Limits...
The maize AAA-type protein SKD1 confers enhanced salt and drought stress tolerance in transgenic tobacco by interacting with Lyst-interacting protein 5.

Xia Z, Wei Y, Sun K, Wu J, Wang Y, Wu K - PLoS ONE (2013)

Bottom Line: ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions.Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco.This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Henan Agricultural University, Zhengzhou, PR China. xiazl08@163.com

ABSTRACT
ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of AAA proteins involved in response to salt and drought stresses in plants are largely unknown. In this study, a putative SKD1 (suppressor of K(+) transport growth defect 1) ortholog from Zea mays (ZmSKD1), which encodes a putative AAA protein, was isolated. The transcript levels of ZmSKD1 were higher in aerial tissues and were markedly up-regulated by salt or drought stress. Over-expression of ZmSKD1 in tobacco plants enhanced their tolerances not only to salt but to drought. Moreover, reactive oxygen species accumulations in ZmSKD1 transgenic lines were relative less than those in wild-type plants during salt or PEG-induced water stress. The interaction between ZmSKD1 and NtLIP5 (Lyst-Interacting Protein 5 homolog from Nicotiana tabacum) was confirmed by both yeast two-hybrid and immuno-precipitation assays; moreover, the α-helix-rich domain in the C-terminus of ZmSKD1 was identified to be required for its interaction with NtLIP5 using truncation mutations. Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco. This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

Show MeSH

Related in: MedlinePlus

ZmSKD1 interacts with NtLIP5 in yeast and in planta.A ZmSKD1 interacts with NtLIP5 in YTH system. Yeast colonies containing pGBKT7-SKD1/pGADT7-LIP5 grew well on the selective medium (SD/−Ade/−His/−Leu/−Trp) and turned blue in the β-galactosidase assays as did yeast colonies containing pGBKT7-T/pGADT7-p53, which was used as the positive control, whereas yeast transformed with pGBKT7-SKD1/pGADT7 or pGBKT7/pGADT7-LIP5 used as negative controls were unable to grow. B ZmSKD1 interacts with NtLIP5 in coimmunoprecipitation assays in Nicotiana benthamiana. Different cell lysates (HA-LIP5, Myc-SKD1, or HA-LIP5 and Myc-SKD1) were immunoprecipitated with anti-Myc or anti-HA antibodies. These immunoprecipitates were then separated by SDS-PAGE and immunoblotted with anti-HA or anti-Myc antibodies. The numbers on the left show the molecular masses of marker proteins in kilodaltons. (a) Samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA (upper) or anti-Myc antibody (lower). (b) Samples were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc (upper) or anti-HA antibody (lower).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3722157&req=5

pone-0069787-g006: ZmSKD1 interacts with NtLIP5 in yeast and in planta.A ZmSKD1 interacts with NtLIP5 in YTH system. Yeast colonies containing pGBKT7-SKD1/pGADT7-LIP5 grew well on the selective medium (SD/−Ade/−His/−Leu/−Trp) and turned blue in the β-galactosidase assays as did yeast colonies containing pGBKT7-T/pGADT7-p53, which was used as the positive control, whereas yeast transformed with pGBKT7-SKD1/pGADT7 or pGBKT7/pGADT7-LIP5 used as negative controls were unable to grow. B ZmSKD1 interacts with NtLIP5 in coimmunoprecipitation assays in Nicotiana benthamiana. Different cell lysates (HA-LIP5, Myc-SKD1, or HA-LIP5 and Myc-SKD1) were immunoprecipitated with anti-Myc or anti-HA antibodies. These immunoprecipitates were then separated by SDS-PAGE and immunoblotted with anti-HA or anti-Myc antibodies. The numbers on the left show the molecular masses of marker proteins in kilodaltons. (a) Samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA (upper) or anti-Myc antibody (lower). (b) Samples were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc (upper) or anti-HA antibody (lower).

Mentions: A yeast two-hybrid assay was performed to determine whether ZmSKD1 could interact with NtLIP5 in vitro. Combinations of plasmids expressing bait protein BD-ZmSKD1 and prey protein AD-NtLIP5 were transformed into AH109, respectively. It was verified that expression of BD-ZmSKD1 and AD-NtLIP5 was not toxic for AH109 yeast cells and that these fusion proteins did not bind or trans-activate the lacZ reporter gene non-specifically when expressed separately (data not shown). Co-transformation assays showed that independent yeast colonies containing pGBKT7-ZmSKD1and pGADT7-NtLIP5, as well as those containing pGADT7-T and pGBKT7-p53 (positive control), grew well on SD/−Ade/−His/−Leu/−Trp medium and turned blue in the β-galactosidase colony lift filter assay, indicating that there were strong interactions between ZmSKD1and NtLIP5. In contrast, no growth was observed for the negative controls (pGBKT7-ZmSKD1+pGADT7 or pGBKT7+pGADT7-NtLIP5) (Fig. 6A). This suggested that ZmSKD1 has the ability to interact with NtLIP5 in vitro.


