==== Front PLoS Pathog PLoS Pathog plos PLOS Pathogens 1553-7366 1553-7374 Public Library of Science San Francisco, CA USA 37339156 10.1371/journal.ppat.1011463 PPATHOGENS-D-22-02186 Research Article Biology and Life Sciences Organisms Eukaryota Plants Fruits Tomatoes Biology and Life Sciences Mycology Fungal Structure Mycelium Biology and Life Sciences Plant Science Plant Anatomy Leaves Biology and life sciences Genetics Gene expression Gene regulation Small interfering RNA Biology and life sciences Biochemistry Nucleic acids RNA Non-coding RNA Small interfering RNA Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Fungal Pathogens Fusarium Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Fungal Pathogens Fusarium Biology and Life Sciences Mycology Fungal Pathogens Fusarium Biology and Life Sciences Organisms Eukaryota Plants Seedlings Biology and Life Sciences Agriculture Crop Science Crops Fruit Crops Biology and life sciences Genetics Epigenetics RNA interference Biology and life sciences Genetics Gene expression RNA interference Biology and life sciences Genetics Genetic interference RNA interference Biology and life sciences Biochemistry Nucleic acids RNA RNA interference Artificial trans-kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease FolRDR1-RNAi is a potential strategy to control tomato wilt disease https://orcid.org/0000-0003-0741-9261 Ouyang Shou-Qiang Conceptualization Project administration Supervision Writing – original draft Writing – review & editing 1 2 * Ji Hui-Min Investigation Methodology 2 Feng Tao Data curation Investigation 2 Luo Shu-Jie Project administration Software 2 Cheng Lu Investigation Software 2 Wang Nan Investigation Methodology 2 1 College of Life Sciences, Zhejiang Normal University, Jinhua, China 2 College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, China Wang Nian Editor University of Florida Institute of Food and Agricultural Sciences, UNITED STATES The authors have declared that no competing interests exist. * E-mail: sqouyang@zjnu.edu.cn 20 6 2023 6 2023 19 6 e101146318 12 2022 5 6 2023 © 2023 Ouyang et al 2023 Ouyang et al https://creativecommons.org/licenses/by/4.0/ 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. 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. http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 31972351 https://orcid.org/0000-0003-0741-9261 Ouyang Shou-Qiang http://dx.doi.org/10.13039/501100004731 Natural Science Foundation of Zhejiang Province KYZ34423025 https://orcid.org/0000-0003-0741-9261 Ouyang Shou-Qiang 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. PLOS Publication Stagevor-update-to-uncorrected-proof Publication Update2023-06-30 Data AvailabilityThe 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. 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. ==== Body pmcIntroduction 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. 10.1371/journal.ppat.1011463.g001 Fig 1 Knocking out FolRDR1 partially dampens the development of Fol. 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). 10.1371/journal.ppat.1011463.g002 Fig 2 FolRDR1 is essential for pathogenicity in Fol. 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. 10.1371/journal.ppat.1011463.g003 Fig 3 Fol efficiently uptakes fluorescein-labelled dsRNA. 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. 10.1371/journal.ppat.1011463.g004 Fig 4 Application of exogenous dsRNA suppresses the production of conidia and expression level 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). 10.1371/journal.ppat.1011463.g005 Fig 5 External dsRNA was transported in plant tissues. 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). 10.1371/journal.ppat.1011463.g006 Fig 6 Exogenous application of FolRDR1-dsRNA alleviates the development of Fusarium wilt disease in Fol pre-treatment tomato seedlings. 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). (TIF) Click here for additional data file. 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. (TIF) Click here for additional data file. 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. (TIF) Click here for additional data file. S4 Fig The analysis of GO. Knockouting of FolRDR1 mainly affected the metabolic pathway in both KO strains. (TIF) Click here for additional data file. 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. (TIF) Click here for additional data file. 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). (TIF) Click here for additional data file. 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. (TIF) Click here for additional data file. 