|
|
|
| Cloning and Functional Analysis of VdKeR in Verticillium dahliae |
| CHEN Rui, LI Biao, WANG Yuan, HUANG Jia-Feng* |
| College of Agriculture/Key Laboratory of Oasis Agricultural Pest Management and Plant Protection Resources Utilization, Xinjiang Uygur Autonomous Region, Shihezi University, Shihezi 832003, China |
|
|
|
|
Abstract Cotton verticillium wilt caused by Verticillium dahliae is an important soil-borne fungal vascular disease threatening cotton production in China. In previous studies, the transcriptional expression of a gene encoding hypothetical protein in the virulent defoliating V. dahliae V592 strain isolated from cotton (Gossypium hirsutum) was dramatically down-regulated when V592 strain was cultured with cotton roots. To determine the function of the gene, the full length of the gene was cloned from the genome of V592 strain. The target gene knockout plasmid was constructed based on homologous recombination, and its complementation plasmid containing the full length of the target gene encoding region was also constructed, then to generate the target knockout mutant and its complementary strain by Agrobacterium-mediated transformation. The growth phenotype characteristics and pathogenicity to cotton of the gene knockout mutant were assayed. The transcriptional expression of 4 genes involved in virulence in the target gene knockout mutants were measured by reverse transcription qPCR (RT-qPCR). The results showed that the target gene full length was determined to be 1 439 bp and the deduced protein contained 6 tandem Kelch motif and a galactose oxidase domains, and Kelch repeats formed a 6-bladed propeller construction. Thus, the gene was designated as VdKeR (GenBank No. MZ855770) due to the encoding protein containing Kelch repeats. On potato dextrose agar (PDA) media, VdKeR knockout mutant produced more aerial hyphae on the edge of the colony than the wild-type strain V592 and complementary strain, but the colony growth rate of VdKeR knockout mutant exhibited obviously lower than those of V592 strain and complementary strain, indicating that VdKeR gene knockout resulted in increased formation of aerial hyphae and slower growth rate in V. dahliae. VdKeR knockout mutant produced significantly less conidia than those of V592 strain and the complementary strain, suggesting that VdKeR gene knockout affected the conidiation in V. dahliae. In cotton seedling, VdKeR knockout mutant displayed significantly reduced disease severity compared with V592 strain and complementary strain, showing that VdKeR gene knockout led to reduced virulence to cotton. In VdKeR deletion mutants, the other 4 genes involved in virulence including the C subunit gene of cAMP-dependent protein kinase A (VdPKAC1), the G protein β subunit gene (VGB), the necrosis-and ethylene-inducing-proteins (Nep1)-like protein (NLP) family genes VdNLP1 and VdNLP2, the transcriptional expression of which were strongly down-regulated. These results indicated that VdKeR gene played an important role in the growth and pathogenicity and affected the transcriptional expression of other genes involved in virulence in V. dahliae. The results provide basic data for further study on the pathogenic mechanism of Verticillium dahliae.
|
|
Received: 01 February 2021
|
|
|
|
Corresponding Authors:
* jiafeng_huang@163.com
|
|
|
|
[1] 高云, 孟佩, 张婷, 等. 2015. 棉花黄萎病菌VdSge1基因的敲除与功能分析[J]. 棉花学报, 27(04): 346-353.
(Gao Y, Meng P, Zhang T, et al. 2015. Knockout and functional analysis of VdSge1 in Verticillium dahliae[J]. Cotton Science, 27(4): 346-353.
[2] 宋雯, 王春巧, 俞燕, 等. 2019. 棉花黄萎病菌鸟氨酸脱羧酶抗酶蛋白基因VdOAZ的功能分析[J]. 棉花学报, 31(02): 101-113.
(Song W, Wang C Q, Yu Y, et al. 2019.Functional analysis of an ornithine decarboxylase antizyme gene VdOAZ in Verticillium dahliae isolated from cotton[J]. Cotton Science, 31(2): 101-113.
[3] 肖枫, 宋宏涛, 魏群. 2011. 哺乳动物中kelch超家族蛋白的结构与功能[J]. 生物化学与生物物理进展, 38(03): 210-217.
(Xiao F, Song H T, Wei Q. 2011. Structure and function of Kelch superfamily proteins in mammals[J]. Advances in Biochemistry and Biophysics, 38(03): 210-217.)
[4] Adams J, Kelso R, Cooley L.2000. The kelch repeat superfamily of proteins: Propellers of cell function[J]. Trends in Cell Biology, 10(1): 17-24.
[5] Budhwar R, Fang G Q, Hirsch J P.2011. Kelch repeat proteins control yeast PKA activity in response to nutrient availability[J]. Cell Cycle, 10(5): 767-770
[6] Curtis R H C, Singh P, Powers S J.2013. The Arabidopsis F-box/Kelch-repeat protein At2g44130 is upregulated in giant cells and promotes nematode susceptibility[J]. Molecular Plant Microbe Interaction, 26(1): 36-43
[7] Gao F, Zhou B J, Li G Y, et al.2010. A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity[J]. PLOS ONE, 5(12): e15319.
[8] Gould C J, Chesarone-Cataldo M, Alioto S L, et al.2014. Scerevisiae Kelch proteins and Bud14 form stable 520 kDa formin-regulatory complex that controls actin cable assembly and cell morphogenesis[J]. Journal of Biological Chemistry, 289(26): 18290-18301.
[9] Gupta V A, Beggs A H.2014. Kelch proteins: Emerging roles in skeletal muscle development and diseases[J]. Skeletal Muscle, 4(1): 11.
[10] Hoppenau C E, Tran V, Kusch H, et al.2014. Verticillium dahliae VdTHI4, involved in thiazole biosynthesis, stress response and DNA repair functions, is required for vascular disease induction in tomato[J]. Environmental and Experimental Botany, 108(1): 14-22.
