Oomycetes, or water moulds, are fungal-like organisms phylogenetically related to algae. They cause devastating diseases in both plants and animals. Here, we describe seven oomycete species that are emerging or re-emerging threats to agriculture, horticulture, aquaculture and natural ecosystems. They include the plant pathogens Phytophthora infestans, Phytophthora palmivora, Phytophthora ramorum, Plasmopara obducens, and the animal pathogens Aphanomyces invadans, Saprolegnia parasitica and Halioticida noduliformans. For each species, we describe its pathology, importance and impact, discuss why it is an emerging threat and briefly review current research activities.
Selective autophagy is a conserved homeostatic pathway that involves engulfment of specific cargo molecules into specialized organelles called autophagosomes. The ubiquitin-like protein ATG8 is a central player of the autophagy network that decorates autophagosomes and binds to numerous cargo receptors. Although highly conserved across eukaryotes, ATG8 diversified from a single protein in algae to multiple isoforms in higher plants. We present a phylogenetic overview of 376 ATG8 proteins across the green plant lineage that revealed family-specific ATG8 clades. Because these clades differ in fixed amino acid polymorphisms, they provide a mechanistic framework to test whether distinct ATG8 clades are functionally specialized. We propose that ATG8 expansion may have contributed to the diversification of selective autophagy pathways in plants.
Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family—key genes in plant immune systems.
Plant and animal nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins often function in pairs to mediate innate immunity to pathogens. However, the degree to which NLR proteins form signaling networks beyond genetically linked pairs is poorly understood. In this study, we discovered that a large NLR immune signaling network with a complex genetic architecture confers immunity to oomycetes, bacteria, viruses, nematodes, and insects. The network emerged over 100 million years ago from a linked NLR pair that diversified into up to one half of the NLR of asterid plants. We propose that this NLR network increases robustness of immune signaling to counteract rapidly evolving plant pathogens.
High humidity has a strong influence on the development of numerous diseases affecting the above-ground parts of plants (the phyllosphere) in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies have emphasized immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another important step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immunodeficient Arabidopsis thaliana under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (AtMIN7) of HopM1 and in pattern-triggered immunity could not only be used to reconstitute the basic features of bacterial infection, but also exhibited humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere–bacterial interactions.
Poly(ADP-ribosyl)ation, or PARylation, was first described over 50 years ago. Since then, our understanding of the biochemistry and enzymology of this protein modification has significantly progressed. PARylation has long been associated with DNA damage and DNA repair as well as genotoxic stress [1,2]. However, over the last two decades this has expanded to chromatin remodelling, DNA replication, transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism . Most eukaryotes, except yeasts, have genes encoding poly (ADP-ribose) polymerases (PARPs) and poly (ADP-ribose) glycohydrolases (PARGs), and our knowledge on PARylation is primarily based on studies in metazoans. In plants, however, mechanistic understanding of the role of ADP-ribosylation in stress response is still lacking. In this issue of EMBO Reports, Feng et al  identify the first set of PARylated plant proteins and show that in vivo PARylation of one of these proteins, a factor named DAWDLE, is important for its role in plant immunity. See also: B Feng et al
Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease “shaving” of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original “hppK” method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1–16 transmembrane segments. This method allowed us to identify 13 “missing proteins” — proteins with no previous evidence on protein level.
Plants detect conserved molecular patterns of pathogens via cell surface-localized receptors, such as the flagellin receptor kinase FLS2, that initiate effective plant immunity. Activated FLS2 is endocytosed, but the degree to which other receptor kinases exhibit similar spatiotemporal dynamics remains unclear. We show that internalization into a common endosomal pathway after ligand perception is a general phenomenon of the tested receptor kinases, including the danger peptide receptor PEPR1. FLS2 endocytosis is mediated by clathrin and is uncoupled from the regulation of acute pathogen-induced responses, but is involved in steady defenses and contributes to plant immunity against bacterial infection. We propose that clathrin-dependent internalization of ligand-activated receptor kinases into a common endosomal pathway facilitates the responses required for full plant immunity.
Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology.
Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.
Parasitic plants are a constraint on agriculture worldwide. Cuscuta reflexa is a stem holoparasite that infests most dicotyledonous plants. One exception is tomato, which is resistant to C. reflexa. We discovered that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after perception of microbe-associated molecular patterns. We identified the cell surface receptor-like protein CUSCUTA RECEPTOR 1 (CuRe1) as essential for the perception of this parasite-associated molecular pattern. CuRe1 is sufficient to confer responsiveness to the Cuscuta factor and increased resistance to parasitic C. reflexa when heterologously expressed in otherwise susceptible host plants. Our findings reveal that plants recognize parasitic plants in a manner similar to perception of microbial pathogens.
CRISPR-Cas is a recent and powerful edition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of T. pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct wa assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤ 61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.
