Spray-induced Gene Silencing Research Articles

Approaches for the protection of Solanum tuberosum from late blight through the regulation of inf1 and inf4 elicitin genes

Late blight is a disease affecting economically important crops, which is caused by the oomycete Phytophthora infestans. Mainly, fungicides are used against it, however they may harm the environment when used in large quantities. Spray-induced gene silencing (SIGS) can become an alternative to the classical fungicides in the fight against P. infestans. SIGS involves the treatment of plants with double-stranded RNA (dsRNA) which triggers the RNA interference mechanism to suppress translation of the target gene. So, it is possible to suppress the expression of genes.
 We have chosen two P. infestans genes, inf1 and inf4 involved in the different stages of the parasite development. For the production of dsRNA in E. coli HT115 two expression vectors were constructed on the basis of the L4440 plasmid, each carrying a cDNA fragment of these genes between two T7 phage promoters oriented in the opposition to each other.
 To evaluate the protective effect of the dsRNA potato explants were treated with a solution containing dsRNA of one of the genes or their combination. 24 hours later, phytophthora zoospores were inoculated with the registration of the effect after 5 days.
 According to the measurement results, the lesion area was significantly larger in plants treated with water than in the other three experimental groups that were treated with dsRNA.

Identification of Essential Genes for the Establishment of Spray-Induced Gene Silencing-Based Disease Control in Fusarium graminearum.

As resistance to chemical fungicides continues to increase inFusarium graminearum, there is a growing need to develop novel disease control strategies. To discover essential genes that could serve as new disease control targets, we selected essential gene candidates that had failed to be deleted in previous studies. Thirteen genes were confirmed to be essential, either by constructing conditional promoter replacement mutants or by employing a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated editing strategy. We synthesized double-stranded RNAs (dsRNAs) targeting these essential genes and analyzed their protective effects in plants using a spray-induced gene silencing (SIGS) method. When dsRNAs targeting Fg10360, Fg13150, and Fg06123 were applied to detached barley leaves prior to fungal inoculation, disease lesions were greatly reduced. Our findings provide evidence of the potential of essential genes identified by a SIGS method to be effective targets for the control of fungal diseases.

Spray-induced gene silencing as a potential tool to control rubber tree powdery mildew disease

Powdery mildew of rubber tree, caused by the obligate biotrophic fungus Erysiphe quercicola, is a serious disease that occurs in all rubber tree growing regions. The disease is mainly controlled by fungicides. Here, an alternative means for powdery mildew control by spraying double-stranded RNAs (dsRNA), which target E. quercicola genes, was investigated. First, we demonstrated that the conidia of E. quercicola can take-up environmental dsRNA. Furthermore, the spraying of dsRNA targeting E. quercicola genes can effectively reduce both the expression of the target gene of the pathogen and the rubber tree powdery mildew severity. The reduction in disease severity was up to 50 % compared to water-treated leaves. These results suggest that Spray-induced gene silencing (SIGS) of the pathogen genes will provide an additional tool to control rubber tree powdery mildew as well as a method to study the function of genes of E. quercicola.

An RNA interference (RNAi) target with potential to control Dothistroma needle blight

Spray-induced gene silencing (SIGS) involves exogenous spray applications of double-stranded RNA (dsRNA) molecules targeting specific genes, such as those essential to pathogens. This technique has shown great potential for controlling fungal pathogens of horticultural and agricultural plants, but few studies have evaluated its application to forest pathogens. Here we report the first demonstration of exogenous application of dsRNA molecules targeting a gene required for virulence in the foliar pine pathogen Dothistroma septosporum, the causal agent of Dothistroma needle blight (DNB). Constructs expressing sense and antisense dsRNAs were generated targeting a 509 bp fragment of the dothistromin toxin regulatory gene (DsAflR) in D. septosporum. DsAflR is required for the production of dothistromin, a phytotoxin essential for full pathogen virulence. We found that exogenous dsRNA is taken up and the RNA silencing mechanism is functional in D. septosporum through a series of in vitro and in planta trials, such as monitoring the uptake of fluorescently labelled DsAflR-dsRNA into the cell by confocal microscopy and quantifying the extent of gene silencing by quantitative Polymerase Chain Reaction (qPCR). In vitro applications of DsAflR-dsRNA to D. septosporum reduced the accumulation of DsAflR mRNA compared to controls and, in some cases, were found to be statistically significant, despite a high level of variability in the results. Our study also demonstrated a reduction in DNB symptoms on infected pine needles sprayed with DsAflR-dsRNA, which was further supported by a significant reduction in fungal biomass. This preliminary study highlights the potential for SIGS as a future management scheme for controlling the forest pathogen D. septosporum, as well as other forest pathogens.

Sprayable double-stranded RNA mediated RNA interference reduced enzymatic browning of fresh-cut potatoes

Postharvest quality deterioration is the major reason for food loss and waste, which hinder global food security, environmental sustainability and human health. Spray-induced gene silencing (SIGS) is a technique involving spraying double-stranded RNA (dsRNA) onto the plant surface for targeted RNA interference, providing a new solution for pests and disease control in agriculture products. Here, fresh-cut browning, which is a common and visible quality deterioration in potatoes, was taken as the indicator for the exploratory SIGS technology in postharvest quality deterioration control. The polyphenol oxidase (PPO) encoding genes StPPO and StPPOD, and the phenylalanine ammonia-lyase (PAL) encoding gene StPAL2, were identified as important transcripts associated with the browning process in freshly-cut potatoes of the Yunshu 505 cultivar. Inhibition of StPPO and StPAL2 using dsRNA-PPO and dsRNA-PALX resulted in a decrease in the expression levels of the two genes, consequently leading to reduced activities of polyphenol oxidase and phenylalanine ammonia-lyase, as well as a decrease in the brown index. Spraying a mixture of these two dsRNAs demonstrated browning control capabilities similar to those of ascorbic acid. The distribution of dsRNA-Cy5 in cells confirmed its uptake and internalization by fresh-cut potatoes. In addition, the application of dsRNA-MYB12 resulted in a reduction of up to 78 % in tuber StMYB12 levels and decreased StPPO and StPAL1 expression. Spraying dsRNAs effectively reduced fresh-cut browning, highlighting the potential application of SIGS in reducing the browning of fresh-cut potatoes.

Exogenous dsRNA trigger RNAi in Venturia inaequalis resulting in down regulation of target genes and growth reduction.

Venturia inaequalis is an apple scab causing fungal pathogen. It is a highly contagious and destructive pathogen which rapidly spreads infection in the surrounding orchards if not managed. The management and control of disease require multiple fungicides to be sprayed at different development stages of the apple. Persistent applications of fungicides also raises environmental concerns. Here, we demonstrate the potential of using spray induced gene silencing (SIGS) by developing target specific gene constructs for the synthesis of corresponding double-stranded RNA (dsRNA). The exogenous application of dsRNAs was found to reduce mycelial growth and spore formation of V. inaequalis on culture plates. Four genes of V. inaequalis viz. CIN1, CE5, VICE12 and VICE16 which get upregulated during infection, were selected as targets for the development of gene construct expressing the corresponding dsRNA. The effect of exogenously supplied in vitro synthesized dsRNA on V. inaequalis was assessed in culture bioassay experiments with respect to growth, and spore formation. The expression level of the target genes in treated and control fungus was evaluated using quantitative PCR. Fungus treated with VICE12 targeted dsRNA showed maximum reduction in colony size (~ 55%), conidia formation (~ 93%) and expression level of the corresponding gene (2.2 fold), which was followed by CIN1-dsRNA. VICE16-dsRNA treatment was least effective with 32% reduction in growth, the non-significant effect of conidial spore formation and 1.13 fold down regulation of corresponding target gene expression level. The result of this investigation validates the hypothesis that RNAi is evoked in V. inaequalis by exogenously supplied dsRNA and spray induced gene silencing (SIGS) based solutions may reduce burden of fungicide usage on apple crop against apple scab disease in future.

Low Molecular Weight and High Deacetylation Degree Chitosan Batch Alleviates Pathogenesis, Toxin Accumulation, and Fusarium Gene Regulation in Barley Leaf Pathosystem.

Fusarium graminearum is a cosmopolitan fungal pathogen that destroys cereal production, in terms of loss of yield and grain contamination with mycotoxins, worldwide. Chitosan is a natural biopolymer abundant in the environment with proven antifungal properties that also acts as a plant immunity elicitor. Despite a number of articles, there is a lack of systematic comparison of antifungal activity of diverse batches of chitosan. The current study aimed to test the inhibitory effects of a collection of diverse chitosan samples on the growth and production of F. graminearum toxins, validated by changes in the Fusarium transcriptome. Experiments included testing antifungal activity of different chitosan samples, the application of the best performing one in vitro to investigate the impact on F. graminearum growth, followed by analyzing its effect on Fusarium toxins accumulation, and Fusarium transcriptomics in the barley leaf pathosystem. Confirmatory antifungal assays revealed that CS_10, a specific batch of chitosan, retarded Fusarium growth with an application concentration of 200 ppm, significantly reducing toxin synthesis and disease symptoms in Fusarium-inoculated barley leaves. RNA-Seq analysis of F. graminearum in barley leaf pathosystem exposed to CS_10 showed a list of differentially expressed genes involved in redox balance, cell respiration, nutrient transport, cell wall degradation enzymes, ergosterol biosynthesis, and trichothecenes production. The genes functioning in these essential pathways are discussed and assigned as critical checkpoints to control Fusarium infections. The results suggest some important molecular targets in F. graminearum that may be suitable in gene-specific targeting or transgene-free methods, such as spray-induced gene silencing during host-pathogen interactions.

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Spray-Induced Gene Silencing of SsOah1 and SsCyp51 confers protection to Nicotianabenthamiana and Brassicajuncea against Sclerotiniasclerotiorum

Brassicajuncea is a major oilseed, vegetable and condiment crop in the Indian subcontinent. Its yields are compromised by the aggressive broad host range Ascomycete Sclerotiniasclerotiorum, which causes Sclerotinia Stem Rot (SSR). The limited effectiveness of fungicides, the rapid development of resistance to fungicides, and the long-term viability of sclerotia in the soil are crucial bottlenecks in controlling the disease. The discovery of the uptake of environmental RNA by fungal pathogens has opened up avenues for exploiting Spray-Induced Gene Silencing (SIGS) as a tool for mitigating plant diseases. We studied the impact of SIGS-based knockdown of S.sclerotiorumSsOah1 (SS1G_08218) and SsCyp51 (SS1G_04805), involved in virulence and fungal development, respectively, on two hosts- N.benthamiana and B.juncea. A knockdown of the two genes delayed disease initiation, reduced lesion development, and slowed disease progression within B.juncea. However, this silencing was concentration and host-dependent. Further, it also altered fungal hyphal morphology and growth, indicating its utility as a functional genomics tool. Our results identify SsCyp51 as a novel and viable target for SSR control along with SsOah1 and corroborate SIGS as a robust method for crop protection against broad-host-range fungal pathogens.

A CRISPR/Cas9-Based Study of CgloRPCYG, a Gene That Regulates Pathogenicity, Conidial Yield, and Germination in Colletotrichum gloeosporioides

The filamentous fungus Colletotrichum gloeosporioides is the causative agent of one of the most serious diseases that damage plant fruit. In this study, we discovered and experimentally characterized a new gene in Colletotrichum gloeosporioides named CgloRPCYG. The CRISPR/Cas9 knockout mutant and complementary strain of CgloRPCYG were then obtained by polyethylene glycol (PEG)-mediated protoplast transformation to determine the related phenotypes. Compared with the wild-type strain and complementary mutant, the pathogenicity of the CRISPR/Cas9 knockout mutant was significantly decreased, the conidial yield was significantly reduced, and conidial germination was significantly delayed. These data indicate that CgloRPCYG contributes to pathogenicity, conidial yield, and germination in C. gloeosporioides. The successful application of the CRISPR/Cas9 system in C. gloeosporioides also confirms its utility in filamentous fungi for fundamental research and practical application. Furthermore, CgloRPCYG is a potential target gene for use in the development of plant protection technologies, such as spray-induced gene silencing, with the aim of controlling plant anthracnose disease caused by C. gloeosporioides.

Spray-induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control.

Spray-induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double-stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole-fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii-Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis-antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a, lipase 1, and acetyl-CoA oxidase in energy production; and genes involved in manipulation of the plant host via abscisic acid metabolism (9-cis-epoxycarotenoid dioxygenase, xanthoxin dehydrogenase, and a putative abscisic acid G-protein coupled receptor) and secretion of the effector protein, effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator-Vitis vinifera system and tested six successful targets identified using the G. orontii-A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii-A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.

Artificial trans-kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease.

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.

A Combinatorial Nanobased Spray-Induced Gene Silencing Technique for Crop Protection and Improvement.

Recent research reports have shown that plant pests and pathogens have depleted the crop yield widely, which has led to an increased dependence on commercial pesticides and fungicides. Increased usage of these pesticides has also shown adverse effects on the environment, therefore many techniques have been implemented for solving the issue, some of which include using nanobioconjugates, RNA(i), which put into use double-stranded RNAs to inhibit gene expression. A more innovative and eco-friendly strategy includes spray induced gene silencing, which is being increasingly implemented. This review delves into the eco-friendly approach of spray induced gene silencing (SIGS) in combination with nanobioconjugates, which have been used concerning various plant hosts and their pathogens to provide improved protection. Furthermore, nanotechnological advancements have been understood by addressing the scientific gaps to provide a rationale for the development of updated techniques in crop protection.

RNA Interference Past and Future Applications in Plants.

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement.

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

Examining spray-induced gene silencing for pollen beetle control

Pollen beetles are a transcontinental threat to oilseed rape cultivation. Conventional insecticide use has resulted in resistance development, and can furthermore lead to losses of non-target organisms, including those that contribute to natural biological control of pollen beetle populations. Biosafe alternatives to synthetic pesticides must be explored. Double-stranded RNA (dsRNA) targeting pollen beetle, by the RNA interference (RNAi) mode of action, has been previously demonstrated under laboratory conditions. The present study examined, under a whole plant context, the potential for spray-induced gene silencing (SIGS) in pollen beetle. We confirmed translocation of sprayed dsRNA to later-emerging, non-sprayed dietary tissues of oilseed rape, as well as infiltration of sprayed dsRNA to the inner tissues of reproductive buds (pollen beetle’s food source). No effect on survival was observed. The inefficacy of SIGS observed here highlights the requirement for more effective SIGS approaches to pollen beetle management. The translocation of sprayed dsRNA within oilseed rape plants, and the non-significant yet detectable effect on target mRNA downregulation, together suggest potential for future optimization of a SIGS approach against pollen beetle.

Artificial nanovesicles for dsRNA delivery in spray-induced gene silencing for crop protection.

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNAencapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.

Nanoparticle carriers enhance RNA stability and uptake efficiency and prolong the protection against Rhizoctonia solani

Spray-induced gene silencing (SIGS) can inhibit plant diseases by topical application of double- (dsRNA) or single-stranded (sRNA) RNA molecules onto plants to silence virulence-related pathogen genes. However, the on-field application of SIGS is limited by the instability of naked RNA and low RNA uptake by pathogens. Nanoparticles have been used as RNA carriers to enhance RNA silencing. Rice sheath blight caused by Rhizoctonia solani (R. solani) is one of the most devastating fungal diseases in rice (Oryza sativa L.). In this study, we aimed to explore the protective effects of nanoparticle-delivered dsRNA against rice sheath blight. The key pathogenic genes, RsAGO1 and RsAGO2, of R. solani were screened as targets for dsRNA. Chitosan (CS), polyethyleneimine (PEI), protamine, carbon quantum dot (CQD), polyamidoamine (PAMAM), and chitosan/SPc complex (CSC) were selected as dsRNA carriers. All the evaluated nanoparticles could assemble with dsRNA to form nanoparticle-dsRNA complexes, and CQD and CSC showed improved dsRNA load capacity. Particularly, CSC could enhance the stability of dsRNA and cause a 7% reduction in fluorescence intensity after nuclease treatment. CSC and CS effectively enhanced the efficiency of dsRNA uptake by pathogens. Furthermore, CSC could reduce pathogen infection and prolong the protection time of dsRNA by up to 20 days. Overall, this study provides a novel and efficacious SIGS-based strategy for producing RNA-based fungicides.

Host-delivered-RNAi-mediated resistance in bananas against biotic stresses

Both the biotic and abiotic stressors restrict the yield potential of many crops, including bananas. Bananas belong to the genus Musa and are the world’s most popular and widely produced fruit for their nutritional and industrial importance. The demand for bananas is growing each day worldwide. However, different pest infestations are hampering the production of bananas, making it a matter of concern for global food security. Several biotechnological tools and applications including RNA interference (RNAi) have been employed to enhance the biotic stress resistance in plants. The capacity to silence targeted genes at transcriptional and post-transcriptional levels makes the RNAi technique a popular choice for gene knock-down and functional genomics studies in crops. Silencing of different suppressor molecule coding genes through RNAi helps crops to combat the detrimental effects of plant pathogens. The host-induced gene silencing (HIGS) technology, also known as the host-delivered RNAi (HD-RNAi), is nowadays gaining popularity due to its ability to target an array of pathogens, comprising bacteria, nematodes, fungi, viruses, and insects. This methodology is employed to manage disease pest outbreaks in a diverse range of crop species, including bananas. Besides HIGS, virus-induced and spray-induced gene silencing (VIGS and SIGS, respectively) are the potential approaches where RNAi technology is exploited to control plant-pathogenic diseases. The current review emphasizes the different kinds of diseases of bananas and the potential of HD-RNAi, a new-age and promising technology to build a barrier against significant crop and economic loss.

MicroRNA-like RNA Functions Are Required for the Biosynthesis of Active Compounds in the Medicinal Fungus Sanghuangporus vaninii.

miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.