Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity
Abstract
:1. Introduction
2. Results
2.1. Deletion and Complementation of VdTHI20 in V. dahliae
2.2. Radial Mycelial Growth in ∆VdTHI20 Mutants Was Significantly Reduced
2.3. Exogenous Thiamine Recovered the Hyphal Growth of ΔVdTHI20 Mutants
2.4. Knockout of VdTHI20 Reduced Conidial Germination and Production and Caused Abnormal V. dahliae Hyphal Morphology
2.5. Loss of VdTHI20 Resulted in Reduced Tolerance to UV Damage
2.6. VdTHI20 Is Crucial for the Pathogenicity of V. dahliae towards Plants
2.7. Fungal Colonization and Root Infection with the ΔVdTHI20 Mutant Were Impaired
2.8. DsRNA of VdTHI20 Confers Resistance Against V. dahliae in Transgenic N. benthamiana Lines
3. Discussion
4. Materials and Methods
4.1. Fungal Strains, Plant Material and Culture Conditions
4.2. Plasmid Construction and Fungal Transformation
4.3. Confirmation of VdTHI20 Gene Disruption or the Complementation of ΔVdTHI20 and Screening for GFP-tagged Strains
4.4. Growth, Conidia Production, and Germination Assays
4.5. Phenotypic Analysis under Stress Conditions
4.6. Quantitative Real-Time PCR (qRT-PCR)
4.7. Hyphal Growth under Different Concentrations of Exogenous Thiamine
4.8. Microscopic Observation of Initial Infection
4.9. Plasmid Construction and Plant Transformation
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ATMT | Agrobacterium tumefaciens-mediated transformation |
GFP | green fluorescent protein |
RNAi | RNA interference |
HIGS | host induced gene scilencing |
dpi | days post-inoculation |
MM | minimal medium |
wt | wild type |
HMP | 2-methyl-4-amino-5-hydroxymethylpyrimidine |
HET | 4-methyl-5-β-hydroxyethylthiazole |
TPP | phosphorylated thiamine |
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Gene | Protein Function | References |
---|---|---|
THI4 | HET-P synthesis; Stress response and DNA repair | [14,19] |
THI5/THI11/THI12/THI13 | HMP-P synthesis | [10] |
THI6 | Thiamin-phosphate pyrophosphorylase and hydroxyethylthiazole kinase | [15] |
THI10 | Thiamine transport protein | [17] |
THI20 | HMP-PP synthesis and thiamine degradation | [11,12,21] |
THI21/THI22 | HMP-PP synthesis | [11,12] |
THI80 | Thiamin pyrophosphokinase | [16] |
VdThit | Thiamine transport protein; Vegetable growth, reproduction and invasive hyphal growth | [20] |
PHO3 | Thiamin phosphates hydrolysis | [18] |
Primer Name Sequences (5′→3′) | |
---|---|
Thi20-5F | GTACCCAATTCGAATTC ATTGTGTTTGAGGAGGACACCGAT |
Thi20-5R | CAAGACAGCCCGCAAAC TCTCCTTGAGAAAACGAGTGA |
Thi20-3F | CCCAGAATGCACAGGT TGTTGATGTCGGTGTCATCGTC |
Thi20-3R | GACGGTATCGATAAGCTT TGTTGGAAAAAGGTCAGTCAT |
C-TrpC-F | TTGAAGGAGCATTTTTGGGC |
C-TrpC-R | ATCGATGCTTGGGTAGAATAGGT |
C-VdThi20-F | AAAAGTACTATGGCACAGCAGATGGGCCG |
C-VdThi20-R | AAACTGCAGTCTACTGGCACGGGAACATCT |
C-Nos-F | AGATGCCGACCGGGATCCACTT |
C-Nos-R | TTATCTTTGCGAACCCAGGG |
neo-F | GTTTGCGGGCTGTCTTGACG |
neo-R | TACCTGTGCATTCTGGGTAA |
Thi20-J-F | GCGCAGGACACAAAGGGCGT |
Thi20-J-R | AGCGTGCCGTTGCCGAGACC |
noe-J-F | ATGATTGAACAAGATGGATT |
noe-J-R | TCAGAAGAACTCGTCAAGAA |
Thit-F | CTCGTGACTTTATCGGGTTTCT |
Thit-R | GGCGGATGAGCTGGAATTAT |
VdBt-F | TTCCCCCGTCTCCACTTCTTCATG |
VdBt-R | GACGAGATCGT TCATGTTGAACTC |
Nb-actin-F | GGACCTTTATGGAAACATTGTGCTCAGT |
Nb-actin-R | CCAAGATAGAACCTCCAATCCAGACAC |
Vd-F | CCGCCGGTCCATCAGTCTCTCTGTTTATAC |
Vd-R | CGCCTGCGGGACTCCGATGCGAGCTGTAAC |
TransThi20F | GGGGACAAGTTTGTACAAAAAAGCAGGCTCGCAACCATTGTCAAGCACA |
TransThi20R | GGGGACCACTTTGTACAAGAAAGCTGGGTTTCCAGTACACGTTGCCCTC |
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Qin, T.; Hao, W.; Sun, R.; Li, Y.; Wang, Y.; Wei, C.; Dong, T.; Wu, B.; Dong, N.; Wang, W.; et al. Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity. Int. J. Mol. Sci. 2020, 21, 1378. https://doi.org/10.3390/ijms21041378
Qin T, Hao W, Sun R, Li Y, Wang Y, Wei C, Dong T, Wu B, Dong N, Wang W, et al. Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity. International Journal of Molecular Sciences. 2020; 21(4):1378. https://doi.org/10.3390/ijms21041378
Chicago/Turabian StyleQin, Tengfei, Wei Hao, Runrun Sun, Yuqing Li, Yuanyuan Wang, Chunyan Wei, Tao Dong, Bingjie Wu, Na Dong, Weipeng Wang, and et al. 2020. "Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity" International Journal of Molecular Sciences 21, no. 4: 1378. https://doi.org/10.3390/ijms21041378