MICROBIAL ENDOPHYTES: DIVERSITY, FUNCTIONS, AND HOST-ENDOPHYTE INTERACTIONS.pptx

MICROBIAL ENDOPHYTES: DIVERSITY, FUNCTIONS, AND HOST-ENDOPHYTE INTERACTIONS

1. MICROBIAL ENDOPHYTES: DIVERSITY, FUNCTIONS, AND HOST-ENDOPHYTE INTERACTIONS James F. White, Jr . Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA; jwhite3728@ gmail.com

2. What are endophytes ? (Botany): Endophytic /endosymbiotic non-pathogenic microbes (fungi, bacteria or algae) present asymptomatically for all or part of their life cycles in tissues of plants. 2 Fungal hyphae of endophyte in stem tissue of tall fescue grass. (Medical definition): A tumor that grows like a parasite into other tissues.

3. Model System: Epichloë spp. F amily Clavicipitaceae Ascomycetes ( Hypocreales ) Grass endophytes

4. Epichloë E ndophytes are Intercellular in: Leaf Sheaths, Rhizomes, Culms, and Often Seeds. Below is a hypha of a fungal endophyte ( Epichloe coenophiala ) in tall fescue grass .

5. Epichlo ë Intercellular hyphae Conidia Seed-vectored mycelium Stromata ( S permatia , Perithecia , Ascospores )

6. Epichloë Life Cycle Variations TYPE I: Only stromatal reproduction TYPE II: Stromata formed and fungus vectors in seeds TYPE III: No stromata formed* * Even where stromata are not formed there is potential for horizontal spread via conidia formed on leaf blade surfaces.

7. The mycophagous fly, Botanophila spp., transports Epichlo ë spores as it visits the conidial stromata to lay eggs. Life cycle

8. White et. al, 1996 Cryptic Epiphyllous Conidial States (Often Present on Surfaces of Leaf Blades) Are conidia wind or water dispersed? Average number of fungal colonies from leaves of Big Bluegrass -Experiments using conidia produced on agar -Experiments using conidia produced on leaves

9. DEFENSIVE MUTUALISM HYPOTHESIS Keith Clay Tulane University, New Orleans, Louisiana Epichloë endophytes function to defend plants from predation.

10. Epichloë endophytes produce defensive chemistry www.kentucky.edu Ergot alkaloids: neurotoxins Lolitrems : neurotoxins Peramine : insect deterrent Lolines : insecticidal activity

11. Tall fescue Festuca arundinacea Epichloe endophytic hyphae in Stem pith scraping (aniline blue stained) In fescue foot hooves and tails show gangrene (as in ergotism) ‘Fescue foot’ ‘Summer syndrome’ In summer syndrome Animals do not gain weight In summer months. Ergovaline is responsible for Fescue toxicity.

12. Ryegrass staggers caused by Epichloe endophyte That produces Lolitrem B, an indole-diterpene toxin. Ryegrass staggers Lolitrem B

13. In Argentina, the endophyte Epichloe tembladerae has been employed as a weapon of defense against armies or soldiers on horseback.  In what has been termed the "strategy of huecu " indians , soldiers, or bandits fleeing pursuers would purposefully enter zones dominated by Poa huecu grass.  Those employing the strategy of huecu had the knowledge to prevent their horses from consuming the toxic grasses.  However, their pursuers were frequently unfamiliar with huecu grass and would permit feeding by their animals.  The rapid intoxication and death of the animals of the pursuer usually permitted escape ( Nicora , 1978). Nicora , E. G. 1978. Gramíneas . In: Correa, M.N. ( ed ), Flora Patagónica . INTA, Buenos Aires, Argentina. Strategy of huecu (indian word ‘ intoxicator ’ ) In South America (Patagonia) several species Of grasses in genus Poa And Festuca contain Endophytes that produce Ergot alkaloids that are Fatal to horses. ‘ Tembladera ’

14. Sleepy grass = Achnatherum robustum Contains Epichloe endophyte that Produces lysergic acid amide. Lysergic acid amide induces sleep in horses. Sleepy grass campground is in the mountains near Cloudcroft, New Mexico. Sleepy grass

15. Dronk grass (= Melica decumbens ) contains an Epichloe endophyte that induces a staggers toxicosis in animals . Transvaal region, South Africa Melica decumbens

16. Achnatherum inebrians ‘ drunken horse grass ’ Epichloe endophytes

17. Fungal Disease Resistance dollar spot / hard fescue Fungal endophyte = Epichloe festucae The endophyte provides an almost absolute resistance to dollar spot disease.

