NOTE: End date changed to 08/31/2023 per NSSC information (Ed., 6/6/22).

1) Investigate physiological and whole genome transcriptional responses to defense activation in wild-type and immune-deficient tomatoes during spaceflight. Tomatoes will be grown in the Advanced Plant Habitat (APH). We will activate defense responses with a chemical elicitor. At 24 and 48 hours after defense activation, we will harvest tissue and subsequently perform next-generation sequencing to identify genome-wide transcriptional defense responses. In addition, we will use next-generation sequencing to examine the transcriptional response to spaceflight in immune-deficient tomatoes. All plants will be imaged daily to understand the impact of spaceflight on growth rates of immune-activated and immune-deficient tomatoes. All experiments will be performed in parallel on the ground.

2) Determine whether colonization of tomato by the fungal plant pathogen Fusarium oxysporum is impacted by simulated microgravity. We will grow plants in a 2D-ground-based microgravity simulator and inoculate them with Fusarium oxysporum. We will assess tomato plant colonization using histological techniques.

This work will generate key fundamental knowledge of plant-microbe interactions that is important for understanding plant production in space. It is consistent with the goal of the Plant Biology Element in the Space Biology Science Plan 2016-2025.

Specific Aim 1: Determine the effect of spaceflight on genome-wide transcriptomic and physiological defense responses in tomatoes.

i) Performed SVT tests in the NASA Advanced Plant Habitat (APH) with Red Wire team

This year (Year 4), work on Specific Aim 1 focused on conducting the Science Verification Test (SVT) experiments with Red Wire and finalizing protocols for the Experiment Verification Test (EVT) and spaceflight. In years 1 and 2 at Purdue, we optimized conditions (fertilizer, media) for tomato growth in the NASA science carrier. In year 3, we translated this work to NASA Kennedy Space Center (KSC) and conducted pre-SVT experiments with our NASA team. Starting in July 2022 (Year 4), we began working with Red Wire to conduct SVT experiments. In late Oct 2022, we initiated an SVT with Red Wire. However, this experiment was stopped 16 days after initiation (DAI) due to blue-green color developing on the wicks and unhealthy plants. After discussing the reasons for the problem, the team identified two primary possibilities: changes in fertilizer concentrations over time and windspeed strength. In Nov 2022 at Purdue, we tested three different combinations of fertilizer + substrate and identified a slightly different combination that appeared to promote better tomato growth in the science carrier.

We had an opportunity for SVT-1.5 in March 2023. The windspeed was reduced to 0.3 m/s for SVT-1.5. The goal of SVT-1.5 was to observe good germination and healthy, growing tomato plants. SVT-1.5 plants grew well.

Based on these results, we moved forward with SVT-2 in the Ground APH inside an International Space Station Environmental Simulator (ISSES) chamber in May 2023. All Moneymaker (MM) plants grew well during SVT-2, but only 5 NahG plants germinated and were healthy. Leaves were swabbed with salicylic acid (SA) or mock solution and harvested as expected. Frozen leaves were sent to Purdue, and we extracted total RNA. All Moneymaker plants had good quality RNA and had more than 250 µg/ul RNA. Only 2 of 5 NahG plants met the same metrics. Further examination of the data showed that plant size was related to RNA quality and amount. NahG plants germinated several days (typically 2 – 4) after the MM plants. The 2 NahG plants with good quality RNA were of comparable size to the MM plants. We concluded that promoting earlier germination of NahG would result in larger plants that would mitigate the issue of size and low quality/levels of RNA.

Based on our success criteria in our Experimental Research Design (ERD), all parts of SVT-2 met the minimum requirement for ‘acceptable’. However, there was a large difference between the two genotypes. If examining MM plants alone, many categories were ‘excellent’ while those for NahG would have been minimum acceptable or unacceptable.

Moving forward for EVT, we made several changes to promote increased NahG germination. We planted an entire quadrant of NahG (an increase of 8 plants to 9 plants), planted extra seeds to ensure better germination, and started the NahG watering (flood fill) three days before quadrants were planted with MM plants. We passed our EVT readiness review on June 16, and are currently conducting our EVT.

Importantly, our experimental design has built-in redundancy. The central question of our research is: "How does spaceflight impact plant immune responses?" We address this question using a chemical method (treating plants with SA) and a genetic method (NahG plants). Thus, even if a sufficient number of NahG plants do not grow, the experimental design still allows us to answer our question.

Specific Aim 2: Investigate how simulated microgravity affects fungal colonization of tomato plants.