The maize AAA-type protein SKD1 confers enhanced salt and drought stress tolerance in transgenic tobacco by interacting with Lyst-interacting protein 5.

Xia Z, Wei Y, Sun K, Wu J, Wang Y, Wu K - PLoS ONE (2013)

ZmSKD1 interacts with NtLIP5 in yeast and in planta.A ZmSKD1 interacts with NtLIP5 in YTH system. Yeast colonies containing pGBKT7-SKD1/pGADT7-LIP5 grew well on the selective medium (SD/−Ade/−His/−Leu/−Trp) and turned blue in the β-galactosidase assays as did yeast colonies containing pGBKT7-T/pGADT7-p53, which was used as the positive control, whereas yeast transformed with pGBKT7-SKD1/pGADT7 or pGBKT7/pGADT7-LIP5 used as negative controls were unable to grow. B ZmSKD1 interacts with NtLIP5 in coimmunoprecipitation assays in Nicotiana benthamiana. Different cell lysates (HA-LIP5, Myc-SKD1, or HA-LIP5 and Myc-SKD1) were immunoprecipitated with anti-Myc or anti-HA antibodies. These immunoprecipitates were then separated by SDS-PAGE and immunoblotted with anti-HA or anti-Myc antibodies. The numbers on the left show the molecular masses of marker proteins in kilodaltons. (a) Samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA (upper) or anti-Myc antibody (lower). (b) Samples were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc (upper) or anti-HA antibody (lower).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722157&req=5

pone-0069787-g006: ZmSKD1 interacts with NtLIP5 in yeast and in planta.A ZmSKD1 interacts with NtLIP5 in YTH system. Yeast colonies containing pGBKT7-SKD1/pGADT7-LIP5 grew well on the selective medium (SD/−Ade/−His/−Leu/−Trp) and turned blue in the β-galactosidase assays as did yeast colonies containing pGBKT7-T/pGADT7-p53, which was used as the positive control, whereas yeast transformed with pGBKT7-SKD1/pGADT7 or pGBKT7/pGADT7-LIP5 used as negative controls were unable to grow. B ZmSKD1 interacts with NtLIP5 in coimmunoprecipitation assays in Nicotiana benthamiana. Different cell lysates (HA-LIP5, Myc-SKD1, or HA-LIP5 and Myc-SKD1) were immunoprecipitated with anti-Myc or anti-HA antibodies. These immunoprecipitates were then separated by SDS-PAGE and immunoblotted with anti-HA or anti-Myc antibodies. The numbers on the left show the molecular masses of marker proteins in kilodaltons. (a) Samples were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA (upper) or anti-Myc antibody (lower). (b) Samples were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc (upper) or anti-HA antibody (lower).
Mentions: A yeast two-hybrid assay was performed to determine whether ZmSKD1 could interact with NtLIP5 in vitro. Combinations of plasmids expressing bait protein BD-ZmSKD1 and prey protein AD-NtLIP5 were transformed into AH109, respectively. It was verified that expression of BD-ZmSKD1 and AD-NtLIP5 was not toxic for AH109 yeast cells and that these fusion proteins did not bind or trans-activate the lacZ reporter gene non-specifically when expressed separately (data not shown). Co-transformation assays showed that independent yeast colonies containing pGBKT7-ZmSKD1and pGADT7-NtLIP5, as well as those containing pGADT7-T and pGBKT7-p53 (positive control), grew well on SD/−Ade/−His/−Leu/−Trp medium and turned blue in the β-galactosidase colony lift filter assay, indicating that there were strong interactions between ZmSKD1and NtLIP5. In contrast, no growth was observed for the negative controls (pGBKT7-ZmSKD1+pGADT7 or pGBKT7+pGADT7-NtLIP5) (Fig. 6A). This suggested that ZmSKD1 has the ability to interact with NtLIP5 in vitro.

Bottom Line: ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions.Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco.This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Henan Agricultural University, Zhengzhou, PR China. xiazl08@163.com

ABSTRACT
ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of AAA proteins involved in response to salt and drought stresses in plants are largely unknown. In this study, a putative SKD1 (suppressor of K(+) transport growth defect 1) ortholog from Zea mays (ZmSKD1), which encodes a putative AAA protein, was isolated. The transcript levels of ZmSKD1 were higher in aerial tissues and were markedly up-regulated by salt or drought stress. Over-expression of ZmSKD1 in tobacco plants enhanced their tolerances not only to salt but to drought. Moreover, reactive oxygen species accumulations in ZmSKD1 transgenic lines were relative less than those in wild-type plants during salt or PEG-induced water stress. The interaction between ZmSKD1 and NtLIP5 (Lyst-Interacting Protein 5 homolog from Nicotiana tabacum) was confirmed by both yeast two-hybrid and immuno-precipitation assays; moreover, the α-helix-rich domain in the C-terminus of ZmSKD1 was identified to be required for its interaction with NtLIP5 using truncation mutations. Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco. This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

Show MeSH
Related in: MedlinePlus