Functional characterization of CgPBS2, a MAP kinase kinase in Colletotrichum gloeosporioides, using osmotic stress sensitivity as a selection marker
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.
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