In Aim 2, we proposed a series of ground-based experiments to investigate the impact of simulated microgravity on the ability of fungal pathogens to infect tomato roots. In the first year of this project, we redesigned the 2D clinostat to accommodate eight plants as opposed to the original four-plant design. Using our redesigned system, we showed we could grow tomato cultivars to maturity under continuous clinorotation. In years 2 and 3, we successfully demonstrated the infection of Moneymaker tomato plants by Fusarium oxysporum in the enclosed rhizoboxes, and built a second clinostat to enable the simultaneous analysis of plants under clinorotation perpendicular and parallel to the gravity vector. We also optimized the growth conditions on the paired clinostat (clinorotated and upright) and established conditions where the plants were healthy for 15 days on the clinostat. In Year 4, we optimized F. oxysporum inoculations and inoculated two replicates. We are currently growing additional replicates and are cutting sections from roots and stems.

In an exciting new result for Year 4, we have identified changes in stem vascular anatomy due to clinorotation. These defects vary among the two cultivars tested: MM and Hawaii7996 (HA7996), and can be classified as two distinct changes:

1) The xylem protrusions into the pith, evident in the upright control stems, are mostly lost in both cultivars. 2) Xylem protrusions have either expanded into a complete band surrounding pith (in HA7996) or are partially absent (in MM).

These differences are not observed in sections from the root-stem junction, and we are currently processing samples from the root. These anatomical differences have significant implications for plant physiology in spaceflight and the potential of vascular pathogens to colonize the xylem. The anatomical differences we observed are not associated with stem diameter or height differences. However, MM clinorotated plants have a reduced shoot weight as compared to their upright counterparts.

In addition to the work in Specific Aims 1 and 2, in year 4 we worked with our NASA colleagues to develop a final version of the ERD.

1) Investigate physiological and whole genome transcriptional responses to defense activation in wild-type and immune-deficient tomatoes during spaceflight. Tomatoes will be grown in the Advanced Plant Habitat (APH). We will activate defense responses with a chemical elicitor. At 24 and 48 hours after defense activation, we will harvest tissue and subsequently perform next-generation sequencing to identify genome-wide transcriptional defense responses. In addition, we will use next-generation sequencing to examine the transcriptional response to spaceflight in immune-deficient tomatoes. All plants will be imaged daily to understand the impact of spaceflight on growth rates of immune-activated and immune-deficient tomatoes. All experiments will be performed in parallel on the ground.

2) Determine whether colonization of tomato by the fungal plant pathogen Fusarium oxysporum is impacted by simulated microgravity. We will grow plants in a 2D-ground-based microgravity simulator and inoculate them with Fusarium oxysporum. We will assess tomato plant colonization using histological techniques.

This work will generate key fundamental knowledge of plant-microbe interactions that is important for understanding plant production in space. It is consistent with the goal of the Plant Biology Element in the Space Biology Science Plan 2016-2025.

The central hypothesis of this proposal is: ‘Spaceflight and simulated microgravity increase tomato susceptibility to pathogens through altered transcriptional defense responses and increased pathogen colonization.’

The goals of this proposal are twofold: 1) gain fundamental insights into how the activated plant immune system responds in space in a horticultural crop, and 2) gain knowledge of the impact of simulated microgravity on plant colonization and disease development by a pathogenic fungus.

To accomplish these goals, we have two Specific Aims:

Specific Aim 1: Determine the effect of spaceflight on genome-wide transcriptomic and physiological defense responses in tomatoes. Aim 1 Hypothesis: In response to a defense elicitor, spaceflight-grown wild type (WT) plants will have delayed transcriptional defense responses compared to ground controls. Spaceflight grown immune-deficient plants will have altered transcriptional responses and slower plant growth rates compared to ground treated and untreated plants.

To test our hypothesis, we will use RNA-seq analysis to investigate genome-wide defense responses before and after treatment with a defense elicitor in wild type and immune-deficient tomatoes during spaceflight. Differential gene expression will be examined in wild-type tomatoes +/- the defense elicitor and in immune-deficient tomatoes compared to wild-type. We will also monitor growth rate of tomatoes using cameras in the APH before and after treatment with the defense elicitor.

Specific Aim 2: Investigate how simulated microgravity affects pathogen colonization of tomato plants. Aim 2 Hypothesis: Simulated microgravity will cause earlier and increased colonization of tomato by the plant fungal pathogen Fusarium oxysporum.

