MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes

Abstract

Transposition of the tobacco retrotransposon Tto1 is regulated mainly by transcription from the long terminal repeat (LTR). Functional analysis of the LTR showed that the 13-bp motif is a cis-regulatory element involved in activation by tissue culture, wounding, and treatment with elicitors. The 13-bp motif contains a conserved motif (L box) that has been implicated in the expression of phenylpropanoid synthetic genes in response to defense-related stresses. To gain further insight into the regulatory mechanism of the retrotransposon and defense-related genes, cDNAs encoding four different proteins binding to the 13-bp motif have been isolated and characterized. One protein is identical to the previously reported NtMYB1, the RNA for which is induced by virus infection; the others are also MYB-related factors. One of these factors, NtMYB2, was analyzed in detail. NtMYB2 mRNA was induced by wounding and by treatment with elicitors. NtMYB2 activated expression from the promoter with the 13-bp motif and from the promoter of the phenylalanine ammonia lyase gene (Pv-PAL2) in tobacco protoplasts. Overexpression of NtMYB2 cDNA in transgenic tobacco plants induced expression of Tto1 and a PAL gene. Together, these results indicate that NtMYB2 is involved in the stress response of the retrotransposon and defense-related genes.

Figure 1.

Amino Acid Sequences of NtMYB Proteins and Sequence Comparisons among MYB Proteins. (A) Amino acid sequences of NtMYB2, NtMYB3, NtMYB1, and NtMYB4. The R2 and R3 of MYB domain and putative acidic activation domains are indicated by bars. (B) Dendrogram relationships among R2R3 domain of MYB proteins constructed from the matrix of sequence similarities. MYB proteins included in this comparison are D-MYB from Drosophila (Katzen et al., 1985); c-MYB from human (Majello et al., 1986); C1 (ZmMYBC1; Paz-Ares et al., 1987) and P (ZmMYBP; Grotewold et al., 1991) from maize; Am305 (AmMYB305; Jackson et al., 1991) and MIXTA (AmMYBMixta; Noda et al., 1994) from Antirrhinum majus; GL1 (AtMYBGL1; Oppenheimer et al., 1991), AtMYB1 (Shinozaki et al., 1992), AtMYB2 (Urao et al., 1993), AtMYB13 (Kirik et al., 1998a), and AtMYB15 (Quaedvlieg et al., 1996) from Arabidopsis; PhMYB1, PhMYB2, and PhMYB3 from petunia (Avila et al., 1993); GA-MYB from barley (HvMYBGa; Gubler et al., 1995); NtMYB1 from tobacco (Yang and Klessig, 1996); NtMYB2, NtMYB3, and NtMYB4 from tobacco (this study). NtMYBs indicated with (T) and (S) are derived from N. tomentosiformis and N. sylvestris, respectively.

Figure 2.

RNA Gel Blot Analysis of NtMYB2 Transcripts. (A) Each lane was loaded with 20 μg of total RNA from leaf segments harvested at 0, 0.5, 1, 2, 4, and 12 hr after cutting and was then probed with an NtMYB2-specific DNA fragment. The 28S rRNA stained with methylene blue is shown under each RNA gel blot to assure equal loading of RNA. (B) Each lane was loaded with 5 μg of total RNA from flowers, roots, leaves, BY2 suspension-cultured cells, and protoplasts of BY2 and probed with an NtMYB2 gene–specific DNA fragment. (C) Five micrograms of RNA from detached whole leaves that had been treated for 1 or 8 hr with Mes buffer in the absence (Mes) or presence of 1 mg/mL Onozuka cellulase solution (R10), autoclaved Onozuka cellulase solution (R10/H), 100 μM chitin oligomer (Ch), 2.5 μg of xylanase per gram of leaf (Xy), and cycloheximide (CHX) were blotted and probed with an NtMYB2-specific DNA fragment. (D) Five micrograms of RNAs from leaf segments was harvested at 0 and 1 hr after cutting and was probed with an NtMYB2-specific DNA fragment (NtMYB2) or a 3′-UTR. The position of the 18S rRNA is indicated by an arrowhead (1.8 kb).

Figure 3.

