Transcriptome Analysis of Potato Infected with the Necrotrophic Pathogen Alternaria solani.

Potato early blight is caused by the necrotrophic fungus Alternaria solani and can result in yield losses of up to 50% if left uncontrolled. At present, the disease is controlled by chemical fungicides, yet rapid development of fungicide resistance renders current control strategies unsustainable. On top of that, a lack of understanding of potato defences and the quantitative nature of resistance mechanisms against early blight hinders the development of more sustainable control methods. Necrotrophic pathogens, compared to biotrophs, pose an extra challenge to the plant, since common defence strategies to biotic stresses such as the hypersensitive response and programmed cell death are often beneficial for necrotrophs. With the aim of unravelling plant responses to both the early infection stages (i.e., before necrosis), such as appressorium formation and penetration, as well as to later responses to the onset of necrosis, we present here a transcriptome analysis of potato interactions with A. solani from 1 h after inoculation when the conidia have just commenced germination, to 48 h post inoculation when multiple cell necrosis has begun. Potato transcripts with putative functions related to biotic stress tolerance and defence against pathogens were upregulated, including a putative Nudix hydrolase that may play a role in defence against oxidative stress. A. solani transcripts encoding putative pathogenicity factors, such as cell wall degrading enzymes and metabolic processes that may be important for infection. We therefore identified the differential expression of several potato and A. solani transcripts that present a group of valuable candidates for further studies into their roles in immunity or disease development.

Visualising the ionome in resistant and susceptible plant-pathogen interactions.

At the morphological and anatomical levels, the ionome, or the elemental composition of an organism, is an understudied area of plant biology. In particular, the ionomic responses of plant-pathogen interactions are scarcely described, and there are no studies on immune reactions. In this study we explored two X-ray fluorescence (XRF)-based ionome visualisation methods (benchtop- and synchrotron-based micro-XRF [µXRF]), as well as the quantitative inductively coupled plasma optical emission spectroscopy (ICP-OES) method, to investigate the changes that occur in the ionome of compatible and incompatible plant-pathogen interactions. We utilised the agronomically important and comprehensively studied interaction between potato (Solanum tuberosum) and the late blight oomycete pathogen Phytophthora infestans as an example. We used one late blight-susceptible potato cultivar and two resistant transgenic plant lines (only differing from the susceptible cultivar in one or three resistance genes) both in control and P. infestans-inoculated conditions. In the lesions from the compatible interaction, we observed rearrangements of several elements, including a decrease of the mobile macronutrient potassium (K) and an increase in iron (Fe) and manganese (Mn), compared with the tissue outside the lesion. Interestingly, we observed distinctly different distribution patterns of accumulation at the site of inoculation in the resistant lines for calcium (Ca), magnesium (Mg), Mn and silicon (Si) compared to the susceptible cultivar. The results reveal different ionomes in diseased plants compared to resistant plants. Our results demonstrate a technical advance and pave the way for deeper studies of the plant-pathogen ionome in the future.

Pathogen-Mediated Stomatal Opening: A Previously Overlooked Pathogenicity Strategy in the Oomycete Pathogen Phytophthora infestans.

Phytophthora infestans, the most damaging oomycete pathogen of potato, is specialized to grow sporangiophore through opened stomata for secondary inoculum production. However, it is still unclear which metabolic pathways in potato are manipulated by P. infestans in the guard cell-pathogen interactions to open the stomata. Here microscopic observations and cell biology were used to investigate antagonistic interactions between guard cells and the oomycete pathogen. We observed that the antagonistic interactions started at the very beginning of infection. Stomatal movement is an important part of the immune response of potato to P. infestans infection and this occurs through guard cell death and stomatal closure. We observed that P. infestans appeared to manipulate metabolic processes in guard cells, such as triacylglycerol (TAG) breakdown, starch degradation, H2O2 scavenging, and NO catabolism, which are involved in stomatal movement, to evade these stomatal defense responses. The signal transduction pathway of P. infestans-induced stomatal opening likely starts from H2O2 and NO scavenging, along with TAG breakdown while the subsequent starch degradation reinforces the opening process by strengthening guard cell turgor and opening the stomata to their maximum aperture. These results suggest that stomata are a barrier stopping P. infestans from completing its life cycle, but this host defense system can be bypassed through the manipulation of diverse metabolic pathways that may be induced by P. infestans effector proteins.

