars, 1 mm. E The fluoresce signals in root were detected using CMLS 3 days after treatment described above. Vascular bundles were pointed by white arrows. Scale bars, 1 mm.
To verify whether external dsRNA applied on tomato leaf may be effectively transferred in plant tissues, one side leaf was daubed with fluorescein-labeled dsRNA followed by checking the fluoresce signals in undaubed leaf, root and stem. No fluoresce signal was detected in water treated leaf which indicated no background excitation fluorescence in nature tomato leaf. On the other hand, strong fluoresce signals were presented in daubed leaf (Fig 5B). Then, visible fluoresce signals were detected in undaubed leaf 2 days after treating for all three dsRNAs (Fig 5C). Intriguingly, relatively strong fluoresce signals were shown in stem and root compared to leaf. In stem, fluoresce signals were obviously emerged in vascular bundle (Fig 5D), and distributed in the entire root, especially in root hair (Fig 5E). Above results demonstrated that tomato plant effectively took up the external FolRDR1-dsRNAs and transferred to the different tissues.
External application of FolRDR1-dsRNA alleviates the development of tomato wilt disease
To assess whether SIGS attenuate Fol infection, we sprayed the Fol pre-treated 2-week tomato seedling with FolRDR1-dsRNAs (200 ng/mL) at 24 hours after infecting with Fol and scaled the development of Fusarium wilt symptoms. At 15 dpi without spraying treatment, susceptible cultivar Moneymakers showed the initial symptoms of tomato wilt disease with cotyledon chlorosis and wilting. Meanwhile, Moneymakers treated with FolRDR1-dsRNA1 and FolRDR1-dsRNA2, respectively, developed severer Fusarium wilt symptoms with euphylla chlorosis and wilting. However, at 25 dpi, Moneymakers treated with or without GFP-dsRNA as negative controls were gradually died, showing severe symptoms of Fusarium wilt. However, the wilt disease symptoms of Moneymakers treated with FolRDR1-dsRNA1 and FolRDR1-dsRNA2 were significantly alleviated (Fig 6A). By staining for the presence of the fungus within the plant stem and fungal mycelium regeneration, we further observed alleviated infection in Moneymaker treated with FolRDR1-dsRNA1 and FolRDR1-dsRNA2, respectively, as well as less presence of the fungus within the plant stem and fungal mycelium regeneration compared to Fol-treated Moneymaker, while no Fusarium wilt symptoms were observed in resistant cultivar Motelle under infection with all three individual strains (Fig 6B). The results of relative biomass of Fol in different sample further supported above impaired infection between different treatments which correlated with the symptoms of Fusarium wilt (Fig 6C and 6D).
A Two-week tomato seedlings were infected by WT Fol as described previously, followed by spraying FolRDR1-dsRNAs and GFP-dsRNA on the leaves, respectively. Wilt disease symptoms were photographed 2 weeks after inoculation. Front, images were taken from the front of plants. Top, images were taken from the top of plants. B Cotton blue staining results reflect the abundance of Fol in the stem of tomato plants. More intense cotton blue staining correlates with higher levels of Fol (Up panel). The outgrowth of fungi from tomato stems of plants inoculated with the indicated strains on PDA, and images were taken at 2 dpi, respectively (Middle panel). Diseased vascular bundles were checked in longitudinal splitting stem (Pointed by red arrows) (Bottom panel). C Biomass of Fol in according treated plants was detected by PCR. D Biomass of Fol in according treated plants was detected by qPCR. Three biological replicates were used in each experiment. a presents no significant differences (p>0.05),the largest average, d, e present significant differences (P<0.01).
Actually, we performed both pre-treatment (Fig 6) and after-treatment (S11 Fig) with FolRDR1-dsRNAs experiments. The data indicated that no significant difference was shown between these two treatments. Due to the unpredictable development of fusarium wilt disease in field, we think that application in the early stage of disease might reduce the cost of disease control. Based on these results, we concluded that SIGS of FolRDR1-dsRNAs attenuated tomato Fusarium wilt disease under lab conditions.
