Objective Cassava is a globally important food and energy crop whose production has long been severely threatened by Cassava common mosaic virus (CsCMV). Currently, the molecular mechanisms underlying the interaction between CsCMV and its host remain incompletely understood. Previous studies utilizing immunoprecipitation-mass spectrometry (IP-MS) screening identified Nicotiana benthamiana cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 2 (NbGAPC2) as a potential interacting target of the CsCMV coat protein (CsCP). Therefore, building on this foundation, the present study employed multiple experimental systems to further validate the specific interactions between CsCP and NbGAPC2 as well as its homolog in cassava, MeGAPC2, thereby laying the groundwork for elucidating the pathogenic mechanisms of CsCMV.

Method The interactions between CsCP and GAPC2 were systematically investigated using a combination of bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H), and in vitro pull-down assays.

Result BiFC analysis revealed distinct fluorescence complementation signals upon co-expression of CsCP and NbGAPC2 in N. benthamiana leaf cells, indicating an interaction within plant cells. Yeast self-activation assays confirmed the absence of autonomous activation activity for both CsCP, NbGAPC2 and MeGAPC2, and Y2H experiments demonstrated a direct interactions of CsCP with NbGAPC2 and MeGAPC2 in the yeast system. Furthermore, in vitro pull-down assays coupled with Western blot analysis detected specific binding between CsCP and NbGAPC2/MeGAPC2, indicating that these proteins also interact dierectly in vitro.

Conclusion CsCP specifically interacts with NbGAPC2/MeGAPC2 both in vivo and in vitro. Given the pivotal roles of GAPC2 in host defense and metabolic homeostasis, this interaction suggests that CsCMV may disrupt host physiological homeostasis by targeting GAPC2, providing new insights into the pathogenic mechanisms of CsCMV.