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Plant pathogens adapt at speeds that challenge contemporary disease management strategies like the deployment of disease resistance genes. The strong evolutionary pressure to adapt, shapes pathogens' genomes, and comparative genomics has been instrumental in characterizing this process. With the aim to capture genomic variation at high resolution and study the processes contributing to adaptation, we here leverage an innovative, multi-genome method to construct and annotate the first pangenome graph of an oomycete plant pathogen. We expand on this approach by analysing the graph and creating synteny based single-copy orthogroups for all genes. We generated telomere-to-telomere genome assemblies of six genetically diverse isolates of the oomycete pathogen Peronospora effusa, the economically most important disease in cultivated spinach worldwide. The pangenome graph demonstrates that P. effusa genomes are highly conserved, both in chromosomal structure and gene content, and revealed the continued activity of transposable elements which are directly responsible for 80% of the observed variation between the isolates. While most genes are generally conserved, virulence related genes are highly variable between the isolates. Most of the variation is found in large gene clusters resulting from extensive copy-number expansion. Pangenome graph-based discovery can thus be effectively used to capture genomic variation at exceptional resolution, thereby providing a framework to study the biology and evolution of plant pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Skiadas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In plants, epitranscriptomic mark N6-adenine methylation (m6A) is dynamically regulated in response to environmental cues. However, little is known about m6A dynamics under biotic stresses and their role in environmental adaptation. Additionally, current methodologies limit the investigation of m6A dynamics at single-nucleotide resolution on specific RNA molecules. Using Oxford Nanopore Technology direct RNA sequencing and a neural network model, we show transcript-specific dynamics of m6A modification at single-nucleotide resolution during Hyaloperonospora arabidopsidis (Hpa) infection in Arabidopsis (Arabidopsis thaliana). In wild-type seedlings, pathogen infection causes a significant reduction in global m6A ratios, which corresponds with the activation of m6A-modified transcripts. Defect of m6A deposition in the m6A mutant hakai-1 mimics m6A reduction from Hpa infection at ∼70% of sites, resulting in constitutive overexpression of basal defense genes and enhanced resistance against the pathogen. Our results demonstrate that m6A dynamics impact defense response against Hpa, providing a promising target for future crop improvement strategies., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Microscopic imaging for studying plant-pathogen interactions is limited by its reliance on invasive histological techniques, like clearing and staining, or, for in vivo imaging, on complicated generation of transgenic pathogens. We present real-time 3D in vivo visualization of pathogen dynamics with label-free optical coherence tomography. Based on intrinsic signal fluctuations as tissue contrast we image filamentous pathogens and a nematode in vivo in 3D in plant tissue. We analyze 3D images of lettuce downy mildew infection (Bremia lactucae) to obtain hyphal volume and length in three different lettuce genotypes with different resistance levels showing the ability for precise (micro) phenotyping and quantification of the infection level. In addition, we demonstrate in vivo longitudinal imaging of the growth of individual pathogen (sub)structures with functional contrast on the pathogen micro-activity revealing pathogen vitality thereby opening a window on the underlying molecular processes., (© 2024. The Author(s).)