To validate the potential off-target of FolRDR1-dsRNAi, the off-targets were searched and predicted in different species using si-Fi algorithm with splitting FolRDR1-dsRNAi trigger sequence (the complement to the target sequence of the corresponding RNA) into all possible MERs [20]. Our data showed that no off-target of FolRDR1-RNAi in Fusarium oxysporum was predicted, and no off-target was predicted in Solanum lycopersicum (tomato), either (S4 Table). We further generated RNA-seq libraries using water (Mock), GFP-dsRNA (negative control), FolRDR1-dsRNA1 treated tomato seedlings to analyze the transcriptom of tomato. The data indicated that no significant changes were found between SIGS-FolRDR1 and two controls (S10 Fig, and all DEGs of mRNA were listed in S6 Table) (The raw sequence data for this study are available in the National Genomics Data Center with accession no. CRA011174, https://bigd.big.ac.cn/gsa/browse/CRA011174). Taken together, our results supported that FolRDR1 could be considered as a suitable candidate for developing biological agent to control tomato wilt disease.
Discussion
Recently, studies illustrated that spraying dsRNAs/sRNAs targeting essential pathogen genes on plant surfaces afforded efficient crop protection, and RNAi-based SIGS strategy of disease control was potentially sustainable eco-friendly alternative to standard chemical pesticides for controlling agricultural losses caused by pests and pathogens [7,21]. Exogenous dsRNA triggering suppression of gene activity in a homology-dependent manner was firstly discovered in Caenorhabditis elegans [22]. Since then, SIGS was known as a powerful, fast, and environmentally friendly strategy to circumvent the problems in creating GMOs [7,13,23,24].
RDR1 (also termed as Rdp1) localizes to all known heterochromatic loci and is required for sense transgene-induced silencing to generate dsRNA molecules in fungi [25,26]. RDRs have mainly been described to be involved in amplification of RNAi in eukaryotes [18]. In the present study, we found that FolRDR1 mediated the invasion to the host plant tomato, and played as an essential regulator in pathogen development and pathogenicity in Fol. However, no similar biological functions of RDR1 were reported in other fungal so far. Even more intriguing, we found that abolishing of FolRDR1 leaded to mycelia ablation and abnormal sclerotia in Fol (Fig 1A), which highlights the importance to study underlying mechanisms.
Host Arabidopsis cells secreted exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen B. cinerea, and transferred host sRNAs induced silencing of fungal genes critical for pathogenicity [27]. SIGS has been shown to be effective on controlling plant disease initialed by taking up external dsRNA. The uptake efficiency of dsRNA was significantly different among various fungi with exception such as Colletotrichum gloeosporioides [13]. Our present results showed that both fungal pathogen Fol and host plant could uptake FolRDR1/GFP-dsRNAs directly from environment without obvious selectivity (Fig 3), and dsRNAs were transferred in different tissues efficiently (Fig 4). These results promote us speculate that fungal pathogens take up external RNA unselectively but species dependently.
More than intriguing, the fluorescence signals of FolRDR1 /GFP-dsRNAs were dominantly localized in the host plant vascular bundles (Fig 5D and 5E). Germination of dormant spores in soil results in adherence and invasion of plant roots by Fol hypha, subsequently, move from the root cortex to the vascular bundles where microconidia spore are produced and disseminated. Using the vascular bundles as transport corridor, endogenetic hypha spreading to aboveground tissues is critical for disease progression for Fol. The characteristic wilt symptoms appear as a result of severe water stress, mainly due to vessel clogging [28]. Further questions need to be answered how these external dsRNAs are taken up by plant and fungal cells directly from environment traveling across the boundaries between organisms of different taxonomic kingdoms.
Previously, Arabidopsis and barley ectopically expressing a double-stranded RNA (dsRNA) targeting three fungal CYP51 genes significantly enhanced plant resistance to Fusarium graminearum species by disrupting fungal membrane integrity, subsequently, spraying detached barley leaves with a 791-nt long CYP3-dsRNAs that contains complementary sequences to CYP51family members prior to fungal infection could effectively inhibit disease and yield much smaller lesions [14,29]. Similarly, externally applying dsRNAs and small RNAs (sRNAs) targeting Dicer-like protein genes DCL1 and DCL2 of B. cinerea on vegetables, fruits, and flower petals could suppress grey mold disease effectively [12]. Here, applying FolRDR1-dsRNAs that target FolRDR1 on the surface of pre-infected tomato seedling leaves significantly inhibited the development of Fusarium wilt (Fig 6). To develop a successful SIGS-based crop protection strategy, several critical aspects must be considered. Firstly, a reasonable duration of efficacy is desired. Our data revealed that the FolRDR1/GFP-dsRNAs could be detected even at 7 dps of the local sprayed site, suggesting either external RNAs were stable for at least seven days on the surface of the leaves and/or remained stable in the plant cells (Fig 5A). Secondly, off-target is another considered factor for eco-friendly alternative to standard chemical pesticides. By bioinformatics prediction, our data showed that FolRDR1-RNAi resulted in no target-specific either in fungal pathogen or host plant (S2 Table, S8 Fig).
