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Molecular basis of methyl-salicylate-mediated plant airborne defence

Nature volume 622, pages 139–148 (2023)Cite this article

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Abstract

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2,3,4,5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylic acid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylic acid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2–SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid–virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.

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Fig. 1: NAC2 regulates MeSA production to mediate PPC-induced aphid resistance.
Fig. 2: NAC2 is required for SA-directed enhancement of MeSA volatilization.
Fig. 3: The conversion between MeSA and SA is required for NAC2-mediated AD against aphids and plant defence against viruses.
Fig. 4: CMV1a suppresses AD.

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Data availability

All data and materials needed to replicate the work are available. NAC2.1 (Niben101Scf01481g02006), NAC2.2 (Niben101Scf07152g04032), SAMT1 (Niben101Scf05122g00005) and SABP2 (Niben101Scf00034g00012) are available from the N. benthamiana genome database (https://solgenomics.net/). RNA-seq raw data have been deposited at the National Center for Biotechnology Information (NCBI) under BioProject accessions PRJNA851626 (WT), PRJNA851854 (nac2), PRJNA955195 (WT-aphid) and PRJNA955395 (nac2-aphid). Uncropped gel and immunoblotting images are provided in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

We thank L. Kang for his suggestions; X. Li and X. Wang for providing the PVY-GFP and CMV (CMV-Fny and CMVΔ2b, the latter is a CMV 2b gene deletion mutant) infectious clones, respectively; H. Guo for providing the seeds of NahG N. benthamiana; P. Zhao and J. Ye for assistance in measurement of SA amount; D. Li for her assistance in the radiolabelling experiments; and the staff at the Facility Center of Metabolomics and Lipidomics of China National Center for Protein Sciences for GC–MS analysis. This work was supported in part by the National Key R&D Program of China (2022YFD1400800 and 2021YFD1400400), the National Natural Science Foundation of China (32130086, 31920103013 and 31872636), the Young Elite Scientists Sponsorship Program by CAST (2022QNRC001) and the Shuimu Tsinghua Scholar Program.

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Author notes

  1. These authors contributed equally: Qian Gong, Yunjing Wang

Authors and Affiliations

  1. MOE Key Laboratory of Bioinformatics and Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Qian Gong, Yunjing Wang, Linfang He, Fan Huang, Danfeng Zhang, Yan Wang, Xiang Wei & Yule Liu

  2. Tsinghua-Peking Center for Life Sciences, Beijing, China

    Qian Gong, Yunjing Wang, Linfang He, Fan Huang, Danfeng Zhang, Yan Wang & Yule Liu

  3. Protein Research Technology Center, Protein Chemistry and Omics Platform, School of Life Sciences, Tsinghua University, Beijing, China

    Meng Han & Haiteng Deng

  4. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Lan Luo & Feng Cui

  5. State Key Laboratory of North China Crop Improvement and Regulation and College of Horticulture, Hebei Agricultural University, Baoding, China

    Yiguo Hong

  6. Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

    Yiguo Hong

  7. School of Life Sciences, University of Warwick, Coventry, UK

    Yiguo Hong

  8. School of Science and the Environment, University of Worcester, Worcester, UK

    Yiguo Hong

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  1. Qian Gong
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Contributions

Y.L. conceived the research. Q.G., Yunjing Wang and Y.L. designed all of the experiments. Q.G. and Yunjing Wang performed all of the aphid choice bioassays, SA–MeSA competition binding experiments, semi in vivo and in vivo protein degradation experiments, ChIP–qPCR, EMSA, GC–MS analysis experiments, virus infection and analysis of viral RNA and protein experiments, and aphid survival as well as virus-transmission analysis. Yunjing Wang, L.H. and F.H. performed BiFC, yeast-one-hybrid and LCI experiments. Yunjing Wang, D.Z., Yan Wang and X.W. generated transgenic plants. Q.G., L.L. and F.C. performed EPG analysis. Yunjing Wang, M.H. and H.D. performed MS analysis. Q.G., Yunjing Wang, Y.H. and Y.L. analysed data and wrote the paper with input from all of the authors.

Corresponding author

Correspondence to Yule Liu.

