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 color-based 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.

Quantitative, Image-Based Phenotyping Methods Provide Insight into Spatial and Temporal Dimensions of Plant Disease.

Plant disease symptoms exhibit complex spatial and temporal patterns that are challenging to quantify. Image-based phenotyping approaches enable multidimensional characterization of host-microbe interactions and are well suited to capture spatial and temporal data that are key to understanding disease progression. We applied image-based methods to investigate cassava bacterial blight, which is caused by the pathogen Xanthomonas axonopodis pv. manihotis (Xam). We generated Xam strains in which individual predicted type III effector (T3E) genes were mutated and applied multiple imaging approaches to investigate the role of these proteins in bacterial virulence. Specifically, we quantified bacterial populations, water-soaking disease symptoms, and pathogen spread from the site of inoculation over time for strains with mutations in avrBs2, xopX, and xopK as compared to wild-type Xam ∆avrBs2 and ∆xopX both showed reduced growth in planta and delayed spread through the vasculature system of cassava. ∆avrBs2 exhibited reduced water-soaking symptoms at the site of inoculation. In contrast, ∆xopK exhibited enhanced induction of disease symptoms at the site of inoculation but reduced spread through the vasculature. Our results highlight the importance of adopting a multipronged approach to plant disease phenotyping to more fully understand the roles of T3Es in virulence. Finally, we demonstrate that the approaches used in this study can be extended to many host-microbe systems and increase the dimensions of phenotype that can be explored.

Toxoplasma GRA7 effector increases turnover of immunity-related GTPases and contributes to acute virulence in the mouse.

The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant gra7 or rop18 was partially attenuated for virulence in mice, whereas the combined gra7rop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.

Circadian gene Bmal1 regulates diurnal oscillations of Ly6C(hi) inflammatory monocytes.

Circadian clocks have evolved to regulate physiologic and behavioral rhythms in anticipation of changes in the environment. Although the molecular clock is present in innate immune cells, its role in monocyte homeostasis remains unknown. Here, we report that Ly6C(hi) inflammatory monocytes exhibit diurnal variation, which controls their trafficking to sites of inflammation. This cyclic pattern of trafficking confers protection against Listeria monocytogenes and is regulated by the repressive activity of the circadian gene Bmal1. Accordingly, myeloid cell-specific deletion of Bmal1 induces expression of monocyte-attracting chemokines and disrupts rhythmic cycling of Ly6C(hi) monocytes, predisposing mice to development of pathologies associated with acute and chronic inflammation. These findings have unveiled a critical role for BMAL1 in controlling the diurnal rhythms in Ly6C(hi) monocyte numbers.

Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification.

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 µg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.

Guanylate-binding protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii.

IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1(-/-) cells, and decreased virulence of this mutant was compensated in Gbp1(-/-) mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.

The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18.

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii 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 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 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.

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 host-pathogen 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 secreted kinase ROP18 defends Toxoplasma's border.

Toxoplasma gondii is a highly successful parasite capable of infecting virtually all warm-blooded animals by actively invading nucleated host cells and forming a modified compartment where it replicates within the cytosol. The parasite-containing vacuole provides a safe haven, even in professional phagocytes such as macrophages, which normally destroy foreign microbes. In an effort to eliminate the parasite, the host up-regulates a family of immunity-related p47 GTPases (IRGs), which are recruited to the parasite-containing vacuole, resulting in membrane rupture and digestion of the parasite. To avoid this fate, highly virulent strains of Toxoplasma coat the external surface of their vacuole with a secretory serine/threonine kinase, known as ROP18. At this host-pathogen interface, ROP18 phosphorylates and inactivates IRGs, thereby protecting the parasite from killing. These findings reveal a novel molecular mechanism by which the parasite disarms host innate immunity.

Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence.

Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.