Taken together, eukaryotic pathogens, including fungi and oomycetes, cause vast worldwide economic losses in crop annually. Compared to traditional chemical pesticides, our collective data provided solid evidences that FolRDR1-RNAi-SIGS is an advantageous artificial trans-kingdom RNAi-based bio-pesticide to protect tomato from Fusarium wilt disease. Application strategies can be improved by encapsulating with chemical reagents to stabilize the dsRNAs and thus increase the strength and duration of plant protection. Such specific pathogen gene-targeting RNAs represents a new generation of environmentally-friendly fungicides for increasing safety and quality of crop yields to feed the growing population.
Materials and methods
Plant materials, fungal inoculation, measurements of Fol biomass
Two previously described tomato near-isogenic cultivars, susceptible Moneymaker (MM, i2/i2) and resistant Motelle (Mot, I2/I2), were employed in this study [30,31,32]. Briefly, tomato seedlings were grown in long-day conditions (16 hr light/8 hr dark, at 25°C, 65% humidity, photon flux density 40 μmol m-2 s-1) for 2 weeks for pathogen inoculation.
The pathogenic fungal strain is Fusarium oxysporum f. sp lycopersici (race 2, FGSC 9935, Fol). Fol was grown on potato dextrose agar medium (PDA) for 5 days at 28°C with constant light. Spore suspensions were prepared by harvesting cultures in Vogel’s minimal medium at a concentration of 107 spores/mL. Tomato seedlings were removed from soil, and rinsed with tap water roots were inoculated with Fol spores for 30 min. Water treatment was used as a mock control. All experiments were conducted using three biological replicates.
To assess the relative levels of Fol biomass in tomato tissues, genomic DNA was isolated from tomato tissues using CTAB [33]. The rDNA Intergenic Spacer Region (IGS) of Fol was amplified from genomic DNA using qPCR (Primers listed in S1 Table) as a marker to assess relative fungal biomass [2].
Statistics of spore production
All strains were inoculated on PDA medium for 4 days. 5 mm mycelium piece at the edge of the colony was cut and cultivated in 100 mL of Vogel’s liquid medium at 28°C, 200 rpm for 4 days. Spores were collected using three layers of sterile gauze. After spinning down, the number of spores were counted using hemocytometer under the optical microscope. These experiments were repeated three times and three biological replicates in each experiment.
Determination of fungal penetrability
In this experiment, cellophane was used to mock plant cell wall to test the fungal penetrability. The cellophane was cut to a semicircle piece with a radius of 4 cm and placed on the PDA medium plate. 5 mm mycelium piece at the edge of the colony was cut and cultivated in the center of the plate under light at 28°C for 5 days. The colony morphology and mycelium morphology were recorded. These experiments were repeated three times and three biological replicates in each experiment.
Total RNA extraction, Northern blotting, quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the Trizol reagent (#15596026, Invitrogen, CA, USA). Purified RNA was treated with DNase I (Thermo Fisher Scientific, Waltham, Ma, USA). For small RNA gel blots, 40 μg of total RNA was separated on 7 M urea 15% denaturing polyacrylamide gels in Tris/Boric Acid/EDTA (1X TBE), followed by transferring to a nylon N+ membrane. For high molecular weight RNA gel blots, 10 μg of total RNA was resolved by electrophoresis using urea polyacrylamide gel electrophoresis (PAGE) and transferred to anylon N+ membrane. Gene-specific nucleotide probes (Primers listed in S1 Table, and DNA fragments listed in S5 Table). Gene-specific nucleotide probes (Primers listed in S1 Table) were end-labeled using [γ-32P]ATP (#M0201, New England Biolabs, Ipswich, MA; nucleotide probes were labeled according to the manufacturer’s recommendations). Blots were stripped and re-probed using a Sly-18s rRNA nucleotide probe to provide a loading control. All blots were imaged using a PhosphorImager (Molecular Dynamics/GE Life Sciences, Pittsburgh, PA) [30].
For reverse transcriptase-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized from 1 μg of total RNA using the Superscript III First-Strand Synthesis System (#18080051, Thermo Fisher Scientific, Waltham, Ma, USA) according to the manufacturer’s recommendations (Primers listed in S1 Table). Diluted cDNA was used as the template for quantitative RT-PCR (#1708880, Bio-Rad, Philadelphia, PA, USA), using Sly-18s rRNA as the internal control. Differential expression of genes was calculated using the 2-ΔΔCt method [34].