Altitudinal Heterogeneity of UV Adaptation in Phytophthorainfestans Is Associated with the Spatial Distribution of a DNA Repair Gene.

Climate change is considered a major threat to society and nature. UV irradiation is the most important environmental genotoxic agent. Thus, how elevated UV irradiation may influence human health and ecosystems has generated wide concern in the scientific community, as well as with policy makers and the public in general. In this study, we investigated patterns and mechanisms of UV adaptation in natural ecosystems by studying a gene-specific variation in the potato late blight pathogen, Phytophthora infestans. We compared the sequence characteristics of radiation sensitive 23 (RAD23), a gene involved in the nucleotide excision repair (NER) pathway and UV tolerance, in P. infestans isolates sampled from various altitudes. We found that lower genetic variation in the RAD23 gene was caused by natural selection. The hypothesis that UV irradiation drives this selection was supported by strong correlations between the genomic characteristics and altitudinal origin (historic UV irradiation) of the RAD23 sequences with UV tolerance of the P. infestans isolates. These results indicate that the RAD23 gene plays an important role in the adaptation of P. infestans to UV stress. We also found that different climatic factors could work synergistically to determine the evolutionary adaptation of species, making the influence of climate change on ecological functions and resilience more difficult to predict. Future attention should aim at understanding the collective impact generated by simultaneous change in several climate factors on species adaptation and ecological sustainability, using state of the art technologies such as experimental evolution, genome-wide scanning, and proteomics.

Horizontal Gene Transfer and Tandem Duplication Shape the Unique CAZyme Complement of the Mycoparasitic Oomycetes Pythium oligandrum and Pythium periplocum.

Crop protection strategies that are effective but that reduce our reliance on chemical pesticides are urgently needed to meet the UN sustainable development goals for global food security. Mycoparasitic oomycetes such as Pythium oligandrum and Pythium periplocum, have potential for the biological control of plant diseases that threaten crops and have attracted much attention due to their abilities to antagonize plant pathogens and modulate plant immunity. Studies of the molecular and genetic determinants of mycoparasitism in these species have been less well developed than those of their fungal counterparts. Carbohydrate-active enzymes (CAZymes) from P. oligandrum and P. periplocum are predicted to be important components of mycoparasitism, being involved in the degradation of the cell wall of their oomycete and fungal prey species. To explore the evolution of CAZymes of these species we performed an in silico identification and comparison of the full CAZyme complement (CAZyome) of the two mycoparasitic Pythium species (P. oligandrum and P. periplocum), with seven other Pythium species, and four Phytophthora species. Twenty CAZy gene families involved in the degradation of cellulose, hemicellulose, glucan, and chitin were expanded in, or unique to, mycoparasitic Pythium species and several of these genes were expressed during mycoparasitic interactions with either oomycete or fungal prey, as revealed by RNA sequencing and quantitative qRT-PCR. Genes from three of the cellulose and chitin degrading CAZy families (namely AA9, GH5_14, and GH19) were expanded via tandem duplication and predominantly located in gene sparse regions of the genome, suggesting these enzymes are putative pathogenicity factors able to undergo rapid evolution. In addition, five of the CAZy gene families were likely to have been obtained from other microbes by horizontal gene transfer events. The mycoparasitic species are able to utilize complex carbohydrates present in fungal cell walls, namely chitin and N-acetylglucosamine for growth, in contrast to their phytopathogenic counterparts. Nonetheless, a preference for the utilization of simple sugars for growth appears to be a common trait within the oomycete lineage.

What are the Top 10 Unanswered Questions in Molecular Plant-Microbe Interactions?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species.

Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5'-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

Efficient RNA silencing suppression activity of Potato Mop-Top Virus 8K protein is driven by variability and positive selection.