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes, microbe-associated molecular patterns (MAMPs), are sensed by plant cells as nonself molecules via pattern recognition receptors. While MAMPs of bacterial and fungal origin have been identified, reports on oomycete MAMPs are relatively limited. This study aimed to identify MAMPs from an oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato (Solanum tuberosum) as markers, two structurally different groups of elicitors, namely ceramides and diacylglycerols, were identified. P. infestans ceramides (Pi-Cer A, B, and D) induced ROS production, while diacylglycerol (Pi-DAG A and B), containing eicosapentaenoic acid (EPA) as a substructure, induced phytoalexins production in potato. The molecular patterns in Pi-Cers and Pi-DAGs essential for defense induction were identified as 9-methyl-4,8-sphingadienine (9Me-Spd) and 5,8,11,14-tetraene-type fatty acid (5,8,11,14-TEFA), respectively. These structures are not found in plants, but in oomycetes and fungi, indicating that they are microbe molecular patterns recognized by plants. When Arabidopsis (Arabidopsis thaliana) was treated with Pi-Cer D and EPA, partially overlapping but different sets of genes were induced. Furthermore, expression of some genes is upregulated only after the simultaneous treatment with Pi-Cer D and EPA, indicating that plants combine the signals from simultaneously recognized MAMPs to adapt their defense response to pathogens., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae , the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Chloroplasts play a crucial role in plant defense against pathogens, making them primary targets for pathogen effectors that suppress host immunity. This study characterizes the Plasmopara viticola CRN-like effector, PvCRN20, which interacts with DEG5 in the cytoplasm but not with its interacting protein, DEG8, which is located in the chloroplast. By transiently overexpressing in tobacco leaves, we show that PvCRN20 could inhibit INF1- and Bax-triggered cell death. Constitutive expression of PvCRN20 suppresses the accumulation of reactive oxygen species (ROS) and promotes pathogen colonization. PvCRN20 reduces DEG5 entry into chloroplasts, thereby disrupting DEG5 and DEG8 interactions in chloroplasts. Overexpression of VvDEG5 and VvDEG8 induces ROS accumulation and enhances grapevine resistance to P. viticola, whereas knockout of VvDEG8 represses ROS production and promotes P. viticola colonization. Consistently, ectopic expression of VvDEG5 and VvDEG8 in tobacco promotes chloroplast-derived ROS accumulation, whereas co-expression of PvCRN20 counteracted this promotion by VvDEG5. Therefore, DEG5 is essential for the virulence function of PvCRN20. Although PvCRN20 is located in both the nucleus and cytoplasm, only cytoplasmic PvCRN20 suppresses plant immunity and promotes pathogen infection. Our results reveal that PvCRN20 dampens plant defenses by repressing the chloroplast import of DEG5, thus reducing host ROS accumulation and facilitating pathogen colonization., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2 Cala2 effector was unknown. To reveal ATR2 Cala2 , an F 2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2 Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2 Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2 Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2 Emoy2 and ATR2 Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2 Cala2 . Stable and transient expression of ATR2 Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2 Cala2 -recognizing accessions., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Interactions between various microbial pathogens including viruses, bacteria, fungi, oomycetes, and their plant hosts have traditionally been the focus of phytopathology. In recent years, a significant and growing interest in the study of eukaryotic microorganisms not classified among fungi or oomycetes has emerged. Many of these protists establish complex interactions with photosynthetic hosts, and understanding these interactions is crucial in understanding the dynamics of these parasites within traditional and emerging types of farming, including marine aquaculture. Many phytopathogenic protists are biotrophs with complex polyphasic life cycles, which makes them difficult or impossible to culture, a fact reflected in a wide gap in the availability of comprehensive genomic data when compared to fungal and oomycete plant pathogens. Furthermore, our ability to use available genomic resources for these protists is limited by the broad taxonomic distance that these organisms span, which makes comparisons with other genomic datasets difficult. The current rapid progress in genomics and computational tools for the prediction of protein functions and interactions is revolutionizing the landscape in plant pathology. This is also opening novel possibilities, specifically for a deeper understanding of protist effectors. Tools like AlphaFold2 enable structure-based function prediction of effector candidates with divergent protein sequences. In turn, this allows us to ask better biological questions and, coupled with innovative experimental strategies, will lead into a new era of effector research, especially for protists, to expand our knowledge on these elusive pathogens and their interactions with photosynthetic hosts. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Variations in chromosome number are occasionally observed among oomycetes, a group that includes many plant pathogens, but the emergence of such variations and their effects on genome and virulence evolution remain ambiguous. We generated complete telomere-to-telomere genome assemblies for Phytophthora sojae, Globisporangium ultimum, Pythium oligandrum, and G. spinosum. Reconstructing the karyotype of the most recent common ancestor in Peronosporales revealed that frequent chromosome fusion and fission drove changes in chromosome number. Centromeres enriched with Copia-like transposons may contribute to chromosome fusion and fission events. Chromosome fusion facilitated the emergence of pathogenicity genes and their adaptive evolution. Effectors tended to duplicate in the sub-telomere regions of fused chromosomes, which exhibited evolutionary features distinct to the non-fused chromosomes. By integrating ancestral genomic dynamics and structural predictions, we have identified secreted Ankyrin repeat-containing proteins (ANKs) as a novel class of effectors in P. sojae. Phylogenetic analysis and experiments further revealed that ANK is a specifically expanded effector family in oomycetes. These results revealed chromosome dynamics in oomycete plant pathogens, and provided novel insights into karyotype and effector evolution., (© 2024. The Author(s).)

Nonmycorrhizal cluster root-forming species enhance the phosphorus (P) acquisition of mycorrhizal neighbours in P-impoverished megadiverse systems. However, whether mycorrhizal plants facilitate the defence of nonmycorrhizal plants against soil-borne pathogens, in return and via their symbiosis, remains unknown. We characterised growth and defence-related compounds in Banksia menziesii (nonmycorrhizal) and Eucalyptus todtiana (ectomycorrhizal, ECM) seedlings grown either in monoculture or mixture in a multifactorial glasshouse experiment involving ECM fungi and native oomycete pathogens. Roots of B. menziesii had higher levels of phytohormones (salicylic and jasmonic acids, jasmonoyl-isoleucine and 12-oxo-phytodienoic acid) than E. todtiana which further activated a salicylic acid-mediated defence response in roots of B. menziesii, but only in the presence of ECM fungi. We also found that B. menziesii induced a shift in the defence strategy of E. todtiana, from defence-related secondary metabolites (phenolic and flavonoid) towards induced phytohormone response pathways. We conclude that ECM fungi play a vital role in the interactions between mycorrhizal and nonmycorrhizal plants in a severely P-impoverished environment, by introducing a competitive component within the facilitation interaction between the two plant species with contrasting nutrient-acquisition strategies. This study sheds light on the interplay between beneficial and detrimental soil microbes that shape plant-plant interaction in severely nutrient-impoverished ecosystems., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Oomycetes are filamentous organisms that resemble fungi in terms of morphology and life cycle, primarily due to convergent evolution. The success of pathogenic oomycetes lies in their ability to adapt and overcome host resistance, occasionally transitioning to new hosts. During plant infection, these organisms secrete effector proteins and other compounds during plant infection, as a molecular arsenal that contributes to their pathogenic success. Genomic sequencing, transcriptomic analysis, and proteomic studies have revealed highly diverse effector repertoires among different oomycete pathogens, highlighting their adaptability and evolution potential.The obligate biotrophic oomycete Plasmopara viticola affects grapevine plants (Vitis vinifera L.) causing the downy mildew disease, with significant economic impact. This disease is devastating in Europe, leading to substantial production losses. Even though Plasmopara viticola is a well-known pathogen, to date there are scarce reviews summarising pathogenicity, virulence, the genetics and molecular mechanisms of interaction with grapevine.This review aims to explore the current knowledge of the infection strategy, lifecycle, effector molecules, and pathogenicity of Plasmopara viticola. The recent sequencing of the Plasmopara viticola genome has provided new insights into understanding the infection strategies employed by this pathogen. Additionally, we will highlight the contributions of omics technologies in unravelling the ongoing evolution of this oomycete, including the first in-plant proteome analysis of the pathogen., (© 2024. The Author(s).)