[11] Imaizumi T, Schultz T F, Harmon F Q, et al.2005. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J]. Science, 309(5032): 293-297
[12] Imaizumi T, Tran H G, Swartz T E, et al.2003. FKF1 is essential for photoperiodic-specific light signaling in Arabidopsis[J]. Nature, 426(6964): 302-306
[13] Ito N, Keen J N, Knowles P F, et al.1990. Structural analysis of galactose oxidase[J]. Biochemical Society Transactions, 18(5): 931.
[14] Kombrink A, Rovenich H, Shi-Kunne X, et al.2017. Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts[J]. Molecular Plant Pathology, 18(4): 596-608.
[15] Liu T L, Song T Q, Zhang X, et al.2014. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis[J]. Nature Communications, 5: 4686.
[16] Luo X M, Tian T T, Tan X, et al.2020. VdNPS, a nonribosomal peptide synthetase, is involved in regulating virulence in Verticillium dahliae[J]. Phytopathology, 110(8): 1398-1409.
[17] Prag S, Adams J C.2003. Molecular phylogeny of the kelchrepeat superfamily reveals an expansion of BTB/kelch proteins in animals[J]. BMC Bioinformatics, 4(1): 1-20.
[18] Qi X L, Su X F, Guo H M, et al.2016. VdThit, a thiamine transport protein, is required for pathogenicity of the vascular pathogen Verticillium dahliae[J]. Molecular Plant-Microbe Interactions, 29(7): 545-559.
[19] Qin J, Wang K L, Sun L F, et al.2018. The plant-specific transcription factors CBP60g and SARD1 are targeted by a Verticillium secretory protein VdSCP41 to modulate immunity[J]. eLife, 7: e34902.
[20] Rauyaree P, Ospina-Giraldo M D, Kang S, et al.2005. Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae[J]. Current Genetics, 48(2): 109-116.
[21] Santhanam P, Thomma B P H J.2013. Verticillium dahliae Sge1 differentially regulates expression of candidate effector genes[J]. Molecular Plant-Microbe Interactions, 26(2): 249-256
[22] Santhanam P, van Esse H P, Albert I, et al.2013. Evidence for functional diversification within a fungal NEP1-like protein family[J]. Molecular Plant-Microbe Interactions, 26(3): 278-286.
[23] Shi X, Xiang S, Cao J, et al.2019. Kelch-like proteins: Physiological functions and relationships with diseases[J]. Pharmacological Research, 148: 104404.
[24] Tad W, Stephanie K, Flora B.2018. UmTea1, a Kelch and BAR domain-containing protein, acts at the cell cortex to regulate cell morphogenesis in the dimorphic fungus Ustilago maydis[J]. Fungal Genetics and Biology, 121(12): 10-28.
[25] Takeshita N, Higashitsuji Y, Konzack S, et al.2008. Apical sterolrich membranes are essential for localizing cell end markers that determine growth directionality in the filamentous fungus Aspergillus nidulans[J]. Molecular Biology of the Cell, 19(1): 339-351.
[26] Tzima A K, Paplomatas E J, Rauyaree P, et al.2010. Roles of the catalytic subunit of cAMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae[J]. Fungal Genetics and Biology, 47(5): 406-415.
[27] Tzima A K, Paplomatas E J, Tsitsigiannis D I, et al.2012. The G protein β subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae[J]. Fungal Genetics and Biology, 49(4): 271-283.
[28] Wang S, Xing H Y, Hua C L, et al.2016. An improved singlestep cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT) -based genedisruption method for Verticillium dahliae[J]. Phytopathology, 106(6): 645-652.
[29] Wang Y L, Deng C L, Tian L Y, et al.2018. The transcription factor VdHapX controls iron homeostasis and is crucial for virulence in the vascular pathogen Verticillium dahliae[J]. mSphere, 3(5): e400-e418.
[30] Wang Y L, Tian L Y, Xiong D G, et al.2016. The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae[J]. Fungal Genetics and Biology, 88(3): 13-23.
[31] Xiong D G, Wang Y L, Tang C, et al.2015. VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae[J]. Fungal Genetics and Biology, 82(9): 201-212.
[32] Yu J, Li T Y, Tian L Y, et al.2019. Two Verticillium dahliae MAPKKKs, VdSsk2 and VdSte11, have distinct roles in pathogenicity, microsclerotial formation, and stress adaptation[J]. mSphere, 4(4): e00426.
[33] Zhang J, Cui W Y, Abdul Haseeb H, et al.2020. VdNop12, containing two tandem RNA recognition motif domains, is a crucial factor for pathogenicity and cold adaption in Verticillium dahliae[J]. Environmental Microbiology, 22(12): 5387-5401.
[34] Zhang T, Zhang B S, Hua C L, et al.2017. VdPKS1 is required for melanin formation and virulence in a cotton wilt pathogen Verticillium dahliae[J]. Science China. Life Sciences, 60(8): 868-879.
[35] Zhang X, Abrahan C, Colquhoun T A, et al.2017. A proteolytic regulator controlling chalcone synthase stability and flavonoid biosynthesis in Arabidopsis[J]. Plant Cell, 29(5):1157-1174.
[36] Zhao Y L, Zhou T T, Guo H S.2016. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by Verticillium dahliae[J].PLOS Pathogens, 12(7): e1005793.
[37] Zhou B J, Jia P S, Gao F, et al.2012. Molecular characterization and functional analysis of a necrosis- and ethyleneinducing, protein-encoding gene family from Verticillium dahliae[J]. Molecular Plant-Microbe Interactions®, 25(7): 964-975. |
|
|
|
