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Supporting information for Transgenic citrus expressing synthesized
cecropin B genes in the phloem exhibits decreased susceptibility to
Huanglongbing
Xiuping Zou1, 2 , †, Xueyou Jiang 2, 3, lanzhen Xu1, 2, Tiangang Lei1, 2, Aihong Peng1, 2,
Yongrui He1, 2, Lixiao Yao1, 2, Shanchun Chen1, 2,
†
1
Citrus Research Institute, Chinese Academy of Agricultural Sciences/ National Center
for Citrus Variety Improvement, Chongqing 400712, P. R. China.
2
Citrus Research Institute, Southwest University, Chongqing 400716, P. R. China.
3
Guangan Agricultural Bureau, Chongqing 400716, P. R. China
†
Corresponding author
Tel: 86-23-68349001
Fax: 86-23-68349020
e-mail:, [email protected] and [email protected]
Supplemental Table 1 Source of selected promoters and their expression patterns in
transgenic citrus
Promoter
Source
Expression pattern in citrus
Specifc and strong expression
GRP1.8
French bean (Phaseolus vulgaris) in phloem tissue (Xu et al.
2014)
Constitutive expression in citrus
rolC
Agrobacterium rhizogenes
(Xu et al. 2014)
Very weak expression in
RSs1
Rice (Oryza sativa L.)
phloem (Dutt et al. 2012; Xu et
al. 2014)
Specific expression in phloem
PFN2
Rice tungro bacilliform virus
(Christensen et al. 1996)
Promoters were fused with the GUS reporter gene. The gene cassette was delivered into Tarocco blood
orange (Citrus sinensis Osbeck) genome by Agrobacterium-mediated transformation method. Their
expression patterns were determined by GUS staining.
Supplementary Table 2 Primers used in the study
name
GRP-f
GRP-r
sequence（5′→3′）
purpose
CAAGCTTGAAGCCATATCAATGG
TAATAAG
Cloning GRP1.8 promoter with
TGGATCCGTGAAGTGAAGCTGA
HindIII and BamHI sites
AATAGGTTG
g-f
AGCCATATCAATGGTAATAAG
PCR confirmation for the
c-r
TCGACCTAACCAAGAGCTTTAG
integration of transgenes in
e-r
TTAGAGTTCGTCGTGACCAAG
transgenic plants
qCB-f
GCTAAATGGAAAGTTTTCAAG
Quantitative RT-PCR analysis
qCB-r
AAGAGCTTTAGCTTCACCCA
for expression of cecropin B
gene
CtAct-f
CATCCCTCAGCACCTTCC
Quantitative RT-PCR analysis
CtAct-r
CCAACCTTAGCACTTCTCC
of the citrus reference gene
actin
Cla16s-f
GCGCGTATGCAATACGAGCGGCA PCR analysis for Calas
Cla16s-r
GCCTCGCGACTTCGCAACCCAT
pathogen in citrus plants
qCla16s-f
TGAGTGCTAGCTGTTGGGTG
Quantitative real-time PCR
qCla16s-r
CTGCGCGTTGCATCGAATTA
analysis for Calas16S gene
Ct18s-f
AATTGTTGGTCTTCAACGAGGAA Quantitative real-time PCR
Ct18s-r
AAAGGGCAGGGACGTAGTCAA
Note: The sequences of restriction enzyme sites were in italic.
analysis for citrus 18S gene
Supplmentary Table 3 Quantitative PCR analysis of Calas population in transgenic
lines 3 months after graft infection
Bacterial population
Line
(Calas cells µg-1 of citrus DNA)
pGA1
6.40×105±5.92×105 e
pGA5
1.61×104±1.20×104 e
pGA6
9.29×103±6.93×103 f
pGA10
1.61×105±1.21×105 e
pGA12
3.33×104±1.36×104 e
pGA16
5.08×105±4.99×105 e
pGA21
2.47×104±2.43×104 e
pGA25
1.53×105±1.43 ×105 e
pGA27
4.64×105±4.45×105 e
pGC8
4.89×103±3.64×103 f
pGC16
1.92×104±1.62×104 e
pGC17
1.48×107 ±9.00×105 c
pGE3
pGE4
pGE5
pGE6
pGE7
pGE8
3.19×103±4.51×102 f
1.92×105±1.82×105 e
2.77×105±2.70×105 e
5.49×103±4.78×103 f
6.01×104±4.91 ×104 e
1.80×107±2.68×105 b
pGE10
pGE11
pGE12
pGE17
pGN
WT
3.56×106±2.77×105 d
3.05×106±4.13×105 de
2.48×106±3.93×105 de
1.26×107 ±9.15×106 c
2.24×107±8.43×106 a
2.57×107±3.94×106 a
The bacterial population (Calas cells µg-1 of citrus DNA) 3 months after infection was investigated
using qPCR. Standard errors per line were calculated from three plants. Different letters on the upper
right of values represented significantly different from wild type (WT) control by Tukey’s test
(P<0.05).
