A suite of Gateway® compatible ternary expression vectors for functional analysis in Zymoseptoria tritici

Sidhu, Yaadwinder; Chaudhari, Yogesh; Usher, Jane; Cairns, Timothy C.; Csukai, Michael; Haynes, Ken

doi:10.1016/j.fgb.2015.03.021

Cited by 14 publications

(8 citation statements)

References 31 publications

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“…For cytological analyses, plasmid pYSKH-4 was introduced into Z. tritici isolates IPO9415 and CFZ008 using Agrobacterium tumefasciens -mediated transformation (ATMT) according to published protocols ( Marchegiani et al, 2015 ). Plasmid pYSKH-4 contains eGFP expressed under the control of a strong constitutive promoter TEF1 and the ILV1-R gene, conferring resistance to sulfonylurea ( Sidhu et al, 2015 ). Transformants resistant to sulfonylurea were purified by monospore isolation, and their GFP fluorescence was assessed as previously described ( Marchegiani et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

et al. 2022

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Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

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Section: Methodsmentioning

confidence: 99%

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

et al. 2022

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“…Additionally, in order to enable c-terminal epitope-tagging of encoded ORFs using a variety of Destination vectors (26), primers were designed to omit the native stop codon. If expression of the encoded ORFs with a 3’ stop codon is desired, we have developed numerous Destination vectors for this purpose (46).…”

Section: Resultsmentioning

confidence: 99%

“…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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“…also included small vectors that had empty T-DNA regions that were amenable to easy manipulation. These have been updated to allow vector construction by means such as yeast recombination in S. cerevisiae [ 14 – 16 ], the Gateway system [ 17 ] or Golden Gate assembly [ 18 ], making vector construction and deployment simple and amenable to high throughput approaches.…”

Section: Agrobacterium Tumefaciens and How It Transforms Spementioning

confidence: 99%

A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi

Idnurm

Bailey

Cairns

et al. 2017

Fungal Biol Biotechnol

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The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.

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A suite of Gateway® compatible ternary expression vectors for functional analysis in Zymoseptoria tritici

Cited by 14 publications

References 31 publications

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

The Zymoseptoria tritici ORFeome: a functional genomics community resource

A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi

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