Regina Hanlon

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

Infection and genotype remodel the entire soybean transcriptome

BackgroundHigh throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen Phytophthora sojae and to analyze transcriptional modulation as a result of genotypic variation.ResultsWith the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures.ConclusionOur findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.

Functional Genomics and Bioinformatics of the Phytophthora sojae Soybean Interaction

Oomycete plant pathogens such as Phytophthora species and downy mildews cause destructive diseases in an enormous variety of crop plant species as well as forests and native ecosystems. These pathogens are most closely related to algae in the kingdom Stramenopiles, and hence have evolved plant pathogenicity independently of other plant pathogens such as fungi. We have used bioinformatic analysis of genome sequences and EST collections, together with functional genomics to identify plant and pathogen genes that may be key players in the interaction between the soybean pathogen Phytophthora sojae and its host. In P. sojae, we have identified many rapidly diversifying gene families that encode potential pathogenicity factors including protein toxins, and a class of proteins (avirulence or effector proteins) that appear to have the ability to penetrate plant cells. Transcriptomic analysis of quantitative or multigenic resistance against P. sojae in soybean has revealed that there are widespread adjustments in host gene expression in response to infection, and that some responses are unique to particular resistant cultivars. These observations lay the foundation for dissecting the interplay between pathogen and host genes during infection at a whole-genome level.

Remote collection of microorganisms at two depths in a freshwater lake using an unmanned surface vehicle (USV)

Microorganisms are ubiquitous in freshwater aquatic environments, but little is known about their abundance, diversity, and transport. We designed and deployed a remote-operated water-sampling system onboard an unmanned surface vehicle (USV, a remote-controlled boat) to collect and characterize microbes in a freshwater lake in Virginia, USA. The USV collected water samples simultaneously at 5 and 50 cm below the surface of the water at three separate locations over three days in October, 2016. These samples were plated on a non-selective medium (TSA) and on a medium selective for the genus Pseudomonas (KBC) to estimate concentrations of culturable bacteria in the lake. Mean concentrations ranged from 134 to 407 CFU/mL for microbes cultured on TSA, and from 2 to 8 CFU/mL for microbes cultured on KBC. There was a significant difference in the concentration of microbes cultured on KBC across three sampling locations in the lake (P = 0.027), suggesting an uneven distribution of Pseudomonas across the locations sampled. There was also a significant difference in concentrations of microbes cultured on TSA across the three sampling days (P = 0.038), demonstrating daily fluctuations in concentrations of culturable bacteria. There was no significant difference in concentrations of microbes cultured on TSA (P = 0.707) and KBC (P = 0.641) across the two depths sampled, suggesting microorganisms were well-mixed between 5 and 50 cm below the surface of the water. About 1 percent (7/720) of the colonies recovered across all four sampling missions were ice nucleation active (ice+) at temperatures warmer than −10 °C. Our work extends traditional manned observations of aquatic environments to unmanned systems, and highlights the potential for USVs to understand the distribution and diversity of microbes within and above freshwater aquatic environments.

Tracking of a fluorescent dye in a freshwater lake with an unmanned surface vehicle and an unmanned aircraft system

Recent catastrophic events in our oceans, including the spill of toxic oil from the explosion of the Deepwater Horizon drilling rig and the rapid dispersion of radioactive particulates from the meltdown of the Fukushima Daiichi nuclear plant, underscore the need for new tools and technologies to rapidly respond to hazardous agents. Our understanding of the movement and aerosolization of hazardous agents from natural aquatic systems can be expanded upon and used in prevention and tracking. New technologies with coordinated unmanned robotic systems could lead to faster identification and mitigation of hazardous agents in lakes, rivers, and oceans. In this study, we released a fluorescent dye (fluorescein) into a freshwater lake from an anchored floating platform. A fluorometer (fluorescence sensor) was mounted underneath an unmanned surface vehicle (USV, unmanned boat) and was used to detect and track the released dye in situ in real-time. An unmanned aircraft system (UAS) was used to visualize the dye and direct the USV to sample different areas of the dye plume. Image processing tools were used to map concentration profiles of the dye plume from aerial images acquired from the UAS, and these were associated with concentration measurements collected from the sensors onboard the USV. The results of this project have the potential to transform monitoring strategies for hazardous agents, enabling timely and accurate exposure assessment and response in affected areas. Fast response is essential in reacting to the introduction of hazardous agents, in order to quickly predict and contain their spread.

