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.
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We thank Z.-D. Jiang of the Department of Plant Pathology, South China Agricultural University for F. oxysporum strains. This study was supported by grants from the National Science Foundation of China (Grant 32230003) and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28030502).
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Nature Plants thanks Roger Innes, Maria Ladera-Carmona and Abdelhak El Amrani for their contribution to the peer review of this work.
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(a) Schematic description for generation of inverted-repeat RNAi construct. (b) Detection of Th-GFPi transformants by Sourthen blotting. Genomic DNA was digested by restriction enzymes as indicated. The promoter sequence indicated in [(a)] was used as a probe. (c) Detection of Th-GFPi-generated siGFPs by Northern blotting. 3’-end biotin-labeled GFP-specific oligos were used as probes. U6 was detected as loading control. These experiments were repeated independently three times with similar results. (d) The Th-GFPi strains showed normal colony and hypha morphology similar to Th. Scale bars: 10 μm. The experiments in (b), (c) and (d) were repeated independently three times with similar results.
(a) Deletion of each VdPMT gene in V592 and complementation of VdPMT2 in VdΔpmt2 were confirmed by Southern blotting. Genomic DNA was digested by restriction enzymes as indicated. Gene-specific sequences were used as probes. Restriction enzyme sites are labeled in the schematic diagrams. Semi quantitative reverse transcription PCR (RT-PCR) was used to detect the transcripts in corresponding mutant strains. These experiments were repeated independently three times with similar results. (b) Evaluation of disease grades of cotton plants infected by V592, VdΔpmt2 and complementation strain VdΔpmt2/PMT2 at 21 dpi. Disease symptoms of cotton plants are shown in Fig. 2b. Disease grades on cotton leaves were classified into five levels of severity of disease symptoms during fungal invasion: 0 = no visible wilting or yellowing symptoms, 1 = one or two cotyledons wilted or dropped off, 2 and 3 = one or two true leaves wilted or dropped off, 4 = all of the leaves dropped off or the whole plant died. Data are presented as mean ± SEM, n = 9 pot plants.
Extended Data Fig. 3 Detection of Th-Pmt2i transformants and their effect on interference with V592 growth.
(a) Schematic description for generation of Pmt2i constructs. (b) Detection of Th-Pmt2i transformants by Southern blotting. Genomic DNA was digested by the restriction enzymes as indicated. The promoter sequence (indicated in Extended Data Fig. 1a) was used as a probe. Restriction enzyme sites are labeled in the schematic diagram. (c) Th-Pmt2i strains showed normal colony and hypha morphology similar to Th. Scale bars: 10 μm. (d) VdAGO proteins are not required for Th-Pmt2i-inhibited V592 growth. Inhibition zones were observed for Th-Pmt2i on either single deletion mutant, VdΔago1 or VdΔago2, or double deletion mutant, VdΔago1/2. (e) Detection of double delection mutant VdΔago1/2 by Southern blotting. VdAGO1 was further deleted in VdΔago2. Genomic DNA was digested by restriction enzymes as indicated. The specific sequence of resistance screening gene NAT was used as probe. Restriction enzyme sites are labeled in the schematic diagram. RT-PCR was used to detect the transcripts in corresponding mutant strains. (f) Original figure of qPCR curves. Primers used for amplification of V592-specific (V) and fungal ITS region (F) were listed in Supplementary Data 1. Soil samples and time points are shown in different color curves. At least three biological replicates were performed and the representative curves were presented. The percentages of V to F (V/F) representing the relative biomass of V592 in total fungi were shown in Fig. 3c. (g) TEM image of extracellular particles from the culture supernatant of Th-Pmt2i−1. The experiments in (b)-(g) were repeated independently three times with similar results.
Extended Data Fig. 4 Th-Pmt2i−1 offered effective cotton protection against V592 infection in natural soil.
(a) Effect of Th-Pmt2i−1 on cotton protection against V592 in non-sterile soil. (b) Disease grade analysis of Verticillium wilting in [(a)] at 40 dps. These experiments were repeated independently three times with similar results. Disease grades were evaluated from 12 pots of total 48 plants for each inoculum. Data are presented as mean ± SEM, n = 12 pot plants.
(a) Schematic description for generation of Pmt2iFo construct. (b) Detection of Th-Pmt2i transformants by Southern blotting. Genomic DNA was digested by the restriction enzymes as indicated. The promoter sequence was used as a probe. Restriction enzyme sites are labeled in the schematic diagram. (c) Th-Pmt2iFo strains showed normal colony and hypha morphology similar to Th. Scale bars: 10 μm. The experiments in (b) and (c) were repeated independently three times with similar results.
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Wen, HG., Zhao, JH., Zhang, BS. et al. Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens. Nat. Plants 9, 1409–1418 (2023). https://doi.org/10.1038/s41477-023-01507-9