Cultivated sweet watermelon ( Citrullus lanatus ) is an important vegetable crop for millions of people around the world. There are limited sources of resistance to economically important diseases within C . lanatus , whereas C. amarus has a reservoir of traits that can be exploited to improve C . lanatus for resistance to biotic and abiotic stresses. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis , is an emerging threat to watermelon production. We screened 122 C . amarus accessions for resistance to CDM over two tests (environments). The accessions were genotyped by whole-genome resequencing to generate 2,126,759 single nucleotide polymorphic (SNP) markers. A genome-wide association study was deployed to uncover marker-trait associations and identify candidate genes underlying resistance to CDM. Our results indicate the presence of wide phenotypic variability (1.1 to 57.8%) for leaf area infection, representing a 50.7-fold variation for CDM resistance across the C . amarus germplasm collection. Broad-sense heritability estimate was 0.55, implying the presence of moderate genetic effects for resistance to CDM. The peak SNP markers associated with resistance to P. cubensis were located on chromosomes Ca03, Ca05, Ca07, and Ca11. The significant SNP markers accounted for up to 30% of the phenotypic variation and were associated with promising candidate genes encoding leucine-rich repeat receptor-like protein kinase and the WRKY transcription factor. This information will be useful in understanding the genetic architecture of the P. cubensis-Citrullus spp. patho-system as well as development of resources for genomics-assisted breeding for resistance to CDM in watermelon.

Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α-ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI-4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain-containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI-4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI-4 via the CACGTG element, leading to inhibition of VvGLYI-4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI-4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI-4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG-associated ROS homeostasis through a salicylic acid-mediated defense pathway., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Kiwifruit vine decline syndrome (KVDS) is a serious soil-borne disease that degrades the fine roots of both Actinidia chinensis var. deliciosa and var. chinensis . The disease seems to be the result of an interaction between several soil-borne pathogens, mostly oomycetes, and waterlogging. This work investigates the pathogenicity of the oomycete Phytopythium chamaehyphon recently isolated from roots of diseased plants. Pathogenicity was tested in 6-month-old and 1-year-old plants that, after inoculation, were flooded up to three times to induce symptom appearance. Leaf wilting and root rot typical of KVDS was observed in all the plants inoculated with P. chamaehyphon strain KD-15 (PCHA) and in all the positive controls potted in a mix of peat and soils collected in KVDS-affected orchards, while negative controls remained symptomless. Disease development on 6-month-old plants was characterized by unusual degradation of the not-lignified collar, occurring even in absence of flooding. Conversely, on 1-year-old plants, symptoms faithfully reproduced KVDS dynamics observed in orchard. This work confirmed the pathogenicity of P. chamaehyphon and raised new questions about the actual role of waterlogging in KVDS etiology.

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection.

Europe is the historical cradle of viticulture, but grapevines (Vitis vinifera) have been increasingly threatened by pathogens of American origin. The invasive oomycete Plasmopara viticola causes downy mildew, one of the most devastating grapevine diseases worldwide. Despite major economic consequences, its invasion history remains poorly understood. We analyzed a comprehensive dataset of ∼2,000 samples, collected from the most important wine-producing countries, using nuclear and mitochondrial gene sequences and microsatellite markers. Population genetic analyses revealed very low genetic diversity in invasive downy mildew populations worldwide and little evidence of admixture. All the invasive populations originated from only one of the five native North American lineages, the one parasitizing wild summer grape (V. aestivalis). An approximate Bayesian computation-random forest approach allowed inferring the worldwide invasion scenario of P. viticola. After an initial introduction into Europe, invasive European populations served as a secondary source of introduction into vineyards worldwide, including China, South Africa, and twice independently, Australia. Only the invasion of Argentina probably represents a tertiary introduction, from Australia. Our findings provide a striking example of a global pathogen invasion resulting from secondary dispersal of a successful invasive population. Our study will also help designing quarantine regulations and efficient breeding for resistance against grapevine downy mildew., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola , is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid's (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola . In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera , the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola . Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Successful plant defence against microbial pathogens is based on early recognition and fast activation of inducible responses. Key mechanisms include detection of microbe-associated molecular patterns by membrane-localized pattern recognition receptors that induce a basal resistance response. A well-described model of such responses to pathogens involves the interactions between Solanaceae plants and proteinaceous elicitors secreted by oomycetes, called elicitins. It has been hypothesized that the formation of oligomeric structures by elicitins could be involved in their recognition and activation of defensive transduction cascades. In this study, we tested this hypothesis using several approaches, and we observed differences in tobacco plant responses induced by the elicitin β-cryptogein (β-CRY) and its homodimer, β-CRYDIM. We also found that the C-terminal domain of elicitins of other ELI (true-elicitin) clades plays a significant role in stabilization of their oligomeric structure and restraint in the cell wall. In addition, covalently cross-linking β-CRYDIM impaired the formation of signalling complexes, thereby reducing its capacity to elicit the hypersensitive response and resistance in the host plant, with no significant changes in pathogenesis-related protein expression. By revealing the details of the effects of β-CRY dimerization on recognition and defence responses in tobacco, our results shed light on the poorly understood role of elicitins' oligomeric structures in the interactions between oomycetes and plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

As an evolutionarily conserved pathway, mitogen-activated protein kinase (MAPK) cascades function as the key signal transducers that convey information by protein phosphorylation. Here we identified PlMAPK2 as one of 14 predicted MAPKs encoding genes in the plant pathogenic oomycete Peronophythora litchii . PlMAPK2 is conserved in P. litchii and Phytophthora species. We found that PlMAPK2 was up-regulated in sporangium, zoospore, cyst, cyst germination and early stage of infection. We generated PlMAPK2 knockout mutants using the CRISPR/Cas9 method. Compared with wild-type strain, the PlMAPK2 mutants showed no significant difference in vegetative growth, oospore production and sensitivity to various abiotic stresses. However, the sporangium release was severely impaired. We further found that the cleavage of the cytoplasm into uninucleate zoospores was disrupted in the PlMAPK2 mutants, and this developmental phenotype was accompanied by reduction in the transcription levels of PlMAD1 and PlMYB1 genes. Meanwhile, the PlMAPK2 mutants exhibited lower laccase activity and reduced virulence to lychee leaves. Overall, this study identified a MAPK that is critical for zoosporogenesis by regulating the sporangial cleavage and pathogenicity of P. litchii , likely by regulating laccase activity.

