Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (16): 3451-3460.doi: 10.3864/j.issn.0578-1752.2021.16.008

• PLANT PROTECTION • Previous Articles     Next Articles

Screening and Function of Plant Immune Proteins from Bacillus velezensis LJ02

WEI YanXia1(),LI ZhuoRan1,ZHANG Bin1,YUAN YuJin1,YU WeiWei1,CHANG RuoKui2(),WANG YuanHong1()   

  1. 1Department of Horticulture, Tianjin Agricultural University, Tianjin 300384
    2College of Engineering and Technology, Tianjin Agricultural University, Tianjin 300384
  • Received:2020-12-03 Accepted:2021-01-11 Online:2021-08-16 Published:2021-08-24
  • Contact: RuoKui CHANG,YuanHong WANG E-mail:1486981814@qq.com;changrl@163.com;wangyh@tjau.edu.cn

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Abstract:

【Objective】Bacillus velezensis LJ02 induces the immune response of cucumbers and other crops. The objective of this study is to screen and identify the immune proteins of LJ02, and further analyze the immune signalling pathways by verifying their functions.【Method】The LJ02 fermentation broth was precipitated with ammonium sulfate precipitation method, and the crude proteins of LJ02 were obtained by centrifugation. Then crude proteins were gel chromatographed and separated by high performance liquid chromatography (HPLC) to collect protein components at different peaks. The immune components were tested with tobacco mosaic virus (TMV) to obtain the immune components in plant. Liquid-phase mass spectrometry (LC-MS) detection and analysis revealed that the component F-23 contained flagellin (FlgLJ02). Purified recombinant protein FlgLJ02 produced from Escherichia coli expression system was infiltrated into tobacco leaves and its immune function was verified by hypersensitive reaction (HR) and immune resistance analysis. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect salicylic acid (SA) and ethylene (ET) key synthetase genes ICS1, PAL, ACS1 and immune-related resistance genes NPR1, PR-1, EIN2 and EIN3. The relative expression of its immune-related resistance genes was tested to identify the Flg LJ02-induced immune signal transduction pathway.【Result】Sixty exocrine protein components (from F-1 to F-60) of LJ02 were separated from HPLC, and components F-20, F-23, F-41, F-44 had strong immune effect against TMV in tobacco, among which, component F-23 showed the most significant anti-TMV effect, with an inhibition rate of 81.7%. Further mass spectrometry analysis found that this component contained flagellin FlgLJ02 and other 6 substances. The FlgLJ02expression vector was constructed, and then transformed into E. coli BL21, the cells were broken after inducing the expression of Flg LJ02. The crude protein was eluted with Ni column purification, further dialyzed, then injected FlgLJ02 into tobacco leaves, hypersensitive reactions appeared about 24 h. Tobacco leaves was infiltrated with 50, 100 and 200 μg·mL -1 FlgLJ02, and then TMV was inoculated 24 hours post FlgLJ02inoculation (hpi). The inhibition rate of FlgLJ02against TMV was 65.6%, 76.1% and 88.1%, respectively. The qRT-PCR determined that the expressions of SA and ET via defense-related genes ICS1, PAL, NPR1, PR-1, ACS1, EIN2, and EIN3 in tobacco plants were significantly up-regulated in 24-48 h.【Conclusion】The Flg LJ02 secreted by the B. velezensis LJ02 activates the SA and ET signalling immune pathway, thereby improving plants disease resistance to TMV.

Key words: Bacillus velezensis, flagellin, immune protein, Nicotiana tabacum var. xanthi, tobacco mosaic virus (TMV)

Table 1

Primers for qRT-PCR of relevant expressed genes"

登录号 ID 基因名称Gene name 正向引物 Forward primer (5′-3′) 反向引物 Reverse primer (5′-3′)
X12737 PR-1 GCTGCTAAGGCCGTTGAGAT ACATCCAACACGAACCGAGT
AF480488 NPR1 CGGCGGCATGACTGAATTTT TTAGCGTCGGCGAAGTAGTC
AB289452 PAL GGAGAACCAAGAACGGTGGT CTGCCCTTGTCCCTGATTGT
HQ693477 EIN2 CCACCACAAGCAAAACAGGG GGTTGCCATCTCCACGTCTT
AY740529 ICS1 GCTCGTAGCACCAGAGTTGT TCCAATGAATGCCGCTGACT
AF247568.1 EIN3 TAATGCGATTCCGGGCAAGA CCAACGGAAATCGCCTCTGA
NM_001326261.2 ACS1 AGGGCCATTGCCAACTTTCA AGTTGCACCACCAGCCATAA
U60495 β-actin ATGCCTATGTGGGTGACGAAG TCTGTTGGCCTTAGGGTTGAG

Fig. 1

Inhibition rate of different effective protein components against TMV Different lowercase letters on the bars indicate significance difference using one-way analysis of variance (P≤0.05). The same as Fig. 4-B"

Table 2

Mass spectrometry results of proteins in F-23"

名称
Name		 序列
Sequence		 匹配率
Score (%)		 氨基酸数目
Number of amino acid (aa)		 蛋白分子量
Molecular weight (kD)
鞭毛蛋白Flagellin (Fragment) TAIDTVSSER 1.80 306 32.6
肌苷5′-单磷酸脱氢酶Inosine-5′-monophosphate dehydrogenase VIEFPNSSK 1.79 488 52.9
未知蛋白Uncharacterized protein LGAESI 1.34 327 36.7
TetR 家族转录调节因子
TetR family transcriptional regulator		 VILL 0 385 44.8
P型ATP酶 ATPase P LAAAAETGSEHPLGEAIVSGAEK 0 811 86.6
未知蛋白Uncharacterized protein IANDDQINEIIPSEWENVYLYAEIL 0 161 19.2
UPF0348 protein AX282_03230 FLNL 0 415 47.1

Fig. 2

FlgLJ02 protein expression and purification"

Fig. 3

FlgLJ02 HR response on tobacco leaves"

Fig. 4

FlgLJ02 induced resistance against TMV in tobacco"

Fig. 5

Relative expression of key genes in SA immune signalling pathway after FlgLJ02 induction"

Fig. 6

Relative expression of key genes in ET immune signalling pathway after FlgLJ02 induction"

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