CLCuMuV-Induced Autophagy Restricts Viral Accumulation in the MED Cryptic Species of Bemisia tabaci

Autophagy is a conserved cellular degradation pathway that plays important roles in insect-virus interactions. In the MED cryptic species of Bemisia tabaci, cotton leaf curl Multan virus (CLCuMuV) can persist upon acquisition, yet this vector fails to transmit the virus. However, whether autophagy contributes to this antiviral response in MED whiteflies remains unknown. In this study, we show that CLCuMuV acquisition activates autophagy in MED whiteflies, as evidenced by increased LC3-I to LC3-II conversion, enhanced LC3-positive puncta formation in the gut, and dynamic transcriptional regulation of multiple autophagy-related genes. Functional investigations using RNAi-mediated silencing of Atg3 and Atg9, two essential autophagy genes, revealed that their knockdown elevated viral DNA accumulation by 1.4-fold at 48 h post-acquisition. Consistently, pharmacological blockade of autophagic flux with bafilomycin A1 led to a 1.0-, 2.2-, and 1.0-fold increase in viral loads across 24, 48, and 72 h post-acquisition, respectively, whereas treatment with rapamycin, an autophagy inducer, decreased viral DNA accumulation by 46.4% and 33.3% relative to control levels at 24 and 48 h post-acquisition, respectively. Together, these loss- and gain-of-function experiments demonstrate that autophagy functions as a host restriction mechanism that limits CLCuMuV persistence in MED whiteflies, providing molecular insights into the immune competence of this non-vector species.