Publications from The Sainsbury Laboratory
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bioRXiv: Convergent gene loss in aquatic plants predicts new components of plant immunity and drought response (2019)

bioRXiv: Convergent gene loss in aquatic plants predicts new components of plant immunity and drought response (2019) | Publications from The Sainsbury Laboratory | Scoop.it

The transition of plants from sea to land sparked an arms race with pathogens. The increased susceptibility of land plants is largely thought to be due to their dependence on micro-organisms for nutrients; the ensuing co-evolution has shaped the plant immune system. By profiling the immune receptors across flowering plants, we identified species with low numbers of NLR immune receptors. Interestingly, four of these species represent distinct lineages of monocots and dicots that returned to the aquatic lifestyle. Both aquatic monocot and dicot species lost the same well-known downstream immune signalling complex (EDS1-PAD4). This observation inspired us to look for other genes with a similar loss pattern and allowed us to predict putative new components of plant immunity. Gene expression analyses confirmed that a group of these genes was differentially expressed under pathogen infection. Excitingly, another subset of these genes was differentially expressed upon drought. Collectively, our study reveals the minimal plant immune system required for life under water, and highlights additional components required for the life of land plants.

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New Phytologist: Commentary: Convergence of cell‐surface and intracellular immune receptor signalling (2019)

Plant immune responses are initiated by recognition of pathogen invasion through immune receptors. The pathogen sensing system is mainly composed of two structurally different proteins that are located on different subcellular compartments. One is the plasma membrane‐localized pattern recognition receptors (PRRs) that detect pathogen‐associated molecular patterns (PAMPs) (Boutrot & Zipfel, 2017). PRRs perceive extracellular PAMPs to activate PAMP‐triggered immunity (PTI). In turn, adapted pathogens interfere with or modulate host signalling by virulence factors (called effectors) for successful infection. The other is intracellular nucleotide‐binding domain and leucine‐rich repeat proteins (NLRs) that recognize these effectors (Jones et al., 2016). Activation of an NLR induces a robust immune response called effector‐triggered immunity (ETI), which is often accompanied by hypersensitive response (HR) cell death. Animals encode both plasma membrane and intracellular immune receptors, which share similar structures with plant PRRs and NLRs for recognition of PAMPs; but plant and animal immune receptors evolved independently (Ronald & Beutler, 2010; Jones et al., 2016). Interestingly, although PRRs and NLRs are structurally different and localize in distinct subcellular compartments, they share substantial downstream signalling, such as Ca2+, mitogen‐activated protein kinase (MAPK), reactive oxygen species (ROS), and phytohormone signalling as well as massive transcriptional reprogramming (Peng et al., 2018). However, it is not known how PRRs and NLRs activate similar signalling outputs. In this issue of New Phytologist, Kadota et al. (2019, pp. 2160–2175) investigated protein phosphorylation dynamics upon NLR activation by phosphoproteomics. By comparing with previously published phosphoproteomics data for PTI, they discovered that phosphorylation occurred in the same residues of an NADPH oxidase, RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), which is activated during both PTI and ETI. Thus, Kadota et al. (2019) precisely defined a signal convergent point between PRR and NLR signalling at the molecular level.

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bioRxiv: The Arabidopsis thaliana pan-NLRome (2019)

bioRxiv: The Arabidopsis thaliana pan-NLRome (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Disease is both among the most important selection pressures in nature and among the main causes of yield loss in agriculture. In plants, resistance to disease is often conferred by Nucleotide-binding Leucine-rich Repeat (NLR) proteins. These proteins function as intracellular immune receptors that recognize pathogen proteins and their effects on the plant. Consistent with evolutionarily dynamic interactions between plants and pathogens, NLRs are known to be encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define the majority of the Arabidopsis thaliana species-wide 'NLRome'. From NLR sequence enrichment and long-read sequencing of 65 diverse A. thaliana accessions, we infer that the pan-NLRome saturates with approximately 40 accessions. Despite the high diversity of NLRs, half of the pan-NLRome is present in most accessions. We chart the architectural diversity of NLR proteins, identify novel architectures, and quantify the selective forces that act on specific NLRs, domains, and positions. Our study provides a blueprint for defining the pan-NLRome of plant species.