The discovery of new mechanisms of resistance and natural bioactive molecules could be two of the possible ways to reduce fungicide use in vineyard and assure an acceptable and sustainable protection against Plasmopara viticola, the grapevine downy mildew agent. Emission of volatile organic compounds (VOCs), such as terpenes, norisoprenoids, alcohols and aldehydes, is frequently induced in plants in response to attack by pathogens, such as P. viticola, that is known to cause a VOCs increment in cultivars harboring American resistance traits. In this study, the role of leaf VOCs in the resistance mechanism of two resistant cultivars (Mgaloblishvili, a pure Vitis vinifera cultivar, and Bianca, an interspecific hybrid) and the direct antimicrobial activity of four selected VOCs have been investigated. The leaf VOCs profiles, analyzed through solid-phase microextraction gas chromatography-mass spectrometry analysis, as well as the expression of six terpene synthases (TPSs), were determined upon pathogen inoculation. In both cultivars, the expression pattern of six TPSs increased soon after pathogen inoculation and an increment of nine VOCs has been detected. While in Mgaloblishvili VOCs were synthesized early after P. viticola inoculation, they constituted a late response to pathogen in Bianca. All the four terpenes (farnesene, nerolidol, ocimene and valencene), chosen according to the VOC profiles and gene expression analysis, caused a significant reduction (53-100%) in P. viticola sporulation. These results support the role of VOCs into defense mechanisms of both cultivars and suggest their potential role as a natural and eco-friendly solution to protect grapevine from P. viticola., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Plasmopara viticola , the casual oomycete of grapevine downy mildew, could cause yield loss and compromise berry quantity. Previously, we have identified several PvRXLR effectors that could suppress plant immunity to promote infection and disease development. In this study, the role of effector, PvRXLR53, in plant-microbe interaction was investigated. PvRXLR53 has several orthologs in other oomycetes and contains a functional signal peptide. Expression level of PvRXLR53 was already detected upon inoculation, further induced in the early stage after P. viticola inoculation and decreased to low level in the late infection stage in grapevine ( Vitis vinifera 'Cabernet Sauvignon'). PvRXLR53 is localized in both nucleus and cytoplasm. When transiently expressed in Nicotiana benthamiana , PvRXLR53 suppressed oomycete elicitor INF1-triggered programmed cell death and defense gene expression, and Phytophthora capsici -induced reactive oxygen species production (ROS) and eventually resistance to P. capsici . In summary, these findings suggest that P. viticola secretes PvRXLR53 to suppress host immunity from the very early stage of infection.

Plasmopara viticola, the causal organism of grapevine downy mildew, secretes a vast array of effectors to manipulate host immunity. Previously, several cell death-inducing PvRXLR effectors have been identified, but their functions and host targets are poorly understood. Here, we investigated the role of PvRXLR111, a cell death-inducing RXLR effector, in manipulating plant immunity. When coexpressed with other PvRXLR effectors, PvRXLR111-induced cell death was prevented. Transient expression of PvRXLR111 in Nicotiana benthamiana suppressed bacterial flagellin peptide flg22-elicited immune responses and enhanced Phytophthora capsici infection. PvRXLR111 induction in Arabidopsis increased susceptibility to Hyaloperonospora arabidopsidis. PvRXLR111 expression in Pseudomonas syringae promoted bacterial colonization. By immunoprecipitation-mass spectrometry analysis, yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays, it was shown that PvRXLR111 interacted with Vitis vinifera putative WRKY transcription factor 40 (VvWRKY40), which increased VvWRKY40 stability. Transient expression of VvWRKY40 in N. benthamiana inhibited flg22-induced reactive oxygen species burst and enhanced P. capsici infection and silencing NbWRKY40 attenuated P. capsici colonization. These results suggest VvWRKY40 functions as a negative regulator in plant immunity and that PvRXLR111 suppresses host immunity by stabilizing VvWRKY40., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Downy mildew, caused by Plasmopara viticola , is one of the most severe diseases of grapevine ( Vitis vinifera L.). Genetic resistance is an effective and sustainable control strategy, but major resistance genes (encoding receptors for specific pathogen effectors) introgressed from wild Vitis species, although effective, may be non-durable because the pathogen can evolve to avoid specific recognition. Previous transcriptomic studies in the resistant species Vitis riparia highlighted the activation of signal transduction components during infection. The transfer of such components to V. vinifera might confer less specific and therefore more durable resistance. Here, we describe the generation of transgenic V. vinifera lines constitutively expressing the V. riparia E3 ubiquitin ligase gene VriATL156 . Phenotypic and molecular analysis revealed that the transgenic plants were less susceptible to P. viticola than vector-only controls, confirming the role of this E3 ubiquitin ligase in the innate immune response. Two independent transgenic lines were selected for detailed analysis of the resistance phenotype by RNA-Seq and microscopy, revealing the profound reprogramming of transcription to achieve resistance that operates from the earliest stages of pathogen infection. The introduction of VriATL156 into elite grapevine cultivars could therefore provide an effective and sustainable control measure against downy mildew.