To test our hypothesis, tomato plants will be grown in simulated microgravity conditions using a 2D clinostat at the University of Delaware, the Co-I’s institution. As a control, tomatoes will be grown parallel to the gravity vector while rotating clockwise. Tomato plants at the 3-leaf stage will be inoculated with Fusarium oxysporum or water as a control. At 10, 14, and 18 days post inoculation, plants will be removed from the clinostat and a 2 cm section below the root shoot junction will be harvested. Samples will be sent to Purdue for processing and microscopy to observe F. oxysporum colonization within the root tissues. Summary of research accomplishment 09/01/21 – 08/31/22, year 3 Specific Aim 1: Determine the effect of spaceflight on genome-wide transcriptomic and physiological defense responses in tomatoes.

i) Optimized conditions for tomato growth in the NASA Science Carrier This year (year 3), work on Specific Aim 1 focused on finalizing protocols necessary for tomato growth in the APH during spaceflight. In years 1 and 2, we optimized conditions (fertilizer, media) for tomato growth, using similar substrates as in the Advanced Plant Habitat /APH (year 1), and in the NASA science carrier (year 2). In year 3, we identified proper light conditions using a MARS hydro system and the NASA science carrier. We, and the NASA team, subsequently used these conditions to conduct pre-Science Verification Test (SVT) tests at NASA to test our salicylic acid (SA) treatment and leaf harvest protocol. The first test was conducted in December of 2021. Plants grew well and RNA integrity (RIN) values of the extracted RNA were mostly acceptable, and there were challenges with SA application and leaf harvest. We therefore conducted a second pre-SVT test in March 2022 with changes in leaf harvesting and RNA extraction procedures. SA application and leaf harvest was improved in this second pre-test. RNA RIN values improved compared to the first pre-SVT test, but were not excellent. For these tests, we harvested leaf tissue using -80°C cold blocks. Currently, only one question remains – whether harvesting leaf tissue with -160°C blocks would further improve RNA RIN values. Since pre-SVT tests are resource-intensive, at Purdue we are testing whether tissue that has been immediately frozen in liquid N2 (as a proxy for -160°C) will yield improved RIN values. These data should be finished by the end of July 2022, and then we will be ready for the SVT.

For both pre-SVT tests, photographs were taken of plants to test whether Purdue could accurately locate which leaf to swab.

In Year 2 we tested whether seeds would germinate if they were watered several weeks and months after planting, and found 100% germination rates even 4 months after planting. We also found the best configuration for our experiments (three seeds per row). In Year 3, we tested seed sterilization methods and found that using NASA’s hydrochloric acid (HCL) sterilization method provided good germination.

ii) Developing a safe spaceflight protocol for defense elicitation in tomato leaves. The goal of specific aim 1 is to investigate the impact of spaceflight on defense responses during spaceflight. To address this, we will elicit defense responses in space using a chemical elicitor. In Years 1 and 2, we started to test methods of treating tomato leaves with an elicitor. The method needs to work well in space and be easy to perform. In addition, we initiated experiments to test whether the chemical will elicit defense responses in tomatoes grown in the APH conditions. At the end of year 1, we found that swabbing tomato leaves with 5 mM salicylic acid (SA) using a Q-tip enabled expression of a SA response gene. However, the response was not as robust as we had hoped. We hypothesized that this could be due to the side of the leaf that had been swabbed, or the amount of SA applied from the Q-tip.

In year 2, we optimized this. We searched for a better SA application method, we tested applying SA to different sides of tomato leaves, we tested different leaf ages, and we tested higher concentrations of SA. We found that applying 7.5 mM SA to top and bottom of leaf #4 consistently activated the SA marker gene PR1-a. In contrast to 10 mM SA, 7.5 mM SA was not toxic to leaves. In year 3, we harvested RNA from leaves grown in our optimized conditions and treated with 7.5mM SA and found that the RIN score was acceptable for all samples.

In year 1, we found that storing tissue for one month did not alter the RNA quality. In year 2, we tested storing tissue up to four months in storage and found no change in RNA quality. In year 3 we tested whether SA that has been stored for 3 and 6 months would still able to elicit defense responses. Results were positive.

Together, these experiments support our ability to grow tomato plants in the APH during spaceflight and perform our experiment.

Specific Aim 2: Investigate how simulated microgravity affects fungal colonization of tomato plants.