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Extended data figures and tables

Extended Data Fig. 1 NAC2 interacts with CMV1a and is required for plant antiviral defence and MeSA mediated aphid repellence.

a, Representative LC-MS/MS spectrum of peptides in NAC2 protein. A peptide (AGIAQDAFVLCR) is shown. b-d, Confirmation of the CMV1a-NAC2 interaction. In Co-immunoprecipitation (Co-IP) assay (b), cLUC-MYC or CMV1a-MYC was co-expressed with RFP-NAC2 in Nb leaves and analysed 2 days post-infiltration (dpi). Both Bimolecular fluorescence complementation (BiFC) (c) and firefly luciferase complementation imaging (LCI) assays (d) further confirmed CMV1a-NAC2 interaction in Nb leaves. Scale bar = 25 μm. e-g, Viral symptoms in WT and nac2 plants infected with CMV at 6 dpi (e), and the relative accumulation of CMV RNA (f) or CP (g) in CMV-infected systemic leaves of WT or nac2 plants. h-j, Viral symptoms in WT and nac2 plants infected with PVY-GFP at 7 dpi (h), and the accumulation of PVY RNA (i) or CP (j) in PVY-GFP infected systemic leaves of WT or nac2 plants. k-m, Viral symptoms in WT and nac2 plants infected with TMV-GFP at 6 dpi (k), and the relative accumulation of TMV RNA (l) or CP (m) in TMV-GFP infected systemic leaves of WT or nac2 plants. n, o, nac2 plants exhibited higher attractiveness to aphids than WT plants in circular-dish (n) or Y-tube olfactometer bioassays (o). Numerals shown inside each bar present number of choice-making aphids. p, q, nac2 plants smeared with MeSA containing lanolin exhibited similar attractiveness to aphids with WT plants under same treatment in circular-dish bioassay (p) or Y-tube olfactometer bioassays (q). r, s, nac2 plants smeared with lanolin alone or with 3,3-dimethyl-hexane containing lanolin exhibited higher attractiveness to aphids than WT plants under same treatment in circular-dish (r) or Y-tube olfactometer bioassays (s). t, u, Volatile MeSA treatment caused WT plants more repellent to aphids in circular-dish bioassay (t) or Y-tube olfactometer bioassays (u). v, w, No significant difference in aphid repellence between nac2 plants with and without volatile MeSA treatment in circular-dish (v) or Y-tube olfactometer (w) bioassays. x, y, After volatile MeSA treatment followed by ventilation, nac2 plants showed higher attractiveness to aphids than WT plants in circular-dish (x) or Y-tube olfactometer (y) bioassays. f, i, l, Two-sided Student’s t-test, n = 3 biologically independent samples. Data are shown as mean ± s.d. n-y, χ2 test (d.f. = 1). All P values are shown in figure. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 2 GC-MS analysis of VOCs emitted from WT and nac2 plants.

a-d, Direct sequencing of PCR product containing targeted sites in CRISPR/Cas9-edited knockout nac2, samt1, and sabp2 homozygous plants. The rectangular area indicates the start positions at or from which the mutations occurred. It is worth mentioning that there is only one SAMT1 or SABP2 copy in N.benthamiana genome although Nb is allotetraploid. e, GC-MS analysis of VOCs emitted from WT and nac2 plants after 48 h aphid feeding. Wet weight per plant was 1.10 g on average. Identifications based on retention indices and GC-MS: (1) oxalic acid, allyl hexyl ester; (2) bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-; (3) benzene, 1,2,3-trimethyl-; (4) 6-methyl heptanoate; (5) butyl pyruvate; (6) benzene, 1,2,3,5-tetramethyl-; (7) methyl salicylate; (8) tridecane, 4-methyl-; (9) 3,3-dimethyl-hexane; (10) Cyclohexasiloxane, dodecamethyl-; (11) bicyclo[3.1.1]heptane, 6,6-dimethyl-3-methylene-; (12) heptane, 2,2,3,3,5,6,6-heptamethyl-; (13) heptane, 2,3,6-trimethyl-; (14) 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate; (15) phenol, 2,6-bis(1,1-dimethylethyl)−4-(1-methylpropyl)-. f, The mean amounts of volatile MeSA collected from the aphid-attacked plants in GC-MS analysis. g, h, Mass spectrum of putative MeSA produced by plants (g) and the authentic MeSA standard (h). i, j, Comparison of the aerial MeSA concentrations in chambers containing either MeSA dissolved lanolin paste or aphid attacked WT plants by GC-MS analysis. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 3 NAC2 binds to SAMT1 promoter to activate its transcription.