18. What are the functions of Epichloë endophytes ? Defense from herbivory Enhanced abiotic stress tolerance Increased drought tolerance Improved plant growth Epichloë endophytes tightly coevolved with hosts (fungal eukaryote-plant eukaryote symbiosis) How do grasses regulate Epichloë endophytes ? Suppression of sporulation in endophytic hyphae Stimulation of sporulation on elongating tillers to form stromata 3. ????????????????????????????????????????????

19. All plants naturally host endophytes ! Endophytes are everywhere! Some endophytes are bacteria . (Plant E ukaryote-Prokaryote symbioses) Plants control this symbiosis!

20. ‘ Cadushy ’ cactus: Subpilocereus repandus in Bonaire

21. Cadushy fruits

22. Seeds

23. Cadushy seedling

24. Assay for bacterial endophytes involves growth on agarose . Then staining with D iaminobenzidine (DAB)/Aniline blue stain overnight. Brown pigment is Hydrogen peroxide Secreted from root. How DAB Works: 1) Plant cells secrete superoxide Onto intracellular bacteria to degrade them. 2) Plant uses superoxide dismutase to transform Superoxide to water and Hydrogen peroxide. 3) DAB reacts with Hydrogen peroxide to form Brown/red coloration Reactive oxygen secretion is used by all eukaryotes to kill endoparasitic bacteria. It is part of the innate Defensive system of all Eukaryotes. Reactive Oxygen Staining Technique

25. Bacteria in root hairs (Stained in DAB followed by aniline blue).

26. Bacteria in root hairs showing recently divided pairs

27. English Ivy ( Hedera helix ) Root Study

28. Most of the root hairs show bacterial colonization and reactive oxygen activity. With bacteria Without bacteria

29. Bacteria often seen to exit hairs at the tips of the hairs Exiting Bacteria

30. Bacteria appear to concentrate at the elongating tip.

31. Bacterial extrusion areas at primordium tip. Bacteria are evident at each pore.

32. What about crop plants?

33. Tomatoes harbor endophytic microbes that stimulate growth of root hairs in tomato seedlings.

34. Bacterial endophytes colonize seeds

35. Tomato seeds have scales that may be adaptations to vector the endophytic bacteria to seedlings. But bacteria may also be vectored within seeds.

36. Bacterial endophytes modulate seedling development. Without endophytic bacteria root hairs often do not form on roots. Surface disinfection of tomato seeds for 45 mins in 4% NaOCl results in drastic reduction of root hair production in seedlings. Stain: diaminobenzidine followed by aniline blue

37. No antibiotic treatment Streptomycin treated Experiment: All seeds surface disinfected for 20 mins in 4% sodium hypochlorite—then washed. ½ seeds treated with streptomycin (100 mg/L) for 24 hours to inhibit growth of endophytic bacteria. Results: Where bacteria are present in seedlings, tomato seedlings (3-days-old) show root hair formation (arrow); and where antibiotic limits bacterial growth no hairs form. This is additional evidence that bacterial endophytes trigger root hair formation in tomato seedlings. Mode of action: Streptomycin binds to the small 16S rRNA bacterial ribosome and inhibits protein synthesis. Streptomycin treatment of tomato seedlings Bacteria are responsible for triggering root hair elongation.

38. Seedlings typically showed abundant bacteria (arrows) often in recently-divided pairs within root cells.

39. Initials showing internal bacteria (arrow)

40. Growing hairs with internal bacteria showing intense hydrogen peroxide staining around internal bacteria. Often root hair tips with internal bacteria appear swollen.

41. Bacteria appear to exit hairs at the growing root hair tips. Arrows show exit canals.

42. Exiting root hair cells from the elongating hair places bacteria outside the hair along its length.

43. Back to Grasses!

44. Grasses vector symbiotic bacteria on paleas and lemmas

45. Bacterial symbiosis: germinating tall fescue seed showing seed-transmitted bacteria ( Pantoea agglomerans and Pseudomonas sp.; both gamma- Proteobacteria ) *Bacteria are ‘ P roteobacteria ’; Perhaps mostly gamma- and beta- Proteobacteria . Small communities of bacteria of 3-4 species are vectored on seeds. The bacteria that vector on seeds appear to be Important for seedling development and survival.

