A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

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Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

10.1101/772699 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marc-Benjamin Aurin ◽

Michael Haupt ◽

Matthias Görlach ◽

Florian Rümpler ◽

Günter Theißen

Keyword(s):

Plant Pathogens ◽

Ornamental Plants ◽

Effector Proteins ◽

Plant Diseases ◽

Scattering Data ◽

Effector Protein ◽

Homeotic Transformation ◽

Structural Requirements ◽

Floral Organs ◽

Floral Homeotic

SummaryPhytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS-WITCHES’ BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves physical binding to the keratin-like K-domain of MIKC-type proteins, and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here we report, based on biophysical and molecular biological analyses, that SAP54 folds into α-helical structures. We also show that the insertion of helix-breaking mutations disrupts correct folding of SAP54, which interferes with the ability of SAP54 to bind to its target proteins and to cause disease phenotypes in vivo. Surprisingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with literature data this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled-coils, possibly also involving other partners such as RAD23 proteins. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and thus may ultimately help to develop treatments against some devastating plant diseases.SIGNIFICANCE STATEMENTPhytoplasmas are bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins such as SAP54, which leads to the degradation of some floral homeotic proteins. Our study clarifies the structural requirements of SAP54 function and illuminates the molecular mode of interaction and thus may ultimately help to develop treatments against some devastating plant diseases.

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Structural Requirements of the Phytoplasma Effector Protein SAP54 for Causing Homeotic Transformation of Floral Organs

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-20-0028-r ◽

2020 ◽

Vol 33 (9) ◽

pp. 1129-1141

Author(s):

Marc-Benjamin Aurin ◽

Michael Haupt ◽

Matthias Görlach ◽

Florian Rümpler ◽

Günter Theißen

Keyword(s):

Plant Pathogens ◽

Effector Proteins ◽

Plant Diseases ◽

Scattering Data ◽

Effector Protein ◽

Mobility Shift ◽

Homeotic Transformation ◽

Structural Requirements ◽

Floral Organs ◽

Specific Subset

Phytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS WITCHES’ BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves binding to the keratin-like domain of MIKC-type proteins and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here, we report, based on biophysical and molecular biological analyses, that SAP54 folds into an α-helical structure. Insertion of helix-breaking mutations disrupts correct folding of SAP54 and compromises SAP54 binding to its target proteins and, concomitantly, its ability to evoke disease phenotypes in vivo. Interestingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with data from literature, this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled coils. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and may thus ultimately help to develop treatments against some devastating plant diseases.

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The anticipated potential nuclear localization sequence of ‘Candidatus Phytoplasma mali’ SAP11-like protein is required for TCP binding but not for transport into the nucleus

10.1101/2020.05.13.094045 ◽

2020 ◽

Author(s):

Alisa Strohmayer ◽

Timothy Schwarz ◽

Mario Braun ◽

Gabi Krczal ◽

Kajohn Boonrod

Keyword(s):

Plant Pathogens ◽

Leader Sequence ◽

Effector Proteins ◽

Nuclear Localization Sequence ◽

Candidatus Phytoplasma Mali ◽

Localization Sequence ◽

Amino Acid Stretch ◽

Apple Production ◽

Proliferating Cell ◽

Heavy Damage

AbstractThe plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana.

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Molecular mechanisms of host cell manipulation by plant pathogens

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091980 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C801-C801

Author(s):

Richard Hughes ◽

Stuart King ◽

Abbas Maqbool ◽

Hazel McLellan ◽

Tolga Bozkurt ◽

...

Keyword(s):

