High-throughput profiling and analysis of plant responses over time to abiotic stress

Sorghum (Sorghum bicolor (L.) Moench) is a rapidly growing, high-biomass crop prized for abiotic stress tolerance. However, measuring genotype-by-environment (G x E) interactions remains a progress bottleneck. We subjected a panel of 30 genetically diverse sorghum genotypes to a spectrum of nitrogen deprivation and measured responses using high-throughput phenotyping technology followed by ionomic profiling. Responses were quantified using shape (16 measurable outputs), color (hue and intensity), and ionome (18 elements). We measured the speed at which specific genotypes respond to environmental conditions, in terms of both biomass and color changes, and identified individual genotypes that perform most favorably. With this analysis, we present a novel approach to quantifying colorbased stress indicators over time. Additionally, ionomic profiling was conducted as an independent, low-cost, and high-throughput option for characterizing G x E, identifying the elements most affected by either genotype or treatment and suggesting signaling that occurs in response to the environment. This entire dataset and associated scripts are made available through an open-access, user-friendly, web-based interface. In summary, this work provides analysis tools for visualizing and quantifying plant abiotic stress responses over time. These methods can be deployed as a time-efficient method of dissecting the genetic mechanisms used by sorghum to respond to the environment to accelerate crop improvement

Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria. © 2014 Pernas et al

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species

High-throughput profiling and analysis of plant responses over time to abiotic stress

Sorghum (Sorghum bicolor (L.) Moench) is a rapidly growing, high-biomass crop prized for abiotic stress tolerance. However, measuring genotype-by-environment (G x E) interactions remains a progress bottleneck. We subjected a panel of 30 genetically diverse sorghum genotypes to a spectrum of nitrogen deprivation and measured responses using high-throughput phenotyping technology followed by ionomic profiling. Responses were quantified using shape (16 measurable outputs), color (hue and intensity), and ionome (18 elements). We measured the speed at which specific genotypes respond to environmental conditions, in terms of both biomass and color changes, and identified individual genotypes that perform most favorably. With this analysis, we present a novel approach to quantifying colorbased stress indicators over time. Additionally, ionomic profiling was conducted as an independent, low-cost, and high-throughput option for characterizing G x E, identifying the elements most affected by either genotype or treatment and suggesting signaling that occurs in response to the environment. This entire dataset and associated scripts are made available through an open-access, user-friendly, web-based interface. In summary, this work provides analysis tools for visualizing and quantifying plant abiotic stress responses over time. These methods can be deployed as a time-efficient method of dissecting the genetic mechanisms used by sorghum to respond to the environment to accelerate crop improvement

The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii

Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this hostpathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine-rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells upregulate immunity-related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N-terminal arginine-rich domain, is critical to its function as a virulence determinant

The Polymorphic Pseudokinase ROP5 Controls Virulence in <em>Toxoplasma gondii</em> by Regulating the Active Kinase ROP18

<div><p>Secretory polymorphic serine/threonine kinases control pathogenesis of <em>Toxoplasma gondii</em> in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 <em>in vitro</em>, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δ<em>rop5</em> and Δ<em>rop18</em> mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3^−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.</p> </div

ROP5 regulates the kinase activity of ROP18.

