Plant Microbe Interaction

Type of Interaction & Examples

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Plant-Microbe Interactions
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Types of Interactions Symbiotic- endophytic ? - Fungi Mycorrhizae - Actinomycetes - Frankia - Bacteria Rhizobium Associative Pathogenic - Biotrophs - Necrotrophs Biotechnological applications of PMI - PGPR - Biocontrol Future advances in PMI - Molecular biology and genomics

Pl Plant-Microbe Mi b I Interactions i
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Three types of interactions Associative - various bacteria/fungi - synthesize growth regulators - Pseudomonas - pathogen suppression - Azospirillum - N2 fixation - Pseudomonas & Bacilli - mycorrhizal stimulation Symbiotic endophytic ? - mycorrhizae hi - N&P mineralisation i li ti - non-leguminous nodules Frankia-Alnus - N2 fixation - leguminous l i nodules d l Rhi Rhizobium-legumes bi l - N2 fixation fi ti P h Pathogenic i - Biotrophs Bi h - Necrotrophs

Location of interaction

Rhizosphere. Root zone. A very narrow zone (a few mm) around the root. Contains root exudates and microbes. Phyllosphere. Leaf surface as a habitat for microbes rhizosphere is a relatively stable, nutrient-rich environment. Phyllosphere is an extreme environment

Densities of microorganisms in rhizosphere

Decreasing microbial c ob a diversity

A Associative i ti microorganisms i i
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Can affect the mineral nutrition of plants growth and morphology of roots physiology and development of plants availability of nutrients nutrient uptake processes Numerous bacteria and fungi synthesize plant growth substances e e.g. g amino acids acids, vitamins vitamins, growth regulators - may be selective e.g auxins cf. gibberellins by Pine rhizosphere organisms Microorganisms also enhance plant growth through suppression of pathogens and deleterious microbes E g Some Pseudomonas sp. produce HCN and antibiotics E.g. inhibit pathogens

A Associative i ti microorganisms i i
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Free-living microbes can also impact on N & P availability Associative N2 fixing bacteria found in rhizosphere of grasses, corn, wheat e.g. Bacillus sp., Enterobacter sp. Azospirillum sp. Azotobacter A t b t sp. Also found in the ECM of Douglas fir N is limiting factor to tree growth - offers potential for future exploitation - conditions supporting N2 fixation B t i i Bacteria increase P availability il bilit through solubilisation of rock phosphates Uptake p P into mycorrhizal y p plants greater in presence of free-living organisms and mycorrhizae

Endophytes
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All plants are infested with microbes Symptomless

Epiphytes/Endophytes Balanced status of symbiosis Eg. Eg Mycorhizae and Rhizobium

Disease
Pathogens P th Unbalanced status of symbiosis

E d h t ? Endophyte
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An endophyte A d h t is i an endosymbiont, d bi t often ft a bacterium b t i or fungus, that lives within a plant for at least part of its life without causing apparent disease disease. Endophytes may be transmitted either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual) individual).
Endophytes. Live within plant tissues intracellularly
typically commensals or symbionts many strains of Klebsiella are also co-evolved N-fixing endophytic symbionts of grasses. Same Klebsiella strains are fully virulent in animals Salmonella, E.coli are now recognized as commensal endophytes

Rhizosphere p
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Rhizoplane
soil in direct contact with plant root

Endophytes dop ytes

microbes attached to root surface
Increasing organic C Decreasing moisture

Symbiotic organisms M Mycorrhizae hi

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Mycorrhizae Most plants are mycorrhizal: fungus u gus receives ece es C for o g growth, o t , plant greater acquisition of nutrients Mycorrhizal fungi exist as spores/vegetative propagules in root fragments Germination & growth stimulated by root exudates (Bowen, 1994), chemotropism to root via exudates (Tawaraya et al, 1998) 3 types Arbuscular E t Ecto Ericoid

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S bi ti organisms Symbiotic i
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Mycorrhizal plants benefit in several ways greater resistance to pathogens - physical protection, production of toxins, , rhizosphere p modification to inhibit pathogen Better nutrition - P - increased root surface area & access organic P? P uptake also enhanced further in presence of free-living free living organisms