The rhizosphere microbiome of plants is essential for plant growth and health. Recent studies have shown that upon infection of leaves with a foliar pathogen, the composition of the root microbiome is altered and enriched with bacteria that in turn can systemically protect the plant against the foliar pathogen. This protective effect is extended to successive populations of plants that are grown on soil that was first conditioned by pathogen-infected plants, a phenomenon that was coined "the soil-borne legacy." Here we provide a detailed protocol for soil-borne legacy experiments with the model plant Arabidopsis thaliana after infection with the obligate biotrophic pathogen Hyaloperonospora arabidopsidis. This protocol can easily be extended to infection with other pathogens or even infestation with herbivorous insects and can function as a blueprint for soil-borne legacy experiments with crop species.

Downy mildew (DM) is one of the severe biotic threats to sunflower production worldwide. The inciting pathogen, Plasmopara halstedii, could overwinter in the field for years, creating a persistent threat to sunflower. The dominant genes Pl 18 and Pl 20 conferring resistance to known DM races have been previously mapped to 1.5 and 1.8 cM intervals on sunflower chromosomes 2 and 8, respectively. Utilizing a whole-genome resequencing strategy combined with reference sequence-based chromosome walking and high-density mapping in the present study, Pl 18 was placed in a 0.7 cM interval on chromosome 2. A candidate gene HanXRQChr02g0048181 for Pl 18 was identified from the XRQ reference genome and predicted to encode a protein with typical NLR domains for disease resistance. The Pl 20 gene was placed in a 0.2 cM interval on chromosome 8. The putative gene with the NLR domain for Pl 20 , HanXRQChr08g0210051, was identified within the Pl 20 interval. SNP markers closely linked to Pl 18 and Pl 20 were evaluated with 96 diverse sunflower lines, and a total of 13 diagnostic markers for Pl 18 and four for Pl 20 were identified. These markers will facilitate to transfer these new genes to elite sunflower lines and to pyramid these genes with broad-spectrum DM resistance in sunflower breeding.

Direct or indirect recognition of pathogen-derived effectors by plant nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) initiates innate immune responses. The Hyaloperonospora arabidopsidis effector ATR1 activates the N-terminal Toll-interleukin-1 receptor (TIR) domain of Arabidopsis NLR RPP1. We report a cryo-electron microscopy structure of RPP1 bound by ATR1. The structure reveals a C-terminal jelly roll/Ig-like domain (C-JID) for specific ATR1 recognition. Biochemical and functional analyses show that ATR1 binds to the C-JID and the LRRs to induce an RPP1 tetrameric assembly required for nicotinamide adenine dinucleotide hydrolase (NADase) activity. RPP1 tetramerization creates two potential active sites, each formed by an asymmetric TIR homodimer. Our data define the mechanism of direct effector recognition by a plant NLR leading to formation of a signaling-active holoenzyme., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plasmopara viticola is one of the most important pathogens infecting Vitis vinifera plants. The interactions among P. viticola and both susceptible and resistant grapevine plants have been extensively characterised, at transcriptomic, proteomic and metabolomic levels. However, the involvement of plants ionome in the response against the pathogen has been completely neglected so far. Therefore, this study was aimed at investigating the possible role of leaf ionomic modulation during compatible and incompatible interactions between P. viticola and grapevine plants. In susceptible cultivars, a dramatic redistribution of mineral elements has been observed, thus uncovering a possible role for mineral nutrients in the response against pathogens. On the contrary, the resistant cultivars did not present substantial rearrangement of mineral elements at leaf level, except for manganese (Mn) and iron (Fe). This might demonstrate that, resistant cultivars, albeit expressing the resistance gene, still exploit a pathogen response mechanism based on the local increase in the concentration of microelements, which are involved in the synthesis of secondary metabolites and reactive oxygen species. Moreover, these data also highlight the link between the mineral nutrition and plants' response to pathogens, further stressing that appropriate fertilization strategies can be fundamental for the expression of response mechanisms against pathogens.

Pathogens that infect plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen Bremia lactucae by searching the predicted proteome for the WY domain, a structural fold found in effectors that has been implicated in immune suppression as well as effector recognition by host resistance proteins. We predicted 55 WY domain containing proteins in the genome of B. lactucae and found substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis of 21 effectors containing WY domains revealed 11 that elicited cell death on wild accessions and domesticated lettuce lines containing resistance genes, indicative of recognition of these effectors by the host immune system. Only two of the 11 recognized effectors contained the canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens., Competing Interests: The authors have declared that no competing interests exist.

