Publications from The Sainsbury Laboratory
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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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New Phytologist: Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes (2018)

New Phytologist: Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes (2018) | Publications from The Sainsbury Laboratory | Scoop.it
  • The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world‐wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor.
  • Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome.
  • PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes.
  • PenSeq represents a cost‐effective alternative to whole‐genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens.
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PLos One: Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum (2018)

PLos One: Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum (2018) | Publications from The Sainsbury Laboratory | Scoop.it
The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops.
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PLoS Genetics: The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates (2018)

PLoS Genetics: The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates (2018) | Publications from The Sainsbury Laboratory | Scoop.it
Author summary Plants are constantly exposed to a multitude of potential pathogens but remain immune to most of these due to a multilayered immune system. Pathogens have specialized by adapting to certain host plants and their defense barriers. Most of our understanding of plant-pathogen interactions stems from these highly specialized interactions, because they are characterized by qualitative interactions (resistant or susceptible). It has generally been assumed that the genetic and molecular basis of resistance to non-adapted pathogens is fundamentally different, as either no variation exists in a species (complete immunity) or variation encompasses only early pathogen invasion (colonization), but not full susceptibility. We have studied the interaction between the agronomically important fungal stripe rust pathogen (Puccinia striiformis) of wheat and barley with the wild grass species Brachypodium distachyon. Rust infections consist of two stages: colonization of plant tissues followed by a reproductive phase. We identified natural variation for the degree of P. striiformis colonization in different B. distachyon accessions and dissected the genetic architecture controlling resistance at this infection stage. QTLs conferring resistance possessed several characteristics similar to adapted host systems, indicating that resistance to adapted and non-adapted pathogens are not intrinsically different.
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Sci Reports: CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus (2018)

Sci Reports: CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus (2018) | Publications from The Sainsbury Laboratory | Scoop.it

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to Moryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in Moryzae and are likely to be broadly applicable to other fungal species.

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Phytopathology: Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates (2018)

Phytopathology: Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates (2018) | Publications from The Sainsbury Laboratory | Scoop.it

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the UK. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.

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Trends Plant Sci: Pathogens Suppress Host Transcription Factors for Rampant Proliferation (2018)

Trends Plant Sci: Pathogens Suppress Host Transcription Factors for Rampant Proliferation (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Root pathogen Verticillium dahliae deploys an effector called VdSCP41 into plants to disrupt the functions of SARD1 and CBP60g, two central transcriptional regulators of plant immunity. This provides new tools to dissect transcriptional regulation of tissue-specific immunity in the root and to understand dynamic interactions between plants and root-associated microorganisms.

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Nature: Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling (2018)

Nature: Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Multicellular organisms use cell-surface receptor kinases to sense and process extracellular signals. Many plant receptor kinases are activated by the formation of ligand-induced complexes with shape-complementary co-receptors1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat receptor kinases (LRR-RKs) to control immunity, growth and development2. Here we report key regulatory events that control the function of BAK1 and, more generally, LRR-RKs. Through a combination of phosphoproteomics and targeted mutagenesis, we identified conserved phosphosites that are required for the immune function of BAK1 in Arabidopsis thaliana. Notably, these phosphosites are not required for BAK1-dependent brassinosteroid-regulated growth. In addition to revealing a critical role for the phosphorylation of the BAK1 C-terminal tail, we identified a conserved tyrosine phosphosite that may be required for the function of the majority of Arabidopsis LRR-RKs, and which separates them into two distinct functional classes based on the presence or absence of this tyrosine. Our results suggest a phosphocode-based dichotomy of BAK1 function in plant signalling, and provide insights into receptor kinase activation that have broad implications for our understanding of how plants respond to their changing environment.

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Plant Biotech J: Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen‐fixing rhizobial symbiosis (2018)

Plant Biotech J: Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen‐fixing rhizobial symbiosis (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFRMedicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.

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Curr Opin Plant Biol: Regulation of pattern recognition receptor signalling by phosphorylation and ubiquitination (2018)

Curr Opin Plant Biol: Regulation of pattern recognition receptor signalling by phosphorylation and ubiquitination (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Our understanding of how plant innate immunity is triggered and regulated has seen tremendous progress over the last decade, with many important players identified in the model systems Arabidopsis thaliana and Oryza sativa (rice). Identification of these components has come from both genetic screens as well as from proteomics approaches. While genetic approaches are powerful tools of discovery to identify key components in a signalling pathway, the application of genetics is limited when dealing with redundancy or when mutations cause lethal phenotypes. This is where the complementary strength of proteomics has brought major advances. With the advancement in technology in the field of proteomics, not only the proteins involved in innate immune signalling and responses have been identified, but also the posttranslational modifications (PTMs) that these proteins carry have been mapped in more intricate detail and shown to be functionally relevant in both genetic and biochemical terms. Here we discuss the most recent progress in pattern recognition receptor (PRR) signalling with a focus on phosphorylation and ubiquitination.

