1. Maintaining Relationships on Long Duration Space Missions Gary W. Stutte* ESC/Vencore Kennedy Space Center, FL, USA gary.w.stutte@nasa.gov ISLSWG Workshop: Bioregenerative Life Support, Turin 18-19 May, 2015 (presented by Raymond Wheeler)

2. Microgravity environment increases susceptibility to pathogens Soybean seedlings from day 7 harvest. In both spaceflight and ground-growth locations, seedling roots inoculated with P. sojae are brown in color and the tissues are macerated. Uninoculated roots were not discolored or macerated (not shown). From Ryba-White, et al.2001. Plant Cell Physiol. 42;657-664.

3. Increasing rhizosphere complexity increases resistance to pathogen infection in hydroponic systems Increasing the diversity of rhizosphere reduces the ability of plant and human associated pathogens to survive on wheat roots. (Jenkins et al., 2000; Morales et al., 1996; Roberts et al., 2004)

4. Plant/Microbe Mutualisms are critical to survival on Earth and may play similar role for long duration space missions. Understanding the nodulation process in legumes and its genetic machinery may have broad implications for decreasing resupply costs on long duration space missions in improving agriculture, and reducing dependence on chemical nitrogen fertilizers. Legumes provide up to 20% of the protein in our diets though direct consumption and are important crops for indirect consumption. Little research on plant / microbe interactions in microgravity exists.

5. CO 2 CH 2 O N2N2 NH 3 ATP + e- Plant-reduced-C is exchanged for bacteria-reduced-N

6. Symbiotic Nodulation in a Reduced Gravity Environment (SyNRGE) 6 Payload Specialist Rex Walheim in Space shuttle middeck during STS-135 with BRIC- SyNRGE preparing to fix tissue prior to return to Earth. BRIC containing PDFU’s with Mt/Sm Actuation Tool Mid-deck stowage location of 6 BRIC units

7. Medicago truncatula Sinorhizobium meliloti

8. Effects of microgravity on growth of M. truncatula cv. Jemalong A17 (Enod11::gus) inoculated with two strains of S. meliloti.

9. M. truncatula (Enod1::Gus) inoculated with S. meliloti ABS7 with a hemi::LacZ marker. The stained area indicates site of Enod11:gus gene activation in the etiolated M. truncatula root. M. truncatula (Enod11::gus) inoculated with S. meliloti ABS7 with a hemA::LacZ marker. The stained area indicates site of S. meliloti infection in the etiolated M. truncatula root. Localization of S. meliloti infection of M. truncatula roots and activation of ENOD11 gene in µg necessary for nodule formation, and subsequent biological nitrogen fixation. Localization of S. Meliloti ABS7 in M. truncatula root. Localization of Enod 11 gene activation in M. truncatula root.

10. H o : Microgravity exposure reduces the susceptibility of the host plant (M. truncatula) to form nodules 4 18 1614 8 12 6 M. truncatula Jemalong 17 (Enod11::Gus) growth and nodule formation M. truncatula cv Jemalong 17 (Enod11::gus) germinated in microgravity and inoculated with S.meliloti ABS7 cultured in microgravity at 18 days after inoculation. Roots of M. truncatula were inoculated within 8 hours of landing, and cultured on buffered nodulation media (BNM), which contains no carbon or nitrogen source in Nunc™ 4-well plates. Nodules

11. Effect growth in microgravity on the number of seedlings that formed nodules on two strains of M. truncatula 18 days after inoculation with two strains of S. meliloti. FL = Flight treatments at µ g; GC=Ground controls at 1 g Sm = S. meliloti strain

12. MUTUALISM IN A REDUCED GRAVITY ENVIRONMENT (MURGE): PIRIFORMOSPORA INDICA: ARABIDOPSIS THALIANA INTERACTIONS IN MICROGRAVITY Gary Stutte and Mike Roberts, co-investigators, CSS Dynamac

13. Piriformospora indica, an endophytic fungus of the Sebacinaceae family. P. indica is capable of colonizing the roots of a wide variety of plant species, including non- mycorrhizal hosts like A. thaliana, and promoting plant growth in a manner similar to arbuscular mycorrhizal fungi (AMF). Unlike most AMF, P. indica is not an obligate plant symbiont and can be grown from spores or hyphae in axenic culture in the absence of a host.

14. Pi spores

15. P. indica shows strong biostimulatory effect on a number of species LETTUCE: More uniform germination, increased root branching, larger leaves, and 55% increased in seedling biomass observed. Lettuce Control Inoculated Tomato Control Inoculated TOMATO: More uniform germination, increased root branching, denser root hairs, and 15% increased in seedling biomass observed.

16. Biostimulatory effect of P. indica retained, but reduced in magnitude under simulated microgravity conditions. Medicago truncatula P. indica increased number of roots (102%), total root length (88%) over controls at 1 g. Root number was increased by 51% and root length by 48% over controls under simulated µ g. Hayes, Stutte, McKeon-Bennett, and Murray. 2014. Grav. Space Res. 2:21-33.

18. SyNRGE3 launched on SpaceX in September, 2014. Plant were returned after ~ 2 weeks after landing, but hardware malfunctions made it impossible to make conclusions from experiment. A reflight is currently scheduled for SpaceX 8 (NET 2 September, 2015) Eight M. truncatula plants were launched that had been inoculated with either S. meliloti or P. indica SyNRGE3 sponsored by Space Florida/Nanoracks ISS Research Competition

19. Conclusions The spaceflight environment imposes a unique set of stresses upon both plants and microbes. There are significant beneficial relationships that can arise when plant / microbe mutualisms develop. The development of mutualisms can occur in microgravity, but ‘strength’ of those associations appears to be reduced. Currently not known whether this is due to direct or indirect, effects of microgravity.

20. Thank You SyNRGE; STS-135 SyNRGE 3 ; SpX 4 Kennedy Space Center, FL Ray Wheeler Jeff Richards Michele Birmele Larry Koss Michael Roberts LaShelle Spencer Janelle Caro Mary Hummerick Gioia Massa LIT, Limerick, IE Fergal Barry Michelle Bennett Martin Hayes Adam Shinners Paschal Meechan Patrick Murray Siobhan Moane Space Florida Tony Gannon Ryan Kobrick NASA’s Space Biology Research Program FP7 Marie Curie Research Program NanoRacks/Space Florida ISS Research Competition

21. Questions? Gary.W.Stutte@nasa.gov