WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis

Abstract

WRKY transcription factors are key players in the plant immune response, but less is known about their involvement in antiviral defense than about their roles in defense against bacterial or fungi pathogens. Here, we report that Arabidopsis thaliana WRKY DNA-binding protein 8 (WRKY8) has a role in mediating the long-distance movement of crucifer-infecting tobacco mosaic virus (TMV-cg). The expression of WRKY8 was inhibited by TMV-cg infection, and mutation of WRKY8 accelerated the accumulation of TMV-cg in systemically infected leaves. Quantitative RT-PCR analysis showed that the expression of ABA insensitive 4 (ABI4) was reduced and the expression of 1-aminocyclopropane-1-carboxylic acid synthase 6 (ACS6) and ethylene response factor 104 (ERF104) was enhanced in the systemically infected leaves of wrky8. Immunoprecipitation assays demonstrated that WRKY8 could bind selectively to putative W-boxes of the ABI4, ACS6, and ERF104 promoters. Furthermore, TMV-cg infection enhanced WRKY8 binding to the ABI4 promoter but reduced the binding of WRKY8 to the ACS6 and ERF104 promoters, indicating that regulation of ABI4, ACS6, and ERF104 by WRKY8 is at least partially dependent on TMV-cg. Exogenous applications of abscisic acid (ABA) reduced the systemic accumulation of TMV-cg. Mutations in ABA deficient 1, ABA deficient 2, ABA deficient 3, or abi4 accelerated systemic TMV-cg accumulation. In contrast, exogenous application of aminocyclopropane-1-carboxylic acid enhanced the systemic accumulation of TMV-cg, but mutations in acs6, erf104, or an octuple acs mutant inhibited systemic TMV-cg accumulation. Our results demonstrate that WRKY8 is involved in the defense response against TMV-cg through the direct regulation of the expression of ABI4, ACS6, and ERF104 and may mediate the crosstalk between ABA and ethylene signaling during the TMV-cg-Arabidopsis interaction.

Keywords: ABA signaling; ET signaling; WRKY8 regulation; virus movement.

Fig. 1.

Mutation of wrky8 affects the accumulation of TMV-cg in systemically infected leaves. The third, fourth, and fifth true leaves of 26-d-old wild-type and wrky8 mutants were inoculated with TMV-cg (5 µg/mL solution in 5 mM sodium phosphate, pH 7.5). RNA samples were prepared from systemically infected leaves of six plants inoculated with TMV-cg for 3, 4, 5, 6, and 7 d, respectively, and were probed with a TMV-cg coat protein (CP) cDNA fragment. Ethidium bromide-stained rRNA was used as a loading control. These experiments were repeated three times with similar results. FL, full length.

Fig. 2.

Induced expression of WRKY8. (A) GUS staining of WRKY8 in leaves locally infected with buffer or TMV-cg at 0, 1, 2, 3, 4, and 5 dpi. (B) Expression of WRKY8 in leaves locally infected with buffer or TMV-cg at 0, 1, 2, 3, 4, and 5 dpi. (C) Expression of WRKY8 after treatment with ABA (time course of 0, 1, 4, 8, 24, and 48 h). (D) Expression of WRKY8 in TMV-cg systemically infected leaves at 3, 4, 5, 6, and 7 dpi. These experiments were repeated three times with similar results.

Fig. 3.

The role of ABA in defense against TMC-cg infection. (A) Expression of ABA1, ABA2, and ABA3 in wild-type leaves systemically infected by TMV-cg at 3, 5, and 7 dpi. (B) Expression of ABI4 in wild-type leaves systemically infected by TMV-cg at 3, 5, and 7 dpi. (C) Expression of ABI4 in wild-type and wrky8 leaves systemically infected by TMV-cg at 6 dpi. (D) ABA delays the accumulation of TMV-cg in systemically infected leaves. Three leaves of 26-d-old wild-type plants treated or not treated with 100 µM ABA were inoculated with TMV-cg. Leaf collection, RNA isolation, and RNA blot analysis of TMV-cg CP were performed as in Fig. 1. (E) Mutation of abi4, aba1, aba2, or aba3 promotes the accumulation of TMV-cg in systemically infected leaves. Inoculation, leaf collection, RNA isolation, and RNA blot analysis of TMV-cg CP were performed as in Fig. 1. These experiments were repeated three times with similar results.

Fig. 4.

Expression of PR2, ACS6, and ERF106. (A) Expression of PR2 in wild-type and wrky8 leaves systemically infected by TMV-cg at 3, 4, 5, 6, and 7 dpi. (B) Expression of PR1 in wild-type and wrky8 leaves systemically infected by TMV-cg at 6 dpi. (C) Expression of ACS6 in wild-type and wrky8 leaves systemically infected by TMV-cg a t6 dpi. (D) Expression of ERF104 in wild-type and wrky8 leaves systemically infected by TMV-cg at6 dpi. These experiments were repeated at least twice with similar results.

Fig. 5.

The role of ET in defense against TMC-cg infection. (A) Expression of ACS6 in wild-type leaves systemically infected dpi by TMV-cg at 3, 4, 5, 6, and 7. (B) Expression of ERF104 in wild-type leaves systemically infected by TMV-cg at 3, 4, 5, 6, and 7 dpi. (C) ACC promotes the accumulation of TMV-cg in systemically infected leaves. Three leaves of 26-d-old wild-type plants treated or not treated with 100 µM ACC were inoculated with TMV-cg. Leaf collection, RNA isolation, and RNA blot analysis of TMV-cg CP were performed as in Fig. 1. (D) Mutation of acs6 or erf104 inhibits the accumulation of TMV-cg in systemically infected leaves. Inoculation, leaf collection, RNA isolation, and RNA blot analysis of TMV-cg CP were performed as in Fig. 1. (E) Mutation of eight acs genes (acs1/2/4/5/6/7/9/11) or ein2 inhibits the accumulation of TMV-cg in systemically infected leaves. Inoculation, leaf collection, RNA isolation, and RNA blot analysis of TMV-cg CP were performed as in Fig. 1. These experiments were repeated at least twice with similar results.

Fig. 6.

ABI4, ACS6, and ERF104 are direct targets of WRKY8. (A–C) (Upper) Schematic representations of the ABI4, ACS6, and ERF104 promoter regions containing W-box clusters. Only prefect W-boxes (T/CTGACC/T, black bar) are depicted. The diagram indicates the number and relative position of the W-boxes in the respective promoters relative to the ATG start codon. In promoter fragment names, the prefix ”p” indicates promoter. Pink lines indicate the sequences detected by ChIP assays. (Lower) ChIP assays were performed with chromatin prepared from HA-WRKY8 plants infected b TMV-cg at 0, 6, and 7 dpi using an anti-HA antibody (IP) or preimmune serum (no Ab) as a negative control. ChIP results are presented as a percentage of input DNA, SDs were calculated from three technical repeats. One representative experiment is presented. Fig. S3 shows other two ChIP results. The experiments were repeated three times with similar results.

Fig. 7.

A simplified model for the function of WRKY8 during TMV-cg–Arabidopsis interaction. Wounding or ABA treatment induces WRKY8 expression, whereas infection by TMV-cg inhibits WRKY8 expression. WRKY8 participates in the TMV-cg defense response by both activating the expression of ABI4 and repressing ET-related genes such as ACS6 and ERF104 during TMV-cg–Arabidopsis interaction.