Extended Data Fig. 7: CMV1a re-localizes and degrades NAC2 by 26S-proteasome system, and CMV1a^G983D possesses a weakened aphid repellence.

a, CMV1a but not CMV1a^G983D 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 CMV1a^G983D-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, CMV1a^G983D-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 CMV1a^G983D-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 CMV1a^G983D-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 CMV1a^G983D suppressed NAC2-mediated activation of the SAMT1 promoter. Photographs were taken at 48 hpi. k, Transgenic plants expressing CMV1a, but not CMV1a^G983D, 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 CMV1a^G983D, 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 (1a^G983D-mE) plants exhibited higher attractiveness than WT-R (1a^G983D-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