Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides

doi:10.13560/j.cnki.biotech.bull.1985.2025-0543

Abstract

Abstract:

Objective The bZIP transcription factor CsFcr3 was cloned from Colletotrichum siamense, the predominant species responsible for Colletotrichum leaf disease (CLD) in rubber trees. This study characterized its sequence and structural features and elucidated its biological role in regulating the sensitivity of C. siamense to six different fungicides. These findings establish a foundational understanding of resistance regulatory mechanisms in pathogenic fungi and provide a theoretical framework for the scientific and efficient management of fungal diseases in rubber tree cultivation. Method The bZIP transcription factor CsFcr3, having high sequence homology with the fluconazole resistance protein Fcr3 from Candida albicans, was cloned from Colletotrichum siamense using homology-based cloning. Comprehensive sequence and structural analyses were conducted to characterize this gene. Real-time quantitative PCR (RT-qPCR) was employed to profile the expression patterns of CsFcr3 under fungicide stress. Additionally, the gene knockout of CsFcr3 was performed by homologous recombination and PEG-mediated protoplast transformation, and the changes in sensitivity of the knockout mutants to different fungicides were analyzed. Result The open reading frame (ORF) of CsFcr3 encompassed 1 101 base pairs, encoding a protein of 336 amino acids with a conserved bZIP_Yap domain. RT-qPCR results demonstrated significant upregulation of CsFcr3 expression under stress induced by demethylation inhibitor (DMI) fungicides (difenoconazole, tebuconazole, prochloraz) and quinone outside inhibitor (QoI) fungicides (pyraclostrobin). Three CsFcr3 knockout mutants (ΔCsFcr3-3, ΔCsFcr3-15, and ΔCsFcr3-16) were successfully generated. Compared to the wild-type strain, the ΔCsFcr3s presented significantly reduced sensitivity to fluconazole, tebuconazole, and prochloraz, increased sensitivity to pyraclostrobin, and no significant differences in sensitivity to difenoconazole and fludioxonil. Conclusion The bZIP transcription factor CsFcr3, which contains conserved motifs and structural domains typical of the bZIP family, is cloned from C. siamense. Phylogenetic analysis reveals its close homology to the Saccharomyces cerevisiae Yap3p protein. Functional characterization demonstrates that CsFcr3 primarily regulates C. siamense sensitivity to DMI and QoI fungicides, exhibiting no significant regulatory role in response to pyrrolnitrin-class fungicides.

Key words: anthracnose, Colletotrichum siamense, Hevea brasiliensis, bZIP transcription factor, fungicide, sensitivity

YANG Hong, ZHANG Chao, XUE Wen-xuan, HUANG Wei-yuan, LIN Chun-hua, WANG Li-feng. Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides[J]. Biotechnology Bulletin, 2025, 41(12): 342-350.

Figures/Tables 7

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Usage
UF	ACAACAAGCGTTTAGCGTCTC	扩增目的基因上臂
UR	AGATGTGGGGCACTGTGGCGTTGGCACGGTCGCGCGATGTTGTCCTGGAAGAG	扩增目的基因上臂
DF	TATTGCACGGGAATTGCATGCTCTCACCAGCCCCCTCTCTCCCCAGTG	扩增目的基因下臂
DR	GATGCCTAAGCCTGCAAAC	扩增目的基因下臂
InF	TCACTGGTGGATTGCCGCTTAG	转化子验证
InR	TGTGCTGTGGTGGTTGCTGAG	转化子验证
OUF	TGTCTTGTCTTGCCTGCAAC	转化子验证
OUR	AAAGAACTCGGCAGTAAGCTTG	转化子验证
S2F	GGCGGTGCTATCCTTCCCGTGTT	扩增氯嘧磺隆抗性基因
S2R	GTGAGAGCATGCAATTCCCGTGCAATA	扩增氯嘧磺隆抗性基因
qCsFcr3_F	ATGGACTTCTCCCACCAGTA	RT-qPCR
qCsFcr3_R	CGGCAGCGACGAGCTCCTGTAG	RT-qPCR
qCsActin_F	GATTTGGCACCACACCTTCTACA	内参
qCsActin_R	TCTCTGTTGGACTTGGGGTTGAT	内参

Fig. 1 Full-length cloning of the CsFcr3 geneA: PCR amplification of the CsFcr3 gene. B: Nucleotide sequence and derived amino acids of CsFcr3. M: DL5000 DNA Marker. Fcr3: PCR amplification product of CsFcr3 gene. CK: Negative control

Fig. 2 Sequence characterization analysis of CsFcr3

Fig. 3 Phenotype (A) and inhibition rate (B) of HN08 in fungicide treatmentDifferent letters indicate significant differences at P < 0.05, the same below

Fig. 4 Expression analysis of CsFcr3 under fungicide stress

Fig. 5 Construction process (A) and PCR verification (B) of the CsFcr3 gene knockout mutant

Fig. 6 Sensitivity analysis of ΔCsFcr3s to 6 fungicidesA: Phenotype of ΔCsFcr3s under fungicide stress. B: Inhibition rate of ΔCsFcr3s under fungicide stress, *P < 0.05, **P < 0.01

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