46. Bacterial Endophytes Seed transmitted Common in diverse plants I ntracellular and intercellular Improve plant biotic and abiotic stress tolerance Colonize soil and suppress plant pathogenic fungi Improve plant growth Modulate root development (stimulate gravitropic response and root hair development) Improve nutrient absorption into roots

47. Figure 1. Roots of axenically grown Arabidopsis and tomato were incubated with E coli or yeast expressing green fluorescent protein (GFPE. coli or GFPyeast). “Rhizophagy” Do plant roots consume bacteria to obtain nutrients? ‘ Turning the Table: Plants Consume Microbes as a Source of Nutrients’ Paungfoo-Lonhienne C et al. 2010. Turning theTable: Plants Consume Microbes as a Source of Nutrients. PLoS ONE 5(7): e11915, doi:10.1371/journal.pone.0011915

48. B acterial Biofilm F ormation Z one (Root exudates: butyric, acetic, lactic acids, carbohydrates stimulate f ormation of biofilms containing w alled bacterial cells.) Bacterial Intracellular E ntry Zone (Organic acids, carbohydrates not produced or may be absorbed by r oot meristem. This triggers bacteria to become intracellular in meristem cells) Bacterial E xit Zone (Bacteria stimulate elongation of root hairs and exit at the tips o f hairs where walls are thin. Bacteria reform cell walls once o utside root hair.) Bacteria Enter Root Cells ( Periplasmic Space) Carrying Nutrients From Soil Bacteria Recharge with Nutrients in the Rhizosphere RHIZOPHAGY CYCLE Nutrients Extracted from Bacterial L-forms Through Oxidation by Superoxide Produced by NADPH Oxidases on Root Cell Plasma Membranes Bacteria Exit Root Hairs Exhausted of Nutrients What is the hypothesized ‘ rhizophagy cycle’? meristem Definition: The rhizophagy cycle is a process whereby plants obtain nutrients from bacteria that alternate between an intracellular endophytic phase and a free-living soil phase. Bacteria acquire soil nutrients in the free-living soil phase; nutrients are extracted from bacteria oxidatively in the intracellular endophytic phase. Bacteria in root parenchyma near root tip meristem (DAB and aniline blue stain). Isotopic N tracking experiments using tall fescue grass suggest that 30% of the nutrients absorbed by roots could come from bacteria involved in the rhizophagy cycle (see White et al. 2015). Bacteria emerging from root h air tip of millet seedling. James White; Rutgers University (11/20/2017)

49. FUNCK-JENSEN, D. & HOCKENHULL, J. 1984. Root exudation, rhizosphere microorganisms and disease control. Växtskyddsnotiser 48: 3-4, 49-54. Marschner , H., 1995. Mineral Nutrition of Higher Plants, 2nd edn ., Academic Press, London. Root exudation zones determined by 14C experiments. Plants manipulate bacteria by cultivating bacteria in The root exudate zone near tip of root. Secretion of exudates in a zone proximal to root tip meristems facilitates Their entry into cells of the meristem.

50. Short-chain fatty acids predominate in microbial zones around roots KOO, B.J.; CHANG, A.C.; CROWLEY, D.E. & PAGE, A.L. Characterization of organic acids recovered from rhizosphere of corn grown on Biosolids treated media. Comm. Soil Sci. Plant Anal., 37:871-887, 2006 Lactic, acetic, and butyric acids were predominant in solutions recovered from the plant media and collectively accounted for 0.65 to 0.75 of the COO(-) mole fraction.

51. Some Organic Acids (e.g., Butyric Acid) Function As Signal Molecules for Bacteria Butyric acid has been shown to function in regulating virulence in some bacteria--including Salmonella. Presence of butyric acid reduces expression of virulence genes in Salmonella (see attached article Sun and O'Riordan , 2013). In the absence of butyric acid virulence genes are expressed--and Salmonella becomes pathogenic. This is the signal molecule role of butyric acid. We find that plant endophytic pseudomonads and other bacteria respond in a similar way to butyric acid. High levels of butyric acid inhibit invasion endophytically into plant roots--but in absence of butyric acid the bacteria become intracellular endophytes in plant root cells. Butyric acid may be a common regulator of virulence in many bacteria . Sun and O’Riordan . Adv Appl Microbiol. 2013 ; 85: 93–118. doi:10.1016/B978-0-12-407672-3.00003-4.

