Differential roles of three FgPLD genes in regulating development and pathogenicity in Fusarium graminearum

doi:10.1016/j.fgb.2017.10.007

Fungal Genetics and Biology

Volume 109, December 2017, Pages 46-52

https://doi.org/10.1016/j.fgb.2017.10.007 Get rights and content

Highlights

•
Deletion of FgPLD1, but not FgPLD2 or FgPLD3, affected hyphal growth, conidiation, and perithecium formation.
•
The Δpld1 mutant exhibited reduced deoxynivalenol (DON) production and virulence in plant infection.
•
Three FgPLD proteins have the same subcellular localization.

Abstract

Phospholipase D (PLD) is an important phospholipid hydrolase that plays critical roles in various biological processes in eukaryotic cells. However, little is known about its functions in plant pathogenic fungi. In this study, we identified three FgPLD genes in Fusarium graminearum that are homologous to the Saccharomyces cerevisiae Spo14 gene. We constructed deletion mutants of all three FgPLD genes using homologous recombination. Deletion of FgPLD1 (Δpld1), but not FgPLD2 or FgPLD3, affected hyphal growth, conidiation, and perithecium formation. The Δpld1 mutant showed reduced deoxynivalenol (DON) production and virulence in flowering wheat heads and corn silks. Furthermore, three FgPLD proteins have the same subcellular localization and localize to the cytoplasm in F. graminearum.

Taken together, these results indicate that FgPLD1, but not FgPLD2 or FgPLD3, is important for hyphal growth, sexual or asexual reproduction, and plant infection.

Introduction

Fusarium head blight (FHB), caused by Fusarium graminearum (teleomorph Gibberella zeae), is an economically devastating disease of wheat and barley (Goswami and Kistler, 2004, Mclain and Dolan, 1997) in many parts of the world, including China (Zhang et al., 2011). Infection of cereal crops by F. graminearum can cause severe yield losses during epidemic years (Dean et al., 2012, Goswami and Kistler, 2004). This fungus produces trichothecene mycotoxins that are harmful to human and animal health (Dean et al., 2012, Mcmullen et al., 2007, Pestka and Smolinski, 2005). Deoxynivalenol (DON) is one of the most abundant trichothecenes produced by the fungus and is also a virulence factor during wheat infection (Desjardins et al., 1993, Proctor et al., 1994).

Phospholipases constitute a heterogeneous group of enzymes including phospholipases A (PLA), B (PLB), C (PLC), and D (PLD), which are distinguished by the site of phospholipid cleavage. PLD hydrolyzes the phosphodiester bond in the phospholipid backbone to yield phosphatidic acid (PA) and a free headgroup (Cummings et al., 2002, Mcdermott et al., 2004). PLD families typically have a highly conserved hallmark, the catalytic H_xK_(x)4D (HKD) motif, which is involved in catalysis (Jenkins and Frohman, 2005).

It has been reported that PLD is involved in many cellular functions in fungi. In Saccharomyces cerevisiae, Spo14/PLD1 has been demonstrated to mediate vesicle fusion, sporulation, and meiosis (Nakanishi et al., 2006, Rose et al., 1995, Waksman et al., 1996). In Candida albicans, CaPLD1 functions as the switch from axial-yeast growth to polarized growth and is required for full virulence (Dolan et al., 2004, Mclain and Dolan, 1997). In Aspergillus nidulans, three PLD isoforms have been identified. The pldA deletion mutant (ΔpldA) did not cause any apparent growth defects, but Ca²⁺-dependent PLD activity against phosphatidylethanolamine was significantly reduced in the mutant (Hong et al., 2003). Another PLD deletion mutant did not alter hyphal growth rate and conidial morphological characteristics, whereas it attenuated the virulence of A. fumigatus in mice (Li et al., 2012). Similar results were also obtained in Purpureocillium lilacinum in which deletion of the PLD gene significantly reduced virulence in Meloidogyne incognita and had no significant effect on hyphal growth, conidium morphology, or spore germination (Yang et al., 2015). However, the roles of PLD in plant pathogenic fungi have not been studied.