The complicated interplay of plant-pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Downy mildew, caused by Plasmopara viticola, is a major disease that affects grapevines, and a few resistance (R) genes have been identified thus far. In order to identify R genes, we investigated F 1 progeny from a cross between the downy mildew-resistant Vitis amurensis 'Shuang Hong' and the susceptible Vitis vinifera 'Cabernet Sauvignon'. The P. viticola-resistance of the progeny varied continuously and was segregated as a quantitative trait. Genotyping-by-sequencing was used to construct linkage maps. The integrated map spanned 1898.09 cM and included 5603 single nucleotide polymorphisms on 19 linkage groups (LGs). Linkage analysis identified three quantitative trait loci (QTLs) for P. viticola resistance: 22 (Rpv22) on LG 02, Rpv23 on LG15, and Rpv24 on LG18. The phenotypic variance contributed by these three QTLs ranged from 15.9 to 30.0%. qRT-PCR analysis of R-gene expression in these QTLs revealed a CC-NBS-LRR disease resistance gene RPP8, two LRR receptor-like serine/threonine-protein kinases, a serine/threonine-protein kinase BLUS1, a glutathione peroxidase 8, an ethylene-responsive transcription factor ERF038, and a transcription factor bZIP11 were induced by P. viticola, and these genes may play important role in P. viticola response., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Downy mildew, powdery mildew, and grey mold are some of the phytopathological diseases causing economic losses in agricultural crops, including grapevine, worldwide. In the current scenario of increasing global warming, in which the massive use of agrochemicals should be limited, the management of fungal disease has become a challenge. The knowledge acquired on candidate resistant (R) genes having an active role in plant defense mechanisms has allowed numerous breeding programs to integrate these traits into selected cultivars, even though with some limits in the conservation of the proper qualitative characteristics of the original clones. Given their gene-specific mode of action, biotechnological techniques come to the aid of breeders, allowing them to generate simple and fast modifications in the host, without introducing other undesired genes. The availability of efficient gene transfer procedures in grapevine genotypes provide valid tools that support the application of new breeding techniques (NBTs). The expertise built up over the years has allowed the optimization of these techniques to overexpress genes that directly or indirectly limit fungal and oomycetes pathogens growth or silence plant susceptibility genes. Furthermore, the downregulation of pathogen genes which act as virulence effectors by exploiting the RNA interference mechanism, represents another biotechnological tool that increases plant defense. In this review, we summarize the most recent biotechnological strategies optimized and applied on Vitis species, aimed at reducing their susceptibility to the most harmful fungal and oomycetes diseases. The best strategy for combating pathogenic organisms is to exploit a holistic approach that fully integrates all these available tools.

Downy mildew caused by Hyaloperonosporabrassicae ( H. brassicae ) leads to up to 90% of the crop yield loss in Chinese cabbage in China. A transcriptome analysis was carried out between a resistant line (13-13, R) and a susceptible line (15-14, S) of Chinese cabbage in response to H. brassicae . The NOISeq method was used to find differentially expressed genes (DEGs) between these two groups and GO and KEGG were carried out to find R genes related to downy mildew response of Chinese cabbage. qRT-PCR was carried out to verify the reliability of RNA-seq expression data. A total of 3,055 DEGs were screened out from 41,020 genes and clustered into 6 groups with distinct expression patterns. A total of 87 candidate DEGs were identified by functional annotation based on GO and KEGG analysis. These candidate genes are involved in plant-pathogen interaction pathway, among which 54 and 33 DEGs were categorized into plant-pathogen interaction proteins and transcription factors, respectively. Proteins encoded by these genes have been reported to play an important role in the pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) processes of disease responses in some model plants, such as Arabidopsis, rice, tobacco, and tomato. However, little is known about the mechanisms of these genes in resistance to downy mildew in Chinese cabbage. Our findings are useful for further characterization of these candidate genes and helpful in breeding resistant strains.

The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis , which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant's Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA ( Hpa sRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of Hpa sRNA-induced target suppression in Arabidopsis. The significant role of Hpa sRNAs together with At AGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block Hpa sRNAs, that both exhibited enhanced resistance. Hpa sRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility., Competing Interests: FD, AT, JR, SK, RP, TS, AT, AK, EK, RH, AW No competing interests declared, (© 2020, Dunker et al.)

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta., Competing Interests: The authors have declared that no competing interests exist.

The ability to secrete effector proteins that can enter plant cells and manipulate host processes is a key determinant of what makes a successful plant pathogen. Here, we review intracellular effectors from filamentous (fungal and oomycete) phytopathogens and the host proteins and processes that are targeted to promote disease. We cover contrasting virulence strategies and effector modes of action. Filamentous pathogen effectors alter the fates of host proteins that they target, changing their stability, their activity, their location, and the protein partners with which they interact. Some effectors inhibit target activity, whereas others enhance or utilize it, and some target multiple host proteins. We discuss the emerging topic of effectors that target negative regulators of immunity or other plant proteins with activities that support susceptibility. We also highlight the commonly targeted host proteins that are manipulated by effectors from multiple pathogens, including those representing different kingdoms of life., (© 2020 The Author(s).)

Mgaloblishvili, a Vitis vinifera cultivar, exhibits unique resistance traits against Plasmopara viticola , the downy mildew agent. This offers the unique opportunity of exploring the molecular responses in compatible and incompatible plant-pathogen interaction. In this study, whole transcriptomes of Mgaloblishvili, Pinot noir (a V. vinifera susceptible cultivar), and Bianca (a resistant hybrid) leaves, inoculated and non-inoculated with the pathogen , were used to identify P. viticola effector-encoding genes and plant susceptibility/resistance genes. Multiple effector-encoding genes were identified in P. viticola transcriptome, with remarkable expression differences in relation to the inoculated grapevine cultivar. Intriguingly, five apoplastic effectors specifically associated with resistance in V. vinifera . Gene coexpression network analysis identified specific modules and metabolic changes occurring during infection in the three grapevine cultivars. Analysis of these data allowed, for the first time, the detection in V. vinifera of a putative P. viticola susceptibility gene, encoding a LOB domain-containing protein. Finally, the de novo assembly of Mgaloblishvili, Pinot noir, and Bianca transcriptomes and their comparison highlighted novel candidate genes that might be at the basis of the resistant phenotype. These results open the way to functional analysis studies and to new perspectives in molecular breeding of grapevine for resistance to P. viticola ., Competing Interests: The authors declare no conflict of interest.

