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RNA processing defects in chloroplasts were previously associated with increased plasmodesmata (PD) permeability. However, the underlying mechanisms for such association are still unknown. To provide insight into this, we silenced the expression of chloroplast-located INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) RNA helicase in Nicotiana benthamiana leaves and determined an increase in PD permeability which is caused by a reduction of PD callose deposition. Moreover, the silencing of two other nuclear genes encoding chloroplastic enzymes involved in RNA processing, RH3 , and CLPR2, also increased PD permeability accompanied by reduced callose accumulation at PD. In addition, we quantified the plastidic hydrogen peroxide levels using the chloroplast-targeted fluorescent sensor, HyPer, in ISE2 , RH3, and CLPR2 silenced N. benthamiana leaves. The levels of chloroplastic hydrogen peroxide were not correlated with the increased cell-to-cell movement of the marker protein GFP2X. We, therefore, propose that defects in chloroplast RNA metabolism mediate PD gating by suppressing PD callose deposition, and hydrogen peroxide levels in the organelles are not directly linked to this process. • The VIGS silencing of ISE2, RH3 and CLPR2 (nuclear-encoded chloroplastic-targeted genes directly and indirectly involved in RNA processing) increases the intercellular propagation via plasmodesmata (PD) of GFP2X but not GFP1X in N. benthamiana leaves. • Reduced callose accumulation at individual PD-pit fields revealed by aniline blue staining explained the increased PD permeability observed in N. benthamiana. • Hydrogen peroxide accumulation in chloroplasts revealed by HyPer did not show a correlation to the increased PD permeability. [ABSTRACT FROM AUTHOR]