Research ArticleVIROLOGY

Viruses mobilize plant immunity to deter nonvector insect herbivores

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Science Advances  21 Aug 2019:
Vol. 5, no. 8, eaav9801
DOI: 10.1126/sciadv.aav9801
  • Fig. 1 Whitefly-transmitted begomoviruses enhance resistance to CBM on infected plants.

    (A) Phenotype of cotton leaves infested with whiteflies or CBM after 5 days. Red arrows indicate CBM. Scale bars, 4 mm. (B) Numbers of live whiteflies infested on healthy cotton and the CLCuMuV complex (CA + β)–infected cotton. (C) Survival rates of CBM larvae infested on healthy cotton, CA + β–infected cotton, and Bt transgenic cotton (BD18). (D) Larval weight of CBM infested on healthy cotton, CA + β–infected cotton, and Bt transgenic cotton. (E) Larval weight of CBM infested on healthy Nicotiana benthamiana (Nb) plants and Nb plants infected with the TYLCCNV complex (TA + β) or with the TYLCCNV complex harboring mutant βC1 (TA + mβ). (F and G) Larval weight of CBM infested on wild-type Nb and transgenic Nb plants ectopically expressing βC1 (F) or Col-0 plants and Arabidopsis plants ectopically expressing βC1 (βC1-1/At and βC1-3/At) (G). (H) Daily number of eggs laid per female whitefly on wild-type Arabidopsis and the transgenic βC1 expression lines. (I) Pupa numbers of whiteflies present on wild-type Arabidopsis and the transgenic βC1 expression lines. (B to G) Bars represent means ± SD (n = 10). (H and I) Bars represent means ± SD (n = 8) (*P < 0.05 and **P < 0.01, Student’s t test). Photo credit: Pingzhi Zhao, Chinese Academy of Sciences.

  • Fig. 2 Begomovirus-encoded βC1 proteins interact with plant WRKY20 proteins for differentiated resistance against herbivores.

    (A) Interaction of CLCuMuV βC1 (βC1-C) with GhWRKY20 homologs (GhWRKY20-1 and GhWRKY20-2) and TYLCCNV βC1 with AtWRKY20 in a yeast two-hybrid system. (B) BiFC analysis of βC1 proteins interaction with WRKY20 homologous proteins. Scale bars, 50 μm. (C and D) Daily number of eggs laid per female whitefly on vector control cotton and VIGS cotton silenced for GhWRKY20 (C) or on Arabidopsis Col-0 and wrky20 mutant plants (D). (E) Pupa numbers of whiteflies present on Arabidopsis Col-0 and wrky20 mutant plants. (F) Daily number of eggs laid per female whitefly on Arabidopsis Col-0 and overexpressing AtWRKY20 plants. (G to I) Larval weight of CBM reared on vector control cotton and GhWRKY20 VIGS cotton (G) on Col-0 and wrky20 mutant plants (H) or on Col-0 and plants overexpressing AtWRKY20 (I). (C to F) Bars represent means ± SD (n = 8). Means with different letters (a, b, and c) are significantly different [P < 0.05, one-way analysis of variance (ANOVA) along with Duncan’s multiple range test]. (G to I) Bars represent means ± SD (n = 10) (*P < 0.05 and **P < 0.01, Student’s t test).

  • Fig. 3 The βC1 interaction partners WRKY20 and MYC2 form a negative regulation feedback loop.

    (A) Phenotypes of 6-week-old Arabidopsis leaves after CBM infestation for 12 hours. (B) Nonconsumed leaf areas were recorded after CBM infestation for 12 hours on Arabidopsis leaves. Bars represent means ± SD (n = 6). (C) Relative expression levels of AtMYC2 and AtWRKY20 in Col-0 treated with methyl JA (MeJA). (D) Relative expression level of AtWRKY20 in Col-0 and myc2-1 plants treated with MeJA. (E) Relative expression levels of AtMYC2, AtMYC3, and AtMYC4 in Col-0 and wrky20-1 plants after 6 hours of MeJA treatment. (F) AtMYC2 promoter activity in YFP-expressing and YFP-AtWRKY20–expressing leaves by β-glucuronidase (GUS) staining and AtWRKY20 promoter activity in YFP-expressing and YFP-AtMYC2–expressing leaves by GUS staining. (G) Total GSs content of 6-week-old Arabidopsis plants. Bars represent means ± SD (n = 4). Fw, fresh weight. (H) Growth rate of CBM reared on Arabidopsis plants. Bars represent means ± SD (n = 10). (B, G, and H) Means with different letters (a, b, c, d, and e) are significantly different (P < 0.05, one-way ANOVA along with Duncan’s multiple range test). (C to E) Bars represent means ± SD (n = 3) (**P < 0.01, Student’s t test). Photo credit: Pingzhi Zhao, Chinese Academy of Sciences.

  • Fig. 4 βC1 regulates WRKY20-mediated GS biosynthesis in a vascular-specific manner.

