| Citation: |
YAN Xin, GAO Linying, HU Menghui, SHAO Miaomiao, HOU Yuxia. Enhanced inhibitory effect of mutations of key amino acids in the disease-resistance protein CkPGIP1 on Verticillium dahliae[J]. Chinese Journal of Pesticide Science, 2024, 26(5): 922-931. DOI: 10.16801/j.issn.1008-7303.2024.0075
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The polygalacturonase inhibitory protein (CkPGIP1) of Cynanchum komarovii can effectively inhibit the activity of polygalacturonase (VdPG1) of Verticillium dahliae. To further enhance the disease resistance of CkPGIP1, this study started with site-directed mutagenesis of the disease-resistant protein CkPGIP1 to investigate its enhanced inhibitory effect on V. dahliae. Using overlapping extension polymerase chain reaction (PCR) technology, mutant variants T152VCkPGIP1, D202SCkPGIP1, and I250KCkPGIP1 of CkPGIP1 were successfully obtained. The corresponding prokaryotic expression vectors were constructed for the induction expression and purification of CkPGIP1 and its mutant proteins. The inhibitory effect of the mutant proteins on VdPG1 was determined by 3,5-dinitrosalicylic acid (DNS) method and agarose radial diffusion method. The results of DNS assay showed that the mutant proteins effectively inhibited the activity of VdPG1 in a dose-dependent manner. Among them, the inhibitory effect of D202SCkPGIP1 on VdPG1 was most significant, followed by T152VCkPGIP1, and the inhibitory effect of I250KCkPGIP1 was not obvious. The results of agarose radial diffusion were consistent with those of DNS assay. D202SCkGIP1-treated V. dahliae showed the most obvious decrease in the halo area of VdPG1, indicating that D202SCkPGIP1 had the most significant inhibitory activity. Further study revealed that the mutant proteins could inhibit the expansion of V. dahliae on cotton leaves, confirming that the key amino acid mutations in CkPGIP1 enhanced its inhibitory effect on V. dahliae.
| [1] |
KLOSTERMAN S J, ATALLAH Z K, VALLAD G E, et al. Diversity, pathogenicity, and management of Verticillium species[J]. Annu Rev Phytopathol, 2009, 47: 39-62. doi: 10.1146/annurev-phyto-080508-081748
|
| [2] |
ZHU Y T, ZHAO M, LI T T, et al. Interactions between Verticillium dahliae and cotton: pathogenic mechanism and cotton resistance mechanism to Verticillium wilt[J]. Front Plant Sci, 2023, 14: 1174281. doi: 10.3389/fpls.2023.1174281
|
| [3] |
DE LORENZO G, D’OVIDIO R, CERVONE F. The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi[J]. Annu Rev Phytopathol, 2001, 39: 313-335. doi: 10.1146/annurev.phyto.39.1.313
|
| [4] |
POWELL A L, VAN KAN J, TEN HAVE A, et al. Transgenic expression of pear PGIP in tomato limits fungal colonization[J]. Mol Plant Microbe Interact, 2000, 13(9): 942-950. doi: 10.1094/MPMI.2000.13.9.942
|
| [5] |
ABRAMOVITCH R B, ANDERSON J C, MARTIN G B. Bacterial elicitation and evasion of plant innate immunity[J]. Nat Rev Mol Cell Biol, 2006, 7(8): 601-611.