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PNAS: Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida) (2019)

PNAS: Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida) (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Most plants resist most plant pathogens. Barley resists wheat-infecting powdery mildew races (and vice versa), and both barley and wheat resist potato late blight. Such “nonhost” resistance could result because the pathogen fails to suppress defense or triggers innate immunity due to failure to evade detection. Albugo candida causes white rust on most Brassicaceae, and we investigated Arabidopsis NHR to Brassica -infecting races. Transgressive segregation for resistance in Arabidopsis recombinant inbred lines revealed genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. Some of these NLR-encoding genes confer resistance to white rust in Brassica sp. This genetic method thus provides a route to reveal resistance genes for crops, widening the pool from which such genes might be obtained.

Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust ( Albugo candida ) races that infect the crop species Brassica juncea and Brassica oleracea . We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance ( R ) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea . Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens.

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Nature Microbiology: News & Views - Old fungus, new trick (2019)

Nature Microbiology: News & Views - Old fungus, new trick (2019) | Publications from The Sainsbury Laboratory | Scoop.it
A secreted effector from the plant pathogenic fungus Ustilago maydis has evolved to acquire a new function that contributes to the unique lifestyle of this species, highlighting the utility of using comparative genetic analyses to address current questions in plant–microorganism interactions.
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New Phytologist: Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors (2019)

New Phytologist: Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long timescales without macroscopic symptoms. However, how plant‐pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown.

 

Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well‐studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi.

 

The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of them, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection.

 

Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long‐lived infection of its perennial hosts.

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BMC Bioinformatics: Rapid fine mapping of causative mutations from sets of unordered, contig-sized fragments of genome sequence (2019)

BMC Bioinformatics: Rapid fine mapping of causative mutations from sets of unordered, contig-sized fragments of genome sequence (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Traditional Map based Cloning approaches, used for the identification of desirable alleles, are extremely labour intensive and years can elapse between the mutagenesis and the detection of the polymorphism. High throughput sequencing based Mapping-by-sequencing approach requires an ordered genome assembly and cannot be used with fragmented, un-scaffolded draft genomes, limiting its application to model species and precluding many important organisms.

 

We addressed this gap in resource and presented a computational method and software implementations called CHERIPIC (Computing Homozygosity Enriched Regions In genomes to Prioritise Identification of Candidate variants). We have successfully validated implementation of CHERIPIC using three different types of bulk segregant sequence data from Arabidopsis, maize and barley, respectively.

 

CHERIPIC allows users to rapidly analyse bulk segregant sequence data and we have made it available as a pre-packaged binary with all dependencies for Linux and MacOS and as Galaxy tool.

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New Phytologist: Dude, where is my mutant? Nicotiana benthamiana meets forward genetics (2019)

New Phytologist: Dude, where is my mutant? Nicotiana benthamiana meets forward genetics (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Nicotiana benthamiana, a solanaceous species native to Australia, is one of the most commonly used model plant organisms. It was initially used by the virology community because of its hyper‐susceptibility to plant viruses. Later, this feature was exploited with the development of viral vectors that can express foreign genes and the establishment of virus‐induced gene silencing (VIGS), which enabled knocking down endogenous plant genes. Benth (or Benthi), as it is colloquially known, was further popularized by the development of agroinfiltration, a method that enabled transient protein expression in plants. Agroinfiltration has been extensively used in cell biology, biochemistry, protein–protein interaction analyses and other in planta studies (Goodin et al., 2008). However, genetic analyses of N. benthamiana remained limited due to its allotetraploid nature and incomplete sequence of the 3.1 Gb genome. In this issue of New Phytologist, Schultink et al. (pp. 1001–1009) used a forward genetic screen in N. benthamiana to determine that NbZAR1, an orthologue of the Arabidopsis thaliana NLR (nucleotide‐binding domain and leucine‐rich repeat domain‐containing) ZAR1, is responsible for the perception of XopJ4 from the tomato bacterial pathogen Xanthomonas perforans. Although forward genetic screens using gene silencing have been performed in N. benthamiana, Schultink et al. are among the first to use a chemical mutagenesis‐based screen to dissect plant biological processes. Together with CRISPR genome editing and improved genomics resources, this study ushers in a new era of forward and reverse genetic analyses for this much‐cherished model plant system (Fig. 1).


