Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

Sidhu, Yaadwinder; Cairns, Timothy C.; Chaudhari, Yogesh; Usher, Jane; Talbot, Nicholas J.; Studholme, David J.; Csukai, Michael; Haynes, Ken

doi:10.1016/j.fgb.2015.04.015

Cited by 22 publications

(21 citation statements)

References 39 publications

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“…DNA assemblies were conducted with the In-Fusion HD Cloning Kit (Takara BIO) following the manufacturer’s instructions. To increase the homologous recombination efficiency, we first inactivated the ZtKu70 ( Mycgr3G85040 or Zt09_3_00215 ) gene in the1E4 strain using the plasmid pGEN-YR- ΔZtKu70 (Sidhu et al, 2015), containing a geneticin resistance gene cassette (also known as G418), as a selectable marker. To disrupt the Z. tritici so gene ( ZtSo ), 1 Kb size of both flanking regions were amplified from the 1E4 genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

The role of vegetative cell fusions in the lifestyle of the wheat fungal pathogenZymoseptoria tritici

Francisco¹,

Zwyssig²,

Palma‐Guerrero³

2020

Preprint

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26The ability of fungal cells to undergo cell fusion allows them to maximize their overall 27 fitness. In this study, we characterized the role of the so gene orthologous in 28Zymoseptoria tritici and the biological contribution of vegetative cell fusions in the 29 lifestyle of this latent necrotrophic fungus. Firstly, we show that Z. tritici undergoes 30 self-fusion between distinct cellular structures and its mechanism is dependent on the 31 initial cell density. Next, the deletion of ZtSo resulted in the loss of cell-to-cell 32 communication affecting both hyphal and germlings fusion. We show that Z. tritici 33 mutants for MAP kinase-encoding ZtSlt2 (orthologous MAK-1) and ZtFus3 34 (orthologous MAK-2) genes also fail to undergo self-stimulation and self-fusion, 35demonstrating the functional conservation of this signaling mechanism across 36 species. Additionally, the DZtSo mutant was severely impaired in melanization, which 37 leads us to identify a trade-off between fungal growth and melanization. Though it has 38 been proposed that So is a scaffold protein for MAP kinase genes from the CWI 39 pathway, its deletion did not affect the cell wall integrity of the fungus. Finally, we 40 demonstrated that anastomose is dispensable for pathogenicity, but essential for the 41 fruiting body development and its absence abolish the asexual reproduction of Z. tritici. 42Taken together, our data show that ZtSo is required for fungal development, while 43 vegetative cell fusions are essential for fungal fitness. 44 45 46 47 48 49 50

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Section: Methodsmentioning

confidence: 99%

The role of vegetative cell fusions in the lifestyle of the wheat fungal pathogenZymoseptoria tritici

Francisco¹,

Zwyssig²,

Palma‐Guerrero³

2020

Preprint

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“…Z. tritici strain IPO323 (Kema and van Silfhout, 1997) was used as wild type (WT). CPR constructs were transformed into Z. tritici strain HLS1000 (IPO323 Dku70::G418 R ) which offers increased frequency of homologous recombination due to inactivation of KU70 (Sidhu et al, 2015a). Z. tritici strains were grown at either 18°C or 25°C for 3-6 days on Yeast Peptone Dextrose (YPD, Bowler et al, 2010), Potato Dextrose (PD, Acumedia-Neogen, Lansing, Michigan, USA) and Minimal Medium (MM-Zt + NO 3 , Supplementary Table 1).…”

Section: Culture Media and Strainsmentioning

confidence: 99%

“…EGFP ORF was transferred from pEntry-EGFP into pYSKH27 (Sidhu et al, 2015b) using the Gateway Ò LR reaction to give pYSKH51. For promoter replacement based on Z. tritici NIA1 (CPR), a 1092 bp region upstream of the NIA1 start codon (chromosome 10: 589,688), was amplified using primers pZtNiA FWD/REV (Supplementary Table 2) and introduced into pC-HYG-YR (Sidhu et al, 2015a) digested with EcoRI and BamHI, using homologous recombination in S. cerevisiae (Collopy et al, 2010) to construct pC-HYG-pZtNIA1-YR. To replace the first 500 bp of the Z. tritici BGS1 promoter upstream of its start codon (chromosome 11: 1,342,357), we constructed pYSKH101 as follows. A 1200 bp left flank (LF) region (chromosome 11:1,340,656-1,341,856) upstream of the BGS1 promoter was amplified using primers ZtBGS1-LF-F/R (Supplementary Table 2).…”

Section: Construction Of Atmt Binary Vectorsmentioning

confidence: 99%

Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters

Marchegiani¹,

Sidhu²,

Haynes³

et al. 2015

Fungal Genetics and Biology

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“…Recently, there has been a community-wide effort to develop numerous tools, techniques, and resources for Z. tritici . This research toolkit includes mutants in the non-homologous end joining pathway for highly efficient gene targeting (41), optimization of conditional expression systems (42), a range of fluorescent translational gene fusion for sub-cellular localization studies (43, 44), optimized virulence assays (45), and a suite of Gateway ® Destination vectors (46, 47). These Gateway ® destination vectors have been validated using a pilot Gateway ® Entry library to generate 32 over-expression mutants, demonstrating the role of a fungal specific transcription factor for in vitro hyphal growth (48).…”

Section: Introductionmentioning

confidence: 99%

The Zymoseptoria tritici ORFeome: a functional genomics community resource

Chaudhari

Cairns

Sidhu

et al. 2019

Preprint

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Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.

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Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

Cited by 22 publications

References 39 publications

The role of vegetative cell fusions in the lifestyle of the wheat fungal pathogenZymoseptoria tritici

The role of vegetative cell fusions in the lifestyle of the wheat fungal pathogenZymoseptoria tritici

Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters

The Zymoseptoria tritici ORFeome: a functional genomics community resource

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