a, Emitter and receiver plants were placed on two trays at a distance of 30 cm from each other in an interplant communication assay set-up in open-air environment. Each emitter was fed with fifty virus-free or viruliferous M. persicae aphids or no aphids before the emitters and receivers were incubated in same cage (100 cm × 70 cm × 70 cm) made by gauze. After 3 days, the receiver plants were taken out for further experiments. b, NbNAC2 and AtNAC2 share similar conserved motifs. c, RFP-tagged NAC2 showed nuclear localization, scale bar = 20 μm. d, e, RT-qPCR quantification of relative SAMT1 mRNA levels in nac2 and WT plants (d) or leaves transiently over-expressing HA-NAC2 and HA-nLUC (e). f, Transient expression assays. NAC2 activates luciferase reporter gene transcription under the control of the SAMT1 promoter (SAMT1pro) in Nb leaves. Photograph was taken at 48 h post-infiltration (hpi). g, In planta ChIP-qPCR. HA-NAC2, but not HA-nLUC, specifically binds to the SAMT1 promoter DNA. Chromatin from plants expressing HA-nLUC or HA-NAC2 were immunoprecipitated and amplified with promoter-specific primers. h, Yeast one-hybrid assay. Yeast cells were co-transformed with an effector vector containing the SAMT1pro cloned into pHis2 vector and a prey vector encoding NAC2 cloned into pGADT7. i, In vitro EMSA. Hot probe is the biotin-labelled NAC2-binding motif DNA of SAMT1pro, cold probe or cold mutant probe is the unlabelled NAC2-binding motif DNA of SAMT1pro or its mutant DNA. j, Transient over-expression of NAC2 increases MeSA production in plants, the samples were collected at 48 hpi. d, e, g, j, Two-sided Student’s t-test, n = 3 biologically independent samples. Data are shown as mean ± s.d.; n.s., no statistical significance. All P values are shown in figure. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 4 RNA-seq transcriptome analysis of WT and nac2 plants.

a, Comparison of WT and nac2 plants (without any treatment) RNA-seq sequences on the reference genome. The Phred quality score Q20 (99% base call accuracy) and Q30 (99.9% base call accuracy) were used to measure the quality of RNA sequencing. b, Hierarchical clustered heat map of 90 differential expressed genes (DEGs, 24-up genes and 66-down genes) based on the log2 (fold change) in transcript levels of WT and nac2 plants. c, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 90 DEGs in (b), the module with the most enriched genes is “metabolism” (green colour, marked by red font). d, Gene ontology (GO) analysis of 90 DEGs in (b), the “biological process” module (green colour) with the most enriched genes is “metabolism process” (marked by red font). e, Phylogenetic analysis of SAMT1 proteins from various plant species. NtSAMT1, Nicotiana tabacum SAMT1 (GenBank ID: FJ015052); SlSAMT1, Solanum lycopersicum SAMT1 (GenBank ID: NM_001247880); AtBSMT1, Arabidopsis thaliana BSMT1 (Tair ID: AT3G11480); AtGAMT1 (Tair ID: AT4G26420); AtIAMT1 (Tair ID: AT5G55250). f, Comparison of WT (aphid) and nac2 (aphid) plants (under aphid attack) RNA-seq sequences on the reference genome. g, Hierarchical clustered heat map of 157 DEGs (100-up genes and 57-down genes) based on the log2(fold change) in transcript levels of WT (aphid) and nac2 (aphid) plants. h, KEGG pathway analysis of 157 DEGs in (g), the module with the most enriched genes is “metabolism” (green colour, marked by red font). i, GO analysis of 157 DEGs in (g), the “biological process” module (green colour) with the most enriched genes is “metabolism process” (marked by red font). All DEGs were identified according to the transcripts per million reads (TPM) (log2 (foldchange) ≥ 1 or ≤ −1 and P ≤ 0.05).