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). (TIF) Click here for additional data file. 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). (TIF) Click here for additional data file. 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). (TIF) Click here for additional data file. 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). (TIF) Click here for additional data file. S1 Table Primers used in this study. (DOC) Click here for additional data file. S2 Table List of all DEGs miRNA. (XLSX) Click here for additional data file. S3 Table List of all predicted targets of miRNA. (XLSX) Click here for additional data file. S4 Table Prediction of FolRDR1 off-target transcripts. (DOC) Click here for additional data file. S5 Table Sequences of 30-nt DNA fragments which uniformly distributed in the predicted regions in the CDS of FolRDR1. (DOC) Click here for additional data file. S6 Table The list of all expressed genes. (XLS) Click here for additional data file. 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. 10.1371/journal.ppat.1011463.r001 Decision Letter 0 Ding Shou-Wei Section Editor Wang Nian Academic Editor © 2023 Ding, Wang 2023 Ding, Wang https://creativecommons.org/licenses/by/4.0/ 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. Submission Version0 4 Feb 2023 Dear Prof. Ouyang, Thank you very much for submitting your manuscript "Artificial trans‐kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts. Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Nian Wang Academic Editor PLOS Pathogens Shou-Wei Ding Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 *********************** Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #1: In the current manuscript, Ouyang and colleagues showed that RDR1 of the plant-pathogen Fusarium oxysporum is required for penetrability, sporulation and virulence on tomato. Further on, the authors utilized SIGS to silence rdr1 and demonstrated that silenced fungi display similar phenotypes as the rdr1 KO strains. Additionally, the authors used RT-PCR and labeled –dsRNA to show that RDR1-directed dsRNA is stable and can be systemically transported in fungal hyphae and the tomato plant host. Last, the authors showed that spraying tomato seedling with RDR1-directed dsRNA inhibited fungal infection and suggested that such treatment might be utilized to control fusarium wilt in the future. The described experiments are well designed, the manuscript is well written and the results provided in this manuscript will appeal to the readers of PLOS Pathogens. However, I do have some comments about specific experiments that should be addressed. Reviewer #2: Tomato wilt disease caused by Fusarium oxysporum f.sp. Lycopersici (Fol) is a severe fungal disease that threatens global tomato production. In the paper, they first characterize the FolRDR1 (RNA-dependent RNA polymerase 1) gene and find that it is essential for the development and pathogenicity of Fol. In this manuscript, a biocontrol agent FolRDR1-RNAi-SIGS was developed and can alleviate the development of tomato wilt disease in the lab without off-target of FolRDR1-RNAi in tomato, which is significant progress for the biocontrol of Fusarium wilt disease. The data support the claims in the paper. The manuscript is well-organized and written. I only have one major comment. Reviewer #3: This study characterized the function of FolRDR1 (RNA-dependent RNA polymerase 1) in Fusarium oxysporum development and pathogenicity, and applied Spray-Induced Gene Silencing (SIGS) strategy to control the tomato wilt disease. The authors successfully adopted SIGS strategy in the tomato- F. oxysporum pathosystem, however, it’s not novel enough as the SIGS strategy has been successfully used in multiple pathosystems including the barley-F. graminearum pathosystem. The title of the manuscript is “Artificial trans‐kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease”, however, the experiments were about the taken up of external dsRNAs by Fol or tomato to induce RNAi, directly taking up of dsRNA from tomato by Fol has not been experimentally demonstrated. Although this study has the potential to provide new insight into the management of tomato Fusarium wilt disease, the manuscript in the current format lacks important information and requires extensive modifications before a new submission. ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #1: -All main figures – all pictures are in poor quality and it's really difficult to judge the plate and plant phenotypes since it is all blurry. -The choice of RDR1 as the silencing target in Fol is very peculiar and not well explained. Why would the authors want to silence a component of the cell silencing machinery? Wouldn't it hurt the efficiency of the silencing itself? Why was this gene silenced and not other housekeeping gene that is required for survival? In addition, the characterization of the rdr1 mutants appear to be detached from the putative cellular function of this gene. It will be more appropriate to check whether gene silencing is affected in this mutant as well. -Figure 6D – According to this figure the authors showed that the amount of Fol in Fol-infected plants was only three folds of the mock infected plants (that are not supposed to contain any Fol at all!!!) which seems unreasonable. How specific are the IGS primers? Is it possible that they are also amplifying other fungal endophytes in the plant? Can the authors repeat this experiments with a different set of primers? -Figure 5 – the presence of the sdRNA in the stem and the root should be monitored by RT-PCR or qRT-PCR as well. Reviewer #2: • In the paper, the authors claim that the application of FolRDR1-dsRNAs on the surface of pre-infected tomato seedling leaves significantly inhibited the development of Fusarium wilt. Since FolRDR1-RNAi-SIGS works like a bio-pesticide, what are results of applying FolRDR1-dsRNAs before Fol infection? Whether it can provide better protection in the lab? Reviewer #3: 1. The knockout experiments indicated that the FolRDR1 is required for the vegetative growth and asexual reproduction in Fol (Figure 1-2). As the authors mentioned that RDRs interact with RNAi machinery and sense transgene-induced silencing to generate dsRNA molecules in fungi (Line 95-103, 266-269), whether the function of RNA silencing pathway and accumulation of sRNA was affected in the FolRDR1-KO strains? Koch et al. (2016) demonstrated that the F. graminearum DCL1 protein is required for exogenous dsRNA processing and intact fungal silencing machinery was required for SIGS in F. graminearum. If the FolRDR1 is essential for sRNA accumulation, when FolRDR1 was silenced by exogenous FolRDR1-dsRNA, this would in turn interfere with the own RNAi process of Fol. In order to ensure the efficiency of SIGS, the function of dsRNA targeted genes in the pathogen RNAi silencing process should be carefully considered. I suggest that small RNA sequencing analysis or northern blot should be carried out to explore the function of FolRDR1 and confirm the intact RNAi machinery of the FolRDR1-KO strains. 2. Line 120-125. The authors found that FolRDR1 was highly conservative in different F. oxysporum races, it is necessary to understand its conservation across species for a functional important protein. And the authors should make clear that how many RDR genes are present in the genome of Fol, and why FolRDR1 was chose for knockout. 3. The authors proved that artificial FolRDR1-dsRNAs can be taken up by Fol, and the mRNA level of RDR1 is repressed (Fig3). I suggest using Northern blot to detect the corresponding RDR1-sRNAs accumulated in the fungal mycelia to demonstrate that the dsRNA were processed into sRNAs in Fol. 4. The fluoresce signals observed in the undaubed leaves were weak and uneven in fig5C, the brighter spots look like autofluorescence from damaged leave cells. The quality of the pictures should be improved. In the stem vascular tissue (Fig5D), whether fluorescence signals in phloem and xylem can be distinguished with higher magnification. In addition, why the fluoresce signals were rich in the root hair in fig5E? And most importantly, whether the FolRDR1-sRNAs accumulated in tomato roots after taking up the FolRDR1-dsRNAs from leaves? 5. Spray of FolRDR1-dsRNAs alleviated the development of tomato wilt disease in fig6. However, the authors just observed the development of tomato wilt disease, did not monitor the accumulation of the FolRDR1-sRNAs in the infected tomato, neither detect the transcript level of FolRDR1 gene during infection. And I think it’s important to verify that the FolRDR1-dsRNAs or the FolRDR1-sRNAs were taken up by the Fol, and processed in the mycelia to induce the RNAi in the pathogen. Only then can it be termed cross-kingdom RNAi. 6. Many results lack experimental support in the Materials and methods section and the experimental conditions are not well described. Some examples, (1) Line 170, 179, 210, 230, 376-377, and 380-382, the concentration or the amount of the dsRNAs used? (2) Line 198-202, what’s the experimental conditions set to explore the optimal treatment in fig4D and fig4E. (3) How long the Fol took to produce conidia? And how to assess the production of the conidia in fig1D and fig4C? (4) Line 229-231, How long a gap should take to spray the dsRNA after inoculation of Fol? (5) For all the inoculation experiments, how many tomato seedlings were inoculated in one experiment, there was no statistics on the severity of the disease in fig 2C and fig6. ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #1: -lines 227-255/figure 6 – how long after dsRNA treatment were the plants infected with Fol? -How long after spraying with dsRNA were the leaves collected for RNA-seq analysis? dsRNA was previously suspected to act as an immune elicitor. The fact that neither dsGFP nor dsRDR1 caused any significant transcriptional changes in the plant is a bit surprising, unless the samples were collected long after spray application. -Please provide a table of all the DEGs which were identified in the RNAseq analyses and their fold change. -All figure legends – the number of times the experiments were repeated and the number of biological replicates in each experiment should be stated in the figure legends. -Fig 2C – please split this section into three: Fig 2C - tomato pictures, Fig 2D – stem staining, Fig 2E –outgrowth assay. -Figure S1B – why tomato actin was used for the tree construction? Is it a mistake? -Figure S2B – The top (WT) and bottom (mutants) bands in the left gel are too close together and it's hard to really distinguish the size difference. Please repeat the experiment the run the gel longer. -Table S2 - Please change the table title from "Table S1" to "Table S2" -Line 145 – sentence is not clear. Please rephrase it. -Line 212- remove the word "at" -Line 235-236 – please rephrase the sentence -Line 285-286 - please rephrase the sentence Reviewer #2: 1) Please label the statistical analysis results in Figures 4C, D, E, and 6D. 2) In Figure 6A, the 15 d next to the bottom panel should be 25 d. 3) Line 238-241, please analyze the interpret the data in Figure 6B-D. 4) Please add the catalog number for the agents used in work. 5) Line 305 should be seedling leaves. Reviewer #3: 1. Line 231-238, in the text the symptoms were described at 7 dpi and 25 dpi, while it was 15d in the figure 6A, and 2 weeks in the figure legend (Line 588). The descriptions must be consistent. 2. FolRDR1-dsRNA1 and FolRDR1-dsRNA2 was designed as the first half and second half of the RDR1 gene respectively according to Fig S5, I suggest that the location of the qRT-PCR primers should be labeled on the sequence to demonstrate the reliability of the transcription level of RDR1 detected by qRT-PCR. ********** PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: Guiyan Huang Figure Files: While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols 10.1371/journal.ppat.1011463.r002 Author response to Decision Letter 0 Submission Version1 4 Apr 2023 Attachment Submitted filename: Response to comments-PPATHOGENS-D-22-02186.pdf Click here for additional data file. 10.1371/journal.ppat.1011463.r003 Decision Letter 1 Ding Shou-Wei Section Editor Wang Nian Academic Editor © 2023 Ding, Wang 2023 Ding, Wang https://creativecommons.org/licenses/by/4.0/ 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. Submission Version1 24 Apr 2023 Dear Prof. Ouyang, Thank you very much for submitting your manuscript "Artificial trans‐kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. The reviewers commended the thorough effort in addressing the questions of 3 reviewers. Please address the request by Reviewer 3 on the trans‐kingdom RNAi in Fol. In addition, both reviewers 2 and 3 had some other minor issues that need to be addressed. Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments. Sincerely, Nian Wang Academic Editor PLOS Pathogens Shou-Wei Ding Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 *********************** The reviewers commended the thorough effort in addressing the questions of 3 reviewers. Please address the request by Reviewer 3 on the trans‐kingdom RNAi in Fol. In addition, both reviewers 2 and 3 had some other minor issues that need to be addressed. Reviewer Comments (if any, and for reference): Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #1: The authors properly addressed all issues that were raised in the first review cycle. The following small cosmetic adjustments should by added in the proof prior to publication: *All figure legends - Please change "biological replicates in each…" to "biological replicates were used in each…" *Figures 6 and S8– the sentence "presents the largest average, de present significance…" is unclear. Please rephrase. *Figure S2C - "C" marking is missing. 