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A Cdc42 homolog in Colletotrichum gloeosporioides regulates morphological development and is required for ROS-mediated plant infection

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Abstract

The Rho GTPase Cdc42 is conserved in fungi and plays a key role in regulating polarity establishment, morphogenesis and differentiation. In this study, we identified an ortholog of Cdc42, CgCdc42, and functionally characterized it to determine the role of Cdc42 in the development and pathogenicity of Colletotrichum gloeosporioides, a causal agent of poplar anthracnose. Targeted deletion of CgCdc42 resulted in reduced vegetative growth and dramatic morphological defects, including the formation of elongated conidia and abnormally shaped appressoria. Moreover, CgCdc42 deletion mutants were less virulent on poplar leaves than were wild type. Appressoria formed by ΔCgCdc42 mutants were morphologically abnormal and present in lower numbers on poplar leaves than were those formed by wild type. However, an ROS scavenging assay indicated that the ΔCgCdc42 mutants maintained wild type pathogenicity in the absence of ROS despite having fewer appressoria than wild type, suggesting that the ΔCgCdc42 mutants were deficient in their tolerance of ROS. Additionally, we also found that the distribution of ROS was different after the deletion of CgCdc42, the ΔCgCdc42 mutants were hypersensitive to H2O2, and transcriptional analysis revealed that CgCdc42 is involved in the regulation of ROS-related genes. Furthermore, loss of CgCdc42 caused defects in cell wall integrity and an uneven distribution of chitin. These data collectively suggest that CgCdc42 plays an important role in the regulation of vegetative growth, morphological development, cell wall integrity and ROS-mediated plant infection in C. gloeosporioides.

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Acknowledgements

We acknowledge the excellent technical support by YONGLIN WANG and XIN XU, We are grateful to YINGMEI LIANG for her help with some supplemental experiments and linguistic support, we further thank LONGYAN TIAN, PUHUIZHONG HE and XIAOLIN ZHANG for their helpful advice. The research was supported by National Natural Science Foundation of China (31470647), Research Fund for the Doctoral Program of Higher Education of China (20130014110004), and Project of Universities in Beijing Supported by Beijing Government (2050205).

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  1. The Key Laboratory for Silviculture and Conservation of Ministry of Education, College of Forestry, Beijing Forestry University, No.35, Qinghua Eastern Road, Haidian District, Beijing, 100083, China

    Xiaolian Wang, Xin Xu, Yingmei Liang, Yonglin Wang & Chengming Tian

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Fig. S1

. Sequence analysis of CgCdc42 and its homologs from other fungi. a Amino acid sequences of CgCdc42 in C. gloeosporioides and Cdc42 homologs in other fungi were aligned by ClustalX 2.1. Red frame indicate the main effector domain. b Phylogenetic tree of CgCdc42 and its homologs in U. maydis (AAM73880), C. trifolii (AAK31624), M. grisea (AAF73431), S. cerevisiae (KZV09474), A. niger (CAK48851), Glomerella cingulata (AAD00177), and A. gossypii (NP_986573.1). The phylogenetic tree was constructed by MEGA 7.0 with full-length protein sequences and neighbor-joining with 1000 bootstrap replicates. (PPTX 1684 KB)

Fig. S2

. Generation of the CgCdc42 deletion mutants in C. gloeosporioides. The split-marker method was used for the replacement of CgCdc42. A hygromycin cassette (1.4 kb) replaced the fragment of CgCdc42 (1.1 kb). Screening of ΔCgCdc42 mutants with a the primers External-CgCdc42for/External-CgCdc42rev and b the primers Internal-CgCdc42for/Internal- CgCdc42rev. c Southern blot of the wild type strain and three independent ΔCgCdc42 mutants (ΔCgCdc42-51, ΔCgCdc42-92, and ΔCgCdc42-11) in C. gloeosporioides. A 500 bp fragment was amplified from the 5′ flanking sequence of CgCdc42 and used as probe to confirm the deletion of CgCdc42. DNA samples were digested with BamHI. Hybridization revealed a 4.3 kb fragment in the wild type and a 3.4 kb fragment in mutants. d Complementation of CgCdc42 and confirmation of ΔCgCdc42 mutants using PCR. (PPTX 945 KB)

Fig. S3

. The unicellular conidium divides and one of the two cells develops a germ tube before the formation of the appressorium in C. gloeosporioides. Conidia inoculated on onion epidermis (5 hpi) from four strains were stained with CFW for 1 min in dark, the letter S indicates a septum between two cells of the conidia. Bar = 10 μm. (PPTX 2627 KB)

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Wang, X., Xu, X., Liang, Y. et al. A Cdc42 homolog in Colletotrichum gloeosporioides regulates morphological development and is required for ROS-mediated plant infection. Curr Genet 64, 1153–1169 (2018). https://doi.org/10.1007/s00294-018-0833-9

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