Functional Analysis Of Acyrthosiphon Pisum Salivary Gland Proteins Armet And ACE In Aphid-plant Interaction

The interactions between aphids and host plants are highly complex. In the long process of evolution, host plants gradually formed constitutive and inducible defense mechanisms to cope with the aphids, while the aphids decrease the impact of plant defense by feeding and proliferation. Aphids secrete proteins by salivary glands to regulate plant defense response. These saliva proteins function as effectors and are probably involved in aphid-plant interactions directly. However, how saliva proteins influenc aphid’s feeding behavior and induce plant’s defense reactions are still not clear.The pea aphid Acyrthosiphon pisum is the unique aphid whose genome has been sequenced until now. Operation on pe aphid is comparatively easy because of its big body size. There are42genes predicted to be highly expressed in aphid salivary glands. Only9genes have annotations, including Armet and ACE genes. Armet exists broadly in animal species. Armet takes part in endoplasmic reticulum stress response intracellularly and functions as neurotrophic factor in mammalian. ACE is angiotensin-converting enzyme, regulating blood pressure in mammalian and development, reproduction, immunity, and neural system in insects. These two genes are highly expressed in aphid salivary glands. Whether these two genes are involved in aphid-plant interactions remains unknown. In this study, the effects of Armet and ACE on the feeding of aphids on plants are explored from molecular biology, electrophysiology, and plant reactions. The main results are as follows:(1) The pea aphid Armet gene encodes174amino acid residues. The protein contains eight conserved cysteines, a signal peptide at N-terminal, and an endoplasmic reticulum retentional signal, KEEL, at C-terminal. The promoter region contains two endoplasmic reticulum stress response elements (ERSE). Armet is upregulated in aphids in response to endoplasmic reticulum stress and its ERSEs of promoter also make such response. The transcriptional factors ATF6and NY-F take part in the response of Armet to endoplasmic reticulum stress. These results indicate that the intracellular function of Armet as response to endoplasmic reticulum stress is conserved among mammalian and insects.(2) Compared to feeding on artificial diet, the pea aphid requires more Armet when feeding on plants. The survival of aphids on plants significantly decreases after the expression of Armet is knocked down while no change happens in aphid survival on artificial diet. Electronic penetration graph technique shows that the feeding behavior of aphids on plants is demaged after the expression of Armet is knocked down, shown as shorter passive ingestion time and longer probing time. When Armet protein is infiltrated to tobacco, the expression of3033tobacco genes varies according to the transcriptomic analysis. Among these genes,144genes in pathogen-plant interaction pathway are upregulated, some of which has been verified using real-time PCR.(3) Three ACE genes are found in the pea aphid genome. ACE1and ACE2are predicted to be secretory proteins while ACE3is non-secretory protein. The expression of ACE1and ACE2is higher in salivery glands than in other organs. ACE3has highest expression in ovaries. Compared to feeding on artificial diet, the pea aphid requires more ACE2. When the three ACEs are knocked down one by one, the survival, feeding, and reproduction of aphids are not influenced. However, simultaneous knockdown of ACE1and ACE2significantly decreases the survival of aphids on plants, but the passive ingestion time increases shown by electronic penetration graph technique.This study explores the effects of two salivary gland proteins on the feeding of aphids on plants. The results have theoretical and application significance on understanding the adaptation of aphids to plants, breeding of new insect-resistant cultivars, and developing new aphid control strategies.