Mechanistic Study on ChiC-mediated Regulation Mechanism of Tomato Resistance to Botrytis cinerea

doi:10.13560/j.cnki.biotech.bull.1985.2025-1242

Abstract

Abstract:

Objective Gray mold caused by Botrytis cinerea poses a serious threat to the production and postharvest safety of tomato and other protected horticultural crops, while exploitable disease-resistance gene resources in plants remain limited. In this study, focusing on chitinase, we aim to elucidate the regulatory mechanism of the chitinase synthesis gene ChiC in tomato resistance to gray mold, thereby providing a basis for understanding the role of chitinases in tomato responses to B. cinerea. Method Using tomato as the experimental material, the ChiC knockout mutants were generated by CRISPR/Cas9 technology, and ChiC overexpression lines were obtained via Agrobacterium-mediated transformation. B. cinerea inoculation assays were performed on these genetic materials, and disease phenotypes and lesion areas on fruits and leaves from different genetic backgrounds were analyzed to verify the role of ChiC in disease resistance. Meanwhile, RT-qPCR analysis was performed to examine the expression variations of defense genes in different tomato genotypes after B. cinerea infection. Result Sequence and structural analyses revealed that the tomato ChiC gene encodes a chitinase consisting of 376 amino acids, which belongs to the GH18 subfamily. Quantitative analyses showed that infection of tomato fruits by B. cinerea significantly induces ChiC expression. Disease assays demonstrated that, compared with wild-type plants, the chic mutant plants have increased susceptibility of both fruits and leaves to B. cinerea, whereas ChiC overexpression plants demonstrate markedly enhanced disease resistance. Further gene expression analysis revealed that in ChiC overexpression plants, the expression of defense-related genes such as ERF.C3, PR-STH2d, and PR-STH2c were significantly upregulated following B. cinerea infection. Conclusion The tomato ChiC gene positively regulates tomato resistance to B. cinerea, and its mode of action may involve directly degrading fungal cell walls and enhancing plant immune responses through the induction of defense gene expression.

Key words: tomato, gray mold, Botrytis cinerea, chitinase, ChiC, CRISPR/Cas9, disease resistance, defense-related gene

LI Ya-ni, HAN Hong-yu, GENG Meng-shuang, MI Ruo-lan, Wang Wei-qi, Yu Wen-jing, MENG Xian-wen, LI Chuan-you. Mechanistic Study on ChiC-mediated Regulation Mechanism of Tomato Resistance to Botrytis cinerea[J]. Biotechnology Bulletin, 2026, 42(3): 255-262.

Figures/Tables 5

Table 1 Primers for RT-qPCR used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
ACTIN2-qRT-F	TTGCTGACCGTATGAGCAAG
ACTIN2-qRT-R	GGACAATGGATGGACCAGAC
ChiC-qRT-F	TGGACTTGATCTTGATTGGGAA
ChiC-qRT-R	CCGGATAGTTCAAACCATTGAC
ERF.C3-qRT-F	GAATTCGTCGTCTAGCTATGGA
ERF.C3-qRT-R	GAACCTCTCATAGCAAATGCAG
PR-STH2c-qRT-F	AATCCTTCTGTCTACGCTTGAA
PR-STH2c-qRT-R	TTAACATCACAACACGTTCACG
PR-STH2d-qRT-F	AATAAGAATCAGGTCCACACGT
PR-STH2d-qRT-R	CACACAACTCAACAAAAAGCAC

Fig. 1 Structure of tomato ChiC and its expression patternsA: Phylogenetic tree of chitinase genes from tomato and Arabidopsis. B: ChiC protein domains. C: Three-dimensional structure of ChiC. D: Relative expression of the ChiC gene in different tissues (IMG: immature green; Br: breaker; Br7: 7 d after breaker stage). E: Expression patterns of ChiC before and after B. cinerea infection in the fruits. F: Expression patterns of ChiC before and after B. cinerea infection in leaves. The values in the bar charts are presented as mean ± standard error (n=3). Different lowercase letters indicate significant differences at P<0.05 level. The same below