Via Kamoun Lab @ TSL
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New Phytologist: Autoimmunity and effector recognition in Arabidopsis thaliana can be uncoupled by mutations in the RRS1‐R immune receptor (2018)

New Phytologist: Autoimmunity and effector recognition in Arabidopsis thaliana can be uncoupled by mutations in the RRS1‐R immune receptor (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Plant nucleotide‐binding leucine‐rich repeat (NLR) disease resistance proteins recognize specific pathogen effectors and activate a cellular defense program. In Arabidopsis thaliana (Arabidopsis) Resistance to Ralstonia solanacearum 1 (RRS1‐R) and Resistance to Pseudomonas syringae 4 (RPS4) function together to recognize the unrelated bacterial effectors PopP2 and AvrRps4. In the plant cell nucleus, the RRS1‐R/RPS4 complex binds to and signals the presence of AvrRps4 or PopP2.

 

The exact mechanism underlying NLR signaling and immunity activation remains to be elucidated. Using genetic and biochemical approaches we characterized the intragenic suppressors of sensitive to low humidity 1 (slh1), a temperature‐sensitive auto‐immune allele of RRS1‐R.

 

Our analyses identified 5 amino acid residues that contribute to RRS1‐RSLH1 auto‐activity. We investigated the role of these residues in the RRS1‐R allele by genetic complementation and found that C15 in the TIR domain and L816 in the LRR domain were also important for effector recognition. Further characterization of the intragenic suppressive mutations located in the RRS1‐R TIR domain revealed differing requirements for RRS1‐R/RPS4‐dependent autoimmunity and effector‐triggered immunity.

 

Our results provide novel information about the mechanisms that, in turn, hold an NLR protein complex inactive and allow adequate activation in the presence of pathogens.

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Nature Protocols: Speed breeding in growth chambers and glasshouses for crop breeding and model plant research (2018)

Nature Protocols: Speed breeding in growth chambers and glasshouses for crop breeding and model plant research (2018) | Publications from The Sainsbury Laboratory | Scoop.it

‘Speed breeding’ (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants’ daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber–based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.

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Molecular Plant Pathology: Kinase activity of SOBIR1 and BAK1 is required for immune signalling (2018)

Molecular Plant Pathology: Kinase activity of SOBIR1 and BAK1 is required for immune signalling (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Leucine‐rich repeat‐receptor‐like proteins (LRR‐RLPs) and LRR‐receptor‐like kinases (LRR‐RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR‐RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR‐RLK Suppressor Of BIR1‐1/EVERSHED (SOBIR1/EVR) to form signalling‐competent receptor complexes. Ligand perception by LRR‐RLPs initiates recruitment of the co‐receptor BRI1‐Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR‐RLP/SOBIR1 complex, thereby activating LRR‐RLP‐mediated immunity. We employed phosphorylation analysis of in planta‐produced proteins, live‐cell imaging, gene silencing, and co‐immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses upon its overexpression in planta, is highly phosphorylated. Moreover, in addition to kinase activity of SOBIR1 itself, kinase‐active BAK1 is essential for AtSOBIR1‐induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered upon perception of Avr4, from the extracellular pathogenic fungus Cladosporium fulvum, by the tomato LRR‐RLP Cf‐4, depends on kinase‐active BAK1. We argue that, besides trans‐autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling‐competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP‐mediated immunity.

