Galaxy as a Platform for Identifying Candidate Pathogen Effectors

AbstractPlants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

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Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

Plant Cell Reports ◽

10.1007/s00299-021-02671-y ◽

2021 ◽

Author(s):

Johannes W. Debler ◽

Bernadette M. Henares ◽

Robert C. Lee

Keyword(s):

Broad Spectrum ◽

Faba Bean ◽

Fluorescent Protein ◽

Transient Gene Expression ◽

Low Frequency ◽

Effector Proteins ◽

Field Pea ◽

Grain Legume ◽

Pathogen Effectors ◽

Green Fluorescent

Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

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Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Genetics ◽

10.1093/genetics/iyaa026 ◽

2020 ◽

Vol 217 (2) ◽

Cited By ~ 1

Author(s):

Antony V E Chapman ◽

Matthew Hunt ◽

Priyanka Surana ◽

Valeria Velásquez-Zapata ◽

Weihui Xu ◽

...

Keyword(s):

Powdery Mildew ◽

Fungal Pathogens ◽

Novel Mutation ◽

Exome Capture ◽

Resistance Locus ◽

Hordeum Vulgare L ◽

Powdery Mildew Fungus ◽

Pathogen Effectors ◽

Cell Processes ◽

Disease Resistances

Abstract Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1ΔKL308–309), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.

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First Insights into the Effect of Mycorrhizae on the Expression of Pathogen Effectors during the Infection of Grapevine with Plasmopara viticola

Sustainability ◽

10.3390/su13031226 ◽

2021 ◽

Vol 13 (3) ◽

pp. 1226

Author(s):

Ana Cruz-Silva ◽

Andreia Figueiredo ◽

Mónica Sebastiana

Keyword(s):

Mycorrhizal Fungi ◽

Arbuscular Mycorrhizal ◽

Plasmopara Viticola ◽

Vitis Vinifera L ◽

Wine Production ◽

Grapevine Downy Mildew ◽

Pathogen Effectors ◽

Rxlr Effectors ◽

Mycorrhizal Inoculation ◽

And Control

Grapevine (Vitis vinifera L.), widely used for berry and wine production, is highly susceptible to the pathogenic oomycete Plasmopara viticola, the etiological agent of grapevine downy mildew disease. The method commonly used to prevent and control P. viticola infection relies on multiple applications of chemical fungicides. However, with European Union goals to lower the usage of such chemicals in viticulture there is a need to develop new and more sustainable strategies. The use of beneficial microorganisms with biocontrol capabilities, such as the arbuscular mycorrhizal fungi (AMF), has been pointed out as a viable alternative. With this study, we intended to investigate the effect of AMF colonization on the expression of P. viticola effectors during infection of grapevine. Grapevine plants were inoculated with the AMF Rhizophagus irregularis and, after mycorrhizae development, plants were infected with P. viticola. The expression of P. viticola RxLR effectors was analyzed by real-time PCR (qPCR) during the first hours of interaction. Results show that pre-mycorrhizal inoculation of grapevine alters the expression of several P. viticola effectors; namely, PvRxLR28, which presented decreased expression in mycorrhizal plants at the two time points post-infection tested. These results suggest that the pre-inoculation of grapevine with AMF could interfere with the pathogen’s ability to infect grapevine by modulation of pathogenicity effectors expression, supporting the hypothesis that AMF can be used to increase plant resistance to pathogens and promote more sustainable agriculture practices, particularly in viticulture.

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The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

PLoS Pathogens ◽

10.1371/journal.ppat.1009368 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009368

Author(s):

Juan Carlos De la Concepcion ◽

Josephine H. R. Maidment ◽

Apinya Longya ◽

Gui Xiao ◽

Marina Franceschetti ◽

...

Keyword(s):

Molecular Mechanisms ◽

Allelic Variation ◽

Immune Recognition ◽

In Planta ◽

Pathogen Effectors ◽

Binding Interface ◽

Recognition Specificity ◽

Blast Fungus ◽

Resistance Specificity ◽

Mechanistic Basis

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.

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Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

10.1101/2021.03.15.435521 ◽

2021 ◽

Author(s):

Cian Duggan ◽

Eleonora Moratto ◽

Zachary Savage ◽

Eranthika Hamilton ◽

Hiroaki Adachi ◽

...

Keyword(s):

Plasma Membrane ◽

Immune Responses ◽

Subcellular Localization ◽

Plant Pathogen ◽

Coiled Coil ◽

Dynamic Changes ◽

Pathogen Effectors ◽

Mediate Response ◽

Potato Famine ◽

Irish Potato Famine

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post- activation during pathogen infection remains poorly known. Here we show that NRC4, from the 'NRC' solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N- terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

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Pathogen effectors: What do they do at plasmodesmata?

Molecular Plant Pathology ◽

10.1111/mpp.13142 ◽

2021 ◽

Author(s):

Arya Bagus Boedi Iswanto ◽

Minh Huy Vu ◽

Sharon Pike ◽

Jihyun Lee ◽

Hobin Kang ◽

...

Keyword(s):

Pathogen Effectors

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Emerging Concepts in Effector Biology of Plant-Associated Organisms

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-2-0115 ◽

2009 ◽

Vol 22 (2) ◽

pp. 115-122 ◽

Cited By ~ 408

Author(s):

Saskia A. Hogenhout ◽

Renier A. L. Van der Hoorn ◽

Ryohei Terauchi ◽

Sophien Kamoun

Keyword(s):

Plant Defense ◽

Plant Pathogen ◽

Plant Tissue ◽

Plant Colonization ◽

Molecular Function ◽

Pathogen Effectors ◽

The Common ◽

Mechanistic Understanding

Plant-associated organisms secrete proteins and other molecules to modulate plant defense circuitry and enable colonization of plant tissue. Understanding the molecular function of these secreted molecules, collectively known as effectors, became widely accepted as essential for a mechanistic understanding of the processes underlying plant colonization. This review summarizes recent findings in the field of effector biology and highlights the common concepts that have emerged from the study of cellular plant pathogen effectors.

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