Cell Death ◽

Crop Production ◽

Plant Pathogens ◽

Molecular Mechanisms ◽

Complex Structure ◽

Effector Proteins ◽

Plant Diseases ◽

Cell Manipulation ◽

Cell Physiology ◽

Structural Studies

An estimated 15% of global crop production is lost to pre-harvest disease every year. New ways to manage plant diseases are required. A mechanistic understanding of how plant pathogens re-program their hosts to enable colonisation may provide novel genetic or chemical opportunities to interfere with disease. One notorious plant parasite is the Irish potato famine pathogen Phytophthora infestans. This pathogen remains a considerable threat to potato/tomato crops today as the agent of late blight. Plant pathogens secrete effector proteins outside of and into plant cells to suppress host defences and manipulate cell physiology. Structural studies have provided insights into effector evolution and enabled experiments to probe function [1-3]. Crystal structures of 4 Phytophthora RXLR-type effectors, which are unrelated in primary sequence, revealed similarities in the fold of these proteins. This fold was proposed to act as a stable scaffold that supports diversification of effectors. Further, molecular modelling has enabled mapping of single-site variants responsible for specialisation of a Phytophthora Cystatin-like effector, revealing how effectors can adapt to new hosts after a "host jump". Structural studies describing how RXLR-effectors interact with host targets are lacking. We have used Y2H/co-IP studies to identify host proteins that interact with P. infestans effectors PexRD2 and PexRD54. PexRD2 interacts with MAPKKKe, a component of plant immune signalling pathways, and suppressed cell death activities of this protein. We used the structure of PexRD2 to design mutants that fail to interact with MAPKKKe, and no longer suppress cell-death activities. We found that PexRD54 interacts with potato homologues of the autophagy protein ATG8. We have obtained a crystal structure for PexRD54 in the presence of ATG8. We are now using X-ray scattering to verify the complex structure in solution prior to establishing the role of this interaction during infection.

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‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.02823-0 ◽

2004 ◽

Vol 54 (4) ◽

pp. 1217-1226 ◽

Cited By ~ 164

Author(s):

Erich Seemüller ◽

Bernd Schneider

Keyword(s):

16S Rdna ◽

Sequence Similarity ◽

Phylogenetic Analyses ◽

Leaf Roll ◽

Species Level ◽

Plant Diseases ◽

Stone Fruit ◽

Apple Proliferation ◽

Rdna Sequences ◽

Candidatus Phytoplasma Mali

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1·0 to 1·5 % of nucleotide positions and were thus below the recommended threshold of 2·5 % for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S–23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99·6 % 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of ‘Candidatus Phytoplasma pyri’.

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Effect of Daytime and Tree Canopy Height on Sampling of Cacopsylla melanoneura, a ‘Candidatus Phytoplasma mali’ Vector

Plants ◽

10.3390/plants9091168 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1168 ◽

Cited By ~ 2

Author(s):

Dana Barthel ◽

Christine Kerschbamer ◽

Bernd Panassiti ◽

Igor Malenovský ◽

Katrin Janik

Keyword(s):

Apple Tree ◽

Tree Canopy ◽

Temperature Threshold ◽

Insect Vector ◽

Apple Proliferation ◽

Candidatus Phytoplasma Mali ◽

Cacopsylla Melanoneura ◽

Cacopsylla Pruni ◽

Vector Behavior ◽

Practical Impact

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium ‘Candidatus Phytoplasma mali’, the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in ‘Candidatus Phytoplasma mali’ transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

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Harnessing Effector-Triggered Immunity for Durable Disease Resistance

Phytopathology ◽

10.1094/phyto-03-17-0086-rvw ◽

2017 ◽

Vol 107 (8) ◽

pp. 912-919 ◽

Cited By ~ 10

Author(s):

Meixiang Zhang ◽

Gitta Coaker

Keyword(s):

Disease Resistance ◽

Plant Pathogens ◽

Domain Architecture ◽

Effector Proteins ◽

Plant Diseases ◽

Sources Of Resistance ◽

Pathogen Effectors ◽

Pathogen Effector ◽

Effector Triggered Immunity ◽

Durable Disease Resistance

Genetic control of plant diseases has traditionally included the deployment of single immune receptors with nucleotide-binding leucine-rich repeat (NLR) domain architecture. These NLRs recognize corresponding pathogen effector proteins inside plant cells, resulting in effector-triggered immunity (ETI). Although ETI triggers robust resistance, deployment of single NLRs can be rapidly overcome by pathogen populations within a single or a few growing seasons. In order to generate more durable disease resistance against devastating plant pathogens, a multitiered strategy that incorporates stacked NLRs combined with other sources of disease resistance is necessary. New genetic and genomic technologies have enabled advancements in identifying conserved pathogen effectors, isolating NLR repertoires from diverse plants, and editing plant genomes to enhance resistance. Significant advancements have also been made in understanding plant immune perception at the receptor level, which has promise for engineering new sources of resistance. Here, we discuss how to utilize recent scientific advancements in a multilayered strategy for developing more durable disease resistance.