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bioRxiv: N-terminal β-strand underpins biochemical specialization of an ATG8 isoform (2018)

bioRxiv: N-terminal β-strand underpins biochemical specialization of an ATG8 isoform (2018) | Publications from The Sainsbury Laboratory | Scoop.it
ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation followed by mass spectrometry, to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand -and, in particular, a single amino acid polymorphism- underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein′s ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal β-strand shapes the ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
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New Phytologist: Albugo candida race diversity, ploidy and host‐associated microbes revealed using DNA sequence capture on diseased plants in the field (2018)

New Phytologist: Albugo candida race diversity, ploidy and host‐associated microbes revealed using DNA sequence capture on diseased plants in the field (2018) | Publications from The Sainsbury Laboratory | Scoop.it
  • Physiological races of the oomycete Albugo candida are biotrophic pathogens of diverse plant species, primarily the Brassicaceae, and cause infections that suppress host immunity to other pathogens. However, A. candida race diversity and the consequences of host immunosuppression are poorly understood in the field.
  • We report a method that enables sequencing of DNA of plant pathogens and plant‐associated microbes directly from field samples (Pathogen Enrichment Sequencing: PenSeq). We apply this method to explore race diversity in A. candida and to detect A. candida‐associated microbes in the field (91 A. candida‐infected plants).
  • We show with unprecedented resolution that each host plant species supports colonization by one of 17 distinct phylogenetic lineages, each with an unique repertoire of effector candidate alleles. These data reveal the crucial role of sexual and asexual reproduction, polyploidy and host domestication in A. candidaspecialization on distinct plant species. Our bait design also enabled phylogenetic assignment of DNA sequences from bacteria and fungi from plants in the field.
  • This paper shows that targeted sequencing has a great potential for the study of pathogen populations while they are colonizing their hosts. This method could be applied to other microbes, especially to those that cannot be cultured.
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Current Biology: Quick Guide: Appressoria (2019)

Current Biology: Quick Guide: Appressoria (2019) | Publications from The Sainsbury Laboratory | Scoop.it

What are appressoria? Appressoria are specialised infection structures used by many disease-causing microorganisms to breach the outer surface of a host plant or animal, and thereby gain entry to internal tissues. Appressoria are made by a wide range of disease-causing microbes — and even by parasitic plants — and they come in many different shapes and sizes. They are, however, best known in plant pathogenic fungi, in which appressoria have been most intensively studied because of their importance to some of the most devastating diseases affecting world agriculture. Appressoria are necessary for rusts, powdery mildews and blast diseases, which affect the major cereal crops of the world, as well as devastating oomycete diseases like potato late blight.

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Nature Biotech: Resistance gene cloning from a wild crop relative by sequence capture and association genetics (2019)

Nature Biotech: Resistance gene cloning from a wild crop relative by sequence capture and association genetics (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.

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Mol Plant Path: Pyricularia graminis‐tritici is not the correct species name for the wheat blast fungus: response to Ceresini et al. (MPP 20:2) - (2019)

Mol Plant Path: Pyricularia graminis‐tritici is not the correct species name for the wheat blast fungus: response to Ceresini et al. (MPP 20:2) - (2019) | Publications from The Sainsbury Laboratory | Scoop.it

In a review article published in this issue of Molecular Plant Pathology, Ceresini et al. (2019) wrongly treat the wheat blast fungus as a new species, Pyricularia graminis‐tritici (Pygt), following the proposal of Castroagudin et al. (2016). Despite the host specificity implied by the name Pygt, the proposed species concept includes isolates that cause major disease epidemics on finger millet and turf grasses (Castroagudin et al., 2016, 2017). These authors also conclude, based on little evidence, that ‘the hypothesis of grass‐specific populations for the overall Pyricularia oryzae species complex is falsified’. In addition, they stress that the rice blast fungus, which they describe as P. oryzae, ‘may not provide a suitable model for understanding the biology of Pygt’. All of these conclusions are misinformed and have serious consequences. International quarantine regulations are needed to block the movement of this fearsome seed‐borne blast fungus through the trade of seed or grain. The Pygt designation magnifies the challenge by grouping the dangerous, highly aggressive wheat pathogens from South America and Bangladesh, which are readily distinguishable from other P. oryzae lineages, with non‐wheat pathogens that are already found worldwide and are not known to be virulent on wheat or rice. Careful biological analysis of wheat blast host–pathogen interactions has clearly shown that studies of other host‐adapted forms of the fungus are relevant to an understanding of wheat blast and the development of new methods for disease control. Here, we summarize the overwhelming evidence that supports the alternative, internationally recognized designation of Pyricularia oryzae (synonym Magnaporthe oryzae; Zhang et al.2016) as a single species divided into host‐adapted lineages with limited primary host ranges. We also delineate the errors that led Ceresini et al. (2019) to their false conclusions. The same discussion applies to a second recently published review on the same topic (Ceresini et al.2018).