Downy mildew (DM) is a major disease of maize that causes significant yield loss in subtropical and tropical regions around the world. A variety of DM strains have been reported, and the resistance to them is polygenically controlled. In this study, we analyzed the quantitative trait loci (QTLs) involved in resistance to Peronosclerospora sorghi (sorghum DM), P. maydis (Java DM) , and Sclerophthora macrospora (crazy top DM) using a recombinant inbred line (RIL) from a cross between B73 (susceptible) and Ki11 (resistant), and the candidate genes for P. sorghi , P. maydis , and S. macrospora resistance were discovered. The linkage map was constructed with 234 simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) markers, which was identified seven QTLs (chromosomes 2, 3, 6, and 9) for three DM strains. The major QTL, located on chromosome 2, consists of 12.95% of phenotypic variation explained (PVE) and a logarithm of odds (LOD) score of 14.12. Sixty-two candidate genes for P. sorghi , P. maydis , and S. macrospora resistance were obtained between the flanked markers in the QTL regions. The relative expression level of candidate genes was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using resistant (CML228, Ki3, and Ki11) and susceptible (B73 and CML270) genotypes. For the 62 candidate genes, 15 genes were upregulated in resistant genotypes. Among these, three ( GRMZM2G028643 , GRMZM2G128315 , and GRMZM2G330907 ) and AC210003.2_FG004 were annotated as leucine-rich repeat (LRR) and peroxidase (POX) genes, respectively. These candidate genes in the QTL regions provide valuable information for further studies related to P. sorghi , P. maydis , and S. macrospora resistance., Competing Interests: The authors declare no conflict of interest.

Six components of partial resistance (RCs) were studied in 15 grapevine varieties with partial resistance to Plasmopara viticola: (i) infection frequency (IFR, proportion of inoculation sites showing sporulation), (ii) latent period (LP50, degree-days between inoculation and appearance of 50% of the final number of sporulating lesions), (iii) lesion size (LS, area of single lesions in mm 2 ), (iv) production of sporangia (SPOR, number of sporangia produced per lesion, and SPOR', number of sporangia produced per mm 2 of lesion), (v) infectious period (IP, number of sporulation events on a lesion), and (vi) infectivity of sporangia (INF, infection efficiency of sporangia produced on resistant varieties). Artificial inoculation monocycle experiments were conducted for a 3-year period on leaves collected at leaf development, flowering, and fruit development. Compared to the susceptible variety 'Merlot', the partially resistant varieties showed reduced IFR, longer LP, smaller LS, fewer SPOR and SPOR', shorter IP, and lower INF. At leaf development, IFR, SPOR, and INF were higher and LP was shorter than at flowering and fruit development. RCs analysis through monocyclic experiments provides reliable assessments of the resistance response of grapevine accessions. The workload required for routine assessment in breeding programs could be reduced by measuring IFR and SPOR, while producing robust results.

The oomycetes are a class of ubiquitous, filamentous microorganisms that include some of the biggest threats to global food security and natural ecosystems. Within the oomycete class are highly diverse species that infect a broad range of animals and plants. Some of the most destructive plant pathogens are oomycetes, such as Phytophthora infestans, the agent of potato late blight and the cause of the Irish famine. Recent years have seen a dramatic increase in the number of sequenced oomycete genomes. Here we review the latest developments in oomycete genomics and some of the important insights that have been gained. Coupled with proteomic and transcriptomic analyses, oomycete genome sequences have revealed tremendous insights into oomycete biology, evolution, genome organization, mechanisms of infection, and metabolism. We also present an updated phylogeny of the oomycete class using a phylogenomic approach based on the 65 oomycete genomes that are currently available., (© 2020 Elsevier Inc. All rights reserved.)

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato ( Solanum tuberosum ) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases. IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples., (Copyright © 2019 American Society for Microbiology.)

The reduction of synthetic fungicides in agriculture is necessary to guarantee a sustainable production that protects the environment and consumers' health. Downy mildew caused by the oomycete Plasmopara viticola is the major pathogen in viticulture worldwide and responsible for up to 60% of pesticide treatments. Alternatives to reduce fungicides are thus utterly needed to ensure sustainable vineyard-ecosystems, consumer health and public acceptance. Essential oils (EOs) are amongst the most promising natural plant protection alternatives and have shown their antibacterial, antiviral and antifungal properties on several agricultural crops. However, the efficiency of EOs highly depends on timing, application method and the molecular interactions between the host, the pathogen and EO. Despite proven EO efficiency, the underlying processes are still not understood and remain a black box. The objectives of the present study were: a) to evaluate whether a continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant resistance primers. To achieve this a custom-made climatic chamber was constructed that enabled a continuous fumigation of potted vines with different EOs during long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility to Plasmopara viticola. Grapevine cuttings were infected with P. viticola and subsequently exposed to continuous fumigation of different EOs at different concentrations, during 2 application time spans (24 hours and 10 days). Experiments were stopped when infection symptoms were clearly observed on the leaves of the control plants. Plant physiology (photosynthesis and growth rate parameters) were recorded and leaves were sampled at different time points for subsequent RNA extraction and transcriptomics analysis. Strikingly, the Oregano vulgare EO vapour treatment during 24h post-infection proved to be sufficient to reduce downy mildew development by 95%. Total RNA was extracted from leaves of 24h and 10d treatments and used for whole transcriptome shotgun sequencing (RNA-seq). Sequenced reads were then mapped onto the V. vinifera and P. viticola genomes. Less than 1% of reads could be mapped onto the P. viticola genome from treated samples, whereas up to 30% reads from the controls mapped onto the P. viticola genome, thereby confirming the visual observation of P. viticola absence in the treated plants. On average, 80% of reads could be mapped onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes. Transcriptomic data clearly showed that the treatment triggered the plant's innate immune system with genes involved in salicylic, jasmonic acid and ethylene synthesis and signaling, activating Pathogenesis-Related-proteins as well as phytoalexin synthesis. These results elucidate EO-host-pathogen interactions for the first time and indicate that the antifungal efficiency of EO is mainly due to the triggering of resistance pathways inside the host plants. This is of major importance for the production and research on biopesticides, plant stimulation products and for resistance-breeding strategies., Competing Interests: The authors have declared that no competing interests exist.

