An Improved Transformation System for Phytophthora cinnamomi Using Green Fluorescent Protein

Dai, Tingting; Xu, Yue; Yang, Xiao; Jiao, Binbin; Qu, Min; Xue, Junxin; Arredondo, Francisco; Tyler, Brett M.

doi:10.3389/fmicb.2021.682754

Cited by 8 publications

(13 citation statements)

References 36 publications

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“…The growth rate was decreased by approximately 28% compared to the growth recorded for the wild-type LT1534 and CK strains ( Figure 2 ). To generate the complemented (C1) strains, we re-introduced the pTOR-GFP cassette ( Dai et al, 2021 ) containing the full-length PcAPT1 coding (ORF) sequence into the Δ Pcapt1 knock-out mutant, qRT-PCR results have also demonstrated that the expression of PcAPT1 gene in the complemented strain ( Supplementary Figure 1 ). Vegetative growth of the complemented strain is comparable to WT and CK ( Figure 2 ).…”

Section: Resultsmentioning

confidence: 99%

A Putative P-Type ATPase Regulates the Secretion of Hydrolytic Enzymes, Phospholipid Transport, Morphogenesis, and Pathogenesis in Phytophthora capsici

et al. 2022

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Phytophthora capsici is an important plant pathogenic oomycete with multiple hosts. The P4-ATPases, aminophospholipid translocases (APTs), play essential roles in the growth and pathogenesis of fungal pathogens. However, the function of P4-ATPase in P. capsici remains unclear. This study identified and characterized PcApt1, a P4-ATPase Drs2 homolog, in P. capsici. Deletion of PcAPT1 by CRISPR/Cas9 knock-out strategy impaired hyphal growth, extracellular laccase activity. Cytological analyses have shown that PcApt1 participates in phosphatidylserine (PS) transport across the plasma membrane. Also, we showed that targeted deletion of PcAPT1 triggered a significant reduction in the virulence of P. capsici. Secretome analyses have demonstrated that secretion of hydrolytic enzymes decreased considerably in the PcAPT1 gene deletion strains compared to the wild-type. Overall, our results showed that PcApt1 plays a pivotal role in promoting morphological development, phospholipid transport, secretion of hydrolytic enzymes, and the pathogenicity of the polycyclic phytopathogenic oomycete P. capsici. This study underscores the need for comprehensive evaluation of subsequent members of the P-type ATPase family to provide enhanced insights into the dynamic contributions to the pathogenesis of P. capsici and their possible deployment in the formulation of effective control strategies.

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Section: Resultsmentioning

confidence: 99%

A Putative P-Type ATPase Regulates the Secretion of Hydrolytic Enzymes, Phospholipid Transport, Morphogenesis, and Pathogenesis in Phytophthora capsici

et al. 2022

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“…The transformation of Phytophthora spp. has been one method used to deduce the function of effectors; however, some species-such as P. cinnamomi-have had limited success in transformation [66,172,173]. The speculated reasons for these limitations are the identification of oomycete promotors and selectable markers to select for transformants [66,172].…”

Section: Techniques Used In the Functional Characterization Of Phytop...mentioning

confidence: 99%

Unraveling Plant Cell Death during Phytophthora Infection

2022

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Oomycetes form a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms, of which several hundred organisms are considered among the most devastating plant pathogens—especially members of the genus Phytophthora. Phytophthora spp. have a large repertoire of effectors that aid in eliciting a susceptible response in host plants. What is of increasing interest is the involvement of Phytophthora effectors in regulating programed cell death (PCD)—in particular, the hypersensitive response. There have been numerous functional characterization studies, which demonstrate Phytophthora effectors either inducing or suppressing host cell death, which may play a crucial role in Phytophthora’s ability to regulate their hemi-biotrophic lifestyle. Despite several advances in techniques used to identify and characterize Phytophthora effectors, knowledge is still lacking for some important species, including Phytophthora cinnamomi. This review discusses what the term PCD means and the gap in knowledge between pathogenic and developmental forms of PCD in plants. We also discuss the role cell death plays in the virulence of Phytophthora spp. and the effectors that have so far been identified as playing a role in cell death manipulation. Finally, we touch on the different techniques available to study effector functions, such as cell death induction/suppression.