Extended Data Fig. 5 NAC2 regulates SAMT1 to fulfill anti-CMV and PVY function.

a-e, Analysis of antiviral role of NAC2 and SAMT1 during CMV infection in nac2, samt1, or nac2/samt1 double mutants. Viral symptoms (a), relative NAC2 mRNA levels in NAC2-silenced WT (b) or NAC2-silenced samt1 plants (c), the accumulation of CMV RNA (d) or CP protein (e), and plant endogenous MeSA amount (f, g) of WT, nac2, samt1, or nac2/samt1 double mutant plants infected with CMV. h-l, Analysis of antiviral role of NAC2 and SAMT1 during PVY-GFP infection in nac2, samt1, or nac2/samt1 double mutants. Viral symptom (h), relative NAC2 mRNA levels in NAC2-silenced WT (i) or NAC2-silenced samt1 plants (j), PVY RNA accumulation (k) or PVY CP (l) of nac2, samt1, or nac2/samt1 double mutant plants infected with PVY-GFP. Plants in panels (a and h) were photographed at 7 days post-inoculation (dpi). NAC2-knockdown (KD) was triggered by the virus-induced gene silencing (VIGS) vector TRV-NAC2 to mimic nac2 mutant while TRV was used as negative control in these experiments. b-d, f, g, i-k, Two-sided Student’s t-test, n = 3 biologically independent samples. Data are shown as mean ± s.d. All P values are shown in figure. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 6 CMV1a suppresses MeSA-mediated AD, and CMV1aG983D impairs its interaction with NAC2.

a, Transgenic CMV1a-MYC or CMV1aG983D-MYC expression Nb plant showed normal growth and development. b, CMV1a-MYC and CMV1aG983D-MYC were detected in transgenic plants by western blot. Rubisco was used as loading control. c, d, Transgenic CMV1a plants exhibited higher attractiveness to aphids than WT plants in circular-dish (c) or Y-tube olfactometer (d) bioassays. e, EPG analysis showed that the number of pd of individual aphids was more in CMV1a plants than WT plants. n = 19 individual aphids. f-h, Accumulation of CMV RNA (f), CP (g) or proportion of living aphids (h) in CMV-carrying aphid-attacked leaves of WT receivers (WT-R) with non-aphid-attacked WT plants as mock emitters (WT-mE), virus-free aphid-attacked WT plants as emitters (WT-AE), non-aphid-attacked transgenic CMV1a plants as mock emitters (1a-mE), or virus-free aphid-attacked transgenic CMV1a plants as emitters (1a-AE). i, j, WT-R (WT-mE) plants exhibited higher attractiveness than WT-R (WT-AE) plants (i), but WT-R plants showed similar attractiveness to aphids when non-aphid-attacked or virus-free aphid-attacked CMV1a plants were used as emitters (1a-mE or 1a-AE) (j) in Y-tube olfactometer bioassays. k, WT-R plants nearby virus-free aphid-attacked CMV1a plants as emitters (1a-AE) exhibited higher attractiveness to WT-R plants close to virus-free aphid-attacked WT plants as emitters (WT-AE). l, LCI assay to show that CMV1a helicase domain (1a-H), but not methyltransferase domain (1a-M), interacts with NAC2. m, AlphaFold-Multimer predicted CMV1a-H-NAC2 interacting complex (parameters: “MMseqs2” and “AlphaFold2-Multimer-v2” pattern). Colours are given based on AlphaFold-Multimer-calculated prediction score: pLDDT. Protein structures with scores over 90 are represented in blue (very high confidence of prediction); scores between 70 and 90 in light blue (high confidence); scores between 50 and 70 in yellow (low), and anything below 50 in orange (very low confidence of prediction). n, Red and blue indicate the CMV1a-H and NAC2, respectively. The stick model represents the potential interacting site between 1a-H and NAC2, this region is predicted with a high confidence score. o, Co-IP assay to show that G983D mutation in CMV1a helicase domain impairs the 1a helicase domain-NAC2 interaction. p, q, Co-IP assay (p) and BiFC assay (q) to show that CMV1aG983D failed to interact with NAC2 in Nb. Scale bar = 25 μm. e, f, Two-sided Student’s t-test. h, One-way ANOVA with Tukey’s multiple comparisons test; letters A-B represent statistically different groups (P < 0.05). f, h, n = 3 biologically independent samples. Data are shown as mean ± s.d.; n.s., no statistical significance. c, d, i-k, χ2 test (d.f. = 1). P values are shown in c-f, i-k; adjusted P values for h are shown in the Source Data. In box plot (e), the centre line represents the median, box edges delimit bottom and top quartiles and whiskers show the highest and lowest data points. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 7 CMV1a re-localizes and degrades NAC2 by 26S-proteasome system, and CMV1aG983D possesses a weakened aphid repellence.

