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. 2021 Mar 16;118(11):e2016673118.
doi: 10.1073/pnas.2016673118.

A class of independently evolved transcriptional repressors in plant RNA viruses facilitates viral infection and vector feeding

Lulu Li  1   2 , Hehong Zhang  1 , Changhai Chen  1 , Haijian Huang  1 , Xiaoxiang Tan  1 , Zhongyan Wei  1 , Junmin Li  1 , Fei Yan  1 , Chuanxi Zhang  1 , Jianping Chen  3 , Zongtao Sun  3
Affiliations

A class of independently evolved transcriptional repressors in plant RNA viruses facilitates viral infection and vector feeding

Lulu Li et al. Proc Natl Acad Sci U S A. .

Abstract

Plant viruses employ diverse virulence strategies to achieve successful infection, but there are few known general strategies of viral pathogenicity and transmission used by widely different plant viruses. Here, we report a class of independently evolved virulence factors in different plant RNA viruses which possess active transcriptional repressor activity. Rice viruses in the genera Fijivirus, Tenuivirus, and Cytorhabdovirus all have transcriptional repressors that interact in plants with the key components of jasmonic acid (JA) signaling, namely mediator subunit OsMED25, OsJAZ proteins, and OsMYC transcription factors. These transcriptional repressors can directly disassociate the OsMED25-OsMYC complex, inhibit the transcriptional activation of OsMYC, and then combine with OsJAZ proteins to cooperatively attenuate the JA pathway in a way that benefits viral infection. At the same time, these transcriptional repressors efficiently enhanced feeding by the virus insect vectors by repressing JA signaling. Our findings reveal a common strategy in unrelated plant viruses in which viral transcriptional repressors hijack and repress the JA pathway in favor of both viral pathogenicity and vector transmission.