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“…This differs to P. sojae, for which an equal ratio of the two enzymes is used [57]. However, this finding is difficult to compare with most other Phytophthora species, as a wide variety of different enzymes have been used [44,46,64]. My digestion protocol gave protoplast yields of approximately 23 million per culture, comparable to P. sojae [57].…”

Section: Protoplast Production Methodsmentioning

confidence: 91%

“…However, these efforts have not come without challenges. There are numerous reports of difficulty with transformation [46,64] Cas9 toxicity [63,65] and low rates of homozygous editing [63,66]. As of February 2023, CRISPR-Cas genome editing has been reported in five Phytophthora species [57,63,[67][68][69] and three other oomycetes [70][71][72] and will undoubtedly continue to provide insights into Phytophthora biology.…”

Section: Crispr-cas Genome Editingmentioning

confidence: 99%

“…Currently, the available plasmids for Phytophthora are integrative -that is, they randomly integrate into the host genome after transformation (Figure 4.1B). Because this process is uncontrolled, it is not guaranteed that the whole plasmid will integrate: in one P. cinnamomi study, an incoming gene was absent in 85% of drug-resistant transformants [64]. Unfortunately, overall transformation efficiencies in Phytophthora are soberingly low compared to the efficiencies obtained with model organisms.…”

Section: P Agathidicida Protoplast Production and Transformationmentioning

confidence: 99%

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Establishing Molecular Biology Tools for Phytophthora agathidicida

Hayhurst¹

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Phytophthora – Greek for “the plant-destroyer” – is a genus of eukaryotic microorganisms that cause devastating plant diseases worldwide. Collectively, Phytophthora species cause billions of dollars in damages to crops and native ecosystems annually. Despite these extensive impacts, Phytophthora research is challenging due to a lack of molecular biology tools. Widely used techniques such as transformation and gene deletion remain challenging to establish in Phytophthora species. However, CRISPR-Cas genome editing was recently achieved in a small number of model Phytophthora species – but these breakthroughs have yet to reach most members of the genus. The pathogen Phytophthora agathidicida is one example. P. agathidicida causes kauri dieback, a disease that threatens kauri, one of our most important tree species in New Zealand. P. agathidicida was formally named in 2015 and is therefore an emergent threat. Consequently, key molecular methods such as transformation and CRISPR-Cas genome editing have yet to be established for this pathogen. The overarching goal of this thesis was to establish molecular biology methods for the study of P. agathidicida. The first aim was to identify a suitable target for genome editing with CRISPR-Cas and design guide RNAs for the target. A putative thymidine kinase gene from the P. agathidicida isolate 3770 genome was selected as a target, and eight guide RNA sequences were designed. All of the guide RNAs successfully directed the digestion of the target gene in vitro. The second aim was to establish a method to produce and transform P. agathidicida protoplasts. An optimised recovery medium was identified using a mock transformation method, which increased protoplast germination rates from 2.4% in a previously used medium to 15% in the new medium. Using the optimised conditions, P. agathidicida was transformed with plasmids encoding Cas nuclease-GFP fusion proteins. Transformants were both fluorescent and antibiotic resistant. The third aim was to express, purify, and characterise the putative thymidine kinase enzyme. Yeast species Pichia pastoris was used as a protein expression host, but neither protein expression nor kinase activity were consistently observed. In this thesis, P. agathidicida was successfully transformed for the first time. Transformation is a huge advancement and will greatly accelerate molecular biology research in P. agathidicida for years to come. In particular, transformation is a key step towards CRISPR-Cas genome editing in P. agathidicida. CRISPR-Cas will not only provide insights into its fundamental biology, but may guide the development of new control strategies for this devastating pathogen.

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An Improved Transformation System for Phytophthora cinnamomi Using Green Fluorescent Protein

Cited by 8 publications

References 36 publications

A Putative P-Type ATPase Regulates the Secretion of Hydrolytic Enzymes, Phospholipid Transport, Morphogenesis, and Pathogenesis in Phytophthora capsici

A Putative P-Type ATPase Regulates the Secretion of Hydrolytic Enzymes, Phospholipid Transport, Morphogenesis, and Pathogenesis in Phytophthora capsici

Unraveling Plant Cell Death during Phytophthora Infection

Establishing Molecular Biology Tools for Phytophthora agathidicida

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