Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (24): 5027-5038.doi: 10.3864/j.issn.0578-1752.2020.24.006

• PLANT PROTECTION • Previous Articles     Next Articles

Prediction and Analysis of Candidate Secreted Proteins from the Genome of Lasiodiplodia theobromae

XING QiKai1(),LI LingXian1,CAO Yang2,ZHANG Wei1,PENG JunBo1,YAN JiYe1,LI XingHong1()   

  1. 1Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Beijing 100097
    2School of Biological Engineering, Dalian University of Technology, Dalian 116024, Liaoning
  • Received:2020-02-14 Accepted:2020-03-20 Online:2020-12-16 Published:2020-12-28
  • Contact: XingHong LI E-mail:qikaixing@163.com;lixinghong1962@163.com

Abstract:

【Objective】Lasiodiplodia theobromae is an important phytopathogenic fungus with a worldwide distribution. This species causes severe Botryosphaeria dieback on a wide range of woody plants, which leads to reduced crop quality and tremendous economic losses. The objective of this study is to predict and analyze the candidate secreted proteins in the genome of L. theobromae, clarify their basic characteristics, so as to lay a foundation for the study of the pathogenic mechanism of secreted proteins in this pathogen.【Method】The signal peptide prediction algorithm SignalP v5.0 and subcellular localization prediction algorithm ProtComp v9.0, transmembrane helix prediction algorithm TMHMM v2.0, GPI-anchoring site prediction algorithm big-PI Fungal Predictor, and subcellular protein location distribution algorithm TargetP v2.0 were used to analyze 12 902 protein sequences of L. theobromae published in the previous study. The basic features including the length of the N-terminal signal peptide, the frequency of amino acid usage and cleavage site of the predicted secreted proteins were statistically analyzed. Based on the homology of the protein sequence, biological function annotation of the predicted secreted proteins was clarified by using BLASTP program. The activity of the signal peptide of the selected secreted proteins was detected by yeast secretion and cell translocation assays. Expression patterns of the selected secreted protein genes during L. theobromae infection were analyzed by qRT-PCR technology.【Result】In this study, 522 secreted proteins were verified, accounting for 4.3% of the total proteins present in the genome of L. theobromae. The lengths of amino acids of secreted proteins were ranged from 101 to 400 aa. The distribution length of signal peptides was from 18 to 20 aa and the largest number was 20 aa. The top frequent amino acid was alanine in the signal peptides, and the most frequently incorporated amino acids were non-polar and hydrophobic, accounting for 60.2% of the total amino acids. Further, the amino acids in the position -3 to -1 in the signal peptides were relatively conserved and the signal peptide cleavage site belonged to A-X-A type, which could be recognized and cleaved by Sp I type peptidase. Among them, 336 secreted proteins were identified with a predictive function, which was mostly enzymatic or virulence-associated protein. Besides, there are differences in terms of molecular weight, isoelectric point, the aliphatic index in the candidate secreted proteins. Finally, the predicted signal peptides of the 9 putative L. theobromae secreted proteins were confirmed to have secretory activity by using a yeast invertase secretion assay. qRT-PCR analysis demonstrated that the expression of selected protein genes was differentially regulated during host infection.【Conclusion】A total of 552 candidate secreted proteins of L. theobromae were predicted by a set of computer algorithms. Lengths of the signal peptides vary greatly and the most frequently are mainly non-polar and hydrophobic amino acids. Secreted proteins characterized in this study can be categorized under enzymes related to the degradation of cell wall components, necrosis induction proteins, and chitin-binding proteins which may play an important role in L. theobromae pathogenetic mechanism.