52. Bacteria entering root epidermal cells in the ‘zone on intracellular colonization’ a t the root tip meristem. A cloud of bacteria (arrows) is seen around the root tip meristem where intracellular colonization is occurring. The blue stain is aniline blue.

53. Bacteria (arrows) colonizing the epidermal cells in the zone of intracellular colonization. Bacteria will enter cells as walled bacteria—but soon lose cell walls after exposure to reactive oxygen (superoxide produced on root cell plasma membranes) .

54. Funk-Jensen D , Hockenhull J (1984) Root exudation, rhizosphere microorganisms and disease control. Växtskyddsnotiser 48: 3-4, 49-54 . Ortiz-Castro R, et al. (2009) The role of microbial signals in plant growth and development. Plant Signaling and Behavior 4: 701-712. Sun Y, O’Riordan M (2013) Regulation of bacterial pathogenesis by intestinal short-chain fatty acids. Pp. 93-113 in Advances in Applied Microbiology. Elsevier. 4. White J (2017) Syntrophic imbalance and the etiology of bacterial endoparasitism diseases. Medical Hypothesis 107C: 14-15. Organic acids are critical to the functioning of the rhizophagy cycle. O rganic acids are secreted in root exudates just behind the root tip meristem to mimic productive anaerobic bacterial biofilms. 1 The organic acids signal to bacteria in the soil that a productive biofilm is present—attracting bacteria to the biofilm. How do plants manipulate bacteria using organic acids in the rhizophagy cycle? - Root exudates contain 20-40% of a plant’s photosynthate in carbohydrates, vitamins, and organic acids. 2 -Many organic acids (e.g., acetic, propionic and butyric acids) are also anaerobic fermentation products of bacteria. 3 -Bacteria remain avirulent and in the biofilm phase as long as their fermentation products remain abundant in the biofilm. 3 The rhizophagy trigger: We hypothesize that organic acids are absorbed from the rhizoplane biofilm by the root tip meristem. The reduction of organic acids in the rhizoplane biofilm mimics a syntrophic imbalance 4 that triggers an up-regulation of virulence genes in the biofilm bacteria 3 and causes bacteria to internally parasitize thin-walled plant cells in the root meristem. This places bacteria in the periplasmic space of plant cells where the cell plasma membranes secrete reactive oxygen (superoxide produced by NADPH oxidases) onto bacteria to extract nutrients from them. Microorganisms Exudates James White (11/20/2017) Rutgers University Tetrads of bacterium Micrococcus luteus (arrows) in the periplasmic space of cells in the root meristem of Rumex crispus . The bacterium still possesses cell walls having just entered cells. On exposure to reactive oxygen bacteria will lose cell walls to form wall-less L-forms. The rhizophagy cycle is a process whereby plants obtain nutrients from bacteria that alternate between an intracellular endophytic phase and a free - living soil phase. Bacteria acquire soil nutrients in the free-living soil phase; nutrients are extracted from bacteria oxidatively in the endophytic phase.

55. Methodology Poa ampla seeds sterilized with 30 min with 4% NaOCl . Seeds placed on agarose media containing 0, 0.5, or 1.0 mM of butyric acid. Seeds inoculated with endoparasitic / endophytic bacterium Pseudomonas fluorescens . After several days seedlings were stained overnight with reactive oxygen stain diaminobenzidine (DAB), then counterstained with aniline blue to visualize bacteria within plant root hairs.

56. Root tip showing long hairs (arrows) in a root that was not treated with butyric acid.

57. 0.5 mM butyric acid treatment showing shorter root hairs due to fewer bacteria entering the root cells at the tip meristem (blue arrow). Note bacterial biofilm around meristem

58. 1 mM butyric acid treatment showing total absence of root hairs due to cessation of intracellular invasion by bacteria. Without intracellular bacteria no root hairs form. We are using butyric acid and derivatives to prevent the symbiosis between Plants and bacteria. We are evaluating whether we can control invasive plants By inhibiting the rhizophagy cycle.

