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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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Hypothetical model of WRKY network in mediating canola responses to S. sclerotiorum and phytohormones. JA/ET-responsive, but not SA-responsive genes were observed in the necrotrophic pathogen S. sclerotiorum-canola interaction [2,5], and these induced BnWRKY genes, such as BnWRKY25, 28, 33, 53 and 75, could potentially activate the downstream JA/ET signaling pathway or cell death. Moreover, BnWRKY70 might also negatively regulate JA/ET signaling pathway while BnWRKY6 possibly modulates the expression of PR1 gene, culminating in cell death, which would benefit the infection and growth of necrotrophic fungi [114,118]. MPK3, one of mitogen-activated protein kinases, was also observed to be induced by S. sclerotiorum in our previous microarray study [5], and MPK3 in Arabidopsis is a positive regulator of AtWRKY22/29 [39]. Solid and dashed arrows represent likely or putatively positive regulation of the downstream targets while open blocks indicate negative regulation of the downstream genes.
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Figure 5: Hypothetical model of WRKY network in mediating canola responses to S. sclerotiorum and phytohormones. JA/ET-responsive, but not SA-responsive genes were observed in the necrotrophic pathogen S. sclerotiorum-canola interaction [2,5], and these induced BnWRKY genes, such as BnWRKY25, 28, 33, 53 and 75, could potentially activate the downstream JA/ET signaling pathway or cell death. Moreover, BnWRKY70 might also negatively regulate JA/ET signaling pathway while BnWRKY6 possibly modulates the expression of PR1 gene, culminating in cell death, which would benefit the infection and growth of necrotrophic fungi [114,118]. MPK3, one of mitogen-activated protein kinases, was also observed to be induced by S. sclerotiorum in our previous microarray study [5], and MPK3 in Arabidopsis is a positive regulator of AtWRKY22/29 [39]. Solid and dashed arrows represent likely or putatively positive regulation of the downstream targets while open blocks indicate negative regulation of the downstream genes.

Mentions: It is possible that several BnWRKY TFs may also be involved in signaling the responses of canola to the pathogens S. sclerotiorum and A. brassicae. For instance, group IIa members have been demonstrated to play both positive and negative roles in plant defense [112,115-117]. The transcript levels of two genes of the group IIa: BnWRKY18, and 40, orthologs of which are known to act as negative regulators of plant defense in Arabidopsis [95], were observed to increase in response to S. sclerotiorum and A. brassicae challenge. For A. brassicae, the differences in transcript abundance between controls and inoculated plants were not statistically significant (Figure 3). In addition, it has been reported that AtWRKY6, one member of the group IIb, acts as a positive regulator of the senescence- and pathogen defense-associated PR1 promoter activity, and is also induced by SA and bacterial infection [114]. Since leaf senescence is often linked to plant defense [118], the induction of BnWRKY6 by S. sclerotiorum, ABA and SA at an early time-point (6 h) but not A. brassicae, may suggest a role in leaf senescence, which is observed very early in the S. sclerotiorum-canola pathosystem (Figure 5).


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)

Hypothetical model of WRKY network in mediating canola responses to S. sclerotiorum and phytohormones. JA/ET-responsive, but not SA-responsive genes were observed in the necrotrophic pathogen S. sclerotiorum-canola interaction [2,5], and these induced BnWRKY genes, such as BnWRKY25, 28, 33, 53 and 75, could potentially activate the downstream JA/ET signaling pathway or cell death. Moreover, BnWRKY70 might also negatively regulate JA/ET signaling pathway while BnWRKY6 possibly modulates the expression of PR1 gene, culminating in cell death, which would benefit the infection and growth of necrotrophic fungi [114,118]. MPK3, one of mitogen-activated protein kinases, was also observed to be induced by S. sclerotiorum in our previous microarray study [5], and MPK3 in Arabidopsis is a positive regulator of AtWRKY22/29 [39]. Solid and dashed arrows represent likely or putatively positive regulation of the downstream targets while open blocks indicate negative regulation of the downstream genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Hypothetical model of WRKY network in mediating canola responses to S. sclerotiorum and phytohormones. JA/ET-responsive, but not SA-responsive genes were observed in the necrotrophic pathogen S. sclerotiorum-canola interaction [2,5], and these induced BnWRKY genes, such as BnWRKY25, 28, 33, 53 and 75, could potentially activate the downstream JA/ET signaling pathway or cell death. Moreover, BnWRKY70 might also negatively regulate JA/ET signaling pathway while BnWRKY6 possibly modulates the expression of PR1 gene, culminating in cell death, which would benefit the infection and growth of necrotrophic fungi [114,118]. MPK3, one of mitogen-activated protein kinases, was also observed to be induced by S. sclerotiorum in our previous microarray study [5], and MPK3 in Arabidopsis is a positive regulator of AtWRKY22/29 [39]. Solid and dashed arrows represent likely or putatively positive regulation of the downstream targets while open blocks indicate negative regulation of the downstream genes.
Mentions: It is possible that several BnWRKY TFs may also be involved in signaling the responses of canola to the pathogens S. sclerotiorum and A. brassicae. For instance, group IIa members have been demonstrated to play both positive and negative roles in plant defense [112,115-117]. The transcript levels of two genes of the group IIa: BnWRKY18, and 40, orthologs of which are known to act as negative regulators of plant defense in Arabidopsis [95], were observed to increase in response to S. sclerotiorum and A. brassicae challenge. For A. brassicae, the differences in transcript abundance between controls and inoculated plants were not statistically significant (Figure 3). In addition, it has been reported that AtWRKY6, one member of the group IIb, acts as a positive regulator of the senescence- and pathogen defense-associated PR1 promoter activity, and is also induced by SA and bacterial infection [114]. Since leaf senescence is often linked to plant defense [118], the induction of BnWRKY6 by S. sclerotiorum, ABA and SA at an early time-point (6 h) but not A. brassicae, may suggest a role in leaf senescence, which is observed very early in the S. sclerotiorum-canola pathosystem (Figure 5).

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