<p>(A) Expression of ROP18 and ROP5 detected by western blotting of parasite lysates with rabbit anti-ROP18 (Rb α-ROP18), rabbit anti-ROP5 (Rb αROP5), and rabbit anti-actin (Rb αACT1) as a loading control. Representative of 3 experiments with similar outcomes. (B) Quantification of ROP18 expression by phosphorimager analysis of western blots, normalized for loading by actin staining. Means ± S.E.M. n = 3 experiments. (C) Immunofluorescence localization of ROP18 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>ΔKu80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. ROP18 was localized based on the C-terminal Ty-1 epitope described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Taylor1" target="_blank">[7]</a> using mAb BB2 (directly conjugated to Alexa Fluor 488, green). The vacuole membrane was stained with polyclonal rabbit anti-GRA7, described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Dunn1" target="_blank">[64]</a>, (secondary: Alexa Fluor 594, red). Scale = 5 microns. (D) Immunofluorescence localization of ROP5 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>Δku80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. The vacuole membrane was labeled with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). ROP5 was labeled with polyclonal Ab for ROP5 (secondary: Alexa Fluor 488, green). Scale = 5 microns. (E) <i>In vitro</i> kinase reaction using the kinase domain of ROP18 (ROP18-KD, 100 ng) and the heterologous substrate dMBP ± recombinant ROP5 (rROP5, 200 ng). (F) <i>In vitro</i> kinase reaction using full length ROP18 (ROP18-FL, 25 ng) and the natural substrate Irgb6 ± recombinant ROP5 (rROP5, 50 ng). Irgb6 was immunoprecipitated (∼10–20 ng/reaction) from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. (G) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with polyclonal rabbit anti-ROP18 (Rb anti-ROP18). Bound (denoted as B) and unbound (denoted as UB) samples were resolved by SDS-PAGE and blotted for ROP18 (Rb anti-ROP18 biotin). (H) <i>In vitro</i> kinase reaction of ROP18 using the heterologous substrate dMBP. ROP18 immunoprecipitations from (G) were incubated with or without substrate in the presence of ³²P ATP. Reactions were resolved by SDS-PAGE and subjected to phosphorimager analysis. (I) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with rabbit anti-ROP18 as in (G). (J) <i>In vitro</i> kinase reaction of ROP18 and a natural substrate Irgb6. ROP18 immunoprecipitations from (I) were incubated with or without substrate in the presence of ³²P ATP. Irgb6 was immunoprecipitated from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. Reactions in E, F, H, and J were carried out in the presence of ³²P ATP, resolved by SDS-PAGE and subjected to phosphorimager analysis. E–J are representative of 3 or more experiments with similar outcomes.</p

ROP5 and ROP18 are required for avoidance of clearance and IRG recruitment.

<p><i>In vitro</i> clearance of parasites in naïve peritoneal macrophages (A) or Gr1⁺ inflammatory monocytes (B) was measured by immunofluorescence microscopy. Cells were stained at 0.5 and 20 h post infection for host cell surface markers (see methods) and the parasite surface marker SAG1. Infection rates at 20 h post infection were normalized to initial infection rates. Means ± S.E.M., n = 3 samples each, from 3 combined experiments. Student's <i>t</i> test, **<i>P</i><0.0005. (C) Immunofluorescence localization of Irgb6 on the parasitophorous vacuole membrane in Gr1⁺ inflammatory monocytes infected for 0.5 h <i>in vitro</i>. The vacuole membrane was visualized by staining with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). Irgb6 was visualized with rabbit anti-Irgb6 (secondary: Alexa Fluor 488, green). Scale = 5 microns. (D) Quantification of Irgb6 localization to the vacuolar membrane in Gr1⁺ monocytes. Mean ± SEM, n = 3 samples each, from 3 combined experiments. Student's <i>t</i> test, *<i>P</i><0.005.</p

ROP5 deficient parasites are controlled in wild type mice and trigger a cytokine response proportional to the parasite load.

<p>(A) <i>In vivo</i> parasite growth was monitored using luciferase expressing wild type (RHΔ<i>ku80</i>) or ROP5 deficient (RHΔ<i>ku80</i>Δ<i>rop5</i>) parasites. CD-1 mice were i.p. injected with either 10⁶ or 10³ parasites and imaged on indicated days. † denotes one or more deaths. Mean values shown per group (n = 5). Representative of 2 experiments with similar outcomes. (B) Analysis of cytokines in serum of infected mice used in (A) (10⁶ inoculum) on days 0 (uninfected), 3, and 5. The levels of IFN-γ, IL-6, MCP-1, IL-10, and TNF-α were determined using a Cytometric Bead Array analyzed on a FACS Canto. IL-12p40 was measured by ELISA. Mean ± S.D., n = 3 animals per group. Student's <i>t</i> test * <i>P</i><0.01. Representative of 2 experiments with similar outcomes.</p