59. BERMUDA GRASS SEEDLING ROOT TIP IN AGAROSE WITHOUT BACTERIA CONSTITUTIVE SECRETION OF REACTIVE OXYGEN IN ROOT TIPS TRIGGER INFECTING BACTERIA TO LOSE CELL WALLS. The brown coloration is due to presence of reactive oxygen. This tissue was stained For 13 hours in diaminobenzidine tetrachloride (DAB) This is another way that plants are m anipulating bacteria in the rhizophagy cycle.

60. TEM of Bacillus subtilis L-forms Photo by Mark Leaver, New Castle University, UK L-forms are bacterial cells that do not form cell walls (also called ‘cell wall deficient bacteria’). L-forms typically are seen inside eukaryotic cells. They are thought to be a mechanism to evade host defense response. L-form bacteria are typically variable in size.

61. Phragmites root stained with diaminobenzidine DAB to visualize reactive oxygen around bacteria (arrows). Reactive oxygen is visualizable as brown or red coloration around bacteria. The reactive oxygen is the result of superoxide produced by NADPH oxidases on the root cell plasma membranes. The reactive oxygen extracts nutrients from the bacteria (mostly pseudomonads) that are symbiotic with Phragmites . Some of the bacteria may be completely degraded/oxidized by reactive oxygen. Other bacteria stimulate root hair elongation and exit the root hair at the elongating tip to reenter soil populations. L-forms are bacterial cells that do not form cell walls (also called ‘cell wall deficient bacteria ’). L-forms typically are seen inside eukaryotic cells. They are thought to be a mechanism to evade host defense response. L-form bacteria are typically variable in size.

62. Pathogen-Associated Molecular Patterns (PAMPs) Detected by Root Cell May Cause Plant Cell to Secrete Additional Reactive Oxygen Onto Bacterium. PAMPs may be composed of fragments of proteins and DNA released from the oxidizing bacterium. We hypothesize that PAMPs may stimulate the root cell to continue secretion of reactive oxygen onto the bacterial L-form. For Concept See: Duran -Flores, D., Heil , M. Extracellular self-DNA as a damage-associated molecular pattern (DAMP) that triggers self-specific immunity induction in plants. Brain Behav . Immun. (2017), https:// doi.org /10.1016/j.bbi.2017.10.010

63. Root hair showing that bacteria locate in the periplasmic space = space between plasma membrane and plant cell wall. In this location the plant plasma membrane NADPH oxidases secrete superoxide onto bacteria (arrows). Root hair stained with DAB shows that r eactive oxygen (orange coloration) Is focused on bacteria. We hypothesize that pathogen-associated molecular patterns (fragments of DNA and Proteins) are detected by the root cell—and the cell responds by continuing secretion of reactive oxygen o nto bacteria.

64. Intracellular bacteria modulate development of seedlings Bacteria trigger the gravitropic response in roots ( function of ACC deaminase reduction of ethylene? ) Bacteria trigger root hair elongation ( function of auxins ? ) Bacteria increase root branching ( function of auxins ? ) Bacteria increase root and shoot elongation ( function of auxins ? )

65. Bermuda grass seedling root in agarose without bacteria showing absence of root hairs Root tip More developed region of seedling root

66. Bermuda grass root containing Pseudomonas endophyte Bacteria (from seed coat) Colonize r oot t ip meristem (enter cells) I ntracellular in root parenchyma Bacteria stimulate root hair formation In root epidermis Bacteria emerge to surface of hair as the h air elongates Proposed route of endophyte colonization of root and reentry to rhizosphere from root hairs Bacteria colonize soil rhizosphere Bacteria acquire nutrients in rhizosphere RHIZOPHAGY CYCLE

67. Pseudomonas sp. (arrows) in Bermuda grass seedling root tip meristem cells. Stained with DAB/aniline blue. Intracellular bacteria are under plant control and kept in check by r eactive oxygen.