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bioRxiv: Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistance (2019)

bioRxiv: Cross-reactivity of a rice NLR immune receptor to distinct effectors from the blast pathogen leads to partial disease resistance (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Unconventional integrated domains in plant intracellular immune receptors (NLRs) can directly bind translocated pathogen effector proteins to initiate an immune response. The rice immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the Magnaporthe oryzae effectors AVR-Pik and AVR-Pia, respectively. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains maintain specificity of recognition, even for structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance towards the mis-matched effector AVR-Pia in planta, and that the Pikp-HMA domain binds AVR-Pia in vitro. The HMA domain from another Pik-1 allele, Pikm, is unable to bind AVR-Pia, and does not trigger a response in plants. The crystal structure of Pikp-HMA bound to AVR-Pia reveals a different binding interface compared to AVR-Pik effectors, suggesting plasticity in integrated domain/effector interactions. This work shows how a single NLR can bait multiple pathogen effectors via an integrated domain, and may enable engineering immune receptors with extended disease resistance profiles.

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MPMI: Widely-conserved attenuation of plant MAMP-induced calcium influx by bacteria depends on multiple virulence factors and may involve desensitization of host pattern recognition receptors (2019)

MPMI: Widely-conserved attenuation of plant MAMP-induced calcium influx by bacteria depends on multiple virulence factors and may involve desensitization of host pattern recognition receptors (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Successful pathogens must efficiently defeat or delay host immune responses, including those triggered by release or exposure of microbe-associated molecular patterns (MAMPs). Knowledge on the molecular details leading to this phenomenon in plant-pathogen interactions is still scarce. We took advantage of the well-established Arabidopsis thaliana-Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) patho-system to explore the molecular prerequisites for the suppression of MAMP-triggered host defense by the bacterial invader. Using a transgenic Arabidopsis line expressing the calcium sensor apoaequorin, we discovered that Pst DC3000 colonization results in a complete inhibition of MAMP-induced cytosolic calcium influx, a key event of immediate-early host immune signaling. A range of further plant-associated bacterial species is also able to prevent, either partially or fully, the MAMP-triggered cytosolic calcium pattern. Genetic analysis revealed that this suppressive effect partially relies on the type III secretion system (T3SS), but cannot be attributed to individual members of the currently known arsenal of Pst DC3000 effector proteins. While the phytotoxin coronatine and bacterial flagellin individually are dispensable for the effective inhibition of MAMP-induced calcium signatures, they contribute to the attenuation of calcium influx in the absence of the bacterial T3SS. Our findings suggest that the capacity to interfere with early plant immune responses is a widespread ability among plant-associated bacteria that at least in Pst DC3000 requires the combinatorial effect of multiple virulence determinants. This may also include the desensitization of host pattern recognition receptors by the prolonged exposure to MAMPs during bacterial pathogenesis.

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PLoS One: Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis (2019)

PLoS One: Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis (2019) | Publications from The Sainsbury Laboratory | Scoop.it

Bacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

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New Phytologist: Diverse NLR immune receptors activate defence via the RPW8‐NLR NRG1 (2018)

New Phytologist: Diverse NLR immune receptors activate defence via the RPW8‐NLR NRG1 (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Most land‐plant genomes carry genes that encode RPW8‐NLR resistance (R) proteins. Angiosperms carry two RPW8‐NLR subclasses: ADR1 and NRG1. ADR1s act as ‘helper’ NLRs for multiple TIR‐ and CC‐NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co‐occurs with TIR‐NLR Resistance (R) genes. We tested if NRG1 is required for signalling of multiple TIR‐NLRs.

Using CRISPR mutagenesis, we obtained an nrg1a‐nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana.