NtMYB2-Mediated trans-Activation Depends on the Binding to the 13-bp Motif. (A) Competition assays for binding of in vitro–translated NtMYB2 to the labeled LTR (−92 to −52; LTR4) probe. EMSA was performed by preincubating 25-fold (×25; left lane under the triangle) or 125-fold (×125; right lane under the triangle) excess amounts (based on the number of binding site) of unlabeled competitor DNA fragments, LTR (−92 to −52) (4), mutant LTR (4m), multimerized wild type (AB), multimerized mutant 13-bp motif (EF), or a multicloning site sequence of pBlueScript SK+ (MCS). Dash, without translate or competitor; N, in vitro translate without NtMYB2 plasmid (pSK-NtMYB2); F, free probe; S, shifted probe. The difference in the sequence between the wild-type (4) and mutant (m) LTR fragments used as unlabeled competitors is shown, highlighting the substituted nucleotides. (B) Structures of the reporter and effector plasmids. pLTR-CAT4, LTR (−96 to +130) promoter fused to the CAT gene; pLTR-CAT5, LTR (−37 to +130) minimal promoter fused to the CAT gene (Hirochika et al., 1996b). pLTR-CAT5AB and pLTR-CAT5EF, 13-bp motif (AB) or mutant (EF; substituted nucleotides are indicated on a black background), were multimerized (×9, nine copies; ×8, eight copies) and inserted upstream of the LTR. Nucleotides in uppercase correspond to the 13-bp motif. The effector plasmid consists of 35S promoter fused to the NtMYB2 cDNA (p35S-NtMYB2). The plasmid consisting of the 35S promoter fused to the GUS gene (p35S-GUS) was used as a control. (C) The effector plasmid (p35S-NtMYB2, NtMYB2) or the control plasmid (p35S-GUS, GUS) was cotransfected with reporter constructs (pLTR-CAT4, LTR4; pLTR-CAT5, LTR5) into tobacco protoplasts. Relative CAT activity (and standard deviation) obtained from three independent experiments is shown. (D) The effector or the control plasmid was cotransfected with reporter constructs (pLTR-CAT5AB, 13AB; pLTR-CAT5EF, 13EF) into tobacco protoplasts. Error bars indicate se.

Figure 4.

Hydroxyl Radical Interference Analysis of NtMYB2 Binding to the 13-bp Motif. (A) Hydroxyl radical–treated LTR probe (−92 to −52), which was asymmetrically labeled with ³²P-dCTP, was incubated with in vitro–translated NtMYB2 and subjected to native PAGE. Shifted DNA (S) was compared with free probe (N) on the sequencing gel. G + A reactions were loaded as a marker (GA). Both DNA strands were analyzed. (B) Summary of nucleotides for which a single-base deletion led to a decrease (open triangle) or failure (closed triangles) of the NtMYB2 binding. Nucleotides in uppercase letters correspond to the 13-bp motif.

Figure 5.

NtMYB2-Mediated trans-Activation of Pv-PAL2 Promoter. (A) EMSA was performed with in vitro–translated NtMYB2 and wild-type (W) Pv-PAL2 promoter probe (−254 to +55) or mutant probes (Lm, Pm, and PLm). Competition assays were performed by preincubating none (–) or 25-fold (left lane under the triangle) or 125-fold (right lane under the triangle) excess amounts of unlabeled competitors, multimerized 13-bp motif (AB), or multimerized mutant motif (EF). Free probe (F) and mobility shift by NtMYB2 complex (S) are indicated. (B) Reporter constructs. Pv-PAL2 (-254 to +55, W) or mutant prompters (Lm, Pm, and PLm) were fused to the CAT coding region. Highlighted letters show the substituted nucleotides. (C) The plasmid consisting of the cauliflower mosaic virus 35S promoter fused to NtMYB2 cDNA (LBM; p35S-NtMYB2 of Figure 3B) or to the GUS coding region (GUS; p35S-GUS of Figure 3B) was cotransfected with reporter constructs (W, Lm, Pm, and PLm) into tobacco protoplasts. Average CAT activity (and standard deviation) obtained from three independent transfection assays is shown.

Figure 6.