Supplementary Fig. 1 Synthetic cecropin B gene and plant expression vectors used for
citrus transformation. a Nucleotide and amino acid sequences of the CB, PR1aCB, and
PR1aCBer genes. For PR1aCB gene, the signal sequence (underlined) from the tobacco
PR1a protein was fused to the CB gene, and for PR1aCBer, ER retention signal sequence
(KDEL) was fused to the 3′ terminus of PR1aCB gene. ET corresponded to the consensus
sequence for efficient translation in plants. b Schematic diagrams of the plant expression
vectors used for transformation. 35S, Cauliflower mosaic virus 35S promoter from
tobacco; gus::npt-II, fusion of β-glucuronidase and neomycin phosphotransferase genes
(for screening of citrus transformants); nos, nos terminator; LB, left border; RB, right
border
Supplementary Fig. 2 Construction of standard equation for quantifying Calas
population in transgenic citrus
Standard equations were developed mainly based on previous work by Tatineni et
al.(2008). Here, the 2× iQTM SYBR Green Supermix (Bio-Rad) was used to quantify
Calas populations by qPCR analysis. A 1,165-bp DNA fragment of the HLB-specific 16s
rDNA was amplified using Cla16s-f/Cla16s-r primers (Table S2), and cloned into pGEM
T-easy vector (Promega Corp.) to construct pCalas16S plasmid. 10 μl 12.2 ng μl-1
pCalas16S plasmid and 10 μl 560 ng μl-1 citrus (Tarocco blood orange) DNA were mixed
together. A series of the mixture ranging from 108 to 2×100 were used to construct the
standard equations for quantifying Calas populations. Calas 16S and citrus 18S genes
were amplified from above the dilutions using qCla16s-f/ qCla16s-r and Ct18s-f /Ct18s-r
primers, respectively (Supplementary Table 2). Then the standard equations for
calculating bacterial cell copies (a) and evaluating citrus genome quality (b) were
generated by blotting the mean Ct values against the nature log concentrations in MS
Excel. Based on the data, the bacterial population per µg citrus DNA were calculated as
the formula:
Calas cells µg-1 citrus DNA= [10^(-0.2718×Ctcalas16S+10.624)/ 10^(0.2749×CtCs18S+4.0531)]×103 (12.7< Ctcalas16S <31.3 and 8.4< CtCs18S <26.5).
Supplementary Fig. 3 GUS expression patterns in Tarocco blood orange (Citrus
sinensis Osbeck) by four different promoters
Histochemical GUS staining as described by Zou et al. (2008) . Leaf and cross stems
from regenerated shoots were incubated in 1 mM 5-bromo-4-chloro-3-indolyl-β-Dglucuronic acid (X-gluc; Sigma, St Louis, MO, USA) reaction buffer at 37 °C for 3 h.
Samples were destained in 75% ethanol for 12 h to remove chlorophyll and other
pigments and then photographed. Ten independent transgenic plants per promoter were
used to investigate GUS expression patterns. The results showed that the GRP1.8
promoter from the French bean (Phaseolus vulgaris) had phloem-specific expression
characteristic in Tarocco blood orange and was the strongest promoter compared with
others.
Supplementary Fig. 4 Western blot analysis of cecropin B protein in transgenic
plants
50 µg soluble protein extracts from fresh veins were probed with a ployclonal antibody
against cecropin B. Lane CB is a positive control with 0.05 µg of synthetic CB peptide
(abcam, Shanghai, China). WT, wild type control.
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