Previously, we investigated the evolution of Potato mop-top virus (PMTV) ORFs. Results indicate that positive selection acts exclusively on an ORF encoding the 8K protein, a weak viral suppressor of RNA silencing (VSR). However, how the extraordinary variability contributes to 8K-mediated RNA silencing suppression remains unknown. Here, we characterized the RNA silencing suppression activity of the 8K protein from seven diverse isolates. We show that 8K encoded by isolate P1 exhibits stronger RNA silencing suppression activity than the 8K protein from six other isolates. Mutational analyses revealed that Ser-50 is critical for these differences. By comparing small RNA profiles we found a lower abundance of siRNAs with U residue at the 5'-terminus after expression of the P1 8K compared to expression of 8K from isolate P125, an isolate with weak VSR activity. These results provide new clues as to the role of positive selection in shaping activities of VSRs.

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

Tolerance and overcompensation to infection by Phytophthora infestans in the wild perennial climber Solanum dulcamara.

Studies of infection by Phytophthora infestans-the causal agent of potato late blight-in wild species can provide novel insights into plant defense responses, and indicate how wild plants might be influenced by recurrent epidemics in agricultural fields. In the present study, our aim was to investigate if different clones of Solanum dulcamara (a relative of potato) collected in the wild differ in resistance and tolerance to infection by a common European isolate of P. infestans. We performed infection experiments with six S. dulcamara genotypes (clones) both in the laboratory and in the field and measured the degree of infection and plant performance traits. In the laboratory, the six evaluated genotypes varied from resistant to susceptible, as measured by degree of infection 20 days post infection. Two of the four genotypes susceptible to infection showed a quadratic (concave downward) relationship between the degree of infection and shoot length, with maximum shoot length at intermediate values of infection. This result suggests overcompensation, that is, an increase in growth in infected individuals. The number of leaves decreased with increasing degree of infection, but at different rates in the four susceptible genotypes, indicating genetic variation for tolerance. In the field, the inoculated genotypes did not show any disease symptoms, but plant biomass at the end of the growing season was higher for inoculated plants than for controls, in-line with the overcompensation detected in the laboratory. We conclude that in S. dulcamara there are indications of genetic variation for both resistance and tolerance to P. infestans infection. Moreover, some genotypes displayed overcompensation. Learning about plant tolerance and overcompensation to infection by pathogens can help broaden our understanding of plant defense in natural populations and help develop more sustainable plant protection strategies for economically important crop diseases.

A preliminary service evaluation of a personality disorder case management service.

AIMS: The aim of this study is to assess the impact of establishing a specialized community personality disorder team on out of area placements, local hospital admissions and out of hours crisis contacts for service users with borderline personality disorder. METHOD: This is a before-after interim evaluation of a new service. We tested, through a paired t-test, whether the intervention generated statistically significant differences over a range of measures of service usage, including out of area placements, local hospital admissions and out of hours crisis contacts. Data from 12 months after the intervention started were compared with the previous 12 months of routine care, to determine the effect on crisis contacts, days in hospital and those returning from/or sent out of area for care. Finally, we have assessed the likelihood to generate cost savings. RESULTS: All service users in out of area placements were repatriated to live in the community locally (100%), and there was a statistically significant decrease in inpatient admissions (80%). This was counterbalanced by a smaller but statistically significant increase in out of hours community crisis contacts (30%), although these reduced over time. CONCLUSION: Reorganizing local care pathways can lead to less out of area placements and less hospital admissions. © 2019 John Wiley & Sons, Ltd.

Draft Genome Sequence for the Tree Pathogen Phytophthora plurivora.

