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. 2021 Mar 4;17(3):e1009370. doi: 10.1371/journal.ppat.1009370

Fig 4. NbMIP1.4b stabilizes NSvc4 in an HSP70 chaperone-independent manner, and silencing NbMIP1s promotes NSvc4 degradation.

Fig 4

(A, B and D) Silencing NbMIP1s impairs the stability of NSvc4. NSvc4-FLAG was co-expressed with NbMIP1-hairpin or GUS-hairpin in N. benthamiana leaves. Total protein was extracted for western blotting at 60 hpi. The polyclonal antibody against NbMIP1s was used to detect NbMIP1s protein level. The relative levels of NSvc4-FLAG to Actin were calculated and normalized by setting the values in GUS-hairpin treatment as 1.0. Meanwhile, some of the leaves were treated with MG132, E64d, 3MA, and DMSO, and then, these leaves were harvested at 6 hpt followed by western blotting. The relative levels of NSvc4-FLAG to Actin were normalized by setting the values in DMSO treatment as 1.0. (C) Expressing NbMIP1.4b stabilized NSvc4. NSvc4-FLAG was co-expressed with NbMIP1.4b-Myc or empty vector in N. benthamiana leaves. Samples were harvested at 60 hpi for western blotting. The expression of NbMIP1.4b-Myc was confirmed by the anti-Myc antibody. The relative levels of NSvc4-FLAG to Actin were normalized by setting the values in empty vector control as 1.0. (E and F) In vivo protein degradation rate assay of NSvc4 upon silencing NbMIP1s. The leaves expressing NSvc4-FLAG with NbMIP1-hairpin or NbMIP1-hairpin and NbATG8-hairpin were treated with CHX. The samples were harvested at 0 h and 2 h after treatment. Total protein was extracted for western blotting. The relative levels of NSvc4-FLAG to Actin were normalized by setting the values at 0 h as 1.0. (G) The relative levels of NSvc4-FLAG from Fig 2C–2F, and Fig 4E and 4F, were used to plot a histogram. Student’s t-test was performed, and asterisks denote significant differences between different treatments (two-sided, *P < 0.05, n.s., not significant). (H) Confocal images of NSvc4-eGFP and RFP-NbATG8f1 co-expressed with NbMIP1.4b-Myc or NbMIP1-hairpin. NSvc4-eGFP and RFP-NbATG8f1 were co-expressed with NbMIP1.4b, empty vector, NbMIP1-hairpin, or GUS-hairpin in N. benthamiana leaves by agroinfiltration, respectively. Then, the leaves were observed by confocal microscope at 60 hpi. Arrows indicate the NSvc4-eGFP and RFP-NbATG8f1 co-localized punctate structures. Bars, 20 μm. (I) Numbers of NSvc4-eGFP and RFP-NbATG8f1 co-localized punctate spots in NbMIP1.4b-Myc or empty vector-expressed leaves were calculated per cell and analyzed by student’s t-test (two-sided, **P < 0.01). (J) Western blotting analysis of NSvc4-FLAG after TM treatment in N. benthamiana leaves upon silencing NbMIP1s. NSvc4-FLAG, eGFP and NbMIP1-hairpin were co-expressed in N. benthamiana leaves by agroinfiltration. Then, the leaves were treated with TM at 42 hpi. Eighteen hours later, total protein was extracted for western blotting analysis. The relative levels of NSvc4-FLAG and eGFP to Actin were calculated and normalized by setting values in DMSO control as 1.0. (K and L) NbMIP1.4b(ΔHPD) mutant stabilizes NSvc4. The experiment is similar to (4C).