Plant-Microbe Interactions

1. Plant-Microbe Interactions

2. Outline of topics for today and Wednesday • Terminology • Microbial plant pathogens • Coevolution between plants and pathogens • Rhizosphere interactions • General biology of mycorrhizae

3. Terms describing the location of microbial habitats related to plants: • Epiphytic = organisms growing on the surface of photosynthetic organisms • Phylloplane = leaf surface • Phyllosphere = area surrounding the leaf and impacted by it • Rhizoplane = root surface • Rhizosphere = area surrounding the root and impacted by it

4. Profiles of two plant pathogens 1. Agrobacterium tumefaciens

5. Figure A. Large gall formed at the base of the stem of a rose bush. Figure B. A series of galls (arrowheads) along a branch of a grapevine. Profiles of two plant pathogens 1. Agrobacterium

6. Agrobacterium tumefaciens causes crown gall disease of a wide range of dicotyledonous (broad-leaved) plants, especially members of the rose family such as apple, pear, peach, cherry, almond, raspberry and roses.

7. Agrobacterium is significant as a tool to insert foreign DNA into a plant. Basically, the bacterium transfers part of its DNA to the plant, and this DNA integrates into the plant’s genome, causing the production of tumors and associated changes in plant metabolism.

8. Agrios, 1978

9. tumefaciens is a Gram-negative, non-sporing, motile, rod-shaped bacterium, closely related to Rhizobium which forms nitrogen-fixing nodules on clover and other leguminous plants. • Most of the genes involved in crown gall disease are not borne on the chromosome of A. tumefaciens but on a large plasmid, termed the Ti(tumour-inducing) plasmid. In the same way, most of the genes that enable Rhizobium strains to produce nitrogen-fixing nodules are contained on a large plasmid termed the Sym (symbiotic) plasmid. • Thus, the characteristic biology of these two bacteria is a function mainly of their plasmids, not of the bacterial chromosome.

10. The central role of plasmids in these bacteria can be shown easily by "curing" of strains. If the bacterium is grown near its maximum temperature (about 30oC in the case of Agrobacterium or Rhizobium) then the plasmid is lost and pathogenicity (of Agrobacterium) or nodule-forming ability (of Rhizobium) also is lost. However, loss of the plasmid does not affect bacterial growth in culture - the plasmid-free strains are entirely functional bacteria.

11. In laboratory conditions it is also possible to cure Agrobacterium or Rhizobium and then introduce the plasmid of the other organism. Introduction of the Tiplasmid into Rhizobium causes this to form galls; introduction of the Sym plasmid into Agrobacterium causes it to form nodule-like structures, although they are not fully functional.

12. Studies such as these raise many interesting and challenging questions about the nature of bacteria. For example, what does the name of a bacterial species or genus really mean, if the organism can change so drastically by loss or gain of a non-essential plasmid? And how much gene exchange occurs by means of plasmids and other mobile genetic elements within natural populations?

13. It is important to note that only a small part of the plasmid (the T-DNA) enters the plant; the rest of the plasmid remains in the bacterium to serve further roles. When integrated into the plant genome, the genes on the T-DNA code for: • production of cytokinins • production ofindoleacetic acid • synthesis and release ofnovel plant metabolites - the opines and agrocinopines.

14. The plant hormones upset the normal balance of cell growth, leading to the production of galls and thus to a nutrient-rich environment for the bacteria. The opines are unique amino acid derivatives, different from normal plant products, and the agrocinopines similarly are unique phosphorylated sugar derivatives.

15. All these compounds can be used by the bacterium as the sole carbon and energy source, and because they are absent from normal plants they provide Agrobacterium with a unique food source that other bacteria cannot use.

16. tumefaciens has been used extensively for genetic engineering of plants. • This is achieved by engineering selected genes into the T-DNA of the bacterial plasmid in laboratory conditions so that they become integrated into the plant chromosomes when the T-DNA is transferred.

17. Some commercial releases of transgenic plants (from Birch, 1997) Crop and release date Name Company Novel properties Tomato (1994) Flavr Savr Calgene Vine-ripened flavour, shelf life Tomato (1995) Zeneca Consistency of tomato paste CottonPotatoMaize (1996-97) BollgardNewLeafYieldGuard Monsanto Bacillus thuringiensis toxin for insect resistance SoybeanCanola (rape)Cotton (1995-96) Roundup Ready Monsanto Glyphosate herbicide resistance A few of the commercial applications of T-DNA technologies:

18. “However, the complexity of the patent landscape has created by the real and perceived obstacles to the effective use of this technology….” “Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants…” Broothaerts et al., 2005

19. 2. Phytophthora (“plant destroyer”) The genus Phytophthora contains about 35 spp., many are notorious plant pathogens. For example, Phytophthora cinnamomi has destroyed millions of avocado trees in CA and eucalyptus in Australia. Its motile zoospores are attracted to root exudates. It produces resistant spores that can survive up to 6 years in moist soil. Big concern with this pathogen for Port Orford Cedar in the Pacific NW.

20. From http://www.parks.tas.gov.au/veg/phytop/whatis.html

21. Phytophthora infestans is famous because it was responsible for the great potato famines in Ireland in which over a million people died due to starvation.

