Chemosensillum immunolocalization and ligand specificity of chemosensory proteins in the alfalfa plant bug Adelphocoris lineolatus(Goeze)
Liang Sun1, 2, 3, Jing-Jiang Zhou4,Shao-Hua Gu1, Hai-Jun Xiao1, 5, Yu-Yuan Guo1, Ze-Wen Liu3,†, Yong-Jun Zhang1,†
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
2Key Laboratory of Tea Plants Biology and Resources Utilization of Agriculture Ministry, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China
3Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education),College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China
4 Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, UK
5 Institute of Entomology, Jiangxi Agricultural University, Nanchang, 330045, China
† Corresponding authors:
Yong-Jun Zhang PhD. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China E-mail: l.: +86 10 62815929; Fax: +86 10 62816631.
Ze-Wen Liu PhD. Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China E-mail: l.: +86 25 84399051; Fax: +86 25 84399051.
Supporting Information:
Figure S1. Sequence alignment of AlinCSP4-6. The secondary structure elements of AlinCSP4-6, which are based on the three-dimensional structure of MbraCSPA6 (PDB code: 1KX8)75,are shownon the top of the sequences. The disulfide bridges and predictedα-helices are numbered 1 to 2 andα1to α6, respectively. The conserved residues are highlighted inwhitewith a red background,andthose residues with similar physico-chemical properties are shown in a red typeface. The predicted signal peptides are underlined with a black line.
Figure S2. Expression and purification of AlinCSP4-6 proteins. SDS-PAGE analyses of the recombinant AlinCSP4-6 proteins.The protein molecular weight marker (M) indicates (from the top) 170, 130, 95, 72, 55, 43, 34, 26, 17, 10 kDa; Lanes 1-5 represent the non-induced protein, induced protein, inclusion bodies, supernatant, and final purified protein, respectively.
Figure S3. SDS-PAGE and antiserum specificity analysis by western blot analysis. From left to right, SDS-PAGE of purified AlinCSP4-6 (1st panel) and western blot analysis (2nd - 4th panels). The results show that the antisera can clearly distinguish among recombinant AlinCSP4-6 proteinsusing anti-AlinCSP4, anti-AlinCSP5,and anti-AlinCSP6 antibodies thatspecifically react with recombinant AlinCSP4, AlinCSP5, and AlinCSP6, respectively.
Figure S4. Binding curves of 1-NPN and relative Scatchard plotanalysis for three CSP recombinant proteins (AlinCSP1-3). Asolution of two proteins in Tris-buffer (pH 7.4),both at the concentration of2 µM, was titrated with 1 mM solution of 1-NPN in methanol to finalconcentrations ranging from 2 to 16 µM.
Figure S5. Comparison of the immunolocalization of CSP proteins in different antennal sensilla of A. lineolatus. The protein distributions of AlinCSP1-3 were citedGu et al. (2012)13.
Figure S6. Comparison of the binding abilities of AlinCSP1-6recombinant proteins of A. lineolatus to semiochemicals.The Ki values of the AlinCSP1-3 proteins were fromGu et al. (2012)13, “u.d.” indicates that the binding affinities (Ki) were not calculated in study of Gu et al. (2012)13.
Figure S7. Comparison of the EAG responses of A. lineolatus to 41 semiochemicals used in the binding assay. The number of compounds(1-41) was the same as Table1. The EAG values of compounds 3-hexanone, 1-hexanol, trans-2-hexenal,myrcene, β-ionone, β-pinene, limonene, α-phellandrene, (+)-α-pinene, nerolidol, β-caryophyllene, α-humulene, trans-β-farnesene, trans,trans-farnesol were fromSun et al. (2013) 9 and Sun et al. (2014) 14, respectively.
Table S1. Primers used in the qPCR analysis and recombinant AlinCSP4-6 prokaryotic expression.
Primer name / Sequence (5'-3') / Product size(bp)qPCR
AlinCSP4-Sense / CACGGTTTGCGGGAGATG / 117
AlinCSP4-Anti-sense / GATGGGTCGTAGATGGACAGAA
AlinCSP5-Sense / GCCACAGAGTCATCAGGTTCC / 120
AlinCSP5-Anti-sense / CGTTTCGCTTCGTTTCTTCC
AlinCSP6-Sense / AGATCCTGACCAACGAACGG / 162
AlinCSP6-Anti-sense / CCTTCTCGACTCCAGCCTTC
β-actin-Sense / GAAACCTTCAACACACCCGC / 193
β-actin-Anti-sense / GGTAGTCGGTCAAGTCACGG
Protein expression
AlinCSP4-Sense / TGccatggAAATGACGGAAGAAGAATT / 326
AlinCSP4-Anti-sense / TGctcgagTTAGGACTCGATGAAAGCCTT
AlinCSP5-Sense / TGccatggCTTATACAACCCACTACGACTACATTG / 323
AlinCSP5-Anti-sense / TGctcgagTCATGATAACGAGACGTTTCGC
AlinCSP6-Sense / TGccatgg CTGATAAGTACACGGACAAATATG / 341
AlinCSP6-Anti-sense / TGctcgagTTAATATTCGACGGGTTTCCCTT