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Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

Yang B, Jiang Y, Rahman MH, Deyholos MK, Kav NN - BMC Plant Biol. (2009)

Bottom Line: We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes.We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, Canada. byang@ualberta.ca

ABSTRACT

Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.

Results: In this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.

Conclusion: We identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

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Nuclear localization of four BnWRKY proteins. Transgenic (T2) Arabidopsis roots of five-day old seedlings were observed under confocal microscope. Panels A-E represent the subcellular localization of BnWRKY6-sGFP, BnWRKY25-sGFP, BnWRKY33-sGFP, BnWRKY75-sGFP and pCsGFPBT vector control, respectively. In each case, the extreme left panel is GFP fluorescence, the middle bright field and the right represents an overlay of the two images.
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Figure 2: Nuclear localization of four BnWRKY proteins. Transgenic (T2) Arabidopsis roots of five-day old seedlings were observed under confocal microscope. Panels A-E represent the subcellular localization of BnWRKY6-sGFP, BnWRKY25-sGFP, BnWRKY33-sGFP, BnWRKY75-sGFP and pCsGFPBT vector control, respectively. In each case, the extreme left panel is GFP fluorescence, the middle bright field and the right represents an overlay of the two images.

Mentions: The function of a TF normally requires that it is localized in the nucleus, although TFs targeting chloroplasts, mitochondria, or endoplasmic reticulum (ER) have also been identified [79]. To confirm that the BnWRKY TFs we identified are indeed targeted to the nucleus, we selected four BnWRKY genes based on their known functions in mediating defense responses in Arabidopsis [25,45,80-82] for analysis in vivo. We fused the coding regions of BnWRKY6, 25, 33, and 75 to the N-terminus of synthetic green fluorescent protein (sGFP) [83] and expressed them in Arabidopsis under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Analysis of conceptually translated BnWRKY6, 25, and 33 coding sequences revealed the presence of a monopartite nuclear localization signal (NLS) (prediction program of protein localization sites, ), however, no NLS was detected in the translated BnWRKY75 sequence. We analyzed transgenic Arabidopsis seedlings harboring the respective four constructs. In all four cases, green fluorescent signals were observed only in the nucleus (Figure 2A–D). With the control vector alone, GFP signals were distributed in both the cytoplasm and nucleus (Figure 2E). Our results indicate that BnWRKY6, 25, 33, and 75 are indeed nuclear-localized proteins, which is consistent with their predicted function as transcription factors.


Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

Yang B, Jiang Y, Rahman MH, Deyholos MK, Kav NN - BMC Plant Biol. (2009)

Nuclear localization of four BnWRKY proteins. Transgenic (T2) Arabidopsis roots of five-day old seedlings were observed under confocal microscope. Panels A-E represent the subcellular localization of BnWRKY6-sGFP, BnWRKY25-sGFP, BnWRKY33-sGFP, BnWRKY75-sGFP and pCsGFPBT vector control, respectively. In each case, the extreme left panel is GFP fluorescence, the middle bright field and the right represents an overlay of the two images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698848&req=5

Figure 2: Nuclear localization of four BnWRKY proteins. Transgenic (T2) Arabidopsis roots of five-day old seedlings were observed under confocal microscope. Panels A-E represent the subcellular localization of BnWRKY6-sGFP, BnWRKY25-sGFP, BnWRKY33-sGFP, BnWRKY75-sGFP and pCsGFPBT vector control, respectively. In each case, the extreme left panel is GFP fluorescence, the middle bright field and the right represents an overlay of the two images.
Mentions: The function of a TF normally requires that it is localized in the nucleus, although TFs targeting chloroplasts, mitochondria, or endoplasmic reticulum (ER) have also been identified [79]. To confirm that the BnWRKY TFs we identified are indeed targeted to the nucleus, we selected four BnWRKY genes based on their known functions in mediating defense responses in Arabidopsis [25,45,80-82] for analysis in vivo. We fused the coding regions of BnWRKY6, 25, 33, and 75 to the N-terminus of synthetic green fluorescent protein (sGFP) [83] and expressed them in Arabidopsis under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Analysis of conceptually translated BnWRKY6, 25, and 33 coding sequences revealed the presence of a monopartite nuclear localization signal (NLS) (prediction program of protein localization sites, ), however, no NLS was detected in the translated BnWRKY75 sequence. We analyzed transgenic Arabidopsis seedlings harboring the respective four constructs. In all four cases, green fluorescent signals were observed only in the nucleus (Figure 2A–D). With the control vector alone, GFP signals were distributed in both the cytoplasm and nucleus (Figure 2E). Our results indicate that BnWRKY6, 25, 33, and 75 are indeed nuclear-localized proteins, which is consistent with their predicted function as transcription factors.

Bottom Line: We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes.We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, Canada. byang@ualberta.ca

ABSTRACT

Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.

Results: In this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.

Conclusion: We identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

Show MeSH
Related in: MedlinePlus