Keywords: antiviral defense; jasmonic acid; plant viruses; transcriptional repressor; vector feeding.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Identification of a group of independently evolved viral transcriptional repressors in plant RNA viruses. (A) Diagrams of the reporter and a series of effectors (SRBSDV SP8, RBSDV P8, RSV P2, and RSMV M) used in the GD system. The reporter gene LUC is driven by a concatenated (5×) GAL4 promoter, and specifically recognized GD effectors are driven by the CaMV 35S promoter. REN, renilla luciferase, an internal control; LUC, firefly luciferase. (B) Relative LUC activities measured in N. benthamiana cells. The 5×GAL4::LUC reporter was cotransformed with the indicated GD effectors into N. benthamiana leaves and measured at 48 hpi. * indicates a significant difference between samples analyzed by ANOVA at P ≤ 0.05 by Fisher's least significant difference tests.
Fig. 2.
Fig. 2.
SP8 interacts with OsMYC3. (A) Interaction of SP8 with OsMYC3, but not with the OsMYC3 homologs OsMYC2 and OsMYC4, in the Y2H system. The full-length sequences of OsMYC2, OsMYC3, and OsMYC4 were amplified into pGADT7 (AD) and SP8 protein fused with pGBKT7 (BD) vector were cotransformed into the yeast strain AH109. Positive yeast transformants were selected on SD-L-T-H-Ade plates at 30 °C. Photos were taken after 3 days. (B) BiFC assays for potential interaction between SP8 and OsMYC3. SP8-cYFP was transiently coexpressed with OsMYC3-nYFP in N. benthamiana leaves and confocal imaged at 48 hpi. Agro-infiltration with SP8-cYFP and Gus-nYFP vector served as a negative control. (Scale bar, 20 μm.) (C) BiFC assays for potential interaction between SP8 and OsMYC3. SP8-cYFP was transiently coexpressed with OsMYC3-nYFP in N. benthamiana leaves and confocal imaged at 48 hpi. Agro-infiltration with SP8-cYFP and Gus-nYFP vector served as a negative control. (Scale bar, 20 μm.) (D) Co-IP assays to analyze the interactions among SP8 with OsMYC2 and OsMYC3 in vivo. Total proteins were incubated with FLAG beads and detected using anti-myc or anti-flag antibody. The sample coexpressing SP8-flag and GUS-myc was the negative control. (E) Co-IP assay confirms the interaction between OsMYC3 and the conserved NTP domain of SP8 in planta. SP8NTP-myc and OsMYC3-flag were coexpressed in N. benthamiana leaves, and total protein was incubated with FLAG beads and detected by anti-myc and anti-flag antibodies. Coinfiltration of SP8-myc and OsMYC3-flag was the positive control, while SP8NTP-myc cotransformed with flag-GFP served as a negative control. (F) Schematic representation of the deleted variations of OsMYC3 used in the Y2H assay. JID, JAZ interaction domain; TAD, transcriptional activation domain; bHLH, the basic helix–loop–helix domain responsible for homo-/hetero-dimerization. The right panel shows that OsMYC3TAD, but not OsMYC3jid or OsMYC3C, interacts with SP8.
Fig. 3.
Fig. 3.
SP8 suppresses the transcriptional activation of OsMYC3 to attenuate JA signaling. (A) Scheme of the reporter and multiple effectors employed in dual-luciferase transient transcriptional activity assay. The reporter gene LUC was driven by the OsJAZ4 promoter and the effectors shown by the CaMV 35S promoter. REN, renilla luciferase, an internal control; LUC, firefly luciferase. (B) SP8 represses the transcription activity of OsMYC3 on OsJAZ4 promoter. The pOsJAZ4::LUC reporter was cotransformed with the effectors shown into N. benthamiana leaves and measured at 48 hpi. Relative luciferase activities were analyzed by the ratio of LUC/REN, error bars represent SD (n = 6). NS: no significance. (C) Phenotypes of SP8-ox lines (SP8-13 and SP8-26) grown on rice nutrient solution containing 0.1 μM or 1 μM MeJA for 7 days. The root lengths of SP8-ox seedlings were mildly inhibited by MeJA compared with the Nip control. Abbreviation: Nip., Nipponbare, the wild-type rice variety. (D) Quantification of the relative root lengths showing that SP8-ox lines were more insensitive to JA-inhibitory root growth than the control Nip. Error bars represent SD, * in B and D indicates a significant difference between samples analyzed by ANOVA at P ≤ 0.05 in Fisher's least significant difference tests.
Fig. 4.
Fig. 4.