a, CMV1a but not CMV1aG983D partially changed NAC2 localization from nucleus to cytoplasm. b, CMV1a did not alter subcellular localization of RFP. c, d, Nuclear exit signal-tagged RFP-NAC2 (NES-NAC2) changed NAC2 localization to cytoplasm (c) and enhanced 26S-proteasome system-dependent degradation (d). Scale bar = 25 μm in panels (a-c). e, Immunoblot assay of RFP protein levels. f, Immunoblots to show cLUC-MYC, CMV1a-MYC, and CMV1aG983D-MYC protein levels with anti-MYC antibody. g, In vivo assay showing effects of MG132 and the CMV1a-NAC2 interaction on NAC2 protein stability. 100 μM MG132 or an equal volume of DMSO (negative control) was infiltrated into leaves transiently co-expressing RFP-NAC2 with CMV1a-MYC, CMV1aG983D-MYC, or cLUC-MYC for 12 h before harvesting. h, Semi-in vivo assay to show that NAC2 protein stability is ATP-dependent. NAC2 protein levels were analysed with anti-RFP antibody at different times following 100 μM CHX treatment in the presence or absence of 10 mM ATP. i, Semi-in vivo assay to show that MG132 inhibits CMV1a-promoted NAC2 degradation. RFP-NAC2, cLUC-MYC, CMV1a-MYC, or CMV1aG983D-MYC was transiently expressed in Nb leaves and extracted respectively. NAC2 degradation was performed as below: The RFP-NAC2 protein extract was mixed with the cLUC-MYC, CMV1a-MYC, or CMV1aG983D-MYC extracts in a 1:1 volume of 100 μM CHX and 10 mM ATP, in the presence of 100 μM MG132 or an equal volume of control DMSO. j, Effect of CMV1a on expression of luciferase reporter gene driven by the SAMT1 promoter (SAMT1pro). Transient expression assays in Nb leaves to show that CMV1a but not CMV1aG983D suppressed NAC2-mediated activation of the SAMT1 promoter. Photographs were taken at 48 hpi. k, Transgenic plants expressing CMV1a, but not CMV1aG983D, exhibited higher attractiveness to aphids than WT plants in Y-tube olfactometer bioassays. l, m, GC-MS analysis to show that transgenic plants expressing CMV1a, but not CMV1aG983D, emitted less volatized MeSA than WT plants once they were fed with virus-free aphids for 3 days. n, WT-R (WT-mE) plants exhibited higher attractiveness than WT-R (WT-AE) plants, WT-R plants showed similar attractiveness to aphids when non-aphid-attacked or virus-free aphid-attacked CMV1a-expressinig plants were used as emitters (1a-mE or 1a-AE), whilst WT-R (1aG983D-mE) plants exhibited higher attractiveness than WT-R (1aG983D-AE) plants in Y-tube olfactometer bioassays. m, Two-sided Student’s t-test, n = 3 biologically independent samples. Data are shown as mean ± s.d.; n.s., no statistical significance. k, n, χ2 test (d.f. = 1). All P values are shown in figure. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 8 Alignment of helicase domain from aphid and non-aphid transmitted viruses.

BCMNV, Bean common mosaic necrosis virus (Uniprot ID: Q65399); BYMV, Bean yellow mosaic virus (Uniprot ID: P17765); MDMV, Maize dwarf mosaic virus (Uniprot ID: J7II85); PPV, Plum pox potyvirus (Uniprot ID: P13529); PRSV, Papaya ringspot virus (Uniprot ID: A0A1L2DBW1); PVY, Potato virus Y (Uniprot ID: A0A5J6BDG4); PeMV, Pepper mottle virus (Uniprot ID: Q01500); PVMV, Pepper veinal mottle virus (Uniprot ID: A0A6J4A295); SCMV, Sugarcane mosaic virus (Uniprot ID: A0A0K0Y0R3); SMV, Soybean mosaic virus (Uniprot ID: Q90069); TEV, Tobacco etch virus (Uniprot ID: P04517); TuMV, Turnip mosaic virus (Uniprot ID: Q9ICI2); TVBMV, Tobacco vein banding mosaic virus (Uniprot ID: F5A3N8); ZYMV, Zucchini yellow mosaic virus (Uniprot ID: P18479); CLV, Carnation latent virus (Uniprot ID: A0A858Z687); CMV, Cucumber mosaic virus (Uniprot ID: P17769); PSV, Peanut stunt virus (Uniprot ID: P28726); TAV, Tomato aspermy virus (Uniprot ID: P28931); BYDV, Barley yellow dwarf virus (Uniprot ID: P29044); SbDV, Soybean dwarf virus (Uniprot ID: A0A6M8PRM6); AMV, Alfalfa mosaic virus (Uniprot ID: P03589); PEBV, Pea early browning virus (Uniprot ID: Q9WJD8); TRV, Tobacco rattle virus (Uniprot ID: Q9J942); PVX, Potato virus X (Uniprot ID: A0A7H1C8Y4); TMV, Tobacco mosaic virus (Uniprot ID: P03586); TYMV, Turnip yellow mosaic virus (Uniprot ID: P10358).

