Efficient Genome Editing in the Oomycete <i>Phytophthora sojae</i> Using CRISPR/Cas9

Fang, Yufeng; Cui, Linkai; Gu, Biao; Arredondo, Francisco; Tyler, Brett M.

doi:10.1002/cpmc.25

Cited by 91 publications

(97 citation statements)

References 36 publications

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“…Phytophthora sojae is a widespread soil-borne pathogen of soybean. Because of its economic impact, and tractable genetic manipulation [31][32][33], P. sojae has become a model species to study oomycete genetics, biology, and interactions with plants.…”

Section: Plos Geneticsmentioning

confidence: 99%

“…All the strains used in this study are listed in S5 Table. The reference P. sojae isolate P6497 (race 2) used in this study was routinely grown and maintained in cleared V8 media at 25˚C in the dark. Transient gene expression assays based on an optimized polyethylene glycol (PEG) mediated protoplast transformation protocol [31] was applied to examine the nuclear localization of CENP-A. Stable and homokaryotic transformants expressing GFP tagged seq, which were generated by passaging on V8 supplemented with 50 μg/mL G418 (Geneticin, AG Scientific, San Diego, California, USA) for at least 5 times followed by zoospore isolation.…”

Section: P Sojae Culture and Transformationmentioning

confidence: 99%

“…Co-transformation was employed to generate strains expressing both H2B-mCherry and GFP-CENP-A. Transformation was performed as previously described [31]. Sporangia and zoospores were induced by water flooding according to a method described previously [68].…”

Section: P Sojae Culture and Transformationmentioning

confidence: 99%

“…An sgRNA guide sequence whose PAM sequence overlapped with the start codon of CENP-A was selected as the CRISPR/Cas9 target. An oligo annealing strategy was used for assembly of the sgRNA expression cassettes according to previously described methods [31]. HDR templates for CENP-A was assembled using NEBuilder HiFi DNA Assembly (NEB, E2621S).…”

Section: Crispr-mediated Gene Replacementmentioning

confidence: 99%

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Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

et al. 2020

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Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.

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Section: Plos Geneticsmentioning

confidence: 99%

Section: P Sojae Culture and Transformationmentioning

confidence: 99%

Section: P Sojae Culture and Transformationmentioning

confidence: 99%

Section: Crispr-mediated Gene Replacementmentioning

confidence: 99%

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Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

et al. 2020

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“…It is distributed throughout most soybean-growing regions, and causes plant stand reductions and even complete yield losses with damage valued at approximately $1-2 billion each year [3,4]. In addition to an abundance of available genome and transcriptome data [1,5], the recently established clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout system has greatly strengthened functional genomic research in P. sojae [6,7].…”

Section: Introductionmentioning

confidence: 99%

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae

Zhang

et al. 2020

PLoS Pathog

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Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, β, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.

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Multiple point mutations in PsORP1 gene conferring different resistance levels to oxathiapiprolin confirmed using CRISPR–Cas9 in Phytophthora sojae

Miao

Liu

et al. 2020

Pest Management Science

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BACKGROUND: Oxathiapiprolin is among the first commercial oxysterol-binding protein inhibitors (OSBPIs) developed by DuPont Corporation and shows excellent activity against plant-pathogenic oomycetes. Although more than 21 target site mutations have been identified in insensitive oomycetes, only G770V, G839W, and ΔN837 have been verified to confer oxathiapiprolin resistance in Phytophthora capsici or P. sojae. The effect of other mutations on OSBPIs sensitivity requires urgent investigation. RESULTS: P. sojae transformants containing 16 mutations of PsORP1 were recovered using the CRISPR-Cas9 system. Transformants containing L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, or I877Y showed high oxathiapiprolin resistance, with resistant factors (RFs) > 3000. Point mutations S768K, S768I, G770L, G770P, G770A, ΔG818/F819, N837I, and I877F exhibited low resistance, with RFs < 80. Phenotype assays revealed that the most highly resistant transformants showed enhanced or similar pathogenicity, oospore production, and cyst gemination. However, most transformants displayed decreased sporangia and zoospore production compared with parental wild-type P6497.CONCLUSION: This study demonstrated that L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, and I877Y in PsORP1 confer high oxathiapiprolin resistance in P. sojae.

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Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9

Cited by 91 publications

References 36 publications

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae

Multiple point mutations in PsORP1 gene conferring different resistance levels to oxathiapiprolin confirmed using CRISPR–Cas9 in Phytophthora sojae

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