关键词: 麦长管蚜, 唾液蛋白, 植物防御, 效应子, 铁蛋白, RNAi

Abstract: 【Aim】 The aim of this study is to explore the function of the salivary ferritin SaFer1 of Sitobion avenae in the processes of feeding and clarify the effects of SaFer1 on the aphid-plant interactions. 【Methods】 Based on the salivary gland transcriptome data of S. avenae, the full-length cDNA sequence of SaFer1 from S. avenae was cloned, and bioinformatically analyzed. RT-qPCR was used to determine the expression levels of SaFer1 in different developmental stages (1st－4th instar nymphs and wingless adults), different tissues of the 1-day-old wingless adults (head, thorax, abdomen, salivary gland, midgut and embryo), different wing morphs (wingless and winged adults), and the 1st－4th instar nymphs and wingless adults fed on different diets (wheat and artificial diet). With the transient expression system of Nicotiana benthamiana, Agrobacterium tumefaciens was used to mediate the expression of SaFer1 and Bcl-2 associated X protein (BAX) in N. benthamiana leaves, the symptoms of leaf necrosis were observed and used to analyze the functions of SaFer1, and subcellular localization of SaFer1 was observed with the laser scanning confocal microscopy. The yeast secretory system was used to validate the function of the signal peptide of SaFer1. Silencing of SaFer1 in wingless adults was utilized to determine the survival rate and average daily number of aphids produced, and the feeding behavior indexes of the wingless adults on the wheat were detected using the insect electrical penetration graph (EPG) technique. The expression levels of defense-related genes (ROS-related genes NOX and SOD, salicylic acid pathway-related gene PAL, and jasmonic acid pathway-related genes AOS and FAD7) in wheat leaves fed by S. avenae for 3 d after RNAi of SaFer1 were determined by RT-qPCR. 【Results】 The full-length cDNA sequence of SaFer1 (GenBank accession no.: PP760384) of S. avenae was cloned. The cDNA length of SaFer1 is 1 212 bp, and the ORF length is 675 bp, encoding 224 amino acid residues, with the relative molecular mass of 25.5 kD and the isoelectric point of 6.20. The N-terminus of SaFer1 has a signal peptide. SaFer1 had the highest amino acid sequence identity (98.61%) with the unnamed protein (GenBank accession no.: CAI6358877.1) of Macrosiphum euphorbiae. RT-qPCR results showed that SaFer1 was expressed at all developmental stages of S. avenae and is the constitutive expression gene, and was highly expressed in the midgut and salivary glands of the wingless adults. SaFer1 was able to inhibit the leaf necrosis of N. benthamiana induced by BAX. SaFer1 was localized in the leaf cell membrane and nucleus of N. benthamiana. The signal peptide of SaFer1 had secretory activities. Compared to the control group (dsGFP), silencing of SaFer1 caused no significant changes in the survival rates, but resulted in significant decrease in the average daily number of aphids produced by S. avenae. Silencing of SaFer1 resulted in a significant reduction in the duration of E1 waveform, and a significant increase in the duration of waveforms F and G during the feeding process compared to the control group (dsGFP). Silencing of SaFer1 also led to significant upregulation of the expression levels of NOX, SOD, PAL, AOS and FAD7 in wheat leaves compared to the control group (dsGFP). 【Conclusion】 The salivary ferritin SaFer1 of S. avenae was found to be an effector protein, which could help the aphid to feed by inhibiting the host defense responses, enhance the aphid’s fitness, and play vital roles in aphid-host plant interactions.

Key words:  Sitobion avenae, salivary protein, plant defense, effector, ferritin, RNAi