Intracellular NLR (Nucleotide-binding domain and Leucine-rich Repeat-containing) receptors are sensitive monitors that detect pathogen invasion of both plant and animal cells. NLRs confer recognition of diverse molecules associated with pathogen invasion. NLRs must exhibit strict intramolecular controls to avoid harmful ectopic activation in the absence of pathogens. Recent discoveries have elucidated the assembly and structure of oligomeric NLR signalling complexes in animals, and provided insights into how these complexes act as scaffolds for signal transduction. In plants, recent advances have provided novel insights into signalling-competent NLRs, and into the myriad strategies that diverse plant NLRs use to recognise pathogens. Here, we review recent insights into the NLR biology of both animals and plants. By assessing commonalities and differences between kingdoms, we are able to develop a more complete understanding of NLR function.
Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae. AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto. This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance.
Puccinia striiformis f. sp. hordei, the causal agent of barley stripe rust, is a destructive fungal pathogen that significantly affects barley cultivation. A major constraint in breeding resistant cultivars is the lack of mapping information of resistance (R) genes and their introgression into adapted germplasm. A considerable number of R genes have been described in barley to P. striiformis f. sp. hordei, but only a few loci have been mapped. Previously, Chen and Line (1999) reported two recessive seedling resistance loci in the Ethiopian landrace HOR 1428. In this study, we map two loci that confer resistance to P. striiformis f. sp. hordei in HOR 1428, which are located on chromosomes 3H and 5H. Both loci act as additive effect QTLs, each explaining approximately 20% of the phenotypic variation. We backcrossed HOR 1428 to the cv. Manchuria and selected based on markers flanking the RpsHOR128-5H locus. Saturation of the RpsHOR1428-5H locus with markers in the region found KASP marker K_1_0292 in complete coupling with resistance to P. striiformis f. sp. hordei and was designated Rps9. Isolation of Rps9 and flanking markers will facilitate the deployment of this genetic resource into existing programs for P. striiformis f. sp. hordei resistance.
CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.
The innate immune systems of both plants and animals depend on the ability to recognize pathogen-derived molecules and stimulate a defense response. Jones et al. review how that common function is achieved in such diverse kingdoms by similar molecules. The recognition system is built for hair-trigger sensitivity and constructed in a modular manner. Understanding such features could be useful in building new pathways through synthetic biology, whether for broadening disease defenses or constructing new signal-response circuits.
Fungi of the Pucciniales order cause rust diseases, which altogether affect thousands of plant species worldwide and pose major threat to several crops. How rust effectors - virulence proteins delivered into infected tissues to modulate host functions - contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplars and its genome encodes 1,184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of GFP-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth. This article is protected by copyright. All rights reserved.
Somatic Embryogenesis Receptor like Kinases (SERKs) are ligand binding co-receptors that are able to combine with different ligand perceiving receptors such as Brassinosteroid Insensitive 1 (BRI1) and Flagellin-Sensitive 2 (FLS2). Phenotypical analysis of serk single mutants is not straightforward because multiple pathways can be affected, while redundancy is observed for a single phenotype. For example, serk1serk3 double mutant roots are insensitive towards brassinosteroids but have a phenotype different from bri1 mutant roots. To decipher these effects, 4-day-old Arabidopsis roots were studied using microarray analysis. 698 genes, involved in multiple biological processes, were found to be differentially regulated in serk1-3serk3-2 double mutants. About half of these are related to BR signalling. The remainder appears to be unlinked to BRs and related to primary and secondary metabolism. In addition, methionine derived glucosinolate biosynthesis genes are upregulated, which was verified by metabolite profiling. The results also show that the gene expression pattern in serk3-2 mutant roots is similar to that of the serk1-3serk3-2 double mutant roots. This confirms the existence of partial redundancy between SERK3 and SERK1 as well as the promoting or repressive activity of a single co-receptor in multiple simultaneously active pathways.
In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields.
Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae.
Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.
The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VRN1, VRN2, and FT. Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon, yet identified considerable ambiguity surrounding the role of VRN2. To investigate the natural diversity governing flowering time pathways in a non-domesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a SNP-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1. Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional QTLs suggests that greater complexity underlies flowering time in this non-domesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaeceae, as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.
Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors.
Recognition of pathogen-derived molecules by pattern recognition receptors (PRRs) is a common feature of both animal and plant innate immune systems. In plants, PRR signalling is initiated at the cell surface by kinase complexes, resulting in the activation of immune responses that ward off microorganisms. However, the activation and amplitude of innate immune responses must be tightly controlled. In this Review, we summarize our knowledge of the early signalling events that follow PRR activation and describe the mechanisms that fine-tune immune signalling to maintain immune homeostasis. We also illustrate the mechanisms used by pathogens to inhibit innate immune signalling and discuss how the innate ability of plant cells to monitor the integrity of key immune components can lead to autoimmune phenotypes following genetic or pathogen-induced perturbations of these components.
Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8-family interacting motif (AIM) and perturbs host selective autophagy. However, the structural basis of this interaction remains unknown. Here we define the crystal structure of PexRD54, which comprises a modular architecture including five tandem repeat domains, with the AIM sequence presented at the disordered C-terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effectors AIM sequence, and established a model of the full-length PexRD54/ATG8CL complex using small angle X-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.
In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,741 hectares, about 16% of cultivated wheat area in Bangladesh, with yield losses reaching up to 100%. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. Population genomics analyses revealed that the outbreak was caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. We show that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.
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