SP8 disturbs the association between OsMED25 and OsMYC3. (A) Co-IP assay analyzing the interaction between OsMED25 and OsMYC3 in vivo. Total proteins were extracted from N. benthamiana leaves infiltrated with OsMED25-flag and OsMYC3-myc and then incubated with FLAG beads and detected using c-myc antibody. The sample coexpressing GFP-flag and OsMED25-myc is a negative control. (B) Co-IP assay analyzing the interaction between OsMED25 and SP8 in vivo. Total proteins were extracted from N. benthamiana leaves infiltrated with OsMED25-myc and GFP-flag and then incubated with FLAG beads and detected using c-myc antibody. The sample coexpressing GFP-flag and OsMED25-myc is a negative control. (C) BiFC assays to detect interaction between OsMED25 and SP8. OsMED25-cYFP was transiently coexpressed with SP8-nYFP in N. benthamiana leaves and confocal imaged at 48 hpi. Agro-infiltration with OsMED25-cYFP and Gus-nYFP vector served as a negative control. (Scale bar, 50 μm.) (D) SP8 disturbs the OsMED25-OsMYC3 association. Fusion proteins were transiently expressed in leaves of N. benthamiana and observed by confocal microscopy. The YFP signals were reduced in the presence of SP8. (Scale bar, 50 μm.) (E) Interaction between OsMYC3 and OsMED25 was weakened by viral protein SP8. OsMED25-flag combined with OsMYC3-GFP and increasing amounts of SP8-myc were coincubated in leaves of N. benthamiana. The immunoprecipitated fractions were probed with anti-flag, anti-myc, and anti-GFP antibodies. Intrinsic protein levels were evaluated by input in the lower panel. (F) Scheme of LCI assays for coexpression in leaves of N. benthamiana. (G) Results from LCI assays showing that SP8 protein impaired the interaction between OsMYC3 and OsMED25.
Fig. 5.
Fig. 5.
SP8 associates with OsJAZ proteins synergistically to attenuate JA signaling. (A) SP8 interacts with many OsJAZ family proteins in Y2H. BD-SP8 was cotransformed with AD-OsJAZs proteins (OsJAZ1 to OsJAZ15, except OsJAZ2) into yeast strain AH109, and positive transformants were selected on SD-l-T-H-Ade plates at 30 °C. Photos were taken after 3 days. (B) BiFC assays confirming the interaction between SP8 and OsJAZs proteins. SP8-cYFP was agro-infiltrated with OsJAZ4/5/9/11/12-nYFP into N. benthamiana leaves and confocal imaged at 48 hpi. The expression pair SP8-cYFP and Gus-nYFP served as a negative control. (Scale bar, 50 μm.) (C and D) Co-IP analysis of SP8 and OsJAZs proteins including OsJAZ5, OsJAZ9, OsJAZ11, and OsJAZ12 in vivo. Total proteins in C and D were purified by FLAG beads and probed with anti-myc antibody. (E) Diagrams of the reporter and a range of effectors used in the GD system. The reporter gene LUC was driven by the 5×GAL4 promoter and specifically recognized GD effectors by the CaMV 35S promoter. REN, renilla luciferase, an internal control; LUC, firefly luciferase. (F) Overexpression of OsJAZs proteins facilitates the transcriptional repressor activity of SP8. The 5×GAL4::LUC reporter was cotransformed with the indicated GD effectors into N. benthamiana leaves and measured at 48 hpi. (G) Schematic representation of the pOsJAZ4::LUC reporter and various effectors in dual-luciferase transient transcriptional activity assay. (H) SP8 interacts with OsJAZs to synergistically suppress the transcriptional activation of OsMYC3. The pOsJAZ4::LUC reporter was cotransformed with the indicated effectors into N. benthamiana leaves and measured at 48 hpi. Relative luciferase activities in F and H were analyzed by the ratio LUC/REN. Error bars represent SD (n = 6); * indicates a significant difference between samples analyzed by ANOVA at P ≤ 0.05 by Fisher's least significant difference tests.
Fig. 6.
Fig. 6.
Manipulation of the JA pathway by viral transcriptional repressors is conserved in plant viruses. (A) Y2H assays showing the interactions of viral proteins RSV P2 and RSMV M protein with OsMYC transcription factors. Viral proteins were fused with BD while OsMYC transcription factors (OsMYC2, OsMYC3, and OsMYC4) were cloned into AD yeast vectors. All transformants were selected on SD-L-T-H-Ade plates at 30 °C and photographed after 3 days. (B) BiFC assays confirming the interactions of RSV P2 and RSMV M protein with OsMYC3. OsMYC3 and viral proteins P2/M were respectively cloned into cYFP and nYFP vectors and then agro-infiltrated into N. benthamiana leaves together. The samples were imaged by confocal microscopy at 48 hpi. (Scale bar, 50 μm.) (C) Schematic diagrams of the pOsJAZ4::LUC reporter and various effectors. REN, renilla luciferase, an internal control; LUC, firefly luciferase. The reporter was coinfiltrated with OsMYC3 and viral proteins P2/M into N. benthamiana leaves and measured at 48 hpi, while the sample with pOsJAZ4::LUC empty vector was the negative control. Relative luciferase activities were analyzed by the LUC/REN ratio. Error bars represent SD (n = 6); * indicates a significant difference between samples analyzed by ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. (D) BiFC assays confirming the interactions of RSV P2 and RSMV M protein with OsMED25. OsMED25 and viral proteins P2/M were respectively cloned into cYFP and nYFP vectors and then agro-infiltrated into N. benthamiana leaves together. The samples were imaged by confocal microscopy at 48 hpi. (Scale bar, 50 μm.) (E) Co-IP assays to examine the interactions between OsMED25 and RSV P2 in vivo. Total proteins were extracted from N. benthamiana leaves coexpressing OsMED25-myc and P2-flag, the supernatant was precipitated with FLAG beads, and the associated proteins