Construction of FolRDR1 knockout strains
FolRDR1 knockout mutant strains were generated by using the split-marker approach previously described by our laboratory [35]. Briefly, for FolRDR1 knockout vector construction, the upstream flanking sequence, downstream flanking sequence of FolRDR1 and HPH cassette were amplified and purified, followed by transformation into protoplasts of the wild-type strain (Primers listed in S1 Table). The knockout construct was then transformed into protoplasts of Fol. Transformants with the desired genetic changes were identified using site-specific primer pairs (Primers listed in S1 Table).
Synthesis of dsRNA and uptake of fluorescein-labelled dsRNA in vitro
Synthesis of dsRNA in vitro was based on established protocols [13]. Briefly, selected fragments of FolRDR1 were amplified using gene-specific primers and inserted into the pL4440 vector containing double T7 promoter (Primers listed in S1 Table). FolRDR1/GFP-dsRNA was labeled using the fluorescein RNA Labeling Mix Kit following the manufacturer’s instructions (#11685619910, Sigma, St. Louis, MO, USA). For confocal microscopy examination of fluorescein-labelled dsRNA uptake by fungal mycelium, 5 μL of 150 ng/μL fluorescent dsRNA was applied to the PDA medium or the microscope slides surface.
Light microscopy studies
To track the fluorescein-labeled FolRDR1/GFP-dsRNA, plant tissues and fungal materials were collected after dsRNA treatment with the concentration of 200 ng/μL and 150 ng/mL, respectively. Images were taken using a Zeiss LSM 710 confocal microscope with a 63/1.2 NA C-Apochromat oil immersion objective (Zeiss, Oberkochen, Germany). The relative fluorescent density was analyzed usingImage-pro Plus (Media Cybernetics Inc., Shanghai, China).
Construction of sRNA-seq and RNA-seq libraries and analysis
For sRNA-seq, total RNA of the KO-strains FolRDR1-KO-36 (named as FolRDR1-1 in library), FolRDR1-KO-126 (named as FolRDR1-2 in library) and wild type strain (named as Fol-WT in library) were extracted individually using the TRIzol reagent (#15596026; Life Technologies) according to the manufacturer’s recommendations. For each Illumina library, 1 μg total RNA was used, according to the manufacturer’s instructions. The libraries were subsequently sequenced using the Illumina HiSeq 2000 (Biomarker Technologies, Rohnert Park, CA, USA). For RNA-seq, two-week-old tomato seedlings were pre-infected with Fol followed by spraying FolRDR1/GFP-dsRNA with the concentration oxcf 200 ng/μL. Three biological replicates were used, with 5 seedlings for each treatment. The leaves were collected and then frozen immediately in liquid nitrogen. Total RNA was extracted described previously.
For individual Illumina library, raw reads were subjected to quality control (QC). After QC, raw reads were filtered into clean reads. All sequence reads were trimmed to remove the low-quality sequences. The sequence data were subsequently processed using in-house software tool SeqQC V2.2. House-keeping small RNAs including rRNAs, tRNAs, snRNAs and snoRNAs were removed by blasting against GenBank (http://www.ncbi.nih.gov/Genbank) servers. The trimmed reads were then aligned to the Fusarium oxysporum and Solanum lycopersicum reference genome respectively using TopHat v2.0.0 and Bowtie v0.12.5 [36] with default settings. The expression levels of miRNAs or mRNAs were normalized to the reads per million (rpm) value for each individual library.
Statistical analysis
Each result was presented as the mean ± standard deviation (SD) of at least three replicate measurements. Significant differences between treatments were statistically evaluated by SD and one-way analysis of variance (ANOVA) using SPSS 2.0 (Chicago, IL, USA). The data for two specific different treatments were compared statistically by ANOVA, followed by Student’s T-test if the ANOVA result was significant at p < 0.01.
Supporting information
S1 Fig. Amino acid sequence of RDR1 alignment and phylogenetic tree construction among different Fusarium oxysporum races.
A Alignment of amino acid sequence of RDR1 using Pairwise Align Protein. All amino acid sequence of RDR1 were from https://www.ncbi.nlm.nih.gov. B The phylogenetic tree was constructed using MEGA (Molecular Evolutionary Genetics Analysis).
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S2 Fig. Construction of FolRDR1 knockout strains.
A Concise schematic diagram of homologous recombination. B PCR fragments used for homologous recombination. C Diagnostic PCR was used to identify positive clones.
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S3 Fig. The analysis of sRNA-seq using FolRDR1-KO and WT strains.