Fig. 2 Generation of ChiC genetic materials and assays of chitinase activityA: Positions of the gene-editing targets and mutation types in the chic-1 and chic-2 lines. B: ChiC protein domain structure in the chic-1 and chic-2 lines. C: Relative expression of ChiC in overexpression lines. D: Detection of chitinase activity in transgenic materials

Fig. 3 ChiC positively regulates the resistance to B. cinerea in tomatoA: Disease symptoms and quantification of lesion areas in breaker-stage fruits at 3 d after B. cinerea infection. B: Disease symptoms and quantification of lesion areas in red-ripe fruits at 3 d after B. cinerea infection. C: Disease symptoms and quantification of lesion areas in leaves at 3 d after B. cinerea infection. Bar=1 cm

Fig. 4 ChiC modulates the expressions of defense genes in response to B. cinerea infection

References 32

[1]	Wang SQ, Zhang XY, Zhong S, et al. BcatrB mediates pyrimethanil resistance in Botrytis cinerea revealed by transcriptomics analysis [J]. Sci Rep, 2025, 15(1): 11478.
[2]	Williamson B, Tudzynski B, Tudzynski P, et al. Botrytis cinerea: the cause of grey mould disease [J]. Mol Plant Pathol, 2007, 8(5): 561-580.
[3]	Mobasher Amini M, Mirzaei S, Heidari A. A growing threat: Investigating the high incidence of benzimidazole fungicides resistance in Iranian Botrytis cinerea isolates [J]. PLoS One, 2023, 18(11): e0294530.
[4]	Shawky A, Hatawsh A, Al-Saadi N, et al. Revolutionizing tomato cultivation: CRISPR/Cas9 mediated biotic stress resistance [J]. Plants, 2024, 13(16): 2269.
[5]	Arie T, Takahashi H, Kodama M, et al. Tomato as a model plant for plant-pathogen interactions [J]. Plant Biotechnol, 2007, 24(1): 135-147.
[6]	Huang H, Zhao WC, Li CH, et al. SlVQ15 interacts with jasmonate-ZIM domain proteins and SlWRKY31 to regulate defense response in tomato [J]. Plant Physiol, 2022, 190(1): 828-842.
[7]	李杰, 梁郅林, 孙燕, 等. SlSnRK1.2调控番茄抗灰霉病功能分析 [J]. 中国农业科学, 2024(21): 4238-4247.
	Li J, Liang ZL, Sun Y, et al. Functional analysis of SlSnRK1.2 in regulating tomato resistance to grey mould [J]. Sci Agric Sin, 2024(21): 4238-4247.
[8]	Ding ST, Feng SX, Zhou SB, et al. A novel LRR receptor-like kinase BRAK reciprocally phosphorylates PSKR1 to enhance growth and defense in tomato [J]. EMBO J, 2024, 43(23): 6104-6123.
[9]	Li S, Huang YL, Zhao Y, et al. Ripening-induced defence signalling in Botrytis cinerea-infected tomato fruits involves activation of ERF.F4 by a MYC2-NOR/RIN protein complex [J]. Plant Biotechnol J, 2025, 23(9): 4126-4139.
[10]	Wang XW, Gao M, Li HX, et al. SCFSlRAE1 regulates tomato resistance to Botrytis cinerea by modulating SlWRKY1 stability [J]. J Integr Plant Biol, 2025, 67(8): 2167-2183.
[11]	Zhu KY, Merzendorfer H, Zhang WQ, et al. Biosynthesis, turnover, and functions of chitin in insects [J]. Annu Rev Entomol, 2016, 61: 177-196.
[12]	Grover A. Plant chitinases: genetic diversity and physiological roles [J]. Crit Rev Plant Sci, 2012, 31(1): 57-73.
[13]	Khan RS, Iqbal A, Bibi A, et al. Plant chitinases: Types, structural classification, antifungal potential and transgenic expression in plants for enhanced disease resistance [J]. Plant Cell Tissue Organ Cult, 2024, 156(3): 75.
