A constitutive serine protease inhibitor suppresses herbivore performance in tea (Camellia sinensis)

Abstract

Protease inhibitors promote herbivore resistance in diverse plant species. Although many inducible protease inhibitors have been identified, there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance. Here, we identified a serine protease inhibitor, CsSERPIN1, from the tea plant (Camellia sinensis). Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses. In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin. Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores, the tea geometrid and fall armyworm, in tea and Arabidopsis plants, respectively. The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters. Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores, making it a promising candidate for pest prevention in agriculture.

Acknowledgements

We thank Prof. Liang Chen for insightful input and valuable scientific suggestions, Prof. Dr Xinchao Wang, Lu Wang and Yuchun Wang for kindly supplying experimental materials, Xiwang Li and Jianying Jin for looking after the insects and plants. This research was supported by National Natural Science Foundation of China (31272053, 31901898), Central Public-interest Scientific Institution Basal Research Fund (Y2023PT03, 1610212019001), and the Elite Youth Program of Chinese Academy of Agricultural Sciences for Meng Ye.

Author contributions

M.Y. and X.S. designed the research; M.Y., C.L., N.L., C.Y., M.L., Z.X. and S.L. performed experiments; M.Y. and X.S. analysed data and wrote the paper. All authors approved the article.

Data availability

All the relevant data are provided in this article and its supplementary information. The original readcount and fpkm of RNA sequencing data for CsSERPINs and CsLOXs reported in this paper is deposited in Data S1. All the materials supporting the findings of this study are available from the corresponding authors upon request.

Conflict of interests

None declared.

References

[1] Hazarika LK, Bhuyan M, Hazarika BN . Insect pests of tea and their management. Annu Rev Entomol. 2009; 54: 267-84

[2] Singh S, Singh A, Kumar S et al. Protease inhibitors: recent advancement in its usage as a potential biocontrol agent for insect pest management. Insect Sci. 2020; 27: 186-201

[3] Napoleao TH, Albuquerque LP, Santos NDL et al. Insect midgut structures and molecules as targets of plant-derived protease inhibitors and lectins. Pest Manag Sci. 2019; 75: 1212-22

[4] Law RH, Zhang Q, McGowan S et al. An overview of the serpin superfamily. Genome Biol. 2006; 7: 216

[5] Irving JA, Pike RN, Dai W et al. Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering α1-antitrypsin to inhibit cathepsin proteases. Biochemistry. 2002; 41: 4998-5004

[6] Clemente M, Corigliano M, Pariani S et al. Plant serine protease inhibitors: biotechnology application in agriculture and molecular farming. Int J Mol Sci. 2019; 20: 1345

[7] Alvarez-Alfageme F, Maharramov J, Carrillo L et al. Potential use of a serpin from Arabidopsis for pest control. PLoS One. 2011; 6: e20278

[8] Hamza R, Pérez-Hedo M, Urbaneja A et al. Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biol. 2018; 18: 24

[9] Quilis J, Lopez-Garcia B, Meynard D et al. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice. Plant Biotechnol J. 2014; 12: 367-77

[10] Pandey A, Yadav R, Sanyal I . Evaluating the pesticidal impact of plant protease inhibitors: lethal weaponry in the coevolutionary battle. Pest Manag Sci. 2022; 78: 855-68

[11] Dunse KM, Stevens JA, Lay FT et al. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field. Proc Natl Acad Sci U S A. 2010; 107: 15011-5

[12] Rustgi S, Boex-Fontvieille E, Reinbothe C et al. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2017; 114: 2212-7

[13] Damle MS, Giri AP, Sainani MN et al. Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum mill, cv. Dhanashree). Phytochemistry. 2005; 66: 2659-67

[14] Sultana MS, Millwood RJ, Mazarei M et al. Proteinase inhibitors in legume herbivore defense: from natural to genetically engineered protectants. Plant Cell Rep. 2022; 41: 293-305

[15] Sarmento RA, Lemos F, Bleeker PM et al. A herbivore that manipulates plant defence. Ecol Lett. 2011; 14: 229-36

[16] Hartl M, Giri AP, Kaur H et al. Serine protease inhibitors specifically defend Solanum nigrum against generalist herbivores but do not influence plant growth and development . Plant Cell. 2010; 22: 4158-75

[17] Pan Q, Shikano I, Hoover K et al. Pathogen-mediated tritrophic interactions: Baculovirus-challenged caterpillars induce higher plant defenses than healthy caterpillars. J Chem Ecol. 2019; 45: 515-24

[18] Ogran A, Wasserstrom H, Barzilai M et al. Water deficiency and induced defense against a generalist insect herbivore in desert and Mediterranean populations of Eruca sativa. J Chem Ecol. 2021; 47: 768-76

[19] Ye M, Liu M, Erb M et al. Indole primes defence signalling and increases herbivore resistance in tea plants. Plant Cell Environ. 2021; 44: 1165-77

