Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Expression profiling of selected biotrophy-associated ChEC and putative toxin genes by qRT-PCR.

<p>Expression levels are shown relative to the mean expression of actin and α-tubulin. Genes with highest expression <i>in planta</i> are highlighted with colours, indicating distinct waves of effector gene expression. <i>In vitro</i> cell types are: dormant spores (SP), saprotrophic mycelium (MY) and mature appressoria (VA). <i>In planta</i> stages are: unpenetrated appressoria (UA), penetrated appressoria with nascent biotrophic hyphae beneath (PA), biotrophy to necrotrophy switch (SW) and late necrotrophy (LN).</p

Appressorial pores as an interface for focal ChEC delivery.

<p>Transformant appressoria expressing the wave 2 effectors ChEC36:mRFP (A–O) and ChEC6:mRFP (P, Q). Appressoria or penetration sites after removal of appressoria were examined by confocal laser scanning microscopy viewed from above (A, B, E–I, O–Q) or from the side (C, D), and with transmission electron microscopy (J, K) and scanning electron microscopy (L–N). (A–D) Bright field and maximum fluorescence intensity overlay images of appressoria. Black arrows indicate the anticlinal plant cell wall and white arrows the penetration pore. (E) Fluorescence overlay image of an appressorium showing weak peripheral labelling of intracellular structures. (F, G) Fluorescence recorded with identical settings at the base (F) and the center (G) of the appressorium shown in (E). Arrow indicates a fluorescent ring surrounding the brightly fluorescing pore. (H, I) Fluorescence overlays recorded with identical settings focused on appressorial pores (H) or biotrophic hyphae (BH) formed beneath a penetrated appressorium (arrow). (J) Median section through an appressorium viewed with transmission electron microscopy (fixed with glutaraldehyde-osmium tetroxide and embedded in epoxy resin). A penetration hypha evaginates from the pore (P). An additional layer of the appressorial wall (asterisk) forms a thickened ring (arrowheads) around the pore, continuous with the penetration hypha wall. PW, plant cell wall. (K) Immunogold labelling of an appressorial pore (arrow) using antibodies recognizing mRFP (cells fixed in formaldehyde-glutaraldehyde and embedded in acrylic resin). PW, plant cell wall. WD, host cell wall deposits. (L) Scanning electron microscope image showing attached turgid appressorium (A) and collapsed conidium (C) on a leaf surface. (M) Plant-exposed underside of detached appressoria with penetration pores (black arrows) and remnants of extracellular matrix and/or plant cuticle (white arrow). (N, O) Penetration sites from which appressoria were detached completely. (N) The lobed outline of a former appressorium is still visible (arrowheads) with a mark representing the penetration point (arrow). (O) Micrograph series representing different focal planes as fluorescence overlay (top panels) and corresponding black on white conversion of the fluorescence channel (bottom panels), focusing from the penetration point (left) downwards into the plant cell wall (right). Arrow: inserted penetration hypha. (P, Q) Fluorescence overlays focused on the appressorial pore (P) and the underlying plant cell wall (Q). Arrow, anticlinal plant cell wall. Images were recorded at 24 hours post inoculation (hpi) (A–G, K, P, Q), 32 hpi (J, L–O), 40 hpi (H, I). Scale bars: 5 µm (A, H, L, N, O, P) and 2 µm (C, E, M), 1 µm (J), 500 nm (K). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s004" target="_blank">Figure S4</a>.</p

ChECs antagonizing plant cell death and supporting multiplication of plant pathogenic bacteria.