22. The fungus is dispersed by wind-borne sporangia, which are produced on branched hyphae (sporangiophores) that emerge from the stomata of infected leaves in humid conditions sporangia germinate either by releasing zoospores or by producing a hyphal outgrowth.

23. Virtually the entire potato crop was wiped out in a single warm, wet week in the summer of 1846. This event initiated large-scale emigration. Within the decade that followed the population of Ireland dropped from 8 million to 4 million people.

24. Coevolution between plants and pathogens Consider pathogens as part of the biotic environment that exert a strong selective force on populations of plants and animals.

25. Plants have two general responses to pathogen attack: • Passive = Constitutive defenses • Active =Induced defenses

26. Two general plant responses to pathogen attack, from Dickinson & Lucas 1982

27. 1. Passive, constitutive defenses (= defenses that are constantly available • Structural physical e.g. waxy or thickened cuticle, hairy stomates, structures to nurture associations with ants, etc. • Chemical e.g. tannins, terpines, resins, alkaloids, ... many drugs & vices)

28. 2. Active, induced defenses = acquired after the plant is attacked. a. Structural localize responses at the site of entry e.g. necrosis, callose deposition, lignification, abscission layers, tyloses etc. b. Chemical i.e. systemic acquired resistance (SAR) e.g. phytoalexins including polyphenolic, flavonoid or proteinaceous antimicrobial compounds. Salicylic acid plays a role in activating the genes coding for these compounds.

29. What is the relationship between pathogens and genetic diversity in plant populations and species diversity in plant communities?

30. What is the relationship between pathogens and genetic diversity in plant populations and species diversity in plant communities? Microbial parasites, pathogen, and herbivores may be responsible for maintaining a high degree of genetic polymorphism in plant populations and a high degree of species diversity within plant communities.

31. Dan Jansen suggested that pathogen pressures are responsible for maintaining the incredibly diverse tropical forests.

32. Keith Clay and Jim Bever suggest that infection of plants by mutualistic fungi may be a prerequisite for survival and persistence of plant species (i.e. a stabilizing force), but parasitic fungi may prevent plant communities from becoming dominated by one or several species (i.e. destabilizing force).

33. Back to the Red Queen analogy of a coevolutionary arms race between plants and microbial pathogens.

34. Red Queen hypothesis • Genetic systems determining virulence in the pathogen will be paralleled by genes conferring resistance in the host. • This is because any mutation to virulence in a pathogen population will be countered by the selection of hosts able to resist this more aggressive pathogen. • Thus, in a ideal world, we might expect a perpetual stalemate, with host and pathogen populations being closely matched in resistance and virulence. • Hence, over time disease would be neither completely absent nor epidemic.

35. So what happened in Ireland in 1846? Why did P. infestans wipe out virtually all of the potatoes?

36. Disease epidemics often occur when genetic diversity of plant populations is eliminated by human intervention.

37. IV. Rhizosphere interactions

38. Roots exude a tremendous quantity of carbon into the rhizosphere. Why?

39. Microbial biomass C (mg C·g–1 soil dry wt) measured by using substrate-induced respiration method in rhizosphere continuums of three experiments shows that root exudates increase microbial biomass. From Bonkowski et al., 2000

40. Rhizosphere bacteria and fungi generally immobilize nitrogen and phosphorus

41. 3. Grazing by microfauna can influence the whether bacteria and fungi in the rhizosphere mineralize or immobilize the nutrients. “Grazing of microflora by microbivores seems to be a crucial mechanism to maintain the balance in the competition between micro-organisms and plants.” (Bonkowski et al. p. 137)

42. These microshredders, immature oribatid mites, skeletonize plant leaves. This starts the nutrient cycling of carbon, nitrogen, and other elements. Collohmannia sp. Credit: Roy A. Norton, College of Environmental Science & Forestry, State University of New York

43. Interactions between soil bacteria and microfauna “Generally,… protozoa increase mineralization in soil, whereas the effects of bacterial feeding nematodes appear to depend on the status of the populations with nutrients released only if nematode populations decline or are in the presence of nematode predators.” (Bonkowski et al. p.136-137)

44. Respiration by protozoa account for~ 2/3 of all respiration by soil-animals Flagellates, photo by Sarah Spaulding

45. Unlike bacteria and the substrates that they consume, protozoa and their bacterial prey differ little in respect to their C:N:P ratios. Why is this important?

46. Protozoa use only 10 to 40% of the prey carbon for biomass production and excrete the excess N and P. Also, when protozoa die (e.g. in the winter) they release a flush of highly decomposable protozoan tissue.

47. Protozoa are picky eaters and they feed selectively on certain species of bacteria.The species composition of soil communities can impact their ecosystem function (e.g. flagellates, amoebae and ciliates stimulate nitrifying bacteria), which can feedback on plant community composition.

48. Interactions between soil fungi and microfauna Collembola and oribatid mites feed selectively on certain soil fungi. Not all fungi are grazed equally. EARLY DOGMA: Fungivorous microarthropods influence plant growth through grazing on mycorrhizal fungi. However, studies have turned this idea upside-down!

49. A mushroom of Laccaria bicolor fruiting with a white pine seedling. The size of the mushroom indicates that abundant photosynthate must be transported from the needles of the seedling to its root/mycorrhiza system. Photo by Christian Godbout and Andre Fortin

50. Close up of springtails, Folsomia candida *