Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae

Wang, Shumei; Vetukuri, Ramesh R.; Kushwaha, Sandeep; Hedley, Pete E.; Morris, Jenny; Studholme, David J.; Welsh, Lydia; Boevink, Petra C.; Birch, Paul R. J.; Whisson, Stephen C.

doi:10.1111/mpp.13072

Cited by 6 publications

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“…parasitica, and in all life stages in Ph. kernoviae (Blackman et al, 2015;Wang et al, 2021). Their involvement in both infection and regular growth can be explained by the fact that oomycete cell walls contain a high proportion of β-1,3-glucans, and plant cell walls can contain β-1,3-glucans as callose in response to pathogen infection.…”

Section: Discussionmentioning

confidence: 99%

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

et al. 2023

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IntroductionOomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors.MethodsTo gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species.Results and discussionEach of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5’ base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

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Section: Discussionmentioning

confidence: 99%

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

et al. 2023

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“…6.1 Significance of this work Transformation is a powerful tool for molecular biology research. It will enable a wide range of advanced methods to be applied for the study of P. agathidicida: for example, generation of fluorescent strains [49,162], gene silencing [51,52,163], and genome editing using CRISPR-Cas [57,63]. These techniques, in turn, will enable us to visualise infection in planta [162,164], probe the function of secreted effector proteins [57,76,164], and understand the molecular basis of chemotaxis [165][166][167].…”

Section: Discussionmentioning

confidence: 99%

“…It will enable a wide range of advanced methods to be applied for the study of P. agathidicida: for example, generation of fluorescent strains [49,162], gene silencing [51,52,163], and genome editing using CRISPR-Cas [57,63]. These techniques, in turn, will enable us to visualise infection in planta [162,164], probe the function of secreted effector proteins [57,76,164], and understand the molecular basis of chemotaxis [165][166][167]. Transformation-based approaches also circumvent recombinant protein expression, which may be useful in light of the challenges encountered in Chapter 5.…”

Section: Discussionmentioning

confidence: 99%

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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Plant–microbe interactions in the apoplast: Communication at the plant cell wall

2022

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The apoplast is a continuous plant compartment that connects cells between tissues and organs and is one of the first sites of interaction between plants and microbes. The plant cell wall occupies most of the apoplast and is composed of polysaccharides and associated proteins and ions. This dynamic part of the cell constitutes an essential physical barrier and a source of nutrients for the microbe. At the same time, the plant cell wall serves important functions in the interkingdom detection, recognition, and response to other organisms. Thus, both plant and microbe modify the plant cell wall and its environment in versatile ways to benefit from the interaction. We discuss here crucial processes occurring at the plant cell wall during the contact and communication between microbe and plant. Finally, we argue that these local and dynamic changes need to be considered to fully understand plant-microbe interactions

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Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 6 publications

References 95 publications

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

Establishing Molecular Biology Tools for Phytophthora agathidicida

Plant–microbe interactions in the apoplast: Communication at the plant cell wall

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