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N l Non-leguminous i nodules d l
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200 dicotyledonous plants form nonleguminous N2 fixing nodules after infection with actinomycete - Frankia sp., all but 2 are woody and found in temperate regions and tropics Most prominent is Frankia sp. with Alder Ald At present interaction thought to be non specific Role of exudates in infection of legumes by Rhizobium sp. suggests probable role in Frankia-Alder symbiosis but not established H t treatment Heat t t t of f root t exudates d t of f alder prevented Frankia infection suggesting proteins in exudates are involved in symbiosis
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N l Non-leguminous i nodules d l
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Most Frankia -associated plants also form mycorrhizal symbioses - tripartite association Synergistic - both N2 fixation and plant growth increased Under low N & P - mycorrhizae enhance P uptake - benefiting Frankia - Frankia enhance N2 fixation f enhancing root growth and mycorrhizal development Substantial Subs a a N2 fixation a o by Frankia a a po points s to o be benefits e so of p planting a g Alder with other trees Increased knowledge of role root exudates in the symbiosis may lead to development of Frankia symbiosis with other trees

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L Leguminous i nodules d l
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Tropical leguminous trees e.g. Acacia form symbiotic associations i ti with ith Rhi Rhizobium bi Infection of trees - direct entry between root epidermal cell ll walls ll to the h cortical i l cells ll to form nodules Contrast to herbaceous legumes where infection is through root hairs - suggests host specific recognition signals i l recognised i db by Rhizobium from herbaceous legumes are absent from t trees
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P th Pathogenic i microorganisms i i
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Bacterial Agrobacterium tumefaciens crown gall roses, roses fruit trees trees, grape vines Xanthomonas campestris pv. pv campestris - brown rot cabbage and cauliflower leaves. X. campestris pv. oryzae -leaf blight of rice rice. X. campestris pv. citri - citrus canker. Pseudomonas solanacearum. vascular wilt diseases of a range of crops
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P th Pathogenic i microorganisms i i
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Fungal & bacterial Biotrophic pathogens establish intricate feeding relationship with host cells Nectrophic pathogens invade plant tissue aggressively killing host cells Biotrophs Phytophthora >60 species infestans potato blight sojae j soybean y blight g ramorum sudden oak death

y mildew fungi g Ascomycetes y Powdery Sphaerotheca pannosa - roses mors-uvae gooseberry Erwinia graminis cereals and grasses

Rust fungi Basidiomycetes Puccinia graminis cereals and barberry Puccinia p punctiformis thistle rust Melampsoridium betulinum birch rust 17

P th Pathogenic i microorganisms i i
Necrotrophs Root rot fungi Armillaria mellea broadleaf and fruit trees and A. A ostoyae conifers

Heterobasidion annosum - conifers

Heart rot fungi

Ganoderma adspersum, a white-rot fungus - heartrot of beech and other broadleaved trees 18

Biotechnological applications of Pl Plant-Microbe Mi b I Interactions i

PGPR plant growth promoting rhizobacteria Produce plant growth regulators Fix N2 Mineralize P Enhance mycorrhization Suppress/kill pathogens - biocontrol
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Biotechnological applications of Pl Plant-Microbe Mi b I Interactions i

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Mycorrhization Helper Bacteria MHB (mainly Pseudomonas sp. and Bacillus sp.) may be responsible esponsible for fo selection p process ocess First isolated from Douglas fir Laccaria laccata system - found to be fungus specific (Duponnois y , 1990) ) & Garbaye, Promote establishment of L. laccata on wide range trees, but inhibit other mycorrhizae May modify exudates to enhance mycorrhization or stimulate tree to produce different compounds or stimulate mycorrhizal fungi directly (Garbaye, 1994) Specificity of MHBs confirmed under field conditions inoculation offers exiting prospect for future

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Biotechnological applications of Pl Plant-Microbe Mi b I Interactions i

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Phlebiopsis gigantea control of Heterobasidion annosum Most damaging root pathogen of coniferous trees in the Northern hemisphere Spreads progressively by contact of healthy roots with infected roots, causing disease gaps g p ( (A) ) may y have bracket-shaped p fruitbodies of Heterobasidion at their base (C). Fruitbodies release air-borne basidiospores - spread infection to new sites. Occurs typically when trees are felled. Basidiospores land on freshly cut stump surfaces - fungus grows down through stump tissues to the dead roots- then infects roots of adjacent healthy trees. Hyphae of P. P gigantea antagonise the hyphae of H. annosum (and some other fungi) on contact - hyphal interference (G). Any hypha of H. annosum making contact with hypha of P. P gigantea shows rapid, rapid localised disruption: the protoplasm becomes disorganised and its membrane integrity is affected (dye taken up).

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Biotechnological applications of Pl Plant-Microbe Mi b I Interactions i

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Use of pseudomonads to control the take-all fungus, Gaeumannomyces graminis Major root-rot pathