The RNA-binding domain of DCL3 is required for long-distance RNAi signaling.

Small RNA (sRNA)-mediated RNA silencing (also known as RNA interference, or RNAi) is a conserved mechanism in eukaryotes that includes RNA degradation, DNA methylation, heterochromatin formation and protein translation repression. In plants, sRNAs can move either cell-to-cell or systemically, thereby acting as mobile silencing signals to trigger noncell autonomous silencing. However, whether and what proteins are also involved in noncell autonomous silencing have not been elucidated. In this study, we utilized a previously reported inducible RNAi plant, PDSi, which can induce systemic silencing of the endogenous PDS gene, and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity. We confirmed that the C-terminus of DCL3, including the predicted RNA-binding domain, is capable of binding short RNAs. Mutations affecting RNA binding, but not processing activity, reduced systemic PDS silencing, indicating that DCL3 binding to RNAs is required for the induction of systemic silencing. Cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves. Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00124-6.

Advances in the mechanisms and applications of RNA silencing in crop protection.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes, which plays vital roles in plant development and response to biotic and abiotic stresses. The discovery of trans-kingdom RNAi and interspecies RNAi provides a theoretical basis for exploiting RNAi-based crop protection strategies. Here, we summarize the canonical RNAi mechanisms in plants and review representative studies associated with plant-pathogen interactions. Meanwhile, we also elaborate upon the principles of host-induced gene silencing, spray-induced gene silencing and microbe-induced gene silencing, and discuss their applications in crop protection, thereby providing help to establish novel RNAi-based crop protection strategies.

Erratum: Publisher Correction: The RNA-binding domain of DCL3 is required for long-distance RNAi signaling.

[This corrects the article DOI: 10.1007/s42994-023-00124-6.].

An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae.

Accurate determination of protein localization, levels, or protein-protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.

Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens.

Small RNA (sRNA)-mediated trans-kingdom RNA interference (RNAi) between host and pathogen has been demonstrated and utilized. However, interspecies RNAi in rhizospheric microorganisms remains elusive. In this study, we developed a microbe-induced gene silencing (MIGS) technology by using a rhizospheric beneficial fungus, Trichoderma harzianum, to exploit an RNAi engineering microbe and two soil-borne pathogenic fungi, Verticillium dahliae and Fusarium oxysporum, as RNAi recipients. We first detected the feasibility of MIGS in inducing GFP silencing in V. dahliae. Then by targeting a fungal essential gene, we further demonstrated the effectiveness of MIGS in inhibiting fungal growth and protecting dicotyledon cotton and monocotyledon rice plants against V. dahliae and F. oxysporum. We also showed steerable MIGS specificity based on a selected target sequence. Our data verify interspecies RNAi in rhizospheric fungi and the potential application of MIGS in crop protection. In addition, the in situ propagation of a rhizospheric beneficial microbe would be optimal in ensuring the stability and sustainability of sRNAs, avoiding the use of nanomaterials to carry chemically synthetic sRNAs. Our finding reveals that exploiting MIGS-based biofungicides would offer straightforward design and implementation, without the need of host genetic modification, in crop protection against phytopathogens.

Comprehensively Optimizing Fenton Reaction Factors for Antitumor Chemodynamic Therapy by Charge-Reversal Theranostics.

Chemodynamic therapy (CDT) is a highly tumor-specific treatment, while its efficacy is compromised by the intratumoral Fenton reaction efficiency, which is determined by the following reaction factors, including the availability of Fenton ions (e.g., Fe2+), the amount of H2O2, and the degree of acidity. Synchronous optimization of these factors is a big challenge for efficient CDT. Herein, a strategy of comprehensively optimizing Fenton reaction factors was developed for traceable multistage augmented CDT by charge-reversal theranostics. The customized pH-responsive poly(ethylene)glycol-poly(ß-amino esters) (PEG-PAE) micelle (PM) was prepared as the carrier. Glucose oxidase (GOx), Fe2+, and pH-responsive second near-infrared (NIR-II) LET-1052 probe were coloaded by PM to obtain the final theranostics. The activity of metastable Fe2+ remained by the unsaturated coordination with PEG-PAE. Then tumor accumulation and exposure of Fe2+ were achieved by charge-reversal cationization of PEG-PAE, which was further enhanced by a GOx catalysis-triggered pH decrease. Together with the abundant H2O2 generation and pH decrease through GOx catalysis, the limiting factors of the Fenton reaction were comprehensively optimized, achieving the enhanced CDT both in vitro and in vivo. These findings provide a strategy for comprehensively optimizing intratumoral Fenton reaction factors to overcome the intrinsic drawbacks of current CDT.

DeSUMOylation of a Verticillium dahliae enolase facilitates virulence by derepressing the expression of the effector VdSCP8.

The soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, causes vascular wilts in a wide variety of economically important crops. The molecular mechanism of V. dahliae pathogenesis remains largely elusive. Here, we identify a small ubiquitin-like modifier (SUMO)-specific protease (VdUlpB) from V. dahliae, and find that VdUlpB facilitates V. dahliae virulence by deconjugating SUMO from V. dahliae enolase (VdEno). We identify five lysine residues (K96, K254, K259, K313 and K434) that mediate VdEno SUMOylation, and SUMOylated VdEno preferentially localized in nucleus where it functions as a transcription repressor to inhibit the expression of an effector VdSCP8. Importantly, VdUlpB mediates deSUMOylation of VdEno facilitates its cytoplasmic distribution, which allows it to function as a glycolytic enzyme. Our study reveals a sophisticated pathogenic mechanism of VdUlpB-mediated enolase deSUMOylation, which fortifies glycolytic pathway for growth and contributes to V. dahliae virulence through derepressing the expression of an effector.

A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

Hyphopodium-Specific Signaling Is Required for Plant Infection by Verticillium dahliae.

For successful colonization, fungal pathogens have evolved specialized infection structures to overcome the barriers present in host plants. The morphology of infection structures and pathogenic mechanisms are diverse according to host specificity. Verticillium dahliae, a soil-borne phytopathogenic fungus, generates hyphopodium with a penetration peg on cotton roots while developing appressoria, that are typically associated with leaf infection on lettuce and fiber flax roots. In this study, we isolated the pathogenic fungus, V. dahliae (VdaSm), from Verticillium wilt eggplants and generated a GFP-labeled isolate to explore the colonization process of VdaSm on eggplants. We found that the formation of hyphopodium with penetration peg is crucial for the initial colonization of VdaSm on eggplant roots, indicating that the colonization processes on eggplant and cotton share a similar feature. Furthermore, we demonstrated that the VdNoxB/VdPls1-dependent Ca2+ elevation activating VdCrz1 signaling is a common genetic pathway to regulate infection-related development in V. dahliae. Our results indicated that VdNoxB/VdPls1-dependent pathway may be a desirable target to develop effective fungicides, to protect crops from V. dahliae infection by interrupting the formation of specialized infection structures.

Synchronous Interventions of Glucose and Mitochondrial Metabolisms for Antitumor Bioenergetic Therapy.

Hydrogen sulfide (H2 S)-based mitochondrial bioenergetic intervention is an attractive therapeutic modality. However, its therapeutic efficacy is limited owing to metabolic plasticity, which allows tumors to shift their metabolic phenotype between oxidative phosphorylation and glycolysis for energy compensation. To overcome this flexibility, a glycopolymer containing a caged H2 S and hydrogen peroxide (H2 O2 ) dual-donor (1-thio-ß-D-glucose [thioglucose]) is synthesized to wrap glucose oxidase (GOx) for complete depletion of tumorigenic energy sources. The loaded GOx catalyzes the glutathione-activated thioglucose to generate cytotoxic H2 S/H2 O2 , which further induces synergistic defects in mitochondrial function by suppressing cytochrome c oxidase expression and damaging the mitochondrial membrane potential. GOx also blocks glycolysis by depleting endogenous glucose. This synchronous intervention strategy exhibits good anticancer performance, broadening the horizon of antitumor bioenergetic therapy.

Contaminating transfection complexes can masquerade as small extracellular vesicles and impair their delivery of RNA.

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV-producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid-based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid-based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV-mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably-expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.

Verticillium dahliae Secretes Small RNA to Target Host MIR157d and Retard Plant Floral Transition During Infection.

Bidirectional trans-kingdom RNA silencing [or RNA interference (RNAi)] plays a key role in plant-pathogen interactions. It has been shown that plant hosts export specific endogenous miRNAs into pathogens to inhibit their virulence, whereas pathogens deliver small RNAs (sRNAs) into plant cells to disturb host immunity. Here, we report a trans-kingdom fungal sRNA retarding host plant floral transition by targeting a miRNA precursor. From Arabidopsis plants infected with Verticillium dahliae, a soil-borne hemibiotrophic pathogenic fungus that causes wilt diseases in a wide range of plant hosts, we obtained a number of possible trans-kingdom V. dahliae sRNAs (VdsRNAs) by sequencing AGO1-immunoprecipitated sRNAs. Among these, a 24-nt VdsRNA derived from V. dahliae rRNA, VdrsR-1, was shown to be an actual trans-kingdom VdsRNA that targets the miR157d precursor MIR157d, resulting in increased rather than reduced miR157d accumulation in V. dahliae-infected plants. Consistent with the miR157 family in the regulation of vegetative and floral transitions by targeting SPL genes in several plant species, we detected two SPL genes, SPL13A/B, that were notably reduced in V. dahliae-infected and VdrsR-1-expressing plants compared with control plants. Furthermore, V. dahliae-infected and VdrsR-1-expressing plants also displayed delayed vegetative phase change and floral transition compared to control plants. Taken together, we disclosed a novel mode of action for a trans-kingdom fungal sRNA, VdrsR-1, which was secreted into host cells to modulate plant floral transition by employing the miR157d/SPL13A/B regulatory module, leading to prolonged host vegetative growth that would undoubtedly benefit fungal propagation.

Trans-Kingdom RNA Silencing in Plant-Fungal Disease Control.

Trans-kingdom RNA interference (RNAi) has been reported in several plant-fungal pathosystems. Our recent works have demonstrated natural RNAi transmission from cotton plants into Verticillium dahliae, a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues, and successful application of trans-kingdom RNAi in cotton plants to confer Verticillium wilt disease resistance. Here, we provide a detailed protocol of cotton infection with V. dahliae, fungal hyphae recovery from infected cotton stems, and transmitted small RNA detection developed from our previous studies for trans-kingdom RNAi assays.

A fungal effector suppresses the nuclear export of AGO1-miRNA complex to promote infection in plants.

Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1­microRNA (AGO1­miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAi-dependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens.

Exploring the Effectiveness and Durability of Trans-Kingdom Silencing of Fungal Genes in the Vascular Pathogen Verticillium dahliae.

Host-induced gene silencing (HIGS) based on trans-kingdom RNA interference (RNAi) has been successfully exploited to engineer host resistance to pests and pathogens, including fungi and oomycetes. However, revealing the mechanisms underlying trans-kingdom RNAi between hosts and pathogens lags behind applications. The effectiveness and durability of trans-kingdom silencing of pathogenic genes are uncharacterized. In this study, using our transgenic 35S-VdH1i cotton plants in which dsVdH1-derived small RNAs (siVdH1) accumulated, small RNA sequencing analysis revealed that siVdH1s exclusively occur within the double-stranded (ds)VdH1 region, and no transitive siRNAs were produced beyond this region in recovered hyphae of Verticillium dahliae (V. dahliae). Accordingly, we found that VdH1 silencing was reduced over time in recovered hyphae cultured in vitro, inferring that once the fungus got rid of the 35S-VdH1i cotton plants would gradually regain their pathogenicity. To explore whether continually exporting dsRNAs/siRNAs from transgenic plants into recipient fungal cells guaranteed the effectiveness and stability of HIGS, we created GFP/RFP double-labeled V. dahliae and transgenic Arabidopsis expressing dsGFP (35S-GFPi plants). Confocal images visually demonstrate the efficient silencing of GFP in V. dahliae that colonized host vascular tissues. Taken together, our results demonstrate that HIGS effectively triggers long-lasting trans-kingdom RNAi during plant vasculature V. dahliae interactions, despite no amplification or transitivity of RNAi being noted in this soil-borne fungal pathogen.

Antrodia cinnamomea exerts an anti-hepatoma effect by targeting PI3K/AKT-mediated cell cycle progression in vitro and in vivo.

Antrodia cinnamomea is extensively used as a traditional medicine to prevention and treatment of liver cancer. However, its comprehensive chemical fingerprint is uncertain, and the mechanisms, especially the potential therapeutic target for anti-hepatocellular carcinoma (HCC) are still unclear. Using UPLC‒Q-TOF/MS, 139 chemical components were identified in A. cinnamomea dropping pills (ACDPs). Based on these chemical components, network pharmacology demonstrated that the targets of active components were significantly enriched in the pathways in cancer, which were closely related with cell proliferation regulation. Next, HCC data was downloaded from Gene Expression Omnibus database (GEO). The Cancer Genome Atlas (TCGA) and DisGeNET were analyzed by bioinformatics, and 79 biomarkers were obtained. Furtherly, nine targets of ACDP active components were revealed, and they were significantly enriched in PI3K/AKT and cell cycle signaling pathways. The affinity between these targets and their corresponding active ingredients was predicted by molecular docking. Finally, in vivo and in vitro experiments showed that ACDPs could reduce the activity of PI3K/AKT signaling pathway and downregulate the expression of cell cycle-related proteins, contributing to the decreased growth of liver cancer. Altogether, PI3K/AKT-cell cycle appears as the significant central node in anti-liver cancer of A. Cinnamomea.

