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AaERF1 positively regulates the resistance to Botrytis cinerea in Artemisia annua.

Lu X, Jiang W, Zhang L, Zhang F, Zhang F, Shen Q, Wang G, Tang K - PLoS ONE (2013)

Bottom Line: The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast.The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua.The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

View Article: PubMed Central - PubMed

Affiliation: Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.

ABSTRACT
Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

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The DNA binding ability of AaERF1 via GCC box.A. Electrophoretic mobility shift assays on DNA binding of AaERF1. Lane 1: negative controls contain MBP plus labelled GCC probe; lane 2: the MBP–AaERF1 protein plus labelled GCC probe; lane 3: only labelled GCC probe; lane 4: negative controls contain MBP plus labelled mutated GCC probe; lane 5: MBP–AaERF1 plus labelled mutated GCC probe; lane 6: only labelled mutated GCC probe. The protein–GCC probe complex and free probes are indicated respectively by two arrows. B. GCC box binding analysis of AaERF1 using the yeast one-hybrid system. Sketch maps show the construction of vectors used in this experiment. Photographs show the growth behavior of transformants on SD/Trp−Ura−/X-gal medium. Sector 1: p178-4×GCC-LacZ+pB42AD-AaERF1; sector 2: p178+ pB42AD-AaERF1; sector 3: p178-4×GCC-LacZ+pB42AD; sector 4: p178+ pB42AD.
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pone-0057657-g004: The DNA binding ability of AaERF1 via GCC box.A. Electrophoretic mobility shift assays on DNA binding of AaERF1. Lane 1: negative controls contain MBP plus labelled GCC probe; lane 2: the MBP–AaERF1 protein plus labelled GCC probe; lane 3: only labelled GCC probe; lane 4: negative controls contain MBP plus labelled mutated GCC probe; lane 5: MBP–AaERF1 plus labelled mutated GCC probe; lane 6: only labelled mutated GCC probe. The protein–GCC probe complex and free probes are indicated respectively by two arrows. B. GCC box binding analysis of AaERF1 using the yeast one-hybrid system. Sketch maps show the construction of vectors used in this experiment. Photographs show the growth behavior of transformants on SD/Trp−Ura−/X-gal medium. Sector 1: p178-4×GCC-LacZ+pB42AD-AaERF1; sector 2: p178+ pB42AD-AaERF1; sector 3: p178-4×GCC-LacZ+pB42AD; sector 4: p178+ pB42AD.

Mentions: Since the AP2 domain of AaERF1 contained the key amino acids to bind the GCC box, the recombinant MBP-AaERF1 protein was constructed and overexpressed in E. coli BL21, purified, and used to examine the DNA binding ability in vitro. The purified MBP-AaERF1 protein was mixed, respectively, with the labeled wild-type GCC probe or a mutated GCC probe in the binding reaction. The results of EMSA showed that the gel mobility shift was specific to the MBP-AaERF1 protein with the labeled GCC probe (lane 2 in Figure 4A). As expected, there were no shifted bands in the combination of MBP-AaERF1 plus the mutated GCC (mGCC) probe (lane 5 in Figure 4A) and in the negative controls, including MBP with the labeled GCC probe (lane 1) or mGCC probe (lane 4), and only the labeled GCC probe (lane 3) or mGCC probe (lane 6) (Figure 4A). The results demonstrated that AaERF1 was able to bind to the GCC box cis-acting element, but not to the mutated GCC box in vitro.


AaERF1 positively regulates the resistance to Botrytis cinerea in Artemisia annua.

Lu X, Jiang W, Zhang L, Zhang F, Zhang F, Shen Q, Wang G, Tang K - PLoS ONE (2013)

The DNA binding ability of AaERF1 via GCC box.A. Electrophoretic mobility shift assays on DNA binding of AaERF1. Lane 1: negative controls contain MBP plus labelled GCC probe; lane 2: the MBP–AaERF1 protein plus labelled GCC probe; lane 3: only labelled GCC probe; lane 4: negative controls contain MBP plus labelled mutated GCC probe; lane 5: MBP–AaERF1 plus labelled mutated GCC probe; lane 6: only labelled mutated GCC probe. The protein–GCC probe complex and free probes are indicated respectively by two arrows. B. GCC box binding analysis of AaERF1 using the yeast one-hybrid system. Sketch maps show the construction of vectors used in this experiment. Photographs show the growth behavior of transformants on SD/Trp−Ura−/X-gal medium. Sector 1: p178-4×GCC-LacZ+pB42AD-AaERF1; sector 2: p178+ pB42AD-AaERF1; sector 3: p178-4×GCC-LacZ+pB42AD; sector 4: p178+ pB42AD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585223&req=5

pone-0057657-g004: The DNA binding ability of AaERF1 via GCC box.A. Electrophoretic mobility shift assays on DNA binding of AaERF1. Lane 1: negative controls contain MBP plus labelled GCC probe; lane 2: the MBP–AaERF1 protein plus labelled GCC probe; lane 3: only labelled GCC probe; lane 4: negative controls contain MBP plus labelled mutated GCC probe; lane 5: MBP–AaERF1 plus labelled mutated GCC probe; lane 6: only labelled mutated GCC probe. The protein–GCC probe complex and free probes are indicated respectively by two arrows. B. GCC box binding analysis of AaERF1 using the yeast one-hybrid system. Sketch maps show the construction of vectors used in this experiment. Photographs show the growth behavior of transformants on SD/Trp−Ura−/X-gal medium. Sector 1: p178-4×GCC-LacZ+pB42AD-AaERF1; sector 2: p178+ pB42AD-AaERF1; sector 3: p178-4×GCC-LacZ+pB42AD; sector 4: p178+ pB42AD.
Mentions: Since the AP2 domain of AaERF1 contained the key amino acids to bind the GCC box, the recombinant MBP-AaERF1 protein was constructed and overexpressed in E. coli BL21, purified, and used to examine the DNA binding ability in vitro. The purified MBP-AaERF1 protein was mixed, respectively, with the labeled wild-type GCC probe or a mutated GCC probe in the binding reaction. The results of EMSA showed that the gel mobility shift was specific to the MBP-AaERF1 protein with the labeled GCC probe (lane 2 in Figure 4A). As expected, there were no shifted bands in the combination of MBP-AaERF1 plus the mutated GCC (mGCC) probe (lane 5 in Figure 4A) and in the negative controls, including MBP with the labeled GCC probe (lane 1) or mGCC probe (lane 4), and only the labeled GCC probe (lane 3) or mGCC probe (lane 6) (Figure 4A). The results demonstrated that AaERF1 was able to bind to the GCC box cis-acting element, but not to the mutated GCC box in vitro.

Bottom Line: The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast.The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua.The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

View Article: PubMed Central - PubMed

Affiliation: Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.

ABSTRACT
Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

Show MeSH
Related in: MedlinePlus