In Aim 2, we proposed a series of ground-based experiments to investigate the impact of simulated microgravity on the ability of fungal pathogens to infect tomato roots. In the first year of this project, we redesigned the 2D clinostat to accommodate eight plants as opposed to the original four-plant design. Using our redesigned system, we showed that we could grow tomato cultivars to maturity under continuous clinorotation. In year 1, we also acquired the US Department of Agriculture (USDA) permits for Fusarium oxysporum. In year 2, we successfully demonstrated the infection of Moneymaker tomato plants by Fusarium oxysporum in the enclosed rhizoboxes, and build a second clinostat to enable the simultaneous analysis of plants under clinorotation perpendicular and parallel to the gravity vector.

In year 3, we have optimized the growth conditions on the paired clinostat (clinorotated and upright). We have successfully demonstrated that tomato roots respond to simulated microgravity as predicted after 3 weeks of stimuli. These responses include the agravitropic growth of roots and larger shoot biomass (as previously shown for rice plants – Jagtap et al. 2011; data not shown). We are now inoculating plants growing in the clinostat with Fusarium oxysporum.

In addition to the work in Specific Aims 1 and 2, in year 3 we worked with our NASA colleagues to develop a near-final version of the Experiment Requirements Document. This document will be finalized in July 2022.

Reference: Jagtap SS, Dhumal KN, Vidyasagar PB (2011) Effects of slow clinorotation on growth and yield in field grown rice. Gravit Space Biol 25(1):48-50.

1) Investigate physiological and whole genome transcriptional responses to defense activation in wild-type and immune-deficient tomatoes during spaceflight. Tomatoes will be grown in the Advanced Plant Habitat (APH). We will activate defense responses with a chemical elicitor. At 24 and 48 hours after defense activation, we will harvest tissue and subsequently perform next-generation sequencing to identify genome-wide transcriptional defense responses. In addition, we will use next-generation sequencing to examine the transcriptional response to spaceflight in immune-deficient tomatoes. All plants will be imaged daily to understand the impact of spaceflight on growth rates of immune-activated and immune-deficient tomatoes. All experiments will be performed in parallel on the ground.

2) Determine whether colonization of tomato by the fungal plant pathogen Fusarium oxysporum is impacted by simulated microgravity. We will grow plants in a 2D-ground-based microgravity simulator and inoculate them with Fusarium oxysporum. We will assess tomato plant colonization using histological techniques.

This work will generate key fundamental knowledge of plant-microbe interactions that is important for understanding plant production in space. It is consistent with the goal of the Plant Biology Element in the Space Biology Science Plan 2016-2025.

Specific Aim 1: Determine the effect of spaceflight on genome-wide transcriptomic and physiological defense responses in tomatoes.

This year, work on Specific Aim 1 continued to focus on optimizing protocols and documents necessary for tomato growth in the APH during spaceflight. The following was accomplished:

i) Optimized conditions for tomato growth in the NASA Science Carrier. Last year, we optimized conditions (fertilizer, media) for tomato growth, using similar substrates as in the APH. This year, we used the NASA science carrier to ask the following questions:

a. Can we grow healthy tomato plants in the science carrier with NASA substrates? We tested these conditions using a science carrier from NASA and two genotypes of tomato, Moneymaker and NahG, that will be used in spaceflight. We used NASA protocols for seed sterilization and planting. Results were good and we obtained 100% germination for the Moneymaker tomato variety, and nearly 100% for the NahG. These experiments support our ability to grow tomato plants in the APH during spaceflight. Tomatoes are grown at 16/8 hour day length, with 25ºC day/night. Currently, we are optimizing the LED (light-emitting diode) lighting conditions for germination.

b. Will seeds still germinate if they are watered several weeks and months after planting? Because the experiment may need to wait for several weeks or months once at the ISS, we tested whether our seeds would continue to germinate if watered months after planting. We planted four quadrants in the science carrier, and watered just one for each of four months. We had nearly 100% germination rates even 4 months after planting.

c. What is the best growth configuration for the experiments? We found that planting three tomato seeds in each row of each quadrant gave us sufficient experimental replicates, and allowed sufficient leaf growth while not overcrowding neighbor plants.

ii) Developing a safe spaceflight protocol for defense elicitation in tomato leaves. The goal of specific aim 1 is to investigate the impact of spaceflight on defense responses during spaceflight. To address this, we will elicit defense responses in space using a chemical elicitor. Last year, we started to test methods of treating tomato leaves with an elicitor. The method needs to work well in space and be easy to perform in a timely manner. In addition, we initiated experiments to test whether the chemical will elicit defense responses in tomatoes grown in the conditions in (i).