RNA silencing: From discovery and elucidation to application and perspectives.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes. The discovery of natural trans-kingdom RNAi indicated that small RNAs act as signaling molecules and enable communication between organisms in different kingdoms. The phenomenon and potential mechanisms of trans-kingdom RNAi are among the most exciting research topics. To better understand trans-kingdom RNAi, we review the history of the discovery and elucidation of RNAi mechanisms. Based on canonical RNAi mechanisms, we summarize the major points of divergence around RNAi pathways in the main eukaryotes' kingdoms, including plants, animals, and fungi. We review the representative incidents associated with the mechanisms and applications of trans-kingdom RNAi in crop protection, and discuss the critical factors that should be considered to develop successful trans-kingdom RNAi-based crop protection strategies.

The Ghd7 transcription factor represses ARE1 expression to enhance nitrogen utilization and grain yield in rice.

The genetic improvement of nitrogen use efficiency (NUE) of crops is vital for grain productivity and sustainable agriculture. However, the regulatory mechanism of NUE remains largely elusive. Here, we report that the rice Grain number, plant height, and heading date7 (Ghd7) gene genetically acts upstream of ABC1 REPRESSOR1 (ARE1), a negative regulator of NUE, to positively regulate nitrogen utilization. As a transcriptional repressor, Ghd7 directly binds to two Evening Element-like motifs in the promoter and intron 1 of ARE1, likely in a cooperative manner, to repress its expression. Ghd7 and ARE1 display diurnal expression patterns in an inverse oscillation manner, mirroring a regulatory scheme based on these two loci. Analysis of a panel of 2656 rice varieties suggests that the elite alleles of Ghd7 and ARE1 have undergone diversifying selection during breeding. Moreover, the allelic distribution of Ghd7 and ARE1 is associated with the soil nitrogen deposition rate in East Asia and South Asia. Remarkably, the combination of the Ghd7 and ARE1 elite alleles substantially improves NUE and yield performance under nitrogen-limiting conditions. Collectively, these results define a Ghd7-ARE1-based regulatory mechanism of nitrogen utilization, providing useful targets for genetic improvement of rice NUE.

Trans-kingdom RNAs and their fates in recipient cells: advances, utilization, and perspectives.

The phenomenon and potential mechanisms of trans-kingdom RNA silencing (or RNA interference, RNAi) are among the most exciting topics in science today. Based on trans-kingdom RNAi, host-induced gene silencing (HIGS) has been widely applied to create crops with resistance to various pests and pathogens, overcoming the limitations of resistant cultivars. However, a lack of transformation technology in many crops limits the application of HIGS. Here, we describe the various fates of trans-kingdom RNAs in recipient organisms. Based on the assumption that small RNAs can be transferred between the host and its microbiome or among microbiome members, we propose a possible alternative strategy for plant protection against pathogens without the need for crop genetic modification.

Lysozyme Improves the Inhibitory Effects of Panax notoginseng Saponins on Phenotype Transformation of Vascular Smooth Muscle Cells by Binding to Ginsenoside Re.

Panax notoginseng saponins (PNS) have been used to treat cardiovascular diseases for hundreds of years in China. Lysozyme can bind to exogenous compounds and promote their activity. Nevertheless, knowledge of whether there is a synergistic role between lysozyme and PNS is far from sufficient. In this study, we show that the mixture of PNS and lysozyme synergistically inhibited platelet derived growth factor BB (PDGF-BB)-induced vascular smooth muscle cell (VSMC) viability, and in the five main components of PNS, GS-Re, but not GS-Rb1, NG-R1, GS-Rg1, or GS-Rd, reduced VSMC viability by combined application with lysozyme. Next, the supramolecular complexes formed by GS-Re and lysozyme were detected by mass spectrometry, and the binding ability increased with the concentration ratio of GS-Re to lysozyme from 4:1 to 12:1. In the supramolecular complexes, the relative contents of α-helix of lysozyme were increased, which was beneficial for stabilizing the structure of lysozyme. The 12:1 mixture of GS-Re and lysozyme (12.8 µmol/L GS-Re+1.067 µmol/L lysozyme) repressed PDGF-BB-induced VSMC viability, proliferation, and migration, which were associated with the upregulated differentiated markers and downregulated dedifferentiated markers. Finally, in CaCl2-induced rodent abdominal aortic aneurysm (AAA) models, we found that the 12:1 mixture of GS-Re and lysozyme slowed down AAA progression and reversed phenotype transformation of VSMCs. Thus, Gs-Re combined with a small amount of lysozyme may provide a novel therapeutic strategy for vascular remodeling-associated cardiovascular diseases.