Diversity and Ice Nucleation Activity of Microorganisms collected with a Small Unmanned Aircraft System (sUAS) in France and the United States

Many microbes relevant to crops, domestic animals, and humans are transported over long distances through the atmosphere. Some of these atmospheric microbes catalyze the freezing of water at higher temperatures and facilitate the onset of precipitation. We collected microbes from the lower atmosphere in France and the United States with a small unmanned aircraft system (sUAS). 55 sampling missions were conducted at two locations in France in 2014 (an airfield in Pujaut, and the top of Puy de Dôme), and three locations in the U.S. in 2015 (a farm in Blacksburg, Virginia, and a farm and a lake in Baton Rouge, Louisiana). The sUAS was a fixed-wing electric drone equipped with a remote-operated sampling device that was opened once the aircraft reached the desired sampling altitude (40–50 meters above ground level). Samples were collected on agar media (TSA, R4A, R2A, and CA) with and without the fungicide cycloheximide. Over 4,000 bacterial-like colonies were recovered across the 55 sUAS sampling missions. A positive relationship between sampling time and temperature and concentrations of culturable bacteria was observed for sUAS flights conducted in France, but not for sUAS flights conducted in Louisiana. A droplet freezing assay was used to screen nearly 2,000 colonies for ice nucleation activity, and 15 colonies were ice nucleation active at temperatures warmer than −8°C. Sequences from portions of 16S rDNA were used to identify 503 colonies from 54 flights to the level of genus. Assemblages of bacteria from sUAS flights in France (TSA) and sUAS flights in Louisiana (R4A) showed more similarity within locations than between locations. Bacteria collected with sUAS on TSA in France and Virginia were significantly different across all levels of classification tested (P < 0.001 for class, order, family, and genus). Principal Coordinates Analysis showed a strong association between the genera Curtobacterium, Pantoea, and Pseudomonas from sUAS flights in Virginia, and Agrococcus, Lysinibacillus, and Paenibacillus from sUAS flights in France. Future work aims to understand the potential origin of the atmospheric microbial assemblages collected with sUAS, and their association with mesoscale atmospheric processes.

Mutagenesis and selection for oligomycin resistance in soybean (Glycine max L. Merr) suspension culture cells

Soybean suspension culture cells were subjected to mutagenesis with ethyl methane sulfonate and cells resistant to the ATP synthase inhibitor oligomycin were recovered. Suspension cultures showed high levels of resistance over a period of 17 months after mutagenesis. The level of resistance to oligomycin decreased during prolonged growth in the absence of the selection agent. Although mitochondrial inheritance of the resistance phenotype was not tested, this observation is consistent with segregation of resistant and sensitive mitochondria in progeny cells. Cross resistance to venturicidin was also observed which suggested the possible involvement of the mitochondrial ATP synthase subunit 9 gene, however, sequences of ATP synthase subunit 9 cDNA clones from resistant cells were identical to the wild type sequence. In addition, Southern blot analyses of DNA from wild type and resistant cultures did not identify rearrangements. These results indicated that resistance to oligomycin could not be directly attributed to a mutation in the primary gene sequence, an alteration in the RNA editing pattern of transcripts, or gross rearrangements in the region containing and directly adjacent to the ATP synthase subunit 9 gene in the mitochondrial DNA from resistant cultures.

Wind-driven spume droplet production and the transport of Pseudomonas syringae from aquatic environments

Natural aquatic environments such as oceans, lakes, and rivers are home to a tremendous diversity of microorganisms. Some may cross the air-water interface within droplets and become airborne, with the potential to impact the Earth’s radiation budget, precipitation processes, and spread of disease. Larger droplets are likely to return to the water or adjacent land, but smaller droplets may be suspended in the atmosphere for transport over long distances. Here, we report on a series of controlled laboratory experiments to quantify wind-driven droplet production from a freshwater source for low wind speeds. The rate of droplet production increased quadratically with wind speed above a critical value (10-m equivalent 5.7 m/s) where droplet production initiated. Droplet diameter and ejection speeds were fit by a gamma distribution. The droplet mass flux and momentum flux increased with wind speed. Two mechanisms of droplet production, bubble bursting and fragmentation, yielded different distributions for diameter, speed, and angle. At a wind speed of about 3.5 m/s, aqueous suspensions of the ice-nucleating bacterium Pseudomonas syringae were collected at rates of 283 cells m−2 s−1 at 5 cm above the water surface, and at 14 cells m−2 s−1 at 10 cm above the water surface. At a wind speed of about 4.0 m/s, aqueous suspensions of P. syringae were collected at rates of 509 cells m−2 s−1 at 5 cm above the water surface, and at 81 cells m−2 s−1 at 10 cm above the water surface. The potential for microbial flux into the atmosphere from aquatic environments was calculated using known concentrations of bacteria in natural freshwater systems. Up to 3.1 × 104 cells m−2 s−1 of water surface were estimated to leave the water in potentially suspended droplets (diameters <100 µm). Understanding the sources and mechanisms for bacteria to aerosolize from freshwater aquatic sources may aid in designing management strategies for pathogenic bacteria, and could shed light on how bacteria are involved in mesoscale atmospheric processes.

Coordinated Sampling of Microorganisms Over Freshwater and Saltwater Environments Using an Unmanned Surface Vehicle (USV) and a Small Unmanned Aircraft System (sUAS)

Biological aerosols (bioaerosols) are ubiquitous in terrestrial and aquatic environments and may influence cloud formation and precipitation processes. Little is known about the aerosolization and transport of bioaerosols from aquatic environments. We designed and deployed a bioaerosol-sampling system onboard an unmanned surface vehicle (USV; a remotely operated boat) to collect microbes and monitor particle sizes in the atmosphere above a salt pond in Falmouth, MA, United States and a freshwater lake in Dublin, VA, United States. The bioaerosol-sampling system included a series of 3D-printed impingers, two different optical particle counters, and a weather station. A small unmanned aircraft system (sUAS; a remotely operated airplane) was used in a coordinated effort with the USV to collect microorganisms on agar media 50 m above the surface of the water. Samples from the USV and sUAS were cultured on selective media to estimate concentrations of culturable microorganisms (bacteria and fungi). Concentrations of microbes from the sUAS ranged from 6 to 9 CFU/m3 over saltwater, and 12 to 16 CFU/m3 over freshwater (over 10-min sampling intervals) at 50 m above ground level (AGL). Concentrations from the USV ranged from 0 (LOD) to 42,411 CFU/m3 over saltwater, and 0 (LOD) to 56,809 CFU/m3 over freshwater (over 30-min sampling intervals) in air near the water surface. Particle concentrations recorded onboard the USV ranged from 0 (LOD) to 288 μg/m3 for PM1, 1 to 290 μg/m3 for PM2.5, and 1 to 290 μg/m3 for PM10. A general trend of increasing concentration with an increase in particle size was recorded by each sensor. Through laboratory testing, the collection efficiency of the 3D-printed impingers was determined to be 75% for 1 μm beads and 99% for 3 μm beads. Additional laboratory tests were conducted to determine the accuracy of the miniaturized optical particle counters used onboard the USV. Future work aims to understand the distribution of bioaerosols above aquatic environments and their potential association with cloud formation and precipitation processes.