|
| [6] |
MISAS-VILLAMIL J C, VAN DER HOORN R A L. Enzyme-inhibitor interactions at the plant-pathogen interface[J]. Curr Opin Plant Biol, 2008, 11(4): 380-388. doi: 10.1016/j.pbi.2008.04.007
|
| [7] |
WANG R, LU L X, PAN X B, et al. Functional analysis of OsPGIP1 in rice sheath blight resistance[J]. Plant Mol Biol, 2015, 87(1-2): 181-191. doi: 10.1007/s11103-014-0269-7
|
| [8] |
LIU N N, MA X W, ZHOU S H, et al. Molecular and functional characterization of a polygalacturonase-inhibiting protein from Cynanchum komarovii that confers fungal resistance in Arabidopsis[J]. PLoS One, 2016, 11(1): e0146959. doi: 10.1371/journal.pone.0146959
|
| [9] |
WANG X J, ZHU X P, TOOLEY P, et al. Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens[J]. Plant Mol Biol, 2013, 81(4-5): 379-400. doi: 10.1007/s11103-013-0007-6
|
| [10] |
PRABHU S A, SINGH R, KOLKENBROCK S, et al. Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases[J]. J Exp Bot, 2014, 65(17): 5033-5047. doi: 10.1093/jxb/eru266
|
| [11] |
MAULIK A, BASU S. Study of Q224K, V152G double mutation in bean PGIP2, an LRR protein for plant defense: an in silico approach[J]. Proteins, 2013, 81(5): 852-862. doi: 10.1002/prot.24243
|
| [12] |
LECKIE F, MATTEI B, CAPODICASA C, et al. The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability[J]. EMBO J, 1999, 18(9): 2352-2363. doi: 10.1093/emboj/18.9.2352
|
| [13] |
马晓闻. 牛心朴子草逆境胁迫转录组分析与CkNF-YB1、CkEDS1的功能研究[D]. 北京: 中国农业大学, 2016.
MA X W. Transcriptome analysis of stress reponse in Cynanchun komarovii and functional study of the genes CkNF-YB1 and CkEDS1[D]. Beijing: China Agricultural University, 2016.
|
| [14] |
胡晓倩, 石志钰, 朱玉涛, 等. 棉花GhRAR1基因调控棉花抗黄萎病机理的研究[J]. 植物保护, 2023, 49(2): 39-47.
HU X Q, SHI Z Y, ZHU Y T, et al. Mechanism of the cotton GhRAR1 gene regulating the resistance of cotton to Verticillium wilt[J]. Acta Phytophy Sin, 2023, 49(2): 39-47.
|
| [15] |
皇甫海燕. 甘蓝型油菜PGIP2的原核表达、蛋白功能及其遗传转化的研究[D]. 长沙: 湖南农业大学, 2011.
HUANGFU H Y. Research on prokaryotic expression, protein function of Brassica napus PGIP2 and its genetic transformation[D]. Changsha: Hunan Agricultural University, 2011.
|
| [16] |
BENDTSEN J D, NIELSEN H, VON HEIJNE G, et al. Improved prediction of signal peptides: SignalP 3.0[J]. J Mol Biol, 2004, 340(4): 783-795. doi: 10.1016/j.jmb.2004.05.028
|
| [17] |
DIAZ A A, TOMBA E, LENNARSON R, et al. Prediction of protein solubility in Escherichia coli using logistic regression[J]. Biotechnol Bioeng, 2010, 105(2): 374-383. doi: 10.1002/bit.22537
|
| [18] |
WILKINS M R, GASTEIGER E, BAIROCH A, et al. Protein identification and analysis tools in the ExPASy server[J]. Methods Mol Biol, 1999, 112: 531-552.
|
| [19] |
COMBET C, BLANCHET C, GEOURJON C, et al. NPS@: network protein sequence analysis[J]. Trends Biochem Sci, 2000, 25(3): 147-150. doi: 10.1016/S0968-0004(99)01540-6
|
| [20] |
ARNOLD K, BORDOLI L, KOPP J, et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[J]. Bioinformatics, 2006, 22(2): 195-201. doi: 10.1093/bioinformatics/bti770
|
| [21] |
NELSON M D, FITCH D H A. Overlap extension PCR: an efficient method for transgene construction[J]. Methods Mol Biol, 2011, 772: 459-470.