In this study, we identified and functionally characterized three FgPLD proteins in F. graminearum. Analysis of FgPLD sequences revealed that FgPLD1 possesses two HKD domains and a phox homology (PX) domain, whereas the other two FgPLD proteins have only HKD domains and lack a PX domain which presents in FgPLD1. The FgPLD1 deletion mutant (Δpld1), but not deletions of the other two FgPLD genes, showed significant defects in hyphal growth, asexual and sexual reproduction, pathogenicity, and DON production in F. graminearum. Overall, we provided direct evidence of differential roles of the three FgPLD genes in F. graminearum.

Section snippets

Fungal strains and growth conditions

Fusarium graminearum strain SF01-3 (Zhang et al., 2013) was used as the wild-type (WT) strain in this study. All strains were grown on complete medium (CM) at 25 °C for mycelial growth assays. Mycelia were harvested from liquid potato dextrose broth (PDB) and used for genomic DNA extraction. Transformants were selected on TB₃ (3 g yeast extract, 3 g casamino acids, 200 g sucrose, 9 g agar in 1000 mL distilled H₂O) medium (Hou et al., 2002).

Generation of deletion mutants

A split-marker approach (Catlett et al., 2003) was used to

The F. Graminearum genome contains three FgPLD genes

Three putative FgPLD genes, FgPLD1 (FGSG_09917), FgPLD2 (FGSG_01973), and FgPLD3 (FGSG_06175), were identified through a BLASTp search using the S. cerevisiae Spo14 sequence as a query in the F. graminearum genome database (http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiHome.html). Three FgPLD genes are predicted to encode proteins of 1824, 836, and 999 amino acids, respectively. SMART-PFAM analysis revealed that all of these FgPLD proteins contain two conserved HKD

Discussion

In this study, analysis of the F. graminearum genome database revealed the presence of three FgPLD genes, which we identified and analyzed functionally. Among the three FgPLD proteins, FgPLD1 has a PX domain (as well as two HKD domains), whereas the other two FgPLD proteins only have HKD domains and lack a PX domain. Deletion of three FgPLD genes revealed that these genes have different roles. Interestingly, only FgPLD1 is responsible for hyphal growth, conidiation, and virulence. In addition, Δ

Acknowledgments

We thank Drs. Zhonghua Ma and Yun Chen (Zhejiang University, China) for providing plasmids and yeast strain. This work was supported by the National Natural Science Foundation of China (31171806), the Natural Science Foundation of Shandong Province (ZR2017MC020), the Wheat Innovation Team of Shandong Province Modern Agricultural Industry Technology System (SDAIT-01-09), and Funds of Shandong “Double Tops” Program (SYL2017XTTD11).

References (36)

W.C. Colley
Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization
Curr. Biol.
(1997)
S. Hong
Molecular cloning of a phospholipase D gene from Aspergillus nidulans and characterization of its deletion mutants
FEMS Microbiol. Lett.
(2003)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT Method
Methods
(2001)
T.C. Sung
Molecular analysis of mammalian phospholipase D2
J. Biol. Chem.
(1999)
T.C. Sung
Structural analysis of human phospholipase D1
J. Biol. Chem.
(1999)
M. Waksman
Identification and characterization of a gene encoding phospholipase D activity in yeast
J. Biol. Chem.
(1996)
F. Yang
The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum)
Microb. Pathog.
(2015)
J.W. Yu et al.
All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate
J. Biol. Chem.
(2001)
K.S. Bruno
Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea
Eukaryot Cell.
(2004)
N.L. Catlett
Split-marker recombination for efficient targeted deletion of fungal genes
Fungal Genet. Newsl.
(2003)

B. Cavinder

Sexual development and ascospore discharge in Fusarium graminearum

J. Vis. Exp.

(2012)

R. Cummings

Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung

Mol. Cell. Biochem.

