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. 2019 May 27;2(3):e201900385. doi: 10.26508/lsa.201900385

Figure 6. RcCHAD localizes to the nucleus and nucleolus of tobacco cells and co-localizes with EcPPK1 to EcPPK1-generated polyP granules.

Figure 6.

(A) Transient expression of Ubi10p:RcCHAD-mCherry in tobacco leaves reveals a nuclear/nucleolar localization of the fusion protein (top row). Expression of Ubi10p:EcPPK1-mCitrine induces the formation of polyP granules (center row), not observed when using a catalytically impaired version of the enzyme (EcPPK1H435A, H592A, bottom row). Scale bars correspond to 50 μm. Shown are Z-stacks from representative cells from three leaves obtained from three different plants. (B) RcCHAD-mCherry co-localizes with DAPI-stained nuclei and shows a higher intensity in nucleoli (not stained by DAPI). Scale bars correspond to 20 μm. (C) Magnified views of the nuclear localization of RcCHAD-mCherry when expressed in isolation (top row) and its redistribution to EcPPK1-generated polyP granules (bottom row). Scale bars correspond to 20 μm. (D) Western blots using anti-mCherry and anti-mCitrine antibodies reveal that RcCHAD-mCherry (63 kD) and EcPPK1-mCitrine (109 kD) migrated at the expected size in tobacco infiltrated leaves. RuBisCO (detected with Ponceau) is shown below as a loading control.