Key words: Botryosphaeria dieback, Lasiodiplodia theobromae, bioinformatics, secreted protein, signal peptide, expression pattern

Table 1

The primer sequences used for validation of predicted signal peptides"

基因 Gene 正向引物序列 Forward primer (5′-3′) 反向引物序列 Reverse primer (5′-3′)
LT_159 TTTATGAATTCATGGTCAAGGCTTCCACC TAATACTCGAGGGCATCGGTGAAGGTGCAG
LT_188 TTTATGAATTCATGCGTGTTTCGACTCTTC TAATACTCGAGAAAGAAGGTGAAGGTAGAAG
LT_233 TTTATGAATTCATGGTCAAGGTTTCCACC TAATACTCGAGGGTGAAAGTGCAGCTGG
LT_359 TTTATGAATTCATGCCTTCCCTCAAGTC TAATACTCGAGGTTTTCGGCGGCCTGGG
LT_595 TACAGGAATTCATGCGTTCCTCTGCTC GACTGCTCGAGCACGATGTCGAGATCAG
LT_62 CCGGAATTCATGGGCTGGTTTTGGTTC CCGCTCGAGCACGACGGTGATCGTCG
LT_936 TACTAGAATTCATGAAGGCTTCCGGTC CACTTCTCGAGACCGTTGACAGCCTGAC
LT_1541 TTTATGAATTCATGGTGTCCTTCCGCTCTC TAATACTCGAGGAGAGACTGCCTGGCAATC
LT_1698 TTTATGAATTCATGAAGTTCTCTACCACC TAATACTCGAGGTCCTCGGTGACCTCGCC

Table 2

The primer sequences used for qRT-PCR"

基因 Gene 正向引物序列 Forward primer (5′-3′) 反向引物序列 Reverse primer (5′-3′)
LT_159 CCAGCAGGACTACAAGAA CCAGAGGTAGACCAGTTC
LT_188 CTACCTTGCCGACCTTAA GATGATGTTGCCGTTGAA
LT_233 GAGCAGGACTACGAGAAC CGCAGAGGATGTAGATGT
LT_359 CAAGTCTTCCTCCATCCA GATCTGAGCCGAGTTGTA
LT_595 AGATGGTCTGGAAGAACTC CGTACTCGTCAAGGATGT
LT_62 GGAATCAACGACGACTCT CGCACTGTGTTGGTTATG
LT_936 CTACAACGAAATCAGCGAAT ATGGTGGTGGTCTTCTTC
LT_1541 CAACGGCTACTACTACTCTT TTGATGTTCCTGGCACTG
LT_1698 AATGGTGCTCAGTTCTACA AGATGTTGATGAGGAGACC
LT_Actin TCTTCGCTCGAGAAGTCGTA ACAATGGAAGGTCCGCTCTC

Fig. 1

Subcellular localization of 937 proteins with signal peptide in L. theobromae"

Fig. 2

The transmembrane domain analysis of 685 proteins transported to the extracellular space in L. theobromae"

Fig. 3

Length of typical secreted protein sequences in L. theobromae"

Fig. 4

Length distribution of signal peptides of secreted protein in L. theobromae"

Fig. 5

The frequency of amino acids of secreted protein in L. theobromae"

Table 3

The composition and distribution of amino acids at signal peptide cleavage site of secreted proteins in L. theobromae"