1 kb and 2 kb markings in the DNA ladder were accidentally switched. Please fix it. *Line 181 – please change "(Fig 2C)" to "(fig 2C-E)" *Lines 240-251 – As requested, the authors also checked the presence of dsRNA in the stem and root by RT-PCR in addition to the labeled RNA (Fig 5A). This should be addressed in the text as well. Reviewer #2: The author mentioned that they performed both pre-treatment and after-treatment with FolRDR1-dsRNAs experiments and found that no significant difference was shown between these two treatments. Please show the data of pre-treatment with FolRDR1-dsRNAs experiment in the supplementary figures. Reviewer #3: (No Response) ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #1: (No Response) Reviewer #2: (No Response) Reviewer #3: To demonstrate the trans‐kingdom RNAi in Fol, it is necessary not only to prove the RDR1-dsRNAs were taken up by Fol, but also that the dsRNAs were processed into small interfering (si)RNAs. The authors proved that artificial FolRDR1-dsRNAs can be taken up by Fol (Fig3) and tomato (Fig5) using confocal microscopy laser scanner and RT-PCR. But there was no evidence of the accumulation of corresponding RDR1-dsRNA-derived siRNAs. Fig 3E and Fig 5A were updated to show the corresponding mRNA levels of FolRDR1 in the fungal mycelia and FolRDR1-dsRNAs transferred from treated leaves to stems and roots. However, these results did not prove that FolRDR1-dsRNAs were processed into siRNAs to induce RNAi in Fol. The authors may have misunderstood my questions. I suggested using Northern blot to detect the RDR1-dsRNA-derived siRNAs accumulated in the fungal mycelia (Fig3) and in the infected tomato (Fig6). ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #1: (No Response) Reviewer #2: (No Response) Reviewer #3: The authors replied that for all the inoculation experiments, at least 30 tomato seedlings were inoculated for each treatment. However, it was “three biological replicates in each experiment” in the revised figure legends of fig 2 and fig 6. Please be consistent in your description of the experimental data. ********** PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 10.1371/journal.ppat.1011463.r004 Author response to Decision Letter 1 Submission Version2 1 Jun 2023 Attachment Submitted filename: Response to comments-PPATHOGENS-D-22-02186-V2.pdf Click here for additional data file. 10.1371/journal.ppat.1011463.r005 Decision Letter 2 Ding Shou-Wei Section Editor Wang Nian Academic Editor © 2023 Ding, Wang 2023 Ding, Wang https://creativecommons.org/licenses/by/4.0/ 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. Submission Version2 5 Jun 2023 Dear Prof. Ouyang, We are pleased to inform you that your manuscript 'Artificial trans‐kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease' has been provisionally accepted for publication in PLOS Pathogens. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated. IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS. Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Nian Wang Academic Editor PLOS Pathogens Shou-Wei Ding Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens ​orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens orcid.org/0000-0002-7699-2064 *********************************************************** We thank the authors for the diligent effort in addressing the concerns of the reviewers and congratulate the authors for the excellent work. Reviewer Comments (if any, and for reference): Reviewer's Responses to Questions Part I - Summary Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship. Reviewer #3: (No Response) ********** Part II – Major Issues: Key Experiments Required for Acceptance Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions. Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject". Reviewer #3: (No Response) ********** Part III – Minor Issues: Editorial and Data Presentation Modifications Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. Reviewer #3: (No Response) ********** PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #3: No 10.1371/journal.ppat.1011463.r006 Acceptance letter Ding Shou-Wei Section Editor Wang Nian Academic Editor © 2023 Ding, Wang 2023 Ding, Wang https://creativecommons.org/licenses/by/4.0/ 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. 13 Jun 2023 Dear Prof. Ouyang, We are delighted to inform you that your manuscript, "Artificial trans‐kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease," has been formally accepted for publication in PLOS Pathogens. We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication. 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