Quantitative Isotope-Labeled Cross-Linker Proteomics Reveals Developmental Variation in Protein Interactions and Post-Translational Modifications in Diaphorina citri, the Citrus Greening Insect Vector

Acquisition
of the citrus greening bacterial pathogen, “Candidatus Liberibacter asiaticus” by Asian citrus
psyllid (Diaphorina citri) nymphs is
required for efficient tree-to-tree transmission during the adult
stage. Quantitative isotope-labeled protein interaction reporter cross-linkers
were used in parallel with protein quantification using spectral counting
to quantify protein interactions within microbe-enriched cellular
fractions of nymph and adult D. citri. Over 100 unique cross-links were found between five insect histone
proteins, and over 30% of these were more abundant in nymphs compared
to adult insects. Strikingly, some cross-links detected in D. citri proteins were conserved in cross-linking
studies on human cells, suggesting that these protein interaction
topologies were present in the common ancestor prior to divergence
of the arthropods or are subject to convergent evolution. Analysis
of post-translational modifications (PTMs) of cross-linked histones
revealed the presence of acetylated and methylated lysine residues,
which may impact the psyllid chromatin structure and gene expression.
Histone H3 peptides acetylated in the N terminal tail region were
found to be more abundant in nymphs compared to adult insects in two
orthogonal proteomics methods. The insect life stage-specific histone
PTMs and protein interactions represent physical evidence that metamorphosis
is associated with changes in chromatin structure that regulate genome-wide
transcriptional reprograming.

亚洲柑橘木虱（Diaphorina citri）若虫获取柑橘黄龙病病原菌——‘候选种亚洲韧皮部杆菌（Candidatus Liberibacter asiaticus）’，是成虫阶段实现高效树间传播的必要前提。本研究采用同位素标记蛋白相互作用报告交联剂，结合光谱计数法进行蛋白定量，以定量分析木虱若虫与成虫的微生物富集细胞组分中的蛋白相互作用。在5种昆虫组蛋白之间共鉴定到100余个独特的交联位点，其中超过30%的交联在若虫中的丰度显著高于成虫。值得注意的是，在亚洲柑橘木虱蛋白中检测到的部分交联位点在人类细胞的交联研究中同样保守，这表明这些蛋白相互作用拓扑结构早在节肢动物分化前的共同祖先中就已存在，或是经历了趋同进化。对交联组蛋白的翻译后修饰（post-translational modifications, PTMs）分析显示，其中存在乙酰化和甲基化赖氨酸残基，这类修饰可能影响木虱的染色质结构与基因表达。通过两种正交蛋白质组学方法均发现，N端尾部区域发生乙酰化的组蛋白H3肽段在若虫中的丰度显著高于成虫。昆虫生命阶段特异性的组蛋白翻译后修饰与蛋白相互作用，为“变态发育与染色质结构改变相关，而后者调控全基因组转录重编程”这一结论提供了直接的实验依据。