Species from the genus Phytophthora are well represented among organisms causing serious diseases on trees. Phytophthora plurivora has been implicated in long-term decline of woodland trees across Europe. Here we present a draft genome sequence of P. plurivora, originally isolated from diseased European beech (Fagus sylvatica) in Malmö, Sweden. When compared with other sequenced Phytophthora species, the P. plurivora genome assembly is relatively compact, spanning 41 Mb. This is organized in 1,919 contigs and 1,898 scaffolds, encompassing 11,741 predicted genes, and has a repeat content of approximately 15%. Comparison of allele frequencies revealed evidence for tetraploidy in the sequenced isolate. As in other sequenced Phytophthora species, P. plurivora possesses genes for pathogenicity-associated RXLR and Crinkle and Necrosis effectors, predominantly located in gene-sparse genomic regions. Comparison of the P. plurivora RXLR effectors with orthologs in other sequenced species in the same clade (Phytophthora multivora and Phytophthora capsici) revealed that the orthologs were likely to be under neutral or purifying selection.

Sphingolipids regulate neuromuscular synapse structure and function in Drosophila.

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ∼50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.

RNase I regulates Escherichia coli 2',3'-cyclic nucleotide monophosphate levels and biofilm formation.

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2',3'-cNMPs in Escherichia coli and demonstrates that 2',3'-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2',3'-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2',3'-cNMPs.

Genome Sequence Resource for the Oomycete Taro Pathogen Phytophthora colocasiae.

Phytophthora colocasiae is a phytopathogenic oomycete that causes leaf blight and corm rot on taro (Colocasia esculenta), an important staple crop in the tropics. The impact of P. colocasiae is a serious concern for food security in Asian and Oceanic regions. Vietnamese strain 7290 of P. colocasiae was sequenced (Illumina) to assemble a draft genome of 56.6 Mb, comprised of 19,853 scaffolds and 19,984 predicted protein-coding genes. As in other Phytophthora species, P. colocasiae possesses numerous pathogenicity-related genes, such as the RxLR class of effectors. This draft genome sequence of P. colocasiae provides a resource to underpin the first steps in determining the molecular mechanisms of disease development in this pathosystem.

Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc2155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS1380 family transposon.

Proteomic Analysis of Phytophthora infestans Reveals the Importance of Cell Wall Proteins in Pathogenicity.

The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1-3 Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.

Draft genome of the oomycete pathogen Phytophthora cactorum strain LV007 isolated from European beech (Fagus sylvatica).

Phytophthora cactorum is a broad host range phytopathogenic oomycete. P. cactorum strain LV007 was isolated from a diseased European Beech (Fagus sylvatica) in Malmö, Sweden in 2016. The draft genome of P. cactorum strain LV007 is 67.81 Mb. It contains 15,567 contigs and 21,876 predicted protein-coding genes. As reported for other phytopathogenic Phytophthora species, cytoplasmic effector proteins including RxLR and CRN families were identified. The genome sequence has been deposited at DDBJ/ENA/GenBank under the accession NBIJ00000000. The version described in this paper is version NBIJ01000000.

Oxygen-Dependent Globin Coupled Sensor Signaling Modulates Motility and Virulence of the Plant Pathogen Pectobacterium carotovorum.

Bacterial pathogens utilize numerous signals to identify the presence of their host and coordinate changes in gene expression that allow for infection. Within plant pathogens, these signals typically include small molecules and/or proteins from their plant hosts and bacterial quorum sensing molecules to ensure sufficient bacterial cell density for successful infection. In addition, bacteria use environmental signals to identify conditions when the host defenses are weakened and potentially to signal entry into an appropriate host/niche for infection. A globin coupled sensor protein (GCS), termed PccGCS, within the soft rot bacterium Pectobacterium carotovorum ssp. carotovorum WPP14 has been identified as an O2 sensor and demonstrated to alter virulence factor excretion and control motility, with deletion of PccGCS resulting in decreased rotting of a potato host. Using small molecules that modulate bacterial growth and quorum sensing, PccGCS signaling also has been shown to modulate quorum sensing pathways, resulting in the PccGCS deletion strain being more sensitive to plant-derived phenolic acids, which can function as quorum sensing inhibitors, and exhibiting increased N-acylhomoserine lactone (AHL) production. These findings highlight a role for GCS proteins in controlling key O2-dependent phenotypes of pathogenic bacteria and suggest that modulating GCS signaling to limit P. carotovorum motility may provide a means to decrease rotting of plant hosts.