GhWRKY15, a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.), is involved in disease resistance and plant development

Abstract

Background: As a large family of regulatory proteins, WRKY transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. Although several studies have investigated the role of WRKY transcription factors during these processes, the mechanisms underlying the function of WRKY members need to be further explored, and research focusing on the WRKY family in cotton crops is extremely limited.

Results: In the present study, a gene encoding a putative WRKY family member, GhWRKY15, was isolated from cotton. GhWRKY15 is present as a single copy gene, and a transient expression analysis indicated that GhWRKY15 was localised to the nucleus. Additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. Consistently, northern blot analysis showed that GhWRKY15 expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. Furthermore, GhWRKY15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. The GhWRKY15-overexpressing tobacco also exhibited increased RNA expression of several pathogen-related genes, NONEXPRESSOR OF PR1, and two genes that encode enzymes involved in ET biosynthesis. Importantly, increased activity of the antioxidant enzymes POD and APX during infection and enhanced expression of NtAPX1 and NtGPX in transgenic tobacco following methyl viologen treatment were observed. Moreover, GhWRKY15 transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco. Additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of GhWRKY15 transcripts.

Conclusion: Overall, our data suggest that overexpression of GhWRKY15 may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ROS system via multiple signalling pathways in tobacco. It is intriguing that GhWRKY15 overexpression in tobacco affects plant growth and development, especially stem elongation. This finding suggests that the role of the WRKY proteins in disease resistance may be closely related to their function in regulating plant growth and development.

Figure 1

Characterisation of WRKY transcription factors from various species. (A) Identical amino acids are highlighted in blue. The approximately 60-amino acid WRKY domain and the C and H residues in the zinc-finger motif (C-X_4-5-C-X_22-23-H-X₁-H) are marked by the two-headed arrow and triangle, respectively. The short conserved HARF structural motif and the highly conserved amino acid sequence WRKYGQK in the WRKY domain are boxed. (B) Phylogenetic analysis of GhWRKY15 in relation to other plant WRKY transcription factors. The WRKY transcription factors used are as follows: GhWRKY15 (GU207867) and GhWRKY3 (ADO51775) from G. hirsutum, PtWRKY26 (ACV92028), PtWRKY24 (ACV92026), PtWRKY12 (ACV92014) and PtWRKY21 (ACV92023) from P. tomentosa, StWRKY2 (ABU49721) from S. tuberosum, AtWRKY15 (NP_179913), AtWRKY7 (NP_194155), AtWRKY11 (NP_567878), AtWRKY4 (NP_172849), AtWRKY40 (NP_178199), AtWRKY31 (NM_118328), AtWRKY6 (NM_104910), AtWRKY28 (NM_117927), AtWRKY48 (NM_124329), AtWRKY14 (NM_102802), AtWRKY22 (NM_116355), and AtWRKY41 (XP_00287254) from A. thaliana, BnWRKY7 (ACQ76809) from Brassi canapus, CaWRKY2 (ABA56495) and CaWRKY30 (ACJ04728) from Capsicum annuum, HvWRKY10 (ABI13376) from Hordeum vulgare, VvWRKY2 (XP_002264243) from Vitis vinifera, OsWRKY71(AY676927) and OsWRKY1 (AAF23898) from O. sativa and NcWRKY53(ABN79278) from Noccaea caerulescens.

Figure 2

Subcellular localisation of the GhWRKY15::GFP fusion protein. (A) Schematic representation of the 35 S-GhWRKY15::GFP fusion construct and the 35 S-GFP construct. GFP was fused in frame to the C terminus of GhWRKY15. (B) Onion epidermal cells transiently expressing either the 35 S-GhWRKY15::GFP and 35 S-GFP construct were viewed using a confocal laser scanning microscope. The nuclei of the onion cells were visualised using DAPI staining.

Figure 3

Expression of GhWRKY15 in response to different fungal infections and hormone treatments. Approximately one-week-old cotton seedlings were used for all treatments. For the fungal inoculation, the roots of the cotton seedlings were dipped into conidial suspensions of C. gossypii (A), F. oxysporum f. sp. vasinfectum (B) or R. solani (C) (10⁵ conidia/ml). The signalling molecules used were 2 mM SA (D), 100 μM MeJA (E) and ET released from 5 mM ethephon (F). Whole seedling plants were collected for RNA extraction. Ethidium bromide-stained rRNA was included as a loading control.

Figure 4

Enhanced resistance of GhWRKY15-overexpressing tobacco to viruses. (A) Northern blot analysis of the expression levels of GhWRKY15 in T₁ transgenic and WT tobacco under normal conditions. Two leaves were tested for the GhWRKY15 transgenic tobacco. WT: wild-type. (B) Leaf symptoms of tobacco plants infected with TMV (10 days post-inoculation) or CMV (14 days post-inoculation). OE: GhWRKY15-overexpressing tobacco; Mock: mock inoculation; CP: coat proteins. (C) RT-PCR analysis of the expression levels of the CP gene in infected transgenic lines (OE1, OE2 and OE3) and the WT line. (D) TMV and CMV titres in the transgenic lines and the wild-type lines. The data are presented as the mean ± standard error from three independent experiments.