Extended Data Fig. 9 PVY suppresses plant AD by CI-NAC2 interaction.

a, Alignment of helicase domain from multiple aphid- and non-aphid- transmitted viruses. Asterisk (*) indicates that the Glycine (G) amino acid residue is conserved among listed aphid-transmitted viruses. b, c, Accumulation of PVY RNA (b) or CP (c) in PVY-carrying aphid-attacked leaves of WT receivers (WT-R) with virus-free aphid-attacked WT plants as emitters (AE) or with non-aphid-attacked WT plants as mock emitters (mE) when these receiver plants were fed with PVY-containing aphids. d, e, GC–MS analysis of volatized MeSA in WT plants fed with virus-free aphids or PVY-carrying aphids for 3 days. f-h, Accumulation of PVY RNA (f), CP (g) or proportion of living aphids (h) in WT-R plants with PVY-containing aphid-attacked WT plants as emitters (APE) or WT-R (mE) plants when these receiver plants were fed with PVY-containing aphids. i, CMV and PVY infection changed NAC2 localization partially to cytoplasm. Scale bar = 25 μm. j, BiFC assay showing that PVY CI but not its mutant PVY CIG347D or TMV 126KD, interacted with NAC2. Scale bar = 25 μm. k, Immunoblot assay of protein levels in BiFC assay. l, PVY CI but not its mutant PVY CIG347D or TMV 126KD, changed NAC2 localization partially to cytoplasm, scale bar = 25 μm. b, e, f, Two-sided Student’s t-test. h, One-way ANOVA with LSD. b, e, f, h, n = 3 biologically independent samples. Data are shown as mean ± s.d.; n.s., no statistical significance. The same letter A represents no statistical difference between samples (P > 0.05). All P values are shown in figure. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 10 Arms race among emitter and receiver plants, aphids, and viruses.

a, AD defends plants against aphids and viruses. When emitter plants are attacked by aphids, they can sense the aphid sap-sucking action and stimulate biosynthesis of SA that activates the NAC2-SAMT1 module to produce volatile MeSA, neighbouring receiver plants perceive and convert volatile MeSA into SA by SABP2, which acts as the cue to trigger NAC2-SAMT1 module and elicit defence against aphids and viruses. b, Virus and aphid counterdefence. When emitter plants were attacked by viruliferous aphids, some aphid-transmitted viruses utilized their helicase-contained viral protein (for example, CMV1a and PVY CI) to subcellularly re-localize and destabilize NAC2, leading to suppression of NAC2-mediated plant airborne defence to facilitate aphid propagation and virus transmission. The graphical model was created with BioRender.com. In summary, we have exploited interplays among aphid, virus, VOC emitter, and receiver plants in a complexed pathosystem to dissect PPC and AD at genetic and molecular levels. Our study on deciphering AD also lays the groundbreaking work to empower VOCs as a novel bioinspired tool in defence of plants including agricultural and horticultural crops against insect infestation and virus epidemics.

Supplementary information

Supplementary Figure 1

All uncropped blots and gel images.

Reporting Summary

Supplementary Table 1

Differentially expressed genes in WT and nac2 plants.

Supplementary Table 2

Differentially expressed genes in WT (aphid) and nac2 (aphid) plants.

Supplementary Table 3

Primers used in this study.

Source data

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Gong, Q., Wang, Y., He, L. et al. Molecular basis of methyl-salicylate-mediated plant airborne defence. Nature 622, 139–148 (2023). https://doi.org/10.1038/s41586-023-06533-3

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