|
| [22] |
崔华威, 杨艳丽, 黎敬涛, 等. 一种基于Photoshop的叶片相对病斑面积快速测定方法[J]. 安徽农业科学, 2009, 37(22): 10760-10762. doi: 10.3969/j.issn.0517-6611.2009.22.192
CUI H W, YANG Y L, LI J T, et al. A faster method for measuring relative lesion area on leaves based on software photoshop[J]. J Anhui Agric Sci, 2009, 37(22): 10760-10762. doi: 10.3969/j.issn.0517-6611.2009.22.192
|
| [23] |
王晓利, 张军科, 汪勇, 等. 苹果多聚半乳糖醛酸酶抑制蛋白的分离纯化[J]. 西北农业学报, 2008, 17(6): 94-97. doi: 10.3969/j.issn.1004-1389.2008.06.020
WANG X L, ZHANG J K, WANG Y, et al. Isolation and purification of a polygalacturonase-inhibiting protein(PGIP) from apple[J]. Acta Agric Boreali Occidentalis Sin, 2008, 17(6): 94-97. doi: 10.3969/j.issn.1004-1389.2008.06.020
|
| [24] |
孙文秀. 大豆疫霉根腐病菌多聚半乳糖醛酸酶活性对其致病力的影响[J]. 现代农业科学, 2008(2): 42-43.
SUN W X. Effect of activities of polygalacturonase on pathogenicity in Phytophthora sojae[J]. Mod Agric Sci, 2008(2): 42-43.
|
| [25] |
周立, 刘勇, 李建吾, 等. 小麦多聚半乳糖醛酸酶抑制蛋白对几种病原真菌抑制作用的研究[J]. 植物病理学报, 1998, 28(2): 107-112. doi: 10.3321/j.issn:0412-0914.1998.02.003
ZHOU L, LIU Y, LI J W, et al. On the inhibition effect of pgip in wheat to some fungi[J]. Acta Phytopathol Sin, 1998, 28(2): 107-112. doi: 10.3321/j.issn:0412-0914.1998.02.003
|
| [26] |
KING D, BERGMANN C, ORLANDO R, et al. Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase I. Homogalacturonan-polygalacturonase inhibiting protein system[J]. Biochemistry, 2002, 41(32): 10225-10233. doi: 10.1021/bi020119f
|
| [27] |
BENEDETTI M, ANDREANI F, LEGGIO C, et al. A single amino-acid substitution allows endo-polygalacturonase of Fusarium verticillioides to acquire recognition by PGIP2 from Phaseolus vulgaris[J]. PLoS One, 2013, 8(11): e80610. doi: 10.1371/journal.pone.0080610
|
| [28] |
CASASOLI M, FEDERICI L, SPINELLI F, et al. Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein[J]. Proc Natl Acad Sci USA, 2009, 106(18): 7666-7671. doi: 10.1073/pnas.0812625106
|
| [29] |
JANNI M, DI GIOVANNI M, ROBERTI S, et al. Characterization of expressed Pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat[J]. Theor Appl Genet, 2006, 113(7): 1233-1245. doi: 10.1007/s00122-006-0378-z
|
| [30] |
MAULIK A, GHOSH H, BASU S. Comparative study of protein-protein interaction observed in polygalacturonase-inhibiting proteins from Phaseolus vulgaris and Glycine max and polygalacturonase from Fusarium moniliforme[J]. BMC Genomics, 2009, 10(Suppl 3): S19.
|
| [31] |
LIU N N, ZHANG X Y, SUN Y, et al. Molecular evidence for the involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to Verticillium and Fusarium wilts in cotton[J]. Sci Rep, 2017, 7: 39840. doi: 10.1038/srep39840
|
| [32] |
D’OVIDIO R, RAIOLA A, CAPODICASA C, et al. Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects[J]. Plant Physiol, 2004, 135(4): 2424-2435. doi: 10.1104/pp.104.044644
|
| [33] |
XIAO Y, SUN G Z, YU Q S, et al. A plant mechanism of hijacking pathogen virulence factors to trigger innate immunity[J]. Science, 2024, 383(6684): 732-739. doi: 10.1126/science.adj9529
|
| [34] |
KALUNKE R M, TUNDO S, BENEDETTI M, et al. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens[J]. Front Plant Sci, 2015, 6: 146.
|
| [35] |
SPINELLI F, MARIOTTI L, MATTEI B, et al. Three aspartic acid residues of polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris are critical for inhibition of Fusarium phyllophilum PG[J]. Plant Biol, 2009, 11(5): 738-743. doi: 10.1111/j.1438-8677.2008.00175.x
|
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