At the end of last year, we found that swabbing tomato leaves with 5 mM salicylic acid (SA) using a Q-tip was able to elicit expression of a SA response gene. However, the response was not as robust as we had hoped. We hypothesized that this could be due to the side of the leaf that had been swabbed, or the amount of SA applied from the Q-tip.

This year, we aimed to optimize this. We searched for a better SA application method; we tested applying SA to different sides of tomato leaves, we tested different leaf ages, and we tested higher concentrations of SA. We identified a swab from Fisher that worked well. We found that applying 7.5 mM SA to top and bottom of leaf #4 consistently activated the SA marker gene PR1-a. In contrast to 10 mM SA, 7.5 mM SA was not toxic to leaves.

iii) Testing whether leaves stored at -20º or -80ºC freezer would yield similar quality RNA. Tissue for RNA extraction is typically stored in -80ºC conditions. However, because -80ºC space is limited on the ISS, last year we initiated testing whether tissue stored at -20ºC would yield as high quality RNA as that stored in a -80ºC freezer. Last year, we found that storing tissue for one month did not alter the RNA quality. This year, we tested this up to four months in storage and found no change in RNA quality.

iv) Test whether SA that has been stored at RT for several months still elicits defense gene expression. Once in space, the experiment may not be initiated for several weeks or months. We tested whether SA stored at RT for one month was still able to elicit PR-1a gene expression. Results were positive, and we are continuing this year to test SA that has been stored for 3 and 6 months.

Specific Aim 2: Investigate how simulated microgravity affects fungal colonization of tomato plants.

In Aim 2, we proposed a series of ground-based experiments to investigate the impact of simulated microgravity on the ability of fungal pathogens to infect tomato roots. In the first year of this project, we redesigned the 2D clinostat to accommodate eight plants as opposed to the original four plant design. Using our redesigned system, we showed that we could grow tomato cultivars to maturity under continuous clinorotation. In Year 1, we also acquired the USDA (U.S. Department of Agriculture) permits for Fusarium oxysporum.

For Year 2, we have successfully demonstrated the infection of Moneymaker tomato plants by Fusarium oxysporum in the enclosed rhizoboxes. We are currently testing different inoculation strategies to ensure reproducible disease progression under clinorotation.

In addition, we built a second clinostat to enable the simultaneous analysis of plants under clinorotation perpendicular and parallel to the gravity vector. A methods paper is preparation describing the clinostat design and build based on this process.

1) Investigate physiological and whole genome transcriptional responses to defense activation in wild-type and immune-deficient tomatoes during spaceflight. Tomatoes will be grown in the Advanced Plant Habitat (APH). We will activate defense responses with a chemical elicitor. At 24 and 48 hours after defense activation, we will harvest tissue and subsequently perform next-generation sequencing to identify genome-wide transcriptional defense responses. In addition, we will use next-generation sequencing to examine the transcriptional response to spaceflight in immune-deficient tomatoes. All plants will be imaged daily to understand the impact of spaceflight on growth rates of immune-activated and immune-deficient tomatoes. All experiments will be performed in parallel on the ground.

2) Determine whether colonization of tomato by the fungal plant pathogen Fusarium oxysporum is impacted by simulated microgravity. We will grow plants in a 2D-ground-based microgravity simulator and inoculate them with Fusarium oxysporum. We will assess tomato plant colonization using histological techniques.

This work will generate key fundamental knowledge of plant-microbe interactions that is important for understanding plant production in space. It is consistent with the goal of the Plant Biology Element in the Space Biology Science Plan 2016-2025.

This year, work on Specific Aim 1 focused on optimizing protocols and documents necessary for tomato growth in the APH during spaceflight. The following was accomplished:

i) Optimized conditions for tomato growth using similar substrates as in the Advanced Plant Habitat (APH). We first aimed to identify optimal tomato growth conditions using the same substrate and fertilizer as used by NASA in the APH. NASA suggested two different combinations of soil and fertilizer and we identified a combination that was best suited for tomato growth. We will continue going forward with this version of fertilizer. We used the same tomato genotypes that will be grown during spaceflight: Moneymaker and NahG. We used LED (light-emitting diode) lights that were on a 16 hour/8 hour day/night schedule, with a temperature of 25ºC day and night. The substrate was kept very wet during germination. A humidity dome was placed on top of the substrate to prevent evaporation. We had 100% germination and will continue future experiments with these conditions. These experiments support our ability to grow tomato plants in the APH during spaceflight.

ii) Developing a safe spaceflight protocol for defense elicitation in tomato leaves. The goal of specific aim 1 is to investigate the impact of spaceflight on defense responses during spaceflight. To address this, we will elicit defense responses in space using a chemical elicitor and will examine transcriptomic responses to spaceflight in wild-type and immune deficient tomatoes. This year, we tested methods of treating tomato leaves with an elicitor. The method needs to work well in space and be easy to perform in a timely manner. In addition, we initiated experiments to test whether the chemical will elicit defense responses in tomatoes grown in the conditions in (i).