were verified using anti-myc antibody. The sample expressing OsMED25-myc and GFP-flag served as a negative control. (F) Co-IP assays to examine the interactions between OsMED25 and RSMV M protein in vivo. Total proteins were extracted from N. benthamiana leaves coexpressing OsMED25-flag and M-myc, the supernatant was precipitated with FLAG beads, and the associated proteins were verified using anti-myc antibody. The sample expressing M-myc and GFP-flag served as a negative control. (G) Viral proteins P2 and M disturb the association of OsMED25-OsMYC3. Fusion proteins were transiently expressed in leaves of N. benthamiana and observed by confocal microscopy. The YFP signals were reduced in the presence of P2 or M protein. (Scale bar, 100 μm.) (H) Interaction between OsMYC3 and OsMED25 was weakened by viral proteins RSV P2 and RSMV M. OsMED25-flag combined with OsMYC3-GFP and increasing amounts of P2/M-myc were coincubated in leaves of N. benthamiana. The immunoprecipitated fractions were detected by anti-flag, anti-myc, and anti-GFP antibody, respectively. Intrinsic protein levels were evaluated by input in the lower panel. (I) Scheme of LCI assays for coexpression in leaves of N. benthamiana. (J) Results from LCI assays showing that RSV P2 and RSMV M proteins impaired the interaction between OsMYC3 and OsMED25. SP8 acts as a positive control. (K) Schematic diagrams of the reporter and a series of effectors employed in the GD system. The reporter gene LUC driven by the 5×GAL4 promoter specifically recognized GD effectors driven by the CaMV 35S promoter. REN, renilla luciferase, an internal control; LUC, firefly luciferase. (L) RSV P2 and RSMV M protein have transcriptional repressor activity and are aggravated by OsJAZs proteins. The 5×GAL4::LUC reporter was coinfiltrated with the GD-P2/M effectors alone or together with OsJAZs proteins into N. benthamiana leaves and measured at 48 hpi. Data were analyzed by the LUC/REN ratio. Error bars represent SD (n = 6); * indicates a significant difference between samples at P ≤ 0.05 by Fisher's least significant difference tests.
Fig. 7.
Fig. 7.
OsMYC3 positively regulates rice resistance to SRBSDV. (A and D) Symptoms in OsMYC3-ox lines (OsMYC3-14 and OsMYC3-23) (A) and Osmyc3 mutants (Osmyc3-1 and Osmyc3-12) (D) following mock-inoculation or SRBSDV infection. The diseased rice plants were verified by RT-PCR and photographs were taken at 30 dpi. (Scale bar, 10 cm.) (B and E) qRT-PCR results showing the relative expression of viral RNA (three different RBSDV genomic RNA segments S2, S4, and S6) in SRBSDV-infected OsMYC3-ox lines (B) and Osmyc3 mutants (E) at 30 dpi. OsUBQ5 was used as the internal reference gene and data were compared with ZH11 background from three biological replicates in a one-way ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. Abbreviation: S2, Segment 2; S4, Segment 4; S6, Segment 6. (C and F) Western blot to assess the accumulation of SRBSDV P10 in SRBSDV-infected OsMYC3-ox lines (C) and Osmyc3 mutants (F) compared with ZH11 at 30 dpi. Total proteins were extracted from SRBSDV-infected transgenic rice leaves and examined by anti-P10 antibody.
Fig. 8.
Fig. 8.
Blocking of JA signaling promotes vector feeding. (A) Phenotypes of OsMYC3-ox, Osmyc3 mutants, and ZH11 plants 7 days after infestation with five adult WBPHs per seedling. (B) Mortality rate of OsMYC3-ox, Osmyc3 mutants, and ZH11 plants infested by adult WBPHs. For each genotype, ∼20 seedlings were tested, and the mortality was counted at 5 dpi and 7 dpi. Data were analyzed by one-way ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. (C) Phenotypes of Oscoi1-13 mutants and Nip plants 7 days after infestation with five adult WBPHs per rice seedling. (D) Mortality rate of Oscoi1-13 mutants and Nip plants infested by adult WBPHs. For each genotype, ∼20 seedlings were tested, and the mortality was counted at 5 dpi and 7 dpi. Data were analyzed by one-way ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. (E) Total honeydew secreted from each adult WBPH individually fed on OsMYC3-ox, Osmyc3 mutants, Oscoi1-13 mutants, and Nip or ZH11 wild-type plants for about 36 hours. Data were analyzed by one-way ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. (F) Mortality rates of WBPH. For each genotype, ∼30 seedlings were tested with four virus-free WBPHs each, and the mortality was counted at 3 dpi and 5 dpi. (G) Total honeydew secreted from each adult WBPH individually fed on SP8-ox transgenic rice plants or Nip controls for about 36 hours. Data were analyzed by ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests. (H) Total honeydew secreted from each adult WBPH individually fed on healthy and SRBSDV-infected rice plants for about 36 hours. Data were analyzed by ANOVA and evaluated at P ≤ 0.05 by Fisher's least significant difference tests.
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