Background: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wild V. vinifera germplasm. Considering that stilbenes represent the most important class of phytoalexins in the Vitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycete Plasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namely VvMYB14 and VvMYB15, and of genes encoding for chalcone synthases., Results: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup of V. vinifera genotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, following P. viticola inoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought by P. viticola appeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor., Conclusions: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wild V. vinifera germplasm. During the early stages of P. viticola infection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response to P. viticola.

Plant-pathogenic oomycetes include numerous species that are ongoing threats to agriculture and natural ecosystems. Understanding the molecular dialogs between oomycetes and plants is instrumental for sustaining effective disease control. Plants respond to oomycete infection by multiple defense actions including strengthening of physical barriers, production of antimicrobial molecules, and programmed cell death. These responses are tightly controlled and integrated via a three-layered immune system consisting of a multiplex recognition layer, a resilient signal-integration layer, and a diverse defense-action layer. Adapted oomycete pathogens utilize apoplastic and intracellular effector arsenals to counter plant immunity mechanisms within each layer, including by evasion or suppression of recognition, interference with numerous signaling components, and neutralization or suppression of defense actions. A coevolutionary arms race continually drives the emergence of new mechanisms of plant defense and oomycete counterdefense.

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector-target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast-two-hybrid (Y2H) screening. Interaction of the lettuce tail-anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N-terminal NAC domain but depends on the C-terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail-anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC-domain (LsNAC069 Δ NAC ). Relocalization was significantly reduced in the presence of the Ser/Cys-protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co-expression of effectors with LsNAC069 Δ NAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses., (© 2019 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Background: European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance., Results: In this study, Rpv3-mediated defense responses were investigated in Rpv3 + and Rpv3- grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3 + and avrRpv3-, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses., Conclusion: This study used histochemical, transcriptomic and metabolomic analyses of Rpv3 + and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.

Pseudoperonospora humuli is an obligate oomycete pathogen of hop ( Humulus lupulus ) that causes downy mildew, an important disease in most production regions in the Northern Hemisphere. The pathogen can cause a systemic infection in hop, overwinter in the root system, and infect propagation material. Substantial yield loss may occur owing to P. humuli infection of strobiles (seed cones), shoots, and cone-bearing branches. Fungicide application and cultural practices are the primary methods to manage hop downy mildew. However, effective, sustainable, and cost-effective management of downy mildew can be improved by developing early detection systems to inform on disease risk and timely fungicide application. However, no species-specific diagnostic assays or genomic resources are available for P. humuli . The genome of the P. humuli OR502AA isolate was partially sequenced using Illumina technology and assembled with ABySS. The assembly had a minimum scaffold length of 500 bp and an N50 (median scaffold length of the assembled genome) of 19.2 kbp. A total number of 18,656 genes were identified using MAKER standard gene predictions. Additionally, transcriptome assemblies were generated using RNA-seq and Trinity for seven additional P. humuli isolates. Bioinformatics analyses of next generation sequencing reads of P. humuli and P. cubensis (a closely related sister species) identified 242 candidate species-specific P. humuli genes that could be used as diagnostic molecular markers. These candidate genes were validated using polymerase chain reaction against a diverse collection of isolates from P. humuli , P. cubensis , and other oomycetes. Overall, four diagnostic markers were found to be uniquely present in P. humuli . These candidate markers identified through comparative genomics can be used for pathogen diagnostics in propagation material, such as rhizomes and vegetative cuttings, or adapted for biosurveillance of airborne sporangia, an important source of inoculum in hop downy mildew epidemics.

Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha-diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha-diversity in soil and roots, but alpha-diversity in leaves was high compared with most other land uses. In contrast to alpha-diversity, differences in within-land use beta-diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large-scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities., (© 2019 John Wiley & Sons Ltd.)

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved., Competing Interests: The authors have declared that no competing interests exist.

Metacaspase, as hypersensitive response (HR) executors, has been identified in many plant species. Previously, the entire gene family of metacaspase has been uncovered, but there are still questions that remain unclear regarding HR-regulating gene members. In this study, based on metacaspase expression during different grapevine genotypes interacting with Plasmopara viticola, we identified MC2 and MC5 as candidates involved in HR. We overexpressed both metacaspases as GFP fusions in tobacco BY-2 cells to address subcellular localization and cellular functions. We found MC2 located at the ER, while MC5 was nucleocytoplasmic. In these overexpressor lines, cell death elicited by the bacterial protein harpin, is significantly enhanced, indicating MC2 and MC5 mediated defence-related programmed cell death (PCD). This effect was mitigated, when the membrane-located NADPH oxidase was inhibited by the specific inhibitor diphenylene iodonium, or when cells were complemented with methyl jasmonate, a crucial signal of basal immunity. Both findings are consistent with a role of MC2 and MC5 in cell death-related immunity. Using a dual-luciferase reporter system in grapevine cells we demonstrated both MC2 and MC5 promoter alleles from V. rupestris were more responsive to harpin than those from V. vinifera cv 'Müller-Thurgau', while they were not induced by MeJA as signal linked with basal immunity. These findings support a model, where MC2 and MC5 act specifically as executors of the HR.