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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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A bootstrap consensus maximum parsimony tree of WRKY TFs in canola. The phylogenetic tree was based on the amino acid sequences from WRKY domains only. Only the ~60 amino acid residues in the WRKY domain were aligned using ClustalX (v1.83) and were further examined manually for optimal alignment. The parsimony tree was drawn using MEGA4. The percentage of replicate trees is shown on the branches and it is calculated in the bootstrap test (500 replicates) for the associated taxa being clustered together. The two letters N and C after group I represent the N-terminal and the C-terminal WRKY domains of group I proteins, respectively.
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Figure 1: A bootstrap consensus maximum parsimony tree of WRKY TFs in canola. The phylogenetic tree was based on the amino acid sequences from WRKY domains only. Only the ~60 amino acid residues in the WRKY domain were aligned using ClustalX (v1.83) and were further examined manually for optimal alignment. The parsimony tree was drawn using MEGA4. The percentage of replicate trees is shown on the branches and it is calculated in the bootstrap test (500 replicates) for the associated taxa being clustered together. The two letters N and C after group I represent the N-terminal and the C-terminal WRKY domains of group I proteins, respectively.

Mentions: From the 46 canola WRKY genes identified, we were able to extract 53 WRKY domains that were each approximately 60 amino acids in length. In 11 BnWRKY TF proteins, we identified two separate WRKY domains (Additional file 3), and both N- and C-terminal WRKY domains of these proteins were included in the phylogenetic analysis. The WRKY domain amino acid sequences were aligned with each other (Additional file 3) and a consensus maximum parsimony (MP) tree was inferred (Figure 1). Subsequently, we reconstructed a rooted MP tree using a WRKY protein from the world's smallest unicellular green algae Ostreococcus tauri WRKY as the outgroup (Figure 1). This tree demonstrates the polyphyletic nature of BnWRKY TFs, which is consistent with previous studies [22,23,26].


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)

A bootstrap consensus maximum parsimony tree of WRKY TFs in canola. The phylogenetic tree was based on the amino acid sequences from WRKY domains only. Only the ~60 amino acid residues in the WRKY domain were aligned using ClustalX (v1.83) and were further examined manually for optimal alignment. The parsimony tree was drawn using MEGA4. The percentage of replicate trees is shown on the branches and it is calculated in the bootstrap test (500 replicates) for the associated taxa being clustered together. The two letters N and C after group I represent the N-terminal and the C-terminal WRKY domains of group I proteins, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A bootstrap consensus maximum parsimony tree of WRKY TFs in canola. The phylogenetic tree was based on the amino acid sequences from WRKY domains only. Only the ~60 amino acid residues in the WRKY domain were aligned using ClustalX (v1.83) and were further examined manually for optimal alignment. The parsimony tree was drawn using MEGA4. The percentage of replicate trees is shown on the branches and it is calculated in the bootstrap test (500 replicates) for the associated taxa being clustered together. The two letters N and C after group I represent the N-terminal and the C-terminal WRKY domains of group I proteins, respectively.
Mentions: From the 46 canola WRKY genes identified, we were able to extract 53 WRKY domains that were each approximately 60 amino acids in length. In 11 BnWRKY TF proteins, we identified two separate WRKY domains (Additional file 3), and both N- and C-terminal WRKY domains of these proteins were included in the phylogenetic analysis. The WRKY domain amino acid sequences were aligned with each other (Additional file 3) and a consensus maximum parsimony (MP) tree was inferred (Figure 1). Subsequently, we reconstructed a rooted MP tree using a WRKY protein from the world's smallest unicellular green algae Ostreococcus tauri WRKY as the outgroup (Figure 1). This tree demonstrates the polyphyletic nature of BnWRKY TFs, which is consistent with previous studies [22,23,26].

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