氨基酸类型
Type of amino acids
信号肽切割位点-3到3位的氨基酸组成 Frequency of amino acids from -3 to +3 at signal peptide cleavage site of secreted proteins
-3 -2 -1 1 2 3
数量
Amount
百分比
Percentage
(%)
数量
Amount
百分比
Percentage
(%)
数量
Amount
百分比
Percentage
(%)
数量
Amount
百分比
Percentage
(%)
数量
Amount
百分比
Percentage
(%)
数量
Amount
百分比
Percentage
(%)
A 249 47.0 60 11.3 387 73.0 145 27.3 26 4.9 35 6.6
C 13 2.5 6 1.1 2 0.4 18 3.4 6 1.1 21 4.0
D 2 0.4 9 1.7 3 0.6 38 7.2 51 9.6 19 3.6
E 8 1.5 12 2.3 2 0.4 24 4.5 35 6.6 16 3.0
F 1 0.2 26 4.9 5 0.9 12 2.3 6 1.1 20 3.8
G 16 3.0 10 1.9 19 3.6 20 3.8 23 4.3 23 4.3
H 1 0.2 29 5.5 0 0 20 3.8 3 0.6 12 2.3
I 10 1.9 10 1.9 0 0 12 2.3 7 1.3 38 7.2
K 0 0 3 0.6 8 1.5 19 3.6 3 0.6 8 1.5
L 22 4.2 87 16.4 9 1.7 21 4.0 15 1.3 49 9.2
M 0 0 10 1.9 1 0.2 4 0.8 2 0.4 4 0.8
N 1 0.2 22 4.2 5 0.9 14 2.6 21 4.0 22 4.2
P 8 1.5 7 1.3 22 4.2 5 0.9 148 27.9 44 8.3
Q 5 0.9 48 9.1 6 1.1 68 12.8 24 4.5 25 4.7
R 9 1.7 27 5.1 3 0.6 10 1.9 6 1.1 14 2.6
S 56 10.6 68 12.8 36 6.8 30 5.7 44 8.3 40 7.5
T 40 7.5 44 8.3 9 1.7 26 4.9 61 11.5 74 14.0
V 89 16.8 35 6.6 9 1.7 31 5.8 33 6.2 41 7.7
W 0 0 1 0.2 1 0.2 4 0.8 6 1.1 5 0.9
Y 0 0 16 3.0 3 0.6 9 1.7 10 1.9 20 3.8

Table 4

The physicochemical properties of some secreted proteins with functional annotation in L. theobromae"

基因编号
Gene ID
蛋白长度
Length (aa)
分子量
Molecular weight (kD)
等电点
pI
脂肪族氨基酸指数
Aliphatic index
功能注释
Function
evm.model.scaffold_1.1884 265 28.57 4.57 70.38 糖基水解酶Glycosyl hydrolases family
evm.model.scaffold_7.188 334 36.85 4.62 71.89 纤维素酶Cellulase
evm.model.scaffold_6.309 322 36.16 5.96 80.90 水解酶家族Alpha/beta hydrolase family
evm.model.scaffold_3.829 549 59.13 5.16 82.00 羧酸酯酶Carboxylesterase family
evm.model.scaffold_5.474 324 35.76 4.96 83.36 过氧化物酶Peroxidase
evm.model.scaffold_5.945 348 39.24 5.23 69.20 酪氨酸酶Tyrosinase
evm.model.scaffold_13.28 251 26.69 5.34 82.03 角质酶Cutinase
evm.model.scaffold_6.540 395 42.03 5.39 74.63 天冬氨酸蛋白酶Aspartyl protease
evm.model.scaffold_8.302 265 27.76 5.55 65.92 脂肪酶GDSL-like Lipase
evm.model.scaffold_4.1035 581 62.25 4.80 87.04 氧化还原酶GMC oxidoreductase
evm.model.scaffold_3.522 252 26.19 4.13 74.64 果胶酸裂解酶Pectate lyase
evm.model.scaffold_2.202 377 40.24 5.22 73.37 肽酶Peptidase family
evm.model.scaffold_2.1494 201 20.37 4.41 60.45 WSC结构域蛋白WSC domain protein
evm.model.scaffold_11.25 430 45.29 4.01 63.14 PAN结构域蛋白PAN domain protein
evm.model.scaffold_1.947 254 28.05 8.38 57.01 坏死诱导蛋白Necrosis inducing protein
evm.model.scaffold_4.1274 186 19.83 4.43 61.51 LysM结构域蛋白LysM domain protein
evm.model.scaffold_10.213 122 12.15 4.33 111.15 FAD结构域蛋白FAD domain protein
evm.model.scaffold_1.937 247 26.14 5.05 84.45 Cupin结构域蛋白Cupin domain protein
evm.model.scaffold_14.112 246 23.24 3.90 71.14 CFEM结构域蛋白CFEM domain protein
evm.model.scaffold_11.308 412 40.74 5.14 53.98 几丁质结合蛋白Chitin binding protein

Fig. 6

Functional validation of signal peptide of secreted proteins"

Fig. 7

Relative expression level of 9 putative secreted protein genes during L. theobromae infection"

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