(A–B) ConcanamycinA treatment increases the number of autophagosomes in PexRD54 expressing samples.RFP:ATG8CL was transienly coexpressed with PexRD54, PexRD54AIM2 and empty vector controls in N. benthamiana. Two days after infiltration, leaves were treated with concanamycinA (conA) or infiltration buffer and number of autophagosomes was counted 24 hr after treatment. ConA treatment significantly increased the number of autophagosomes in PexRD54 expressing cells (p<0.05), confirming PexRD54 does not block autophagic flux. Scale bar=10 μm. (C) E64D treatment increases ATG8CL protein levels in PexRD54 expressing samples. GFP:ATG8CL, GFP:ATG8IL and GFP:EV were transiently coexpressed with RFP:GUS, RFP:PexRD54 or RFP:PexRD54AIM2 in N. benthamiana leaves and protein levels in total extracts were determined two and 3 days post infiltration (dpi). RFP:PexRD54 increased protein levels of GFP:ATG8CL but not GFP:ATG8IL consistent with stronger binding affinity of PexRD54 to ATG8CL. RFP:PexRD54 did not increase protein levels of GFP:EV, suggesting that protein level increase depends on ATG8CL binding and that PexRD54 does not increase protein levels in general. The samples were also treated with E64d to measure autophagic flux. In RFP:PexRD54 coexpressed 3 dpi samples, E64d treatment increased ATG8CL protein levels even more suggesting PexRD54 does not block autophagic flux. Hence, protein level increase is a result of stimulation of autophagy. The blots were stained with Ponceau stain (PS) to show equal loading.