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The Epichloë festucae Antifungal Protein Efe-AfpA Is Also a Possible Effector Protein Required for the Interaction of the Fungus with its Host Grass Festuca rubra subsp. rubra

Microorganisms ◽

10.3390/microorganisms9010140 ◽

2021 ◽

Vol 9 (1) ◽

pp. 140

Author(s):

Ruying Wang ◽

Simin Luo ◽

Bruce B. Clarke ◽

Faith C. Belanger

Keyword(s):

Disease Resistance ◽

Insect Resistance ◽

Effector Proteins ◽

Antifungal Protein ◽

Grass Species ◽

Effector Protein ◽

Festuca Rubra ◽

Symbiotic Interaction ◽

Red Fescue ◽

Epichloë Festucae

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichloë festucae. Epichloë spp. are endophytes of several cool-season grass species, often conferring insect resistance to the grass hosts due to the production of toxic alkaloids. In addition to insect resistance, a unique feature of the strong creeping red fescue/E. festucae symbiosis is the endophyte-mediated disease resistance to the fungal pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Such disease resistance is not a general feature of other grass/ Epichloë interactions. E. festucae isolates infecting red fescue have an antifungal protein gene Efe-afpA, whereas most other Epichloë spp. do not have a similar gene. The uniqueness of this gene suggests it may, therefore, be a component of the unique disease resistance seen in endophyte-infected red fescue. Here, we report the generation of CRISPR-Cas9 Efe-afpA gene knockouts with the goal of determining if absence of the protein in endophyte-infected Festuca rubra leads to disease susceptibility. However, it was not possible to infect plants with the knockout isolates, although infection was possible with the wild type E. festucae and with complemented isolates. This raises the interesting possibility that, in addition to having antifungal activity, the protein is required for the symbiotic interaction. The antifungal protein is a small secreted protein with high expression in planta relative to its expression in culture, all characteristics consistent with effector proteins. If Efe-AfpA is an effector protein it must be specific to certain interactions, since most Epichloë spp. do not have such a gene in their genomes.

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Microbial antagonists against plant pathogens in Iran: A review

Open Agriculture ◽

10.1515/opag-2020-0031 ◽

2020 ◽

Vol 5 (1) ◽

pp. 404-440 ◽

Cited By ~ 1

Author(s):

Mehrdad Alizadeh ◽

Yalda Vasebi ◽

Naser Safaie

Keyword(s):

Biological Control ◽

Chemical Control ◽

Plant Disease ◽

Plant Pathogens ◽

Plant Diseases ◽

Agricultural Products ◽

Control Measures ◽

Wide Range ◽

Plant Disease Management ◽

Published Research

AbstractThe purpose of this article was to give a comprehensive review of the published research works on biological control of different fungal, bacterial, and nematode plant diseases in Iran from 1992 to 2018. Plant pathogens cause economical loss in many agricultural products in Iran. In an attempt to prevent these serious losses, chemical control measures have usually been applied to reduce diseases in farms, gardens, and greenhouses. In recent decades, using the biological control against plant diseases has been considered as a beneficial and alternative method to chemical control due to its potential in integrated plant disease management as well as the increasing yield in an eco-friendly manner. Based on the reported studies, various species of Trichoderma, Pseudomonas, and Bacillus were the most common biocontrol agents with the ability to control the wide range of plant pathogens in Iran from lab to the greenhouse and field conditions.

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The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Journal of Fungi ◽

10.3390/jof7020086 ◽

2021 ◽

Vol 7 (2) ◽

pp. 86

Author(s):

Bilal Ökmen ◽

Daniela Schwammbach ◽

Guus Bakkeren ◽

Ulla Neumann ◽

Gunther Doehlemann

Keyword(s):

Fungal Pathogens ◽

Plant Pathogens ◽

Expression System ◽

Pathogenic Fungi ◽

Effector Proteins ◽

Mating Partner ◽

Fungal Virulence ◽

Functional Studies ◽

Infection Structures ◽

Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

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