Figure 5

Enhanced resistance of GhWRKY15-overexpressing tobacco to fungi. (A) Leaf symptoms of tobacco plants infected with fungi. The detached leaves in transgenic and wild-type tobacco were inoculated with C. gossypii or P. parasitica suspensions (10⁶ conidia/ml) prepared in 1% glucose, and the leaves were photographed 7 days after inoculation. (B) The diameters of the lesions on the inoculated leaves. The diameters of the lesion spots were recorded using the following scoring system: 0, < 1 mm; 1, 1–2 mm; 2, > 2 mm. (C) The numbers of lesions on the inoculated leaves. The number of lesions per 10 cm² was counted on the inoculated leaves of three independent transgenic and wild-type plants. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

Figure 6

Expression of defence-related genes and ET biosynthesis genes. (A) The expression of defence-related genes and ET biosynthesis genes was examined following TMV infection. Next, qPCR was used to examine the expression of defence-related genes, including PR1, PR2, PR4, PR5 and NPR1, and ET biosynthesis genes, including ACO and ACS genes, in plants 10 days post-infection with TMV. (B) The expression of defence-related genes and ET biosynthesis genes 7 days post infection with a fungus. The actin gene was used to normalise the amount of template in each reaction. The data are presented as the mean ± standard error of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range test.

Figure 7

Expression of GhWRKY15 in tobacco decreased the accumulation of ROS, and MV enhanced GhWRKY15 expression. (A), (B) and (C) show that the expression of GhWRKY15 in tobacco decreased the accumulation of ROS after TMV, CMV or C. gossypii treatment, respectively. The level of H₂O₂ in the tobacco leaves was determined using 1 mg/ml DAB as substrate. The top figure indicates the visualisation of the H₂O₂ accumulation, and the bottom figure shows the microscopic observations of the brown precipitate. (D) MV enhances GhWRKY15 expression. Approximately one-week-old cotton seedlings were used for the 0.5 mM MV treatment. Ethidium bromide-stained rRNA was included as a loading control.

Figure 8

Expression of antioxidant enzymes in transgenic lines. (A) The expression of antioxidant enzymes under normal conditions. (B) The expression of antioxidant enzymes during MV treatment. Also, qPCR analysis was performed to detect the levels of the antioxidant enzymes (NtSOD, NtGPX, NtAPX1, NtAPX2, NtCAT1 and NtCA). Approximately three-week-old transgenic and wild-type tobacco plants were used for the expression analysis. For the MV treatment, the tobacco seedlings were sprayed with 0.5 mM MV and analyzed 6 h after treatment. The data are presented as the mean ± standard error of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

Figure 9

Effect of virus infection on the SOD, POD, CAT and APX activities. (A) and (B) present the SOD, POD, CAT and APX activities 7 days post inoculation with TMV and CMV. The data are presented as the mean ± standard error of three independent experiments. The values indicated by the different letters are significantly different at P < 0.05, as determined using Duncan’s multiple range tests.

Figure 10

Comparison of the growth and development of the transgenic and wild-type tobacco. (A) Seed germination and growth phenotype of transgenic and wild-type tobacco. (B) The growth phenotype of transgenic and wild-type tobacco at approximately 10 weeks. Differences in stem elongation are clearly observable. (C) The height of transgenic and wild-type tobacco from the shooting stage to the flowering stage. (D) Premature flowering of the transgenic plants relative to the wild-type plants. The growth phenotype was photographed at approximately 22 weeks. (E) The phenotype of the bottom leaves of the transgenic and wild-type tobacco at approximately 18 weeks. The figures are a magnification of the red boxes in (E).

Figure 11

Comparison of stems between transgenic and wild-type tobacco. (A) Transverse section of the stems of transgenic and wild-type tobacco at the shooting stage. (B) Vertical section of the stems of transgenic and wild-type tobacco at the shooting stage. (C) Magnification of the red boxes on the left in (A). (D) Magnification of the red boxes on the right in (A). The left and right red boxes primarily indicate cells of the cortex, vascular bundle and pith. Bar: 100 μm. (E) Visual differences in the stems of transgenic and wild-type tobacco at the shooting stage.

Figure 12

Tissue-specific expression of GhWRKY15 and expression analysis of GhWRKY15 in response to abiotic stresses. Total RNA was extracted from the roots (R), stems (S) and leaves (L) for the tissue-specific expression analysis (A). Total RNA was extracted from the leaves at the indicated time points after treatment with cold (4 °C) (B), 200 mM NaCl (C), wounding (D), 15% (w/v) PEG6000 (E), 500 μM GA₃ (F) or 100 μM ABA (G). Ethidium bromide-stained rRNA was included as a loading control.