Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae

Authors: WANG, DAN - Chinese Academy Of Agricultural Sciences; TIAN, LI - Qufu Normal University; ZHANG, DAN-DAN - Chinese Academy Of Agricultural Sciences; SONG, JIAN - Chinese Academy Of Agricultural Sciences; SONG, SHUANG-SHUANG - Qufu Normal University; YIN, CHUN-MEI - Chinese Academy Of Agricultural Sciences; ZHOU, LEI - Chinese Academy Of Agricultural Sciences; LIU, YAN - Qufu Normal University; WANG, BAO-LI - Chinese Academy Of Agricultural Sciences; KONG, ZHI-QIANG - Chinese Academy Of Agricultural Sciences; Klosterman, Steven; LI, JUN-JIAO - Chinese Academy Of Agricultural Sciences; WANG, JIE - Chinese Academy Of Agricultural Sciences; LI, TING-GANG - Chinese Academy Of Agricultural Sciences; ADAMU, SABIU - Chinese Academy Of Agricultural Sciences; SUBBARAO, KRISHNA - University Of California; CHEN, JIE-YIN - Chinese Academy Of Agricultural Sciences; DAI, XIAO-FENG - Chinese Academy Of Agricultural Sciences

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/17/2020
Publication Date: 3/10/2020
Citation: Wang, D., Tian, L., Zhang, D.-D., Song, J., Song, S.-S., Yin, C.-M., Zhou, L., Liu, Y., Wang, B.-L., Kong, Z.-Q., Klosterman, S.J., Li, J.-J., Wang, J., Li, T.-G., Adamu, S., Subbarao, K.V., Chen, J.-Y., Dai, X.-F. 2020. Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae. Molecular Plant Pathology. 21(5):667–685. https://doi.org/10.1111/mpp.12921.
DOI: https://doi.org/10.1111/mpp.12921

Interpretive Summary: The fungus Verticillium dahliae is a plant pathogen that causes the disease known as Verticillium wilt. Because V. dahliae is a broad host range pathogen that affects numerous important crops, discovering and understanding which molecules that the pathogen uses to breech plant defenses is critical to develop new means to control this pathogen. Once plants are infected with V. dahliae there are no successful control measures. In this research, we analyzed the roles of 123 genes from V. dahliae encoding cysteine-rich molecules that are thought to have roles in virulence based on homology to known proteins from related pathogens. Expression analyses of these 123 genes revealed that three of these genes, when expressed in a plant, caused cell death and reactions that are plant immunity specific, suggesting a role of these three proteins encoded by these genes in inciting defense responses against this pathogen. However, the pathogen uses a fourth, cysteine-rich protein discovered previously to suppress immunity that is triggered by the proteins, and this suppression promotes plant colonization by the pathogen.

Technical Abstract: Secreted small cysteine-rich proteins (SCPs) play a critical role in modulating host immunity in plant–pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host–pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP-encoding genes in Nicotiana benthamiana identified three candidate genes involved in host–pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor-like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana . Site-directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27 , VdSCP113 , and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum , although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27 /VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae– plant interaction via an intrinsic virulence function and suppress immunity following infection.