Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi.
Phytophthora cinnamomi
Flow cytometry
Fluorescence microscopy
PEG/CaCl2 transformation
Protoplast
Journal
Protoplasma
ISSN: 1615-6102
Titre abrégé: Protoplasma
Pays: Austria
ID NLM: 9806853
Informations de publication
Date de publication:
04 May 2024
04 May 2024
Historique:
received:
02
11
2023
accepted:
11
04
2024
medline:
4
5
2024
pubmed:
4
5
2024
entrez:
3
5
2024
Statut:
aheadofprint
Résumé
Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.
Identifiants
pubmed: 38702562
doi: 10.1007/s00709-024-01953-y
pii: 10.1007/s00709-024-01953-y
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2024. The Author(s).
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