Efficient Approaches for Generating GFP Fusion and Epitope-Tagging Constructs in Filamentous Fungi

Zhou, Xiaoying; Li, Guotian; Xu, Jin‐Rong

doi:10.1007/978-1-61779-040-9_15

Cited by 127 publications

(166 citation statements)

References 13 publications

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“…The resulting PCR products were co-transformed into S . cerevisiae strain XK1-25 together with Xho I-digested pFL2 vector [37,38]. PGvm2-GFP and PGvm3-GFP fusion constructs were identified by PCR with primers Gvm2-GFP-CF/CR and Gvm3-GFP-CF/CR (S1 Table) and confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali

et al. 2017

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In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. ‘Fuji’ leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes—important virulence factors in V. mali—are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

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Section: Methodsmentioning

confidence: 99%

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali

et al. 2017

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“…A8. The resulting PCR products were co-transformed with XhoIdigested pFL1 (Zhou et al 2011) into S. cerevisiae strain XK1-25 (Bruno et al 2004). The FgSge1-GFP fusion vector was recovered from yeast transformants and inframe fusion was confirmed by sequencing with primer pair GFP-F1?…”

Section: Generation Of the Fgsge1-gfp Fusion Construct And Transformamentioning

confidence: 99%

The potential protein kinase A (Pka) phosphorylation site is required for the function of FgSge1 in Fusarium graminearum

Zhang

et al. 2015

World J Microbiol Biotechnol

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The new transcription factor Sge1 has garnered much attention in filamentous fungi recently, which highlights its role in pathogenicity, conidiation, and the production of secondary metabolites. In this study, we demonstrated that FgSge1 is localized in the nucleus in Fusarium graminearum using fluorescent protein GFP. Mutants containing a T67A mutation within the potential protein kinase A (Pka) phosphorylation site of FgSge1 exhibited a significant decrease in conidiation and dramatically impaired virulence on both wheat head and non-host tomato. These results indicated that the Pka phosphorylation site is required for the function of FgSge1 in F. graminearum. In addition, we characterized the FgSGE1 deletion mutants and found that the mutants showed increased sensitivity to osmotic stress mediated by NaCl or KCl, and to cell wall damaging agent congo red (CR). Real-time PCR assays revealed increased transcription levels of FgSGE1 with the treatment of NaCl or CR, and decreased FgSGE1 transcription in the FgOS-2 deletion mutant ΔFgOs-2. Based on the transcription levels, it can be concluded that FgSge1 is a downstream target of the mitogen-activated protein kinase FgOs-2.

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“…To generate the P GIL1 - GIL1 -GFP fusion construct, the GIL1 gene with its 1.5-Kb upstream promoter sequence were amplified with primer GIL1/F and GIL1/R (Table S1) and cloned into pFL2 [47] by the yeast in vivo homologous recombination approach [48]. The P RP27 - GIL1 -GFP construct was generated with a similar approach by cloning the PCR product amplified with primers GIL1F/RP27 and GIL1R/RP27 (Table S1) into pFL2.…”

Section: Methodsmentioning

confidence: 99%

A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum

Zhang

et al. 2017

IJMS

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Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1, was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δgil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δgil1 mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δgil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum, and is involved in septation and hyphal branching.

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Efficient Approaches for Generating GFP Fusion and Epitope-Tagging Constructs in Filamentous Fungi

Cited by 127 publications

References 13 publications

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali

The potential protein kinase A (Pka) phosphorylation site is required for the function of FgSge1 in Fusarium graminearum

A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum

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