A new transformant selection system for the gray mold fungus Botrytis cinerea based on the expression of fenhexamid-insensitive ERG27 variants

Cohrs, Kim Christopher; Burbank, Joachim; Schumacher, Julia

doi:10.1016/j.fgb.2017.02.001

Cited by 7 publications

(9 citation statements)

References 34 publications

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“…To generate targeted B . cinerea insertion mutants, and to compare NHEJ- and HR-editing frequencies, a fenhexamid resistance cassette (Fen R ) [ 33 ] flanked by 1 kb Bos1 sequences was delivered as a repair template (RT) in addition to the Cas9-RNP targeting Bos1 into protoplasts. Transformants were selected for Fen R or Ipr R .…”

Section: Resultsmentioning

confidence: 99%

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CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

et al. 2020

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CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

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Section: Resultsmentioning

confidence: 99%

“…To generate targeted B. cinerea insertion mutants, and to compare NHEJ-and HR-editing frequencies, a fenhexamid resistance cassette (Fen R ) [33] flanked by 1 kb Bos1 sequences was…”

Section: Targeted Cas9-rnp-mediated Editingmentioning

confidence: 99%

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

et al. 2020

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“…To generate targeted B. cinerea insertion mutants, and to compare NHEJ- and HR-editing frequencies, a fenhexamid resistance cassette (Fen R ) [30] flanked by 1 kb Bos1 sequences was delivered as a repair template (RT) in addition to the RNP targeting Bos1 into protoplasts. Transformants were selected for Fen R or Ipr R .…”

Section: Resultsmentioning

confidence: 99%

“…To generate a RT with 1 kb Bos1 homology flanks and a fenhexamid resistance cassette, pBS-KS(-) was digested with EcoRV and combined by Gibson assembly with two adjacent 1 kb Bos1 homology flanks, using primers TL29 pBS_ol_bos 3.REV/ TL30 pBS_ol_bos 3.FOR and TL31 pBS_ol_bos 1.FOR/ TL32 pBS_ol_bos 1.REV, and a fenhexamid resistance cassette amplified from pNDF-OCT [30] with primers TL33 Fen_ol_bos 2.FOR/TL34 Fen_ol_bos 2.REV. From the resulting plasmid (pBS_Bos1_KO_Fen), Bos1 RT with short homology flanks were amplified with the following primers: TL37_Fen_fw/ TL38_Fen_rev (0 bp); TL65_Bos1_Fen30_fw/ TL66_Bos1_Fen30_rev (30 bp), TL67_Bos1_Fen40_fw/ TL68_Bos1_Fen40_rev (40 bp); TL69_Bos1_Fen60_fw/ TL70_Bos1_Fen60_rev (60 bp).…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

Hahn

Leisen

Bietz

et al. 2020

Preprint

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20CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse 21 organisms, enabling targeted genetic changes to be performed with unprecedented 22 37 research in B. cinerea and other fungi. 38 39 40Botrytis cinerea is a plant pathogenic ascomycete which infects more than a thousand species, 41 triggering gray mold disease which is responsible for over a billion dollars of losses in fruits, vegetables 42 and flowers every year [1]. Due to its worldwide occurrence, great economic importance and non-43 specific necrotrophic lifestyle, it has been ranked as the second most important plant pathogenic 44 fungus [2]. Control of gray mold often requires repeated treatments with fungicides, in particular 45 under high humidity conditions, but rapid adaption and resistance development of B. cinerea has 46 dramatically reduced their efficiency worldwide in many cultures, for example in strawberry fields [3]. 47After germination of a conidium on the plant surface, the fungus penetrates and invades the host, 48 rapidly killing plant cells by releasing a complex mixture of cell wall degrading enzymes, phytotoxic 49 metabolites and proteins, and by tissue acidification [4, 5]. How host cell death is induced is not fully 50 understood, but the invading hyphae seem to trigger the hypersensitive response, a plant-specific type 51 of apoptosis linked to strong defence reactions [6, 7]. Furthermore, B. cinerea releases small RNAs 52 (sRNAs) that can suppress the expression of defence-related genes in its host plants [8]. As a 53 countermeasure, plants also release sRNAs aimed to suppress fungal virulence [9]. To facilitate access 54 to genes or non-coding RNA loci that are important for pathogenesis, a gapless genome sequence of 55 B. cinerea has been published recently [10]. Considerable efforts have been made to generate tools 56 for the genetic manipulation of B. cinerea. Agrobacterium-mediated and protoplast-based 57 transformation have been developed [11][12][13], and several vectors are available which facilitate the 58 generation of mutants and strains expressing fluorescently tagged proteins for cytological studies [14]. 59Nevertheless, the generation of mutants remains time-consuming, partly because of the multinuclear 60 nature of B. cinerea, which requires several rounds of sub-cultivation to achieve homokaryosis. 61 Furthermore, the generation of multiple knock-out mutants is hampered by the lack of marker 62 recycling systems for serial gene replacements, as described in some filamentous fungi [15]. 63The application of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 64 RNA-guided Cas9 endonuclease activity has revolutionized genome editing and greatly facilitated the 65 4 genetic manipulation in a wide range of species [16]. CRISPR/Cas is based on the introduction of double 66 stranded breaks by the Cas9 endonuclease in the genome of an organism. Cas9 targeting occurs by 67 complementary sequences of a single guide RNA (sgRNA), which directs the endonu...

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“…The nourseothricin and hygromycin selection systems have been the most widely used and the most efficient systems for B. cinerea genetic modifications, although recently the resistance to the fungicide fenhexamid has been proposed as a new selection marker ( Cohrs et al, 2017 ). Therefore, the possibility to generate fungal strains with multiple gene deletions is so far limited to only 3 genes that could be manipulated in a single strain.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

García

González

et al. 2017

Front. Plant Sci.

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The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B) were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.

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A new transformant selection system for the gray mold fungus Botrytis cinerea based on the expression of fenhexamid-insensitive ERG27 variants

Cited by 7 publications

References 34 publications

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

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