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,†
1
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
2
Key Laboratory of Tea Plants Biology and Resources Utilization of Agriculture Ministry, Tea
Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China
3
Key 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: yjzhang@ippcaas.cn Tel.: +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: jemunson@njau.edu.cn Tel.: +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 shown on the top of the sequences. The disulfide bridges and predicted
α-helices are numbered 1 to 2 and α1 to α6, respectively. The conserved residues are
highlighted in white with a red background, and those 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 proteins using anti-AlinCSP4, anti-AlinCSP5, and anti-AlinCSP6 antibodies that
specifically react with recombinant AlinCSP4, AlinCSP5, and AlinCSP6, respectively.
Figure S4. Binding curves of 1-NPN and relative Scatchard plot analysis for three CSP
recombinant proteins (AlinCSP1-3). A solution of two proteins in Tris-buffer (pH 7.4), both at
the concentration of 2 µM, was titrated with 1 mM solution of 1-NPN in methanol to final
concentrations 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 cited Gu et al.
(2012)13.
Figure S6. Comparison of the binding abilities of AlinCSP1-6 recombinant proteins of A.
lineolatus to semiochemicals. The Ki values of the AlinCSP1-3 proteins were from Gu 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 Table 1. 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 from Sun 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
qPCR
AlinCSP4-Sense
AlinCSP4-Anti-sense
AlinCSP5-Sense
AlinCSP5-Anti-sense
AlinCSP6-Sense
AlinCSP6-Anti-sense
β-actin-Sense
β-actin-Anti-sense
Protein expression
AlinCSP4-Sense
AlinCSP4-Anti-sense
AlinCSP5-Sense
AlinCSP5-Anti-sense
AlinCSP6-Sense
AlinCSP6-Anti-sense
Sequence (5'-3')
CACGGTTTGCGGGAGATG
GATGGGTCGTAGATGGACAGAA
GCCACAGAGTCATCAGGTTCC
CGTTTCGCTTCGTTTCTTCC
AGATCCTGACCAACGAACGG
CCTTCTCGACTCCAGCCTTC
GAAACCTTCAACACACCCGC
GGTAGTCGGTCAAGTCACGG
TGccatggAAATGACGGAAGAAGAATT
TGctcgagTTAGGACTCGATGAAAGCCTT
TGccatggCTTATACAACCCACTACGACTACATTG
TGctcgagTCATGATAACGAGACGTTTCGC
TGccatgg CTGATAAGTACACGGACAAATATG
TGctcgagTTAATATTCGACGGGTTTCCCTT
Product size
(bp)
117
120
162
193
326
323
341