We first tested the chemical elicitors BTH (a chemical analog of salicylic acid) and salicylic acid (SA; a plant defense hormone). Because BTH is more toxic than SA, we switched to SA for subsequent experiments. In the first experiment we used cotton balls to swab the top and bottom of the leaf of 4-week-old plants. The cotton balls were messy and required several dips in solution to swab both the top and bottom of the leaf. The plants were large and we decided to use smaller plants so that less solution is required.

For the next experiments we only used SA. We treated the leaves of 3-week-old plants with a Q-tip to swab the top and bottom of the leaves. This was much less messy and we were able to wet the leaf more quickly. We extracted RNA from these leaves and were about to test gene expression for SA-marker genes just before Purdue shut down due to COVID-19. In the next steps, we will generate cDNA from the RNA and test for SA-dependent defense gene expression. In addition, we will test different concentrations of SA.

iii) Testing whether leaves stored at -20º or -80ºC freezer would yield similar quality RNA. Tissue for RNA extraction is typically stored in -80ºC conditions if it is not used immediately. However, because -80ºC space is limited on the ISS, we are testing whether tissue stored at -20ºC will yield as high quality RNA as that stored in a -80ºC freezer. To do this, we placed 15 tomato leaves in a -80C freezer and 15 in a -20ºC freezer. We removed tissues one month later and extracted RNA. At this timepoint, no difference was observed between -80º and -20ºC and RNA extracted from fresh leaves. We are continuing to test this monthly during 2020-2021.

Specific Aim 2: Investigate how simulated microgravity affects fungal colonization of tomato plants.

In Aim 2, we proposed a series of ground-based experiments to investigate the impact of simulated microgravity on the ability of fungal pathogens to infect tomato roots. In the first year of this project, we have redesigned the 2D clinostat to accommodate eight plants as opposed to the original four plant design. In the first round of experiments, tomato cultivars Moneymaker and NahG were grown to the 5-leaf stage under continuous clinorotation. Plants germinated and grew well. At the end of the experiments, root systems were examined and found to have agravitropic behavior.

One major outcome of this experiment was the need for a quick-release system for the rhizoboxes to ensure rapid sample acquisition. During future experiments, root systems will be harvested at specific time points after infection with a fungal pathogen. Rapid sample acquisition is needed to ensure results are due to the effects of clinorotation. We modified the rhizobox attachment to the frame to reduce the time to access samples.

A second experiment grew Moneymaker plants to maturity. Plants were successfully grown for three months under continuous clinorotation before experiments were halted due to the COVID-19 pandemic. Consistent with growth in simulated microgravity, shoots biomass was 4X reduced compared to plants grown under standard conditions. These preliminary results support the ability to complete ground-based experiments in Years 2 and 3.

1) Investigate physiological and whole genome transcriptional responses to defense activation in wild-type and immune-deficient tomatoes during spaceflight. Tomatoes will be grown in the Advanced Plant Habitat. We will activate defense responses with a chemical elicitor. At 24 and 48 hours after defense activation, we will harvest tissue and subsequently perform next-generation sequencing to identify genome-wide transcriptional defense responses. In addition, we will use next-generation sequencing to examine the transcriptional response to spaceflight in immune-deficient tomatoes. All plants will be imaged daily to understand the impact of spaceflight on growth rates of immune-activated and immune-deficient tomatoes. All experiments will be performed in parallel on the ground.

2) Determine whether colonization of tomato by the fungal plant pathogen Fusarium oxysporum is impacted by simulated microgravity. We will grow plants in a 2D-ground-based microgravity simulator and inoculate them with Fusarium oxysporum. We will assess tomato plant colonization using histological techniques.

This work will generate key fundamental knowledge of plant-microbe interactions that is important for understanding plant production in space. It is consistent with the goal of the Plant Biology Element in the Space Biology Science Plan 2016-2025.