    (A to G) AtWRKY20 promoter activity in a cotyledon (A), first rosette leaf (B), roots (C and D), inflorescence (E), and siliques (F and G). Scale bars, 1 mm. (H) Resin root slice of the AtWRKY20 promoter: GUS lines. Scale bars, 20 μm. (I and J) GUS staining of the leaves of 6-week-old plants before (I) and after (J) MeJA treatment in AtWRKY20 promoter: GUS lines. Scale bars, 2 mm. (K) Relative expression level of AtMYB122 in 6-week-old Arabidopsis plants after 6 hours MeJA treatment. Bars represent means ± SD (n = 3). (L) Schematic diagram of the AtMYB122 promoter. The small hollow triangles represent W-box motifs in the schematic diagram of the promoters. ATG indicates the coding initiation site of a gene. Three lines under triangles represent various DNA fragments were amplified in ChIP assay. (M) Fold enrichment of YFP-AtWRKY20 associated with DNA fragments of the AtMYB122 promoter in a ChIP assay. Enrichments are referred to the 35S:YFP-AtWRKY20 or 35S:YFP against wild-type Col-0 seedlings. Bars represent means ± SD (n = 4) (*P < 0.05 and **P < 0.01, Student’s t test). (N) Effects of βC1 on the transactivation activity of AtWRKY20 on the AtMYB122 promoter. Bars represent means ± SD (n = 8). (O) Contents of AGS and IGS in leaf veins and nonveins, respectively, of 6-week-old Arabidopsis plants after 6 hours of MeJA treatment. Bars represent means ± SD (n = 4). (K, N, and O) Means with different letters (a, b, c, and d) are significantly different (P < 0.05, one-way ANOVA along with Duncan’s multiple range test). Photo credit: Pingzhi Zhao, Chinese Academy of Sciences.

  • Fig. 5 βC1 interferes with the interaction between WRKY20 and ERF-ORA59.

    (A and B) Relative expression level of AtPDF1.2 (A) or AtORA59 (B) in Arabidopsis plants under MeJA treatment. Bars represent means ± SD (n = 3) (*P < 0.05 and **P < 0.01, Student’s t test). (C) GST pull-down assay. Two micrograms of MBP-AtWRKY20 fusion protein was used to pull down 2 μg of GST fusion proteins. WB, Western blot. (D) BiFC analysis of AtWRKY20 interaction only with AtORA59 but not with AtMYC2 or AtERF1. Scale bars, 50 μm. (E) Effects of βC1 on the interaction of WRKY20 with ORA59 by modified BiFC assay. The EYFP fluorescences were detected after coproduction of cEYFP-AtORA59 + nEYFP-AtWRKY20 (control), βC1 + cEYFP-AtORA59 + nEYFP-AtWRKY20 (βC1), or GUS + cEYFP-AtORA59 + nEYFP-AtWRKY20 (GUS). Scale bars, 50 μm. (F) Quantitative data of EYFP fluorescence intensity show effects of βC1 on the interaction of WRKY20 with ORA59. Bars represent means ± SD (n = 20) (**P < 0.01, Student’s t test). (G) Pull-down protein competition assays. The indicated protein amount of His-βC1 or GST was mixed with 2 μg of GST-AtORA59 and pulled down by 2 μg of MBP-AtWRKY20. Immunoblots were performed using anti-GST antibody to detect the associated proteins. (C and G) Membranes were stained with Coomassie brilliant blue to monitor input protein amount.

  • Fig. 6 The βC1-WRKY20 interaction promotes plant resistance to aphid by activating SA signaling.

    (A) Number of progeny produced by each aphid 9 days after infestation of 6-week-old Arabidopsis plants. Bars represent means ± SD (n = 8). (B to E) Relative expression levels of SA-related genes, AtPAL1 (B), AtSID2 (C), AtPAD4 (D), AtEDS5 (E), or AtPR1 (F) in 6-week-old Arabidopsis plants. Bars represent means ± SD (n = 3). (G) Symptoms of cabbage leaf curl virus (CaLCuV)–infected Arabidopsis plants at 31 days post infection (dpi). Scale bar, 1 cm. (H) Disease index of CaLCuV-infected Arabidopsis plants. Disease indexes is an indicator of the average disease severity on the survey plant samples, which were observed by level of viral symptomatic onset such as obvious yellow and curly leaves in infected plants. (I) Relative CaLCuV virus titer in Arabidopsis plants after 31 dpi. Values are means ± SD (n = 3) (**P < 0.01, Student’s t test). (A to F) Means with different letters (a and b) are significantly different (P < 0.05, one-way ANOVA along with Duncan’s multiple range test). Photo credit: Pingzhi Zhao, Chinese Academy of Sciences.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaav9801/DC1

    Fig. S1. Whitefly vector competes with nonvector CBM on cotton.

    Fig. S2. TYLCCNV βC1 protein interacts with WRKY20 proteins.

    Fig. S3. WRKY20 mediates plant immunity against whitefly.

    Fig. S4. WRKY20 and MYC2 form a negative feedback loop.

    Fig. S5. Plant WRKY20 is a dual-function transcription factor controlling GS biosynthesis.

    Fig. S6. WRKY20 directly targets GS biosynthetic-related genes by binding to their promoters.

    Fig. S7. WRKY20 regulates a JA-mediated GS accumulation in a vascular-specific pattern.

    Fig. S8. βC1 suppresses the WRKY20 activity by interfering with its homodimerization.

    Fig. S9. WRKY20 negatively regulates SA-mediated defense against the green peach aphid.

    Table S1. DNA primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Whitefly vector competes with nonvector CBM on cotton.
    • Fig. S2. TYLCCNV βC1 protein interacts with WRKY20 proteins.
    • Fig. S3. WRKY20 mediates plant immunity against whitefly.
    • Fig. S4. WRKY20 and MYC2 form a negative feedback loop.
    • Fig. S5. Plant WRKY20 is a dual-function transcription factor controlling GS biosynthesis.
    • Fig. S6. WRKY20 directly targets GS biosynthetic-related genes by binding to their promoters.
    • Fig. S7. WRKY20 regulates a JA-mediated GS accumulation in a vascular-specific pattern.
    • Fig. S8. βC1 suppresses the WRKY20 activity by interfering with its homodimerization.
    • Fig. S9. WRKY20 negatively regulates SA-mediated defense against the green peach aphid.
    • Table S1. DNA primers used in this study.

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