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The plant pathogenic bacteriumPseudomonas syringaepv tomato DC3000 (PstDC3000) causes disease in tomato, in the model plantArabidopsis thaliana,and conditionally inNicotiana benthamiana.The pathogenicity ofPstDC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. ThePstDC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliananonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging ofNicotiana benthamianaplants transiently co-expressingHopD1fused to the epitope haemagglutinin (HopD1-HA) withAtNHR2Bfused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.