(2002)

R. Dean

The top 10 fungal pathogens in molecular plant pathology

Mol. Plant Pathol.

(2012)

A.E. Desiardins

Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeae in wheat field tests

Mol. Plant-Microbe Interact.

(1995)

A.E. Desjardins

Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance

Microbiol. Rev.

(1993)

J.W. Dolan

Candida albicans PLD1 activity is required for full virulence

Med. Mycol.

(2004)

R.S. Goswami et al.

Heading for disaster: Fusarium graminearum on cereal crops

Mol. Plant Pathol.

(2004)

R.S. Goswami et al.

Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex on wheat and rice

Phytopathology

(2005)

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Phosphatidylserine decarboxylases (Psds) are enzymes regulating phosphatidylethanolamine biosynthesis in prokaryotes and eukaryotes, and have the central role in lipid metabolism. To date, the functions of Psds in plant pathogenic fungi are not fully understood. In this study, we have characterized two yeast Psd orthologues: FgPsd1 and FgPsd2, in Fusarium graminearum. Our results indicate that FgPsd1 and FgPsd2 are localized in mitochondria and Golgi, respectively. In addition, we have determined that FgPsd1 is a lethal gene and deletion of FgPsd2 resulted in a significant reduction of mycelial growth and conidiation. Futhermore, the FgPsd2 deletion mutant (ΔFgPsd2) is defective in ascospore production and virulence in wheat. Our study has also found that the ΔFgPsd2 mutant is more sensitive to osmotic and oxygen stresses. Moreover, deletion of FgPsd2 reduced the formation of lipid droplets and aggravated autophagy in F. graminearum. In summary, our findings indicate that FgPsd2 is important for mycelial growth, sexual and asexual reproduction, virulence, lipid droplet formation and autophagy in F. graminearum.
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This pathogen not only causes serious loss of wheat yields and reduces the quality of wheat (Dean et al., 2012) but also produces many kinds of mycotoxins, such as deoxynivalenol (DON), that endanger the health of humans and animals (Desjardins and Proctor, 2007). DON is synthesized by enzymes encoded by trichothecene biosynthesis TRI genes that include TRI1, TRI4, TRI5, TRI6, TRI10 and other TRI genes (Brown et al., 2004; Ding et al., 2017; Chen et al., 2019). In the process of plant infection, the mRNA expression levels of the TRI genes increase, resulting in high levels of DON (Ding et al., 2017; Tang et al., 2018; Flynn et al., 2019).
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¹: These authors contributed equally to this work.

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Regular ArticlesDifferential roles of three FgPLD genes in regulating development and pathogenicity in Fusarium graminearum

Highlights

Abstract

Introduction

Section snippets

Fungal strains and growth conditions

Generation of deletion mutants

The F. Graminearum genome contains three FgPLD genes

Discussion

Acknowledgments

Curr. Biol.

FEMS Microbiol. Lett.

Methods

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Microb. Pathog.

J. Biol. Chem.

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea

Eukaryot Cell.

Split-marker recombination for efficient targeted deletion of fungal genes

Fungal Genet. Newsl.

Sexual development and ascospore discharge in Fusarium graminearum

J. Vis. Exp.

Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung

Mol. Cell. Biochem.

The top 10 fungal pathogens in molecular plant pathology

Mol. Plant Pathol.

Reduced virulence of trichothecene-nonproducing mutants of Gibberella zeae in wheat field tests

Mol. Plant-Microbe Interact.

Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance

Microbiol. Rev.

Candida albicans PLD1 activity is required for full virulence

Med. Mycol.

Heading for disaster: Fusarium graminearum on cereal crops

Mol. Plant Pathol.

Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex on wheat and rice

Phytopathology

Regular Articles
Differential roles of three FgPLD genes in regulating development and pathogenicity in Fusarium graminearum