In the library, KO-strains FolRDR1-KO-36 was named as FolRDR1-1, FolRDR1-KO-126 was named as FolRDR1-2, and wild type strain was named as Fol-WT. A Correlation heat map analysis. B, C The abundances of miRNAs declined in both FolRDR1-KO strains.
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S4 Fig. The analysis of GO.
Knockouting of FolRDR1 mainly affected the metabolic pathway in both KO strains.
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S5 Fig. Knockouting FolRDR1 has no effect on the growth of Fol.
A All strains were culture on PDA plate, and photographed at different time points. B The growth curve was generated based on the colony diameter. Front, images were taken from the front of plate. Back, images were taken from the back of plate. Three biological replicates were used in each experiment.
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S6 Fig. Knockouting FolRDR1 has no effect on the response to abiotic stress.
A All strains were culture on PDA plate with different concentration of NaCl (Left). The growth of colony was scaled at different time points, and the growth curve was generated (Right). B All strains were culture on PDA plate with different pH (Left). The growth of colony was scaled at different time points, and the growth curve was generated (Right). C All strains were culture on PDA plate with different concentration of sorbital (Left). The growth of colony was scaled at different time points, and the growth curve was generated (Right). Front, images were taken from the front of plate. Back, images were taken from the back of plate. Three biological replicates were used in each experiment. a presents no significant differences (p>0.05).
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S7 Fig. Construction of FolRDR1-dsRNA expression pL4440 vector containing double T7 promoter.
A Sketch map of FolRDR1-dsRNA1 and FolRDR1-dsRNA2. B Fragments of FolRDR1-dsRNA1, FolRDR1-dsRNA2 and GFP-dsRNA were amplified using gene-specific primers. C Diagnostic PCR was used to identify positive clones.
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S8 Fig. Establishment of heterologous expression dsRNA in E. coli.
A Expression strains of FolRDR1-dsRNA1, FolRDR1-dsRNA2 and GFP-dsRNA were induced using different concentration of IPTG. B Abundance of FolRDR1-dsRNA1, FolRDR1-dsRNA2 and GFP-dsRNA were scaled under different temperature. C Abundance of FolRDR1-dsRNA1, FolRDR1-dsRNA2 and GFP-dsRNA were scaled under different induced time points. D The growth curve of different strains measured by light transmittance (OD = 600 nm). a presents no significant differences (p>0.05), d, e present significant differences (P<0.01).
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S9 Fig. Prediction of efficient siRNAs generated in different regions of FolRDR1.
Efficient siRNAs generated in different regions of FolRDR1 were predicted using siRNA-Finder (Si-Fi). Briefly, the off-target searching pipeline starts with splitting a long RNAi trigger sequence (the complement to the target sequence of the corresponding RNA) into all possible MERs using stringent parameters (stricter strand selection rules plus target site accessibility calculations).
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S10 Fig. Analysis of RNA-seq libraries using water (Mock), GFP-dsRNA (negative control), FolRDR1-dsRNA1 treated tomato seedlings.
The samples were collected at 24 hours after treatment. A The number of DEGs (Different Expressed Gene) in RNA-seq libraries using water (Mock), GFP-dsRNA (negative control), FolRDR1-dsRNA1. B Analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes).
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S11 Fig. Exogenous application of FolRDR1-dsRNA alleviates the development of Fusarium wilt disease in Fol after-treatment tomato seedlings.
A Two-week tomato seedlings were sprayed with FolRDR1-dsRNAs on the leaves respectively, followed by infecting by WT Fol two days later as described previously. Wilt disease symptoms were photographed 2 weeks after inoculation. Front, images were taken from the front of plants. Top, images were taken from the top of plants. B Cotton blue staining results reflect the abundance of Fol in the stem of tomato plants. More intense cotton blue staining correlates with higher levels of Fol (Up panel). The outgrowth of fungi from tomato stems of plants inoculated with the indicated strains on PDA, and images were taken at 2 dpi, respectively (Middle panel). Diseased vascular bundles were checked in longitudinal splitting stem (Pointed by red arrows) (Bottom panel).
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S3 Table. List of all predicted targets of miRNA.
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S4 Table. Prediction of FolRDR1 off-target transcripts.
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S5 Table. Sequences of 30-nt DNA fragments which uniformly distributed in the predicted regions in the CDS of FolRDR1.
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Acknowledgments
We are grateful for the gift of tomato cultivars from Dr. Isgouhi Kaloshian at the University of California, Riverside, US. We appreciate the valuable discussions with Prof. Xiao-Ming Zhang from the Institute of Zoology, Chinese Academy of Sciences. We thank Mr. Sijian Li for cooperation.
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