[14]	郭林霞, 董旋, 赵德刚. 转杜仲几丁质酶基因EuCHIT1番茄提高对灰霉病的抗性 [J]. 植物生理学报, 2016, 52(5): 703-714.
	Guo LX, Dong X, Zhao DG. Transgenic tomato plants expressing a Eucommia ulmoides chitinase gene EuCHIT1 and their resistance to Botrytis cinerea [J]. Plant Physiol J, 2016, 52(5): 703-714.
[15]	Tabei Y, Kitade S, Nishizawa Y, et al. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea) [J]. Plant Cell Rep, 1998, 17(3): 159-164.
[16]	Liu ZH, Yang CP, Qi XT, et al. Cloning, heterologous expression, and functional characterization of a chitinase gene, Lbchi32, from Limonium bicolor [J]. Biochem Genet, 2010, 48(7/8): 669-679.
[17]	Liu JJ, Wen JX, Li JF, et al. Nepenthes chitinase NkChit2b-1 confers broad-spectrum resistance to chitin-containing pathogens and insects in plants [J]. Adv Biotechnol, 2025, 3(2): 12.
[18]	Cao J, Tan XN. Comprehensive analysis of the chitinase family genes in tomato (Solanum lycopersicum) [J]. Plants, 2019, 8(3): 52.
[19]	Ohnuma T, Numata T, Osawa T, et al. A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis [J]. Planta, 2011, 234(1): 123-137.
[20]	Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3 [J]. Nature, 2024, 630(8016): 493-500.
[21]	Lian JJ, Han HY, Zhao JH, et al. In-vitro and in-planta Botrytis cinerea inoculation assays for tomato [J]. Bio Protoc, 2018, 8(8): e2810.
[22]	Du MM, Zhao JH, Tzeng DT, et al. MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato [J]. Plant Cell, 2017, 29(8): 1883-1906.
[23]	Wang T, Zhang HY, Zhu HL. CRISPR technology is revolutionizing the improvement of tomato and other fruit crops [J]. Hortic Res, 2019, 6: 77.
[24]	Zhang JL, Dong DH, Jia CY, et al. Fine-tuning of MYC2-mediated Botrytis defense response by the LBD40/42-CRL3BPM4 module in tomato [J]. Plant Cell, 2025, 37(11): koaf258.
[25]	Vaghela B, Vashi R, Rajput K, et al. Plant chitinases and their role in plant defense: a comprehensive review [J]. Enzyme Microb Technol, 2022, 159: 110055.
[26]	Jabeen N, Chaudhary Z, Gulfraz M, et al. Expression of rice chitinase gene in genetically engineered tomato confers enhanced resistance to Fusarium wilt and early blight [J]. Plant Pathol J, 2015, 31(3): 252-258.
[27]	Li QQ, Yang YY, Bai X, et al. Systematic analysis and functional characterization of the chitinase gene family in Fagopyrum tataricum under salt stress [J]. BMC Plant Biol, 2024, 24(1): 1222.
[28]	Hamid R, Khan M, Ahmad M, et al. Chitinases: an update [J]. J Pharm Bioall Sci, 2013, 5(1): 21-29.
[29]	He TN, Fan JS, Jiao GZ, et al. Bioinformatics and expression analysis of the chitinase genes in strawberry (Fragaria vesca) and functional study of FvChi-14 [J]. Plants, 2023, 12(7): 1543.
[30]	Ali M, Li QH, Zou T, et al. Chitinase gene positively regulates hypersensitive and defense responses of pepper to Colletotrichum acutatum infection [J]. Int J Mol Sci, 2020, 21(18): 6624.
[31]	Xiao YH, Li XB, Yang XY, et al. Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants [J]. Biosci Biotechnol Biochem, 2007, 71(5): 1211-1219.
[32]	Lombardo L, Zelasco S. Biotech approaches to overcome the limitations of using transgenic plants in organic farming [J]. Sustainability, 2016, 8(5): 497.