An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri

Citrus Huanglongbing (HLB), a severe and destructive plant disease caused by the Gram-negative, phloem-limited bacterium “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by Diaphorina citri , has been extensively studied. Previous studies have reported that protein post-translational modifications play a crucial role in D. citri response to C Las infection. However, comprehensive phosphoproteomic profiling of D. citri induced by C Las remains underexplored. In this study, a total of 144 differentially expressed proteins (DEPs) and 997 differentially phosphorylated proteins (DPPs) were identified by 4D label-free quantitative proteomics and phosphoproteomics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEPs were mainly associated with molecular binding, structural constituent of cuticle and cytochrome P450, whereas DPPs were predominately involved in acting and calcium binding. A total of thirteen proteins were selected for parallel reaction monitoring (PRM) analysis to validate the reliability of proteomics. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins: vitellogenin-A1 (Vg-A1), alpha-crystallin A chain (αA- crystallin), facilitated trehalose transporter Tret1 (Tret1), LOC103509854, zinc finger protein 319 (ZFP319), LOC113471498 and Protein argonaute-2 (Ago-2). Furthermore, RNA interference (RNAi)-mediated knockdown of vitellogenin-A1 and alpha-crystallin A chain significantly reduced C Las content in D. citri . In conclusion, this study provides the most comprehensive phosphorylation profiles of D. citri in response to C Las infection and identifies two potential targets implicated in C Las infection.