 

These mutants are compromised in signalling of all TIR‐NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell‐death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss‐of‐function does not compromise the CC‐NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC‐NLRs) function is slightly compromised in an nrg1mutant. Thus, NRG1 is required for full TIR‐NLR function and contributes to the signalling of some CC‐NLRs.

 

Some NRG1‐dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.

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Nature Communications: A downy mildew effector evades recognition by polymorphism of expression and subcellular localization (2018)

Nature Communications: A downy mildew effector evades recognition by polymorphism of expression and subcellular localization (2018) | Publications from The Sainsbury Laboratory | Scoop.it
Plant pathogens have evolved to evade detection by their hosts. Here, Asai et al. show that virulent isolates of the oomycete Hyaloperonospora arabidopsidis can break resistance conferred by the Arabidopsis RPP4 resistance gene via variation in effector expression or subcellular localization.
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Traffic: The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure (2018)

Traffic: The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterise the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.

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PLoS Pathogens: REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement (2018)

PLoS Pathogens: REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thalianaCALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.

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Genome Biology: Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions (2018)

Genome Biology: Phytophthora methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions (2018) | Publications from The Sainsbury Laboratory | Scoop.it
Filamentous plant pathogen genomes often display a bipartite architecture with gene-sparse, repeat-rich compartments serving as a cradle for adaptive evolution. The extent to which this two-speed genome architecture is associated with genome-wide DNA modifications is unknown. We show that the oomycetes Phytophthora infestans and Phytophthora sojae possess functional adenine N6-methylation (6mA) methyltransferases that modulate patterns of 6mA marks across the genome. In contrast, 5-methylcytosine could not be detected in these species. Methylated DNA IP sequencing (MeDIP-seq) of each species reveals 6mA is depleted around the transcription start sites (TSSs) and is associated with lowly expressed genes, particularly transposable elements. Genes occupying the gene-sparse regions have higher levels of 6mA in both genomes, possibly implicating the methylome in adaptive evolution. All six putative adenine methyltransferases from P. infestans and P. sojae, except PsDAMT2, display robust enzymatic activities. Surprisingly, single knockouts in P. sojae significantly reduce in vivo 6mA levels, indicating that the three enzymes are not fully redundant. MeDIP-seq of the psdamt3 mutant reveals uneven 6mA methylation reduction across genes, suggesting that PsDAMT3 may have a preference for gene body methylation after the TSS. Furthermore, transposable elements such as DNA elements are more active in the psdamt3 mutant. A large number of genes, particularly those from the adaptive genomic compartment, are differentially expressed. Our findings provide evidence that 6mA modification is potentially an epigenetic mark in Phytophthora genomes, and complex patterns of 6mA methylation may be associated with adaptive evolution in these important plant pathogens.
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New Phytologist: Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector‐ and PAMP‐triggered immunity in plants (2018)

New Phytologist: Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector‐ and PAMP‐triggered immunity in plants (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Plant immunity consists of two arms: pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI), induced by surface‐localized receptors, and effector‐triggered immunity (ETI), induced by intracellular receptors. Despite the little structural similarity, both receptor types activate similar responses with different dynamics.

To better understand phosphorylation events during ETI, we employed a phosphoproteomic screen using an inducible expression system of the bacterial effector avrRpt2 in Arabidopsis thaliana and identified 109 differentially phosphorylated residues of membrane‐associated proteins upon activation of the intracellular RPS2 receptor.

Interestingly, several RPS2‐regulated phosphosites overlap with sites that are regulated during PTI, suggesting that these phosphosites may be convergent points of both signaling arms. Moreover, some of these sites are residues of important defense components including the NADPH oxidase RBOHD, ABC‐transporter PEN3, calcium‐ATPase ACA8, non‐canonical Gα protein XLG2, and H+‐ATPases. In particular, we found that S343 and S347 of RBOHD are common phosphorylation targets during PTI and ETI. Our mutational analyses showed that these sites are required for the production of reactive oxygen species during both PTI and ETI, and immunity against avirulent bacteria and virulent necrotrophic fungus.

We provide, for the first time, large‐scale phosphoproteome data of ETI thereby suggesting crucial roles of common phosphosites in plant immunity.

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