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Nature Plants: Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen (2018)

Nature Plants: Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Accelerated adaptive evolution is a hallmark of plant–pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture.

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BMC Evol Biol: Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen (2018)

BMC Evol Biol: Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Outbreaks caused by asexual lineages of fungal and oomycete pathogens are a continuing threat to crops, wild animals and natural ecosystems (Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ, Nature 484:186–194, 2012; Kupferschmidt K, Science 337:636–638, 2012). However, the mechanisms underlying genome evolution and phenotypic plasticity in asexual eukaryotic microbes remain poorly understood (Seidl MF, Thomma BP, BioEssays 36:335–345, 2014). Ever since the 19th century Irish famine, the oomycete Phytophthora infestans has caused recurrent outbreaks on potato and tomato crops that have been primarily caused by the successive rise and migration of pandemic asexual lineages (Goodwin SB, Cohen BA, Fry WE, Proc Natl Acad Sci USA 91:11591–11595, 1994; Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10:e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, Deahl KL, Farrer RA, Gilroy EM, Goss EM, et al. PLoS Pathog 8:e1002940, 2012). However, the dynamics of genome evolution within these clonal lineages have not been determined. The objective of this study was to use a comparative genomics and transcriptomics approach to determine the molecular mechanisms that underpin phenotypic variation within a clonal lineage of P. infestans.

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Sci Signal: The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana (2018)

Sci Signal: The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana (2018) | Publications from The Sainsbury Laboratory | Scoop.it
When plant cell walls are damaged by mechanical or biochemical means, the cells initiate adaptive responses to repair the walls and prevent infection. Whereas the cell wall integrity (CWI) maintenance mechanism monitors the status of the cell wall and initiates adaptive cellular responses to damage, pattern-triggered immunity (PTI) induces both cellular and systemic responses that limit infection. Engelsdorf et al . found that osmosensitive alterations in the mechanical properties of the cell wall were important for inducing the adaptive responses to cell wall damage. The receptor-like kinases THE1 and FEI2 and the ion channel MCA1 were required for stimulating CWI-dependent responses to damage. Cell wall damage also induced the expression of host defense peptides that promoted PTI and dampened CWI-dependent processes. The CWI mechanism still elicited damage responses even when PTI was impaired, suggesting that it serves as a failsafe to protect plants even if the immune response is compromised.
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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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Sci Rep: A rust fungal effector binds plant DNA and modulates transcription (2018)

Sci Rep: A rust fungal effector binds plant DNA and modulates transcription (2018) | Publications from The Sainsbury Laboratory | Scoop.it

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thalianaand Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thalianarepressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

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PLoS Genetics: Components of Brachypodium distachyon resistance to nonadapted wheat stripe rust pathogens are simply inherited (2018)

PLoS Genetics: Components of Brachypodium distachyon resistance to nonadapted wheat stripe rust pathogens are simply inherited (2018) | Publications from The Sainsbury Laboratory | Scoop.it
Author summary Plant pathogens are specialists and can colonise only a limited number of plant species (hosts). Pathogen infection of a plant that is not a host of the disease often results in an active plant defense response. This poorly characterised defense response is durable as phytopathogens rarely successfully colonise new hosts. The ability to transfer this resistance to host plants would be highly beneficial in protecting crops from disease. However, this resistance has been difficult to genetically dissect as all members of a plant species are resistant. Here we show that some accessions of the model grass Brachypodium distachyon show some variation in their ability to suppress colonisation by the wheat stripe rust pathogen Puccinia striiformis. Brachypodium is not a host species of wheat stripe rust disease and no accessions are fully susceptible, however, some allow more pathogen growth than others. We have exploited these relatively subtle phenotypic differences to genetically dissect this difference in resistance and identified two Brachypodium loci that contribute increased resistance to the nonadapted wheat stripe rust pathogen.
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Plant Biotech J: The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity (2018)

Plant Biotech J: The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signaling events, defense gene expression, and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2 or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.

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PNAS: Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors (2018)

PNAS: Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Plants and animals carry intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. How NLR receptors activate defense on perceiving pathogen molecules is poorly understood, especially in plants. Some NLRs function in pairs, with one NLR carrying a domain that mimics a pathogen effector target. Effector action on this domain activates the second “helper” NLR. In the Arabidopsis RPS4 and RRS1 pair, RRS1 carries a WRKY transcription factor domain targeted by bacterial effectors AvrRps4 and PopP2. We monitored conformational changes in RPS4–RRS1 during activation and developed a “molecular padlock” to reversibly restrict such changes. This revealed domains within RRS1 required to keep the RRS1–RPS4 complex inactive prior to effector detection, and specific domain–domain interactions whose disruption or modification contributes to defense activation.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R–RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector’s authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY–domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY–domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a “reversibly closed” conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

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New Phytologist: Comparing Arabidopsis receptor kinase and receptor protein‐mediated immune signaling reveals BIK1‐dependent differences (2018)

New Phytologist: Comparing Arabidopsis receptor kinase and receptor protein‐mediated immune signaling reveals BIK1‐dependent differences (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families.