[20] Zhang X, Ran W, Zhang J et al. Genome-wide identification of the TIFY gene family and their expression profiles in response to biotic and abiotic stresses in tea plants (Camellia sinensis). Int J Mol Sci. 2020; 21: 8316

[21] Hu Y, Zhang M, Lu M et al. Salicylic acid carboxyl glucosyltransferase UGT87E7 regulates disease resistance in Camellia sinensis. Plant Physiol. 2022; 188: 1507-20

[22] Xin Z, Li X, Li J et al. Application of chemical elicitor (Z)-3-hexenol enhances direct and indirect plant defenses against tea geometrid Ectropis obliqua. BioControl. 2016; 61: 1-12

[23] Zhang Y-H, Wan SQ, Wang WD et al. Genome-wide identification and characterization of the CsSnRK2 family in Camellia sinensis. Plant Physiol Biochem. 2018; 132: 287-96

[24] Hu Z, Ban Q, Hao J et al. Genome-wide characterization of the C-repeat binding factor (CBF) gene family involved in the response to abiotic stresses in tea plant (Camellia sinensis). Front Plant Sci. 2020; 11: 921

[25] Seth R, Maritim TK, Parmar R et al. Underpinning the molecular programming attributing heat stress associated thermotolerance in tea (Camellia sinensis (L.) O Kuntze). Hortic Res. 2021; 8: 99

[26] Cao Q, Lv W, Jiang H et al. Genome-wide identification of glutathione S-transferase gene family members in tea plant (Camellia sinensis) and their response to environmental stress . Int J Biol Macromol. 2022; 205: 749-60

[27] Saadati F, Bandani AR . Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest. Eurygaster integriceps. J Insect Sci. 2011; 11: 72

[28] Haq SK, Atif SM, Khan RH . Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Arch Biochem Biophys. 2004; 431: 145-59

[29] Puchol Tarazona AA, Maresch D, Grill A et al. Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana. FEBS Lett. 2021; 595: 379-88

[30] van der Hoorn RAL, Klemenčič M . Plant proteases: from molecular mechanisms to functions in development and immunity. J Exp Bot. 2021; 72: 3337-9

[31] Lara P, Ortego F, Gonzalez-Hidalgo E et al. Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco . Transgenic Res. 2000; 9: 169-78

[32] Oppert B, Morgan TD, Hartzer K et al. Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae). Comp Biochem Physiol C Toxicol Pharmacol. 2005; 140: 53-8

[33] Kuwar SS, Pauchet Y, Vogel H et al. Adaptive regulation of digestive serine proteases in the larval midgut of Helicoverpa armigera in response to a plant protease inhibitor . Insect Biochem Mol Biol. 2015; 59: 18-29

[34] Zhu-Salzman K, Zeng R . Insect response to plant defensive protease inhibitors. Annu Rev Entomol. 2015; 60: 233-52

[35] Tamura K, Stecher G, Kumar S . MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38: 3022-7

[36] Wong ML, Medrano JF . Real-time PCR for mRNA quantitation. BioTechniques. 2005; 39: 75-85

[37] Lu QH, Wang Y, Xiong F et al. Integrated transcriptomic and metabolomic analyses reveal the effects of callose deposition and multihormone signal transduction pathways on the tea plant- Colletotrichum camelliae interaction . Sci Rep. 2020; 10: 12858

[38] Johnston KA, Lee MJ, Brough C et al. Protease activities in the larval midgut of Heliothis virescens: evidence for trypsin and chymotrypsin-like enzymes. Insect Biochem Mol Biol. 1995; 25: 375-83

[39] Cruz ACB, Massena FS, Migliolo L et al. Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds . Plant Physiol Biochem. 2013; 70: 61-8

[40] Li H, Guo L, Yan M et al. A rapid and efficient transient expression system for gene function and subcellular localization studies in the tea plant (Camellia sinensis) leaves . Sci Hortic. 2022; 297: 110927

[41] Zhang XR, Henriques R, Lin SS et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006; 1: 641-6

[42] Yang Z, Duan XN, Jin S et al. Regurgitant derived from the tea geometrid Ectropis obliqua suppresses wound-induced polyphenol oxidases activity in tea plants . J Chem Ecol. 2013; 39: 744-51

[43] Benjamini Y, Hochberg Y . Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B-Stat Methodol. 1995; 57: 289-300

[44] Puth M-T, Neuhäuser M, Ruxton GD . Effective use of Pearson’s product-moment correlation coefficient. Anim Behav. 2014; 93: 183-9

[45] Jiang C, Moon DG, Ma J et al. Characteristics of non-volatile metabolites in fresh shoots from tea plant (Camellia sinensis) and its closely related species and varieties . Beverage Plant Res. 2022; 2: 9

[46] Qiao D, Yang C, Guo Y et al. Transcriptome and co-expression network analysis uncover the key genes mediated by endogenous defense hormones in tea plant in response to the infestation of Empoasca onukii Matsuda . Beverage Plant Res. 2023; 3: 4