CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.

Collection of aboveground community and species-specific plant biomass from the Jena Experiment (time series since 2002).

This data set comprises time series of aboveground community plant biomass (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) and species-specific biomass from the sown species of several experiments at the field site of a large grassland biodiversity experiment (the Jena Experiment; see further details below). Aboveground community biomass was normally harvested twice a year just prior to mowing (during peak standing biomass twice a year, generally in May and August; in 2002 only once in September) on all experimental plots in the Jena Experiment. This was done by clipping the vegetation at 3 cm above ground in up to four rectangles of 0.2 x 0.5 m per large plot. The location of these rectangles was assigned by random selection of new coordinates every year within the core area of the plots. The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: individual species for the sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. Sown plant community biomass was calculated as the sum of the biomass of the individual sown species. The data for individual samples and the mean over samples for the biomass measures on the community level are given. Overall, analyses of the community biomass data have identified species richness as well as functional group composition as important drivers of a positive biodiversity-productivity relationship. The following series of datasets are contained in this collection: 1. Plant biomass form the Main Experiment: In the Main Experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). 2. Plant biomass from the Dominance Experiment: In the Dominance Experiment, 206 grassland plots of 3.5 x 3.5 m were established from a pool of 9 species that can be dominant in semi-natural grassland communities of the study region. In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 3, 4, 6, and 9 species). 3. Plant biomass from the monoculture plots: In the monoculture plots the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species like the other experiments in May 2002. All plots were maintained by bi-annual weeding and mowing.

Aboveground community and species-specific plant biomass from the Jena Experiment (Dominance Experiment, year 2008)

This data set contains aboveground community plant biomass (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) and species-specific biomass from the sown species of the dominance experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the dominance experiment, 206 grassland plots of 3.5 x 3.5 m were established from a pool of 9 plant species that can be dominant in semi-natural grassland communities of the study region. In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 3, 4, 6, and 9 species). Plots were maintained by bi-annual weeding and mowing. Aboveground community biomass was harvested twice in May and August 2008 on all experimental plots of the dominance experiment. This was done by clipping the vegetation at 3 cm above ground in two rectangles of 0.2 x 0.5 m per experimental plot. The location of these rectangles was assigned by random selection of coordinates within the central area of the plots (excluding an outer edge of 50cm). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: individual species for the sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material, and remaining plant material that could not be assigned to any category. All biomass was dried to constant weight (70°C, >= 48 h) and weighed. Sown plant community biomass was calculated as the sum of the biomass of the individual sown species. The mean of both samples per plot and the individual measurements are provided in the data file. Overall, analyses of the community biomass data have identified species richness and the presence of particular species as an important driver of a positive biodiversity-productivity relationship.

Aboveground community and species-specific plant biomass from the Jena Experiment (Main Experiment, year 2009)

This data set contains aboveground community biomass in 2009 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) and species-specific biomass from the sown species of the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Aboveground community biomass was harvested twice in 2009 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the main experiment. This was done by clipping the vegetation at 3 cm above ground in three rectangles of 0.2 x 0.5 m per large plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. the central 10 x 15 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: individual species for the sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. Sown plant community biomass was calculated as the sum of the biomass of the individual sown species. The data for individual samples and the mean over samples for all biomass measures are given. Overall, analyses of the community biomass data have identified species richness as well as functional group composition as important drivers of a positive biodiversity-productivity relationship.

Aboveground community and species-specific plant biomass from the Jena Experiment (Main Experiment, year 2010)

This data set contains aboveground community biomass in 2010 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) and species-specific biomass from the sown species of the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Aboveground community biomass was harvested twice in 2010 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the main experiment. This was done by clipping the vegetation at 3 cm above ground in two rectangles of 0.2 x 0.5 m per large plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. the central 10 x 15 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: individual species for the sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. Sown plant community biomass was calculated as the sum of the biomass of the individual sown species. The data for individual samples and the mean over samples for all biomass measures are given. Overall, analyses of the community biomass data have identified species richness as well as functional group composition as important drivers of a positive biodiversity-productivity relationship.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2003)

This data set contains aboveground plant biomass in 2003 (Sown plant community, Weed plant community, and Dead plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2003 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), and detached dead plant material (i.e., dead plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2010)

This data set contains aboveground plant biomass in 2010 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. One of the replicate plots per species was given up after the vegetation period of 2007 for all but the nine species belonging also to the so called dominance experiment in Jena. These nine species are: Alopecurus pratensis, Anthriscus sylvestris, Arrhenatherum elatius, Dactylis glomerata, Geranium pratense, Poa trivialis, Phleum pratense, Trifolium repens and Trifolium pratense.In 2010 plot size was reduced to 1 x 1 m. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2010 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 1 rectangle of 0.2 x 0.5 m per plot. The location of this rectangle was in the center of the plot area. The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2005)

This data set contains aboveground plant biomass in 2005 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2005 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2006)

This data set contains aboveground plant biomass in 2006 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2006 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2007)