68. Bermuda grass seedling root containing Pseudomonas endophyte . All brown spots in roots are intracellular bacteria .

69. Pseudomonas sp. (arrows) in root hairs of Bermuda grass seedling . L-forms shown in hairs.

70. What is the function of root hairs? The traditional answer is that root hairs function to absorb water and nutrients. Although this function is logical since hairs increase absorption surface area of roots, another function is suggested relative to the rhizophagy cycle. Since intracellular bacteria of the rhizophagy cycle trigger root hair development and exit at the tips of elongating hairs, the hairs effectively function to extend bacteria away from the rhizoplane and into the rhizosphere . This encourages bacteria to rejoin rhizosphere populations and acquire soil nutrients. This optimizes functioning of the rhizophagy cycle and increases nutrients acquired by the plant. Later attraction of these soil bacteria to the exudate zone behind the root tip meristem places them in position to reenter plant cells at the meristem where nutrients can be extracted oxidatively from them. From this perspective root hairs have dual functions that maximize nutritional benefits to plants . Root growing in agarose showing extension of root hairs beyond the rhizoplane and the b acterial biofilm on the rhizoplane . biofilm Bacteria emerging from tips of elongating r oot hairs. Stained with nuclear stain Syto 13. Bacteria emerging from root hair tip. Bacteria in hairs are p resent as wall-less L-forms. Bacteria reform their walls after e xiting from the tip o f the hair. James White 11/5/2017 Rutgers University

71. Micrococcus luteus bacteria exiting root hair tips. Spherical L-forms (white arrow) visible within hair beneath plant cell wall, tetrads of cells (black arrows) outside wall, and channels visible through plant cell wall. Host is Rumex crispus seedling.

72. Pseudomonas sp . in root hairs of Bermuda grass seedling. The s maller aniline blue-staining cells (white arrows) are younger; non-staining cells (blue arrows) are degrading L-forms.

73. Pseudomonas sp. in root parenchyma of Bermuda grass seedling . Bacteria in cells t hat do not form root hairs are e ventually degraded completely .

74. Nutrient Absorption Function of the Rhizophagy Cycle: I sotope tracking experiments.

75. 15N-labeled protein absorption experiment: Bacillus amyloliquefaciens grown in 15N-labeled glycine medium Total proteins extract from bacterial cells and freeze dried Proteins mixed with egg albumin at ration of 1:5 Proteins (0.05%) incorporated into 0.7% agarose Tall fescue seeds with and without bacteria were germinated on the labeled protein media Seedling shoots analyzed for incorporation of 15N using Mass Spec Analysis

76. 15N tracking experiment: 15-N labeled protein incorporated into agar. Bacteria Abundant + 15N-labeled protein Bacteria Reduced + 15N-labeled protein

77. 15N-labeled protein absorption experiment: seed disseminated microbes increase labeled protein acquisition by seedlings. Seedlings with bacteria absorb 30% more 15N-labeled nitrogen than seedlings that lack bacteria!

78. What nutrients does the rhizophagy cycle provide? Examples of nutrients that may be extracted from bacteria oxidatively : P roteins  nitrate Nucleic acids  phosphate Cellular cations of potassium  potassium oxide Cellular ions of calcium  calcium oxide Macro- and micro-nutrients  oxidized forms

79. What happens to plants without the rhizophagy cycle?

80. E+ E- 1 . Endophytes removed from rice by surface sterilization. 2. Endophytes ( Pseudomonas spp.) isolated from Phragmites australis inoculated onto seeds to restore development. Rice: Growth Promotion!

81. Rice experiment Endophyte = Pseudomonas sp. (from Phragmites ) E+ E-

82. How Do Endophytes Support Their Plant Hosts? Stimulate plant Development (seedling geotropism and root hairs) Interact with soil pathogens to reduce their growth (alter pathogen behavior) Suppress p athogens on Plant surfaces (antifungal compounds and upregulate defense genes) Increase r esistance to stress (antioxidants) Carry nutrients to Plants ( rhizophagy ) Suppress p lant competitors of the host (colonize s eedlings and suppress growth) Deter herbivore feeding (produce s econdary metabolites )

83. Some next steps Confirm diversity of nutrients acquired from bacteria. Evaluate which bacteria function in the rhizophagy cycle. Evaluate efficiency of species of bacteria as a nutrient source. Evaluate whether rhizophagy symbiosis bacteria are host specific—or can bacteria be transferred between hosts.