We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling.

Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23‐signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity.

Our study unveils unexpected differences in two closely‐related receptor systems and reports a new negative role of BIK1 in plant immunity.

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bioRxiv: Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis (2018)

bioRxiv: Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis (2018) | 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: Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL‐INDUCED CELL DEATH1 (2018)

New Phytologist: Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL‐INDUCED CELL DEATH1 (2018) | Publications from The Sainsbury Laboratory | Scoop.it
  • The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and ArabidopsisRADICAL‐INDUCED CELL DEATH1 (RCD1).
  • We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes.
  • We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)‐induced defense genes and alters plant growth responses to light. HaRxL106‐mediated suppression of immunity is abolished in RCD1 loss‐of‐function mutants. We report that RCD1‐type proteins are phosphorylated, and we identified Mut9‐like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1‐interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA‐induced defense marker gene expression compared with wild‐type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling.
  • Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.
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Science: Shifting the limits in wheat research and breeding using a fully annotated reference genome (2018)

Science: Shifting the limits in wheat research and breeding using a fully annotated reference genome (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Wheat is one of the major sources of food for much of the world. However, because bread wheat's genome is a large hybrid mix of three separate subgenomes, it has been difficult to produce a high-quality reference sequence. Using recent advances in sequencing, the International Wheat Genome Sequencing Consortium presents an annotated reference genome with a detailed analysis of gene content among subgenomes and the structural organization for all the chromosomes. Examples of quantitative trait mapping and CRISPR-based genome modification show the potential for using this genome in agricultural research and breeding. Ramírez-González et al. exploited the fruits of this endeavor to identify tissue-specific biased gene expression and coexpression networks during development and exposure to stress. These resources will accelerate our understanding of the genetic basis of bread wheat.

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bioRxiv: DeepStomata: Facial Recognition Technology for Automated Stomatal Aperture Measurement (2018)

bioRxiv: DeepStomata: Facial Recognition Technology for Automated Stomatal Aperture Measurement (2018) | Publications from The Sainsbury Laboratory | Scoop.it
Stomata are an attractive model for studying the physiological responses of plants to various environmental stimuli. Of the morphological parameters that represent the degree of stomatal opening, the length of the minor axis of the stomatal pore (the stomatal aperture) has been most commonly used to dissect the molecular basis of its regulation. Measuring stomatal apertures is time consuming and labour intensive, preventing their use in large-scale studies. Here, we completely automated this process by developing a program called DeepStomata, which combines stomatal region detection and pore isolation by image segmentation. The former, which comprises histograms of oriented gradients (HOG)-based stomatal detection and the convolutional neural network (CNN)-based classification of open/closed-state stomata, acts as an efficient conditional branch in the workflow to selectively quantify the pores of open stomata. An analysis of batches of images showed that the accuracy of our automated aperture measurements was equivalent to that of manual measurements, however had higher sensitivity (i,e., lower false negative rate ) and the process speed was at least 80 times faster. The outstanding performance of our proposed method for automating a laborious and repetitive task will allow researchers to focus on deciphering complex phenomena.
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Ann Rev Phytopath: CRISPR Crops: Plant Genome Editing Toward Disease Resistance (2018)

Ann Rev Phytopath: CRISPR Crops: Plant Genome Editing Toward Disease Resistance (2018) | Publications from The Sainsbury Laboratory | Scoop.it

Genome editing by sequence-specific nucleases (SSNs) has revolutionized biology by enabling targeted modifications of genomes. Although routine plant genome editing emerged only a few years ago, we are already witnessing the first applications to improve disease resistance. In particular, CRISPR-Cas9 has democratized the use of genome editing in plants thanks to the ease and robustness of this method. Here, we review the recent developments in plant genome editing and its application to enhancing disease resistance against plant pathogens. In the future, bioedited disease resistant crops will become a standard tool in plant breeding.

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GigaScience: A workflow for simplified analysis of ATAC-cap-seq data in R (2018)

GigaScience: A workflow for simplified analysis of ATAC-cap-seq data in R (2018) | Publications from The Sainsbury Laboratory | Scoop.it

ATAC-cap-seq is a high-throughput sequencing method that combines ATAC-seq with targeted nucleic acid enrichment of precipitated DNA fragment. There are increased analytical difficulties arising from working with a set of regions of interest that may be small in number and biologically dependent. Common statistical pipelines for RNAseq might be assumed to apply but can give misleading results on ATAC-cap-seq data. A tool is needed to allow a non-specialist user to quickly and easily summarise data and apply sensible and effective normalisation and analysis.

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