<p>(A) Infiltration scheme for the transient co-expression assay. Agrobacteria containing constructs for ChEC or YFP expression were mixed with those for cell death-inducer (CDI) expression. Mixtures were infiltrated into opposite sides of <i>N. benthamiana</i> leaves to allow pair-wise comparisons and to take account of leaf-to-leaf variation in necrosis manifestation. Thus, an infiltrated site expressing YFP/ChNLP1 was included as an internal control in every infiltrated leaf, to which the site expressing ChEC/ChNLP1 was compared. (B, C) Examples of infiltration site pairs 8 dpi. ChEC3 abolishes ChNLP1-induced necrosis (B, dotted circle), but a fungal secreted chitinase does not (C). (D) Quantification of cell death-suppressing activity of four wave 2 effectors (ChEC3, 3a, 6, 36), three wave 3 effectors (ChEC89, 34, 13) and an <i>in vitro</i>-expressed effector (ChEC5). Histograms show the proportion of sites expressing ChEC/CDI that displayed reduced necrosis compared to control sites expressing YFP/CDI. *, ** and *** indicate significant difference from the respective chitinase control with and without signal peptide at P<0,02, <0.005 and <0.0002, respectively (Student's t-test). <i>P. infestans</i> effector Avr3a^KI was used as positive control for suppression of INF1-induced cell death. Data represent means of at least three independent experiments, with at least 15 leaves/experiment/co-expression combination (± standard error). (E) Bacterial titers in <i>Arabidopsis</i> Col-0 leaves infected with <i>Pseudomonas syringae</i> pv <i>tomato</i> expressing ChECs as fusions with a bacterial effector mediating delivery into plant cells <i>via</i> type III secretion. <i>Hyaloperonospora arabidopsidis</i> ATR13^Emco5<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643-Sohn1" target="_blank">[27]</a> and YFP were included as positive and negative controls, respectively. Colony forming units were determined 0 and 3 days after spray inoculation. * and ** indicate significant difference from the YFP control at P<0.03 and P<0.0005, respectively. Data represent means of 4 replicates (± standard error).</p

ChECs accumulate in interfacial bodies and diffuse into the host cell wall.

<p>Transformant biotrophic hyphae expressing ChEC34:mRFP (wave 3 effector). (A, B) Bright field micrograph and corresponding maximum fluorescence intensity projection. Arrows: fluorescent foci. (C, D) Biotrophic hypha expressing CHEC34:mRFP viewed with confocal laser scanning microscopy settings optimized to show fluorescence in the penetrated epidermal cell wall (arrows). Arrowheads: unpenetrated wall of the same cell. (E, F) Transmission electron microscopy of a wild-type appressorium that produced a biotrophic hypha underneath with interfacial bodies (arrowheads). Arrows indicate the penetration site of the host cell wall. Cells were fixed with glutaraldehyde-osmium tetroxide and embedded in epoxy resin. (F) Close-up of an interfacial body (white asterisk) located between the plant plasma membrane (black arrowheads) and the fungal cell wall (black asterisk). (G, H) Immunogold cytochemistry using an antibody recognizing mRFP labels (G) interfacial bodies (arrows) or (H) the plant-fungal interface. Cells were fixed in formaldehyde-glutaraldehyde and embedded in acrylic resin. A, appressorium. FC, fungal cytoplasm. PC, plant cytoplasm. V, plant vacuole. BH, biotrophic hypha. (*) Fungal cell wall. Images were recorded at 40 hpi (A, B) and 43 hpi (C–H). Scale bars: 5 µm (A, C), 2.5 µm (E), 500 nm (F, G) and 250 nm (H).</p

ChEC delivery to the biotrophic interface and host apoplast.

<p>Transformant biotrophic hyphae expressing CHEC89:mRFP (wave 3 effector) viewed with confocal laser scanning microscopy. (A) Maximum fluorescence intensity overlay projection of appressoria (arrows) and underlying biotrophic hyphae showing fluorescent foci (arrowheads) on the hyphal surface. (B) Single optical section from (A) showing labelling of the plant cell wall (arrows). (C, D) Mature biotrophic hypha, viewed as in (A and B), showing fluorescence accumulation in hyphal concavities (arrowheads). Arrow: appressorium. (E) Epidermal cell infected by a biotrophic hypha (arrows) showing fluorescence in the apoplastic space (*) enlarged by plasmolysis. Arrowheads demarcate the host plasma membrane. V, vacuole of the host protoplast. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s007" target="_blank">Figure S7</a> for the corresponding bright field image. (F) Unlabelled necrotrophic hypha (arrow) emerging from a biotrophic hypha. Images were recorded at 43 hpi (A–E) and 55 hpi (F). Scale bars: 10 µm (E) and 5 µm (A, C, F). BH, biotrophic hypha. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s006" target="_blank">Figure S6</a>.</p