Figure 2.

Silencing of NbP3IP inhibits autophagy. (a) Silencing of the endogenous NbP3IP gene reduces the number of autophagosomes with or without (upper) E-64d. Confocal images showing NbP3IP-silenced (TRV:NbP3IP) or control (TRV:00) plants transiently expressing CFP-NbATG8f at 48 hpi. 50 μM E-64d was pre-infiltrated at 10 h ahead of imaging. Bars, 10 μm. (b) Relative autophagic activity in NbP3IP-silenced plants. The autophagic activity was calculated in comparison to TRV:00 plants with or without E-64d. Autophagic bodies were counted from approximately 150 cells for each treatment in three independent experiments. Values represent the mean ± SD. Different letters indicate significant difference (ANOVA, P < 0.05). (c) Real-time RT-PCR analysis of the relative expression of ATGs in TRV:NbP3IP plants compared to TRV:00 plants. Values represent means ± SD from three independent experiments. Double asterisks indicate P < 0.01 of significant difference between TRV:00 control plants and TRV:NbP3IP silenced plants. (Student’s t-test, two-sided). (d) Joka2 was accumulated in NbP3IP-silenced plants. Joka2 was detected using Anti-NBR1 antibody. The value represents protein accumulation relative to rubisco, and the relative levels were calculated in relation to the TRV:00 treatment. Tests were repeated three independent times. Representative result was displayed. Anti-NBR1 120 kD