Functional characterization of CgPBS2, a MAP kinase kinase in Colletotrichum gloeosporioides, using osmotic stress sensitivity as a selection marker
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Colletotrichum leaf disease of Hever brasiliensis (rubber tree) caused by C. gloeosporioides is one of the major causes of declining rubber tree yields. Little is known about the fungal molecular characters that are important for pathogenicity on rubber tree and fungicide resistance. In this study, we cloned the CgPBS2 gene, the key component of the Hog1 pathway which controls various aspects of osmoregulation and fungicide resistance in various fungal pathogens, including the causal agent of Colletotrichum leaf disease of rubber tree. We characterized the function of the CgPBS2 gene by reverse genetics. Because the Hog1 pathway plays an important role in stress responses, we obtained a CgPBS2 gene deletion mutant by PEG-mediated transformation of protoplasts after reducing the concentration of sucrose in the screening medium from 1.0 M to 0.2 M. Then, the complemented transformants and GFP-labelled CgPBS2 gene transformants were selected directly under highly hyperosmotic medium (PDA + 1.5 M sorbitol) without using other selectable gene markers. Phenotypic observations showed that the CgPBS2 protein was mainly localized in the conidial cytoplasm of the CgPBS2-GFP transformants. In addition, disruption of CgPBS2 led to sensitivity to hyperosmosis and high salt concentration as well as resistance to the fungicide fludioxonil. No obvious difference in virulence was observed between the null mutant and the wild-type strain. These results provide insights into the role of the CgPBS2 gene in osmotic stress, salt stress and fludioxonil resistance and suggest that osmotic stress sensitivity can be used as a selection marker.
KeywordsCgPBS2 MAP kinase kinase Osmotic stress Selection marker Colletotrichum gloeosporioides Hevea brasiliensis
This research was supported by the National Natural Science Foundation of China (No. 31760499, No.31201468) and the earmarked fund for China Agriculture Research System (No. CARS-33-GW-BC1).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Human participants and animal studies
This research did not involve human participants or animals.
- Alonso-Monge, R., Navarro-Garcia, F., Roman, E., Negredo, A. I., Eisman, B., Nombela, C., & Pla, J. (2003). The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans. Eukaryotic Cell, 2, 351–361.CrossRefPubMedPubMedCentralGoogle Scholar
- Cai, Z. Y., Li, G. H., Lin, C. H., Shi, T., Zhai, L. G., Chen, Y. P., & Huang, G. X. (2013). Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutangenesis. Microbiological Research, 168, 340–350.CrossRefPubMedGoogle Scholar
- Kovar, J. L., Zhang, J., Funke, R. P., & Weeks, D. P. (2002). Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. The Plant Journal, 29(1), 109–117.CrossRefPubMedGoogle Scholar
- Mehrabi, R., Zwiers, L. H., de Waard, M.A., Kema, G.H. (2006). MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola. Molecular Plant-Microbe Interactions, 11, 1262–1269.Google Scholar
- Park, S. H., Choi, E. S., Kim, M. J., Cha, B. J., Yang, M. S., & Kim, D. H. (2004). Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress. Molecular Microbiology, 51, 1267–1277.CrossRefPubMedGoogle Scholar
- Rispail, N., Soanes, D. M., Ant, C., Czajowski, R., Grünler, A., Huguet, R., Perez-Nadales, E., Poli, A., Sartorel, E., Valiante, V., Yang, M., Beffa, R., Brakhage, A. A., Gow, N. A., Kahmann, R., Lebrun, M. H., Lenasi, H., Perez-Martin, J., Talbot, N. J., Wendland, J., & Di Pietro, A. (2009). Comparative genomics of MAP kinase and calcium-calcineurin signaling components in plant and human pathogenic fungi. Fungal Genetics and Biology, 46(4), 287–298.CrossRefPubMedGoogle Scholar