EMSA of the 13-bp Motif Performed with Nuclear Extracts. (A) EMSA experiments were performed with nuclear extracts or in vitro–translated NtMYB2 (0.1 μL) (NtMYB2). Nuclear extracts were prepared from nontreated leaf (L) or wounded leaf incubated for 1 hr (Wounding), and were incubated with ³²P-labeled LTR4 probe (−92 to −37) and 25-fold (×25, left lane under the triangle) or 125-fold (×125, right lane under the triangle) excess amounts of unlabeled wild-type (4) and mutant (m) LTR4 fragment. Preimmune antiserum (1:1000 dilution) (P) or anti-NtMYB2 serum (1:1000 dilution) (I) was added in the binding reaction. Free probe (F), mobility shift (S) by NtMYB2 complex, and the mobility shift (SS and <) by NtMYB2–anti-NtMYB2 antibody complexes are indicated. Dashes indicate the absence of a given component. (B) EMSA experiments were performed with nuclear extracts prepared from nontreated leaf (L) or leaf treated with elicitor (autocleaved Onozuka cellulase solution) for 1 hr (R10/H). Nuclear extracts were incubated with ³²P-labeled LTR4 probe (−92 to −37). Abbreviations as in (A). (C) Differences in sequences of the wild-type (4) and mutant (m) LTR fragments used as nonlabeled competitors. Highlighted CTT shows the substituted nucleotides. (D) Protein gel blot analysis was performed with the affinity-purified anti-NtMYB2 antibody (final concentration, 0.66 μg/mL). Nuclear extracts prepared from nontreated leaf (Leaf), wounded leaf (Leaf/w), elicitor-treated leaf (Leaf/R10/H), or BY2 cell (BY2) as well as from in vitro–translated NtMYB2, NtMYB3, NtMYB1, or NtMYB4 (0.1 μL each) were loaded onto the gel. To assure equal loading of in vitro–translated MYB proteins, these proteins were labeled with ³⁵S-methionine and detected by autoradiography. The purified fusion protein (10 or 50 pg) of glutathione S-transferase with a part of NtMYB2 (amino acid positions from 174 to 231) was loaded as a quantitative marker. An arrowhead indicates the NtMYB2 protein signals.

Figure 7.

Induction of the NtMYB2 Protein and Its Binding Activity by Wounding. (A) EMSA was performed without (Non) or with nuclear extracts prepared from nontreated leaf (Leaf) or wounded leaf incubated for 1, 4, 8, and 24 hr by incubating with the ³²P-labeled LTR4 probe (−92 to −37). EMSA with in vitro–translated NtMYB2 (0.1 μL) was also performed to show mobility shift patterns specific to each protein. Free probe (F) and the mobility shift by NtMYB2 (S) are indicated. (B) Protein gel blot analysis was performed with purified NtMYB2-specific antibody. Nuclear extract prepared from nontreated leaf (Leaf), wounded leaf incubated for indicated times (Wounding), and in vitro–translated full-length NtMYB2 (0.1 μL) (NtMYB2) were analyzed together with 10 or 50 pg of the purified fusion protein of glutathione S-transferase and a part of NtMYB2. An arrowhead indicates the NtMYB2 protein signals. (C) Inhibition of induction of NtMYB2 protein synthesis and NtMYB2 DNA binding activity by cycloheximide (X) treatment. EMSA was performed with nuclear extracts prepared from nontreated leaf (Leaf) or wounded leaf incubated for 1 hr in the absence (Leaf/W) or presence of 0.5 mM CHX (Leaf/W/X) by incubating with the ³²P-labeled LTR4 probe (−92 to −37). EMSA with in vitro–translated NtMYB2 (NtMYB2) was also performed. Free probe (F) and the mobility shift by NtMYB2 (S) are indicated. The CHX treatment inhibited de novo protein synthesis by 85%. (D) Protein gel blot analysis was performed with purified NtMYB2-specific antibody. The nuclear extracts used in (C), 0.1 μL of in vitro–translated NtMYB2 (NtMYB2), and the 10 or 50 pg of purified fusion protein of glutamine S-transferase with a portion of NtMYB2 were analyzed. An arrowhead indicates the NtMYB2 protein signals.

Figure 8.

Overexpression of the NtMYB2 cDNA Driven by the 35S Promoter in Independent Transgenic Tobacco Lines. Each lane was loaded with 5 μg of total RNA from nontreated transgenic (OX1 to OX6) and control tobacco leaf (N) and was probed with NtMYB2, GUS, Tto1, PAL, and F₁-ATP synthase β-subunit (F₁-β)–specific DNA fragments. rRNA was stained with methylene blue.

Figure 9.

Expression of NtMYB2 and GUS Driven by the Promoter Containing Multiple Copies of the 13-bp Motif in Response to Wounding and Treatment with Inhibitors for Protein Synthesis or Protein Phosphatase. Transgenic tobacco plants carrying 13AB-LTR(–37)-GUS were used in which nine copies of the 13-bp motif were fused with the minimal LTR promoter–GUS construct (Takeda et al., 1999). (A) Each lane was loaded with 5 μg of total RNA from wounded leaf incubated in Mes buffer in the absence (Wounding) or presence of 0.5 mM CHX (Wounding+CHX) for 1 hr. The RNA gel blot was hybridized with an NtMYB2-specific or GUS-specific probe. (B) Each lane was loaded with 5 μg of total RNA from detached whole leaves for which the petioles had been dipped into Mes buffer in the absence (Mes) or presence of 0.5 μM okadaic acid (Mes+OA) for the times indicated. The RNA gel blot was hybridized with the NtMYB2-specific or the GUS-specific probe.