This data set contains aboveground plant biomass in 2007 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2007 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2008)

This data set contains aboveground plant biomass in 2008 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. One of the replicate plots per species was given up after the vegetation period of 2007 for all but the nine species belonging also to the so called dominance experiment in Jena. These nine species are: Alopecurus pratensis, Anthriscus sylvestris, Arrhenatherum elatius, Dactylis glomerata, Geranium pratense, Poa trivialis, Phleum pratense, Trifolium repens and Trifolium pratense.In 2008 plot size was reduced to 2.5 x 2.5 m. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2008 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2009)

This data set contains aboveground plant biomass in 2009 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. One of the replicate plots per species was given up after the vegetation period of 2007 for all but the nine species belonging also to the so called dominance experiment in Jena. These nine species are: Alopecurus pratensis, Anthriscus sylvestris, Arrhenatherum elatius, Dactylis glomerata, Geranium pratense, Poa trivialis, Phleum pratense, Trifolium repens and Trifolium pratense.In 2008 plot size was reduced to 2.5 x 2.5 m. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2009 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was in the center of the plot area. The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2002)

This data set contains aboveground plant biomass in 2002 (Sown plant community; measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2002 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. From the harvested biomass only the separated biomass of the sown plant species was kept. All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Aboveground plant biomass from the Jena Experiment (Monocultures, year 2004)

This data set contains aboveground plant biomass in 2004 (Sown plant community, Weed plant community, Dead plant material, and Unidentified plant material; all measured in biomass as dry weight) of the monoculture plots of a large grassland biodiversity experiment (the Jena Experiment). In the monoculture plots the biomass of the sown plant community contains only a single species per plot and this species is a different one for each plot. Which species has been sown in which plot is stated in the plot information table for monocultures (see further details below). The monoculture plots of 3.5 x 3.5 m were established for all of the 60 plant species of the Jena Experiment species pool with two replicates per species. These 60 species comprising the species pool of the Jena Experiment belong to four functional groups (grasses, legumes, tall and small herbs). Plots were sown in May 2002 and are since maintained by bi-annual weeding and mowing. Aboveground plant biomass was harvested twice in 2004 just prior to mowing (during peak standing biomass in early June and in late August) on all experimental plots of the monocultures. This was done by clipping the vegetation at 3 cm above ground in 2 rectangles of 0.2 x 0.5 m per plot. The location of these rectangles was assigned prior to each harvest by random selection of coordinates within the core area of the plots (i.e. excluding an outer edge of 0.5 m). The positions of the rectangles within plots were identical for all plots. The harvested biomass was sorted into categories: sown plant species, weed plant species (species not sown at the particular plot), detached dead plant material (i.e., dead plant material in the data file), and remaining plant material that could not be assigned to any category (i.e., unidentified plant material in the data file). All biomass was dried to constant weight (70°C, >= 48 h) and weighed. The data for individual subsamples (i.e. rectangles) and the mean over samples for all biomass measures are given.

Exploring several nonconventional interactions of ice-binding proteins

Traditionally, ice-binding proteins (IBPs), also known as antifreeze proteins (AFPs), have been defined by two universal activities: ice recrystallization inhibition and thermal hysteresis. However, there remains the possibility IBPs have other complementary functions given the diversity found within this protein group. This thesis explores some of these in both natural and applied settings, in the hopes of furthering our understanding of this remarkable group of proteins. Plant IBPs could function as part of a defensive strategy against ice nucleators produced by certain pathogens. To assess this hypothesis, recombinant IBPs from perennial ryegrass and purple false brome were combined with the ice nucleation protein (INP) from the plant pathogen, Pseudomonas syringae. Strikingly, the plant proteins depressed the freezing point of the bacterial INP, while a fish AFP could not, nor did the INPs have any effect on IBP activity. Thus, the interaction between these two different proteins suggests a role in plant defensive strategies against pathogenic bacteria as another IBP function. In addition, the potential use of hyperactive insect IBPs in organ preservation was investigated. Current kidney preservation techniques involve storing the organ at 4 °C for a maximum of 24 h prior to transplantation. Extending this “safe” time would have profound effects on renal transplants, however, ischemic injury is prevalent when storage periods are prolonged. Experiments described here allowed subzero preservation for 72 h with the addition of a beetle IBP to CryoStasis® solution. Kidneys stored using the traditional technique for 24 h and the method developed here for 72 h showed similar levels of biomarker enzymes, underscoring the potential utility of insect IBPs for future transplant purposes. Finally, IBP function in the freeze-tolerant gall fly, Eurosta solidaginis, was examined. Larvae representing the mid-autumn stage displayed ice-binding activity, suggesting an IBP is being expressed, possibly as a protective measure against freezing damage when fall temperatures can unpredictably drop. IBP activity was also observed in the larvae’s host plant, Solidago spp. Mass spectrometry analysis of ice-affinity purified plant extracts provided three candidate pathogenesis-related proteins that could be responsible for the detected activity, further demonstrating additional functions of IBPs.