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Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes.

Miao J, Frazier T, Huang L, Zhang X, Zhao B - Front Plant Sci (2016)

Bottom Line: CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity.The remaining two genes were found to be homologous to CENH3.Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Virginia TechBlacksburg, VA, USA; Department of Grassland Science, Sichuan Agricultural UniversityYa'an, China.

ABSTRACT
Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

No MeSH data available.


Related in: MedlinePlus

Diagram of eight fragments of the switchgrass histone H3.3 and CENH3, and the phenotype of overexpression of different fragments of switchgrass histone H3.3 and CENH3 in N. benthamiana. (A) PCR primer location for different fragments of switchgrass histone H3; (B) PCR primer location for different fragments of switchgrass CENH3; (C) The chimeric gene contains the N-terminal tail of switchgrass histone H3.3 and the histone-fold domain of CENH3 was constructed by using overlap PCR. Agrobacterium strains expressing different DNA fragments as outlined in (A-C) were inoculated in N. benthamiana and the cell death phenotype were pictured at 4 days post inoculation. (D) 1, Fragment 1-YFP; 2, Fragment 2-YFP; 3, Fragment 3-YFP; 4, Fragment 4-YFP; 5, Fragment 5-YFP; 6, YFP only (negative control); (E) 1, PvCENH3-YFP; 2, Fragment 8-YFP; 3, Fragment 6-YFP; 4, Fragment 7-YFP; 5, YFP only. (F) Western blot to detect switchgrass histone H3-YFP fusion proteins. Different fragments of histone H3-YFP fusion proteins transiently expressed in N. benthamiana plant cells were detected by western blot. 1. Fragment 1-YFP; 2. Fragment 2-YFP; 3. Fragment 3-YFP; 4. Fragment 4-YFP; 5. Fragment 5-YFP; 6. Fragment 6-YFP; 7. Fragment7-YFP; 8.YFP only (negative control); 9. N. benthamiana total proteins (negative control); the western blot membrane was also stained with Ponceau S Staining Solution to show the equal loading of each protein samples.
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Figure 3: Diagram of eight fragments of the switchgrass histone H3.3 and CENH3, and the phenotype of overexpression of different fragments of switchgrass histone H3.3 and CENH3 in N. benthamiana. (A) PCR primer location for different fragments of switchgrass histone H3; (B) PCR primer location for different fragments of switchgrass CENH3; (C) The chimeric gene contains the N-terminal tail of switchgrass histone H3.3 and the histone-fold domain of CENH3 was constructed by using overlap PCR. Agrobacterium strains expressing different DNA fragments as outlined in (A-C) were inoculated in N. benthamiana and the cell death phenotype were pictured at 4 days post inoculation. (D) 1, Fragment 1-YFP; 2, Fragment 2-YFP; 3, Fragment 3-YFP; 4, Fragment 4-YFP; 5, Fragment 5-YFP; 6, YFP only (negative control); (E) 1, PvCENH3-YFP; 2, Fragment 8-YFP; 3, Fragment 6-YFP; 4, Fragment 7-YFP; 5, YFP only. (F) Western blot to detect switchgrass histone H3-YFP fusion proteins. Different fragments of histone H3-YFP fusion proteins transiently expressed in N. benthamiana plant cells were detected by western blot. 1. Fragment 1-YFP; 2. Fragment 2-YFP; 3. Fragment 3-YFP; 4. Fragment 4-YFP; 5. Fragment 5-YFP; 6. Fragment 6-YFP; 7. Fragment7-YFP; 8.YFP only (negative control); 9. N. benthamiana total proteins (negative control); the western blot membrane was also stained with Ponceau S Staining Solution to show the equal loading of each protein samples.

Mentions: Histone H3 proteins have two domains: an N-terminal tail and a histone fold domain (Luger et al., 1997; Malik and Henikoff, 2003). In order to determine the part of the PvH3.3 protein that is essential for triggering cell death in N. benthamiana, we performed a deletion mutagenesis series from both the N- and C-terminal ends. Five different fragments of PvH3.3 (Figure 3A) were used to generate PvH3.3-YFP fusion genes. As shown in Figure 3D, the N-terminal tail (1–43aa) is the part of the protein that maintains the ability to trigger cell death. All of the fragments that contained the N-terminal tail were predominately localized in the nucleus (Figures S4A–C). This suggests that there is an unidentified nuclear localization signal in the N-terminal tail sequence. The fragments that contained solely the PvH3.3 C-terminal histone fold domain, however, lost the ability to localize in the nucleus (Figures S4D,E) and could not trigger cell death in the transformed N. benthamiana plant cells (Figure 3D).


Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes.

Miao J, Frazier T, Huang L, Zhang X, Zhao B - Front Plant Sci (2016)

Diagram of eight fragments of the switchgrass histone H3.3 and CENH3, and the phenotype of overexpression of different fragments of switchgrass histone H3.3 and CENH3 in N. benthamiana. (A) PCR primer location for different fragments of switchgrass histone H3; (B) PCR primer location for different fragments of switchgrass CENH3; (C) The chimeric gene contains the N-terminal tail of switchgrass histone H3.3 and the histone-fold domain of CENH3 was constructed by using overlap PCR. Agrobacterium strains expressing different DNA fragments as outlined in (A-C) were inoculated in N. benthamiana and the cell death phenotype were pictured at 4 days post inoculation. (D) 1, Fragment 1-YFP; 2, Fragment 2-YFP; 3, Fragment 3-YFP; 4, Fragment 4-YFP; 5, Fragment 5-YFP; 6, YFP only (negative control); (E) 1, PvCENH3-YFP; 2, Fragment 8-YFP; 3, Fragment 6-YFP; 4, Fragment 7-YFP; 5, YFP only. (F) Western blot to detect switchgrass histone H3-YFP fusion proteins. Different fragments of histone H3-YFP fusion proteins transiently expressed in N. benthamiana plant cells were detected by western blot. 1. Fragment 1-YFP; 2. Fragment 2-YFP; 3. Fragment 3-YFP; 4. Fragment 4-YFP; 5. Fragment 5-YFP; 6. Fragment 6-YFP; 7. Fragment7-YFP; 8.YFP only (negative control); 9. N. benthamiana total proteins (negative control); the western blot membrane was also stained with Ponceau S Staining Solution to show the equal loading of each protein samples.
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Figure 3: Diagram of eight fragments of the switchgrass histone H3.3 and CENH3, and the phenotype of overexpression of different fragments of switchgrass histone H3.3 and CENH3 in N. benthamiana. (A) PCR primer location for different fragments of switchgrass histone H3; (B) PCR primer location for different fragments of switchgrass CENH3; (C) The chimeric gene contains the N-terminal tail of switchgrass histone H3.3 and the histone-fold domain of CENH3 was constructed by using overlap PCR. Agrobacterium strains expressing different DNA fragments as outlined in (A-C) were inoculated in N. benthamiana and the cell death phenotype were pictured at 4 days post inoculation. (D) 1, Fragment 1-YFP; 2, Fragment 2-YFP; 3, Fragment 3-YFP; 4, Fragment 4-YFP; 5, Fragment 5-YFP; 6, YFP only (negative control); (E) 1, PvCENH3-YFP; 2, Fragment 8-YFP; 3, Fragment 6-YFP; 4, Fragment 7-YFP; 5, YFP only. (F) Western blot to detect switchgrass histone H3-YFP fusion proteins. Different fragments of histone H3-YFP fusion proteins transiently expressed in N. benthamiana plant cells were detected by western blot. 1. Fragment 1-YFP; 2. Fragment 2-YFP; 3. Fragment 3-YFP; 4. Fragment 4-YFP; 5. Fragment 5-YFP; 6. Fragment 6-YFP; 7. Fragment7-YFP; 8.YFP only (negative control); 9. N. benthamiana total proteins (negative control); the western blot membrane was also stained with Ponceau S Staining Solution to show the equal loading of each protein samples.
Mentions: Histone H3 proteins have two domains: an N-terminal tail and a histone fold domain (Luger et al., 1997; Malik and Henikoff, 2003). In order to determine the part of the PvH3.3 protein that is essential for triggering cell death in N. benthamiana, we performed a deletion mutagenesis series from both the N- and C-terminal ends. Five different fragments of PvH3.3 (Figure 3A) were used to generate PvH3.3-YFP fusion genes. As shown in Figure 3D, the N-terminal tail (1–43aa) is the part of the protein that maintains the ability to trigger cell death. All of the fragments that contained the N-terminal tail were predominately localized in the nucleus (Figures S4A–C). This suggests that there is an unidentified nuclear localization signal in the N-terminal tail sequence. The fragments that contained solely the PvH3.3 C-terminal histone fold domain, however, lost the ability to localize in the nucleus (Figures S4D,E) and could not trigger cell death in the transformed N. benthamiana plant cells (Figure 3D).

Bottom Line: CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity.The remaining two genes were found to be homologous to CENH3.Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

View Article: PubMed Central - PubMed

Affiliation: Department of Horticulture, Virginia TechBlacksburg, VA, USA; Department of Grassland Science, Sichuan Agricultural UniversityYa'an, China.

ABSTRACT
Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

No MeSH data available.


Related in: MedlinePlus