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Current Genetics

, Volume 65, Issue 2, pp 591–605 | Cite as

Involvement of the two l-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum

  • Wenchan Chen
  • Lingling Wei
  • Yu Zhang
  • Dongya Shi
  • Weichao Ren
  • Zhihui Zhang
  • Jin Wang
  • Wenyong Shao
  • Xiali Liu
  • Changjun ChenEmail author
  • Qingli GaoEmail author
Original Article
  • 285 Downloads

Abstract

Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD+. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, l-lactate biosynthesis and activity. Additionally, the two l-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of d-/l-lactate dehydrogenase in phytopathogenic fungi.

Keywords

Fusarium graminearum l-Lactate dehydrogenase Development differentiation Deoxynivalenol Pathogenicity 

Notes

Acknowledgements

This research was supported by the National Science Foundation of China (31672065) and Jiangsu Provincial Agricultural Plans [BA2018039, BE2018378 and SXGC (2016) 154].

Supplementary material

294_2018_909_MOESM1_ESM.tif (1.9 mb)
Fig. S1 Generation and identification of FgldhL1 gene deletion mutants. (A) Gene replacement strategy for the FgldhL1 gene. The gene replacement cassette HPH contains the hygromycin resistance gene. Primer binding sites are indicated by arrows (see Table S1 for the primer sequences). (B) PCR strategy to screen ΔFgldhL1 transformants was followed. a) A fragment in size of 2.973-kb amplified by primer pair F9/F10 indicates ΔFgldhL1 integration at the left junction. b) A 2.903-kb fragment amplified primer pair F11/F12 indicates ΔFgldhL1 integration at the right junction. c) A 0.807-kb fragment amplified with primer pair F7/F8 indicates a wild-type gene locus. (C) Southern blot hybridization analysis for the wild-type strain PH-1, ΔFgldhL1, and ΔFgldhL1-C. A 740-bp homologous target gene fragment was used as a probe, and genomic DNA was digested with NcoΙ (TIF 1967 KB)
294_2018_909_MOESM2_ESM.tif (407 kb)
Fig. S2 Generation and identification of FgldhL2 gene deletion mutants. (A) Gene replacement strategy for the FgldhL2 gene. The gene replacement cassette HPH contains the hygromycin resistance gene. Primer binding sites are indicated by arrows (see Table S1 for the primer sequences). (B) PCR strategy to screen ΔFgldhL2 transformants: a) A fragment in size of 2.93-kb amplified by primer pair P7/P8 indicates ΔFgldhL2 integration at the left junction. b) A fragment in size of 2.845-kb amplified by primer pair P9/P10 indicates ΔFgldhL2 integration at the right junction. c) A 0.778-kb fragment amplified with primer pair P5/P6 indicates indicates a wild-type gene locus. (C) Southern blot hybridization analysis for the wild-type strain PH-1, ΔFgldhL2, and ΔFgldhL2-C. A 684-bp homologous target gene fragment was used as a probe, and genomic DNA was digested with NcoΙ (TIF 406 KB)
294_2018_909_MOESM3_ESM.tif (8.8 mb)
Fig. S3 Determination of defective phenotypes of the wild-type strain, deleted- and complemented- mutants in response to the stresses. Cultures of the wild-type strain PH-1, the deletion mutants (ΔFgldhL1 and ΔFgldhL2), and the complemented strains (ΔFgldhL1-C and ΔFgldhL2-C) on PDA with or without 1.2 M NaCl or KCl, 0.05 % SDS, 0.05 % Congo red or 24 mM H2O2. Cultures were photographed after 3 days at 25°C (TIF 8996 KB)
294_2018_909_MOESM4_ESM.tif (1.1 mb)
Fig. S4 Western blot assay of FgldhL1-GFP and FgldhL1-GFP. Mycelial cultures of FgldhL1-GFP and FgldhL2-GFP were recovered from two-day-old liquid CM medium and transferred to liquid MM with either glucose or L-lactate as the sole carbon source, or fresh CM medium for another two-day shaking incubation. Mycelia were then collected, and mycelial extracts were analyzed by immunoblot. Actin was used as an internal reference protein (TIF 1115 KB)
294_2018_909_MOESM5_ESM.tif (226 kb)
Fig. S5 Effect of FgldhL1 and FgldhL2 on the L-Lactate dehydrogenase activity of F. graminearum. Relative expression of FgldhL3(FGSG_10444), FgldhL4(FGSG_05238), FgldhL5(FGSG_04988) and FgldhL6 (FGSG_10227) in wild-type strain and deletion mutants was assessed after 3 days in CM liquid medium. Bars denoted standard errors from three experiments. Values on bars followed by the same letter for each treatment were not significantly different at p= 0.05 (TIF 226 KB)
294_2018_909_MOESM6_ESM.docx (25 kb)
Supplementary material 6 (DOCX 25 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Plant ProtectionNanjing Agricultural UniversityNanjingChina
  2. 2.Plant Protection Station of Pizhou CityPizhouChina
  3. 3.Food Inspection and Testing Institute of Henan ProvinceZhengzhouChina

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