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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

The phenotype of transient expression of different fragments as outlined in Figure 3 of switchgrass H3 in N. tabacum. (A) Transient assay phenotype. Left is non-transgenic plant, right is NtSGT1-RNAi transgenic plant. 1, Fragment 3-YFP; 2, Fragment 6-YFP; 3,YFP only (negative control). (B) Different fragments of histone H3-YFP fusion proteins transiently expressed in NtSGT1-RNAi transgenic N. tabacum and non-transgenic N. tabacum plant cells were detected by western blot. 1, Fragment 3-YFP in transgenic plant; 2, Fragment 6-YFP in transgenic plant; 3, YFP only in transgenic plant; 4, Fragment 3-YFP in non-transgenic plant; 5, Fragment 6-YFP in non-transgenic plant; 6, YFP only in non-transgenic plant; 7, NtSGT1-RNAi transgenic N. tabacum total proteins (negative control); 8, Non-transgenic N. tabacum total proteins (negative control).
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Figure 4: The phenotype of transient expression of different fragments as outlined in Figure 3 of switchgrass H3 in N. tabacum. (A) Transient assay phenotype. Left is non-transgenic plant, right is NtSGT1-RNAi transgenic plant. 1, Fragment 3-YFP; 2, Fragment 6-YFP; 3,YFP only (negative control). (B) Different fragments of histone H3-YFP fusion proteins transiently expressed in NtSGT1-RNAi transgenic N. tabacum and non-transgenic N. tabacum plant cells were detected by western blot. 1, Fragment 3-YFP in transgenic plant; 2, Fragment 6-YFP in transgenic plant; 3, YFP only in transgenic plant; 4, Fragment 3-YFP in non-transgenic plant; 5, Fragment 6-YFP in non-transgenic plant; 6, YFP only in non-transgenic plant; 7, NtSGT1-RNAi transgenic N. tabacum total proteins (negative control); 8, Non-transgenic N. tabacum total proteins (negative control).

Mentions: The cell death phenotype caused by the overexpression of PvH3.3 and PvCENH3 is similar to the hypersensitive response (HR) triggered by the interaction between plant pathogen effectors and cognate plant R genes (Coll et al., 2011). Since the HR-like cell death that is triggered by many R genes requires the function of SGT1, which is a conserved immune signaling component (Peart et al., 2002), we therefore tested whether or not SGT1 is also required for the elicitation of cell death induced by overexpression of PvH3.3 and PvCENH3 in Nicotiana tabacum. Two independent NtSGT1-RNAi transgenic lines (Traore et al., under revision) were used for transient overexpression of PvH3.3 and PvCENH3. As shown in Figure 4A, transient expression of PvH3.3(1−63aa)-YFP and PvCENH3(1−72aa)-YFP triggered cell death phenotypes on the wild type N. tabacum plants but failed to trigger any phenotype on the NtSGT1-RNAi plants. Western blot analysis found that the PvH3.3(1−63aa)-YFP and PvCENH3(1−72aa)-YFP fusion proteins were both expressed in the wild type and the NtSGT1-RNAi transgenic plants (Figure 4B). Therefore, NtSGT1 is essential for promoting histone H3-mediated cell death. Further studies are needed to investigate the role that SGT1 may play in histone gene-mediated cell death.


Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes.

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

The phenotype of transient expression of different fragments as outlined in Figure 3 of switchgrass H3 in N. tabacum. (A) Transient assay phenotype. Left is non-transgenic plant, right is NtSGT1-RNAi transgenic plant. 1, Fragment 3-YFP; 2, Fragment 6-YFP; 3,YFP only (negative control). (B) Different fragments of histone H3-YFP fusion proteins transiently expressed in NtSGT1-RNAi transgenic N. tabacum and non-transgenic N. tabacum plant cells were detected by western blot. 1, Fragment 3-YFP in transgenic plant; 2, Fragment 6-YFP in transgenic plant; 3, YFP only in transgenic plant; 4, Fragment 3-YFP in non-transgenic plant; 5, Fragment 6-YFP in non-transgenic plant; 6, YFP only in non-transgenic plant; 7, NtSGT1-RNAi transgenic N. tabacum total proteins (negative control); 8, Non-transgenic N. tabacum total proteins (negative control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940616&req=5

Figure 4: The phenotype of transient expression of different fragments as outlined in Figure 3 of switchgrass H3 in N. tabacum. (A) Transient assay phenotype. Left is non-transgenic plant, right is NtSGT1-RNAi transgenic plant. 1, Fragment 3-YFP; 2, Fragment 6-YFP; 3,YFP only (negative control). (B) Different fragments of histone H3-YFP fusion proteins transiently expressed in NtSGT1-RNAi transgenic N. tabacum and non-transgenic N. tabacum plant cells were detected by western blot. 1, Fragment 3-YFP in transgenic plant; 2, Fragment 6-YFP in transgenic plant; 3, YFP only in transgenic plant; 4, Fragment 3-YFP in non-transgenic plant; 5, Fragment 6-YFP in non-transgenic plant; 6, YFP only in non-transgenic plant; 7, NtSGT1-RNAi transgenic N. tabacum total proteins (negative control); 8, Non-transgenic N. tabacum total proteins (negative control).
Mentions: The cell death phenotype caused by the overexpression of PvH3.3 and PvCENH3 is similar to the hypersensitive response (HR) triggered by the interaction between plant pathogen effectors and cognate plant R genes (Coll et al., 2011). Since the HR-like cell death that is triggered by many R genes requires the function of SGT1, which is a conserved immune signaling component (Peart et al., 2002), we therefore tested whether or not SGT1 is also required for the elicitation of cell death induced by overexpression of PvH3.3 and PvCENH3 in Nicotiana tabacum. Two independent NtSGT1-RNAi transgenic lines (Traore et al., under revision) were used for transient overexpression of PvH3.3 and PvCENH3. As shown in Figure 4A, transient expression of PvH3.3(1−63aa)-YFP and PvCENH3(1−72aa)-YFP triggered cell death phenotypes on the wild type N. tabacum plants but failed to trigger any phenotype on the NtSGT1-RNAi plants. Western blot analysis found that the PvH3.3(1−63aa)-YFP and PvCENH3(1−72aa)-YFP fusion proteins were both expressed in the wild type and the NtSGT1-RNAi transgenic plants (Figure 4B). Therefore, NtSGT1 is essential for promoting histone H3-mediated cell death. Further studies are needed to investigate the role that SGT1 may play in histone gene-mediated cell death.

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