Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae

Authors: LI, HUAN - Nanjing Forestry University; SHENG, RUO-CHENG - Nanjing Forestry University; ZHANG, CHEN-NING - Nanjing Forestry University; WANG, LI-CHAO - Nanjing Forestry University; LI, MIN - Nanjing Forestry University; WANG, YA-HONG - Nanjing Forestry University; QIAO, YU-HANG - Nanjing Forestry University; Klosterman, Steven; CHEN, JIE-YIN - Chinese Academy Of Agricultural Sciences; KONG, ZHI-QIANG - Chinese Academy Of Agricultural Sciences; SUBBARAO, KRISHNA - University Of California; CHEN, FENG-MAO - Nanjing Forestry University; ZHANG, DAN-DAN - Chinese Academy Of Agricultural Sciences

Submitted to: BMC Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2023
Publication Date: 10/31/2023
Citation: Li, H., Sheng, R.C., Zhang, C.N., Wang, L.C., Li, M., Wang, Y.H., Qiao, Y.H., Klosterman, S.J., Chen, J.Y., Kong, Z.Q., Subbarao, K.V., Chen, F.M., Zhang, D.D. 2023. Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae. BMC Biology. 21. Article 237. https://doi.org/10.1186/s12915-023-01697-w.
DOI: https://doi.org/10.1186/s12915-023-01697-w

Interpretive Summary: Verticillium dahliae is a soilborne plant pathogenic fungus and a cause of Verticillium wilt diseases of plants. This fungus causes disease on well over 200 plant species worldwide, including many agriculturally important crops. In this study, we examined the function of two genes, known as VdZFP1 and VdZFP2, thought to play a role in the gene regulation in V. dahliae. The VdZFP1 and VdZFP2 genes were deleted in the fungus and the growth and pathogenicity were examined relative to the original strain that had not been mutated. The results indicate that VdZFP1 and VdZFP2 in V. dahliae are required to produce normal levels of the long-lived survival structures of V. dahliae, known as microsclerotia. VdZFP1 and VdZFP2 are also required for protection against osmotic stress, contribute to microsclerotia production but are not required for virulence in the plant hosts studied. Insights into the genes necessary for survival and microsclerotia formation in V. dahliae may lead to alternative control approaches to eliminate the fungus or reduce it survival in the soil.

Technical Abstract: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.