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Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans
Zhiping Zhang1, Yingbin Li1, Laixin Luo1
1College of Plant Protection/Beijing Key Laboratory of Seed Disease Testing and Control (BKL-SDTC), China Agricultural University, Beijing, China.
Abstract:
Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.
Insights
The cerato-platanin protein (CmCP) from Ceratocystis manginecans is essential for fungal growth and a key factor in causing mango wilt disease. Deleting the cmcp gene reduced pathogenicity, while its expression induced plant defense responses.
Area of Science:
- Plant Pathology
- Molecular Mycology
- Biochemistry
Background:
- Ceratocystis manginecans causes significant economic losses due to mango wilt.
- Cerato-platanin proteins (CPPs) are implicated in fungal pathogenesis but their role in C. manginecans is unconfirmed.
Purpose of the Study:
- To characterize the cerato-platanin protein (CmCP) from C. manginecans.
- To determine the function of the cmcp gene in fungal development and pathogenicity.
Main Methods:
- Gene editing using CRISPR/Cas-U6-1 to create cmcp deletion mutants (Δcmcp).
- Complementation of mutants (Δcmcp-C) and characterization of wild-type, Δcmcp, and Δcmcp-C strains.
- Heterologous expression of CmCP in Pichia pastoris and subsequent inoculation of Nicotiana tabacum.
Main Results:
- The Δcmcp mutant showed impaired morphology, mycelial growth, and conidial production compared to the wild-type.
- Recombinant CmCP induced severe necrosis and hypersensitive response symptoms in N. tabacum leaves.
- CmCP triggered electrolyte leakage, ROS generation, and upregulation of defense genes (PR-1, PAD3, ERF1, HSR203J, HIN1) in plants.
Conclusions:
- The cmcp gene is crucial for the growth and development of C. manginecans.
- CmCP acts as a major pathogenicity factor contributing to mango wilt disease.
- CmCP elicits plant defense responses, indicating its role in host-pathogen interactions.
