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Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy.

Wang Y, Zheng X, Yu B, Han S, Guo J, Tang H, Yu AY, Deng H, Hong Y, Liu Y - Autophagy (2015)

Bottom Line: Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana.Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy.Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: a Center for Plant Biology ; Beijing , China.

ABSTRACT
Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.

No MeSH data available.


ATG6 interacts with TUB8 in yeast and N. benthamiana. (A) Interaction of NtATG6 with NtTUB8 in yeast. Yeast cells harboring both BD-ATG6 and AD-TUB8 were able to turn blue on X-Gal-containing plate and grow on Leu-deficient medium in the presence of galactose and raffinose. Yeast harboring other control constructs showed no growth or color-changes, on corresponding induction plates. (B) NtTUB8 coimmunoprecipitates with NtATG6. Immunoprecipitation (IP) by anti-HA antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-TUB8 or other control groups. Precipitates were then analyzed by immunoblotting (IB) using anti-MYC (top panel) or anti-HA (middle panel) antibodies. Expression of MYC-tagged proteins was checked with anti-MYC (bottom panel). (C) Firefly luciferase complementation imaging (LCI) assay shows the interaction of ATG6 with TUB8. Coexpression of cLUC-TUB8 with ATG6-nLUC, but not other negative controls, reconstituted LUC activities and generated luminescence in the presence of luciferin. Infiltration areas of various combinations of constructs are indicated by the dashed circle.
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f0001: ATG6 interacts with TUB8 in yeast and N. benthamiana. (A) Interaction of NtATG6 with NtTUB8 in yeast. Yeast cells harboring both BD-ATG6 and AD-TUB8 were able to turn blue on X-Gal-containing plate and grow on Leu-deficient medium in the presence of galactose and raffinose. Yeast harboring other control constructs showed no growth or color-changes, on corresponding induction plates. (B) NtTUB8 coimmunoprecipitates with NtATG6. Immunoprecipitation (IP) by anti-HA antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-TUB8 or other control groups. Precipitates were then analyzed by immunoblotting (IB) using anti-MYC (top panel) or anti-HA (middle panel) antibodies. Expression of MYC-tagged proteins was checked with anti-MYC (bottom panel). (C) Firefly luciferase complementation imaging (LCI) assay shows the interaction of ATG6 with TUB8. Coexpression of cLUC-TUB8 with ATG6-nLUC, but not other negative controls, reconstituted LUC activities and generated luminescence in the presence of luciferin. Infiltration areas of various combinations of constructs are indicated by the dashed circle.

Mentions: To better understand the role of ATG6 in plant autophagy, we performed a yeast 2-hybrid (Y2H) screen of a tomato cDNA library36 using Nicotiana tabacum ATG6 (NtATG6) as bait to identify ATG6-binding host proteins. One clone encoding partial β-tubulin 8 was identified as a putative candidate for ATG6-interacting factor. We cloned its N. tabacum homolog gene and named it NtTUB8. Sequence alignment analysis showed that NtTUB8 shares 96% amino acid identity with TUB8/β-tubulin 8 in Arabidopsis thaliana (AtTUB8) and 98.9% identity with TUB8 in Solanum lycopersicum (SlTUB8) (Fig. S1). To test whether NtTUB8 interacts with NtATG6, we introduced TUB8 into the activation domain (AD) vector and cotransformed it into yeast with a bait vector carrying ATG6 fused with LexA DNA binding domain (BD). As indicated by the galactose (Gal) dependent growth of yeast on Leu-deficient plates and β-galactosidase assays on 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal) containing plates, NtTUB8 interacts with NtATG6 in yeast (Fig. 1A).Figure 1.


Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy.

Wang Y, Zheng X, Yu B, Han S, Guo J, Tang H, Yu AY, Deng H, Hong Y, Liu Y - Autophagy (2015)

ATG6 interacts with TUB8 in yeast and N. benthamiana. (A) Interaction of NtATG6 with NtTUB8 in yeast. Yeast cells harboring both BD-ATG6 and AD-TUB8 were able to turn blue on X-Gal-containing plate and grow on Leu-deficient medium in the presence of galactose and raffinose. Yeast harboring other control constructs showed no growth or color-changes, on corresponding induction plates. (B) NtTUB8 coimmunoprecipitates with NtATG6. Immunoprecipitation (IP) by anti-HA antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-TUB8 or other control groups. Precipitates were then analyzed by immunoblotting (IB) using anti-MYC (top panel) or anti-HA (middle panel) antibodies. Expression of MYC-tagged proteins was checked with anti-MYC (bottom panel). (C) Firefly luciferase complementation imaging (LCI) assay shows the interaction of ATG6 with TUB8. Coexpression of cLUC-TUB8 with ATG6-nLUC, but not other negative controls, reconstituted LUC activities and generated luminescence in the presence of luciferin. Infiltration areas of various combinations of constructs are indicated by the dashed circle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0001: ATG6 interacts with TUB8 in yeast and N. benthamiana. (A) Interaction of NtATG6 with NtTUB8 in yeast. Yeast cells harboring both BD-ATG6 and AD-TUB8 were able to turn blue on X-Gal-containing plate and grow on Leu-deficient medium in the presence of galactose and raffinose. Yeast harboring other control constructs showed no growth or color-changes, on corresponding induction plates. (B) NtTUB8 coimmunoprecipitates with NtATG6. Immunoprecipitation (IP) by anti-HA antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-TUB8 or other control groups. Precipitates were then analyzed by immunoblotting (IB) using anti-MYC (top panel) or anti-HA (middle panel) antibodies. Expression of MYC-tagged proteins was checked with anti-MYC (bottom panel). (C) Firefly luciferase complementation imaging (LCI) assay shows the interaction of ATG6 with TUB8. Coexpression of cLUC-TUB8 with ATG6-nLUC, but not other negative controls, reconstituted LUC activities and generated luminescence in the presence of luciferin. Infiltration areas of various combinations of constructs are indicated by the dashed circle.
Mentions: To better understand the role of ATG6 in plant autophagy, we performed a yeast 2-hybrid (Y2H) screen of a tomato cDNA library36 using Nicotiana tabacum ATG6 (NtATG6) as bait to identify ATG6-binding host proteins. One clone encoding partial β-tubulin 8 was identified as a putative candidate for ATG6-interacting factor. We cloned its N. tabacum homolog gene and named it NtTUB8. Sequence alignment analysis showed that NtTUB8 shares 96% amino acid identity with TUB8/β-tubulin 8 in Arabidopsis thaliana (AtTUB8) and 98.9% identity with TUB8 in Solanum lycopersicum (SlTUB8) (Fig. S1). To test whether NtTUB8 interacts with NtATG6, we introduced TUB8 into the activation domain (AD) vector and cotransformed it into yeast with a bait vector carrying ATG6 fused with LexA DNA binding domain (BD). As indicated by the galactose (Gal) dependent growth of yeast on Leu-deficient plates and β-galactosidase assays on 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal) containing plates, NtTUB8 interacts with NtATG6 in yeast (Fig. 1A).Figure 1.

Bottom Line: Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana.Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy.Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts.

View Article: PubMed Central - PubMed

Affiliation: a Center for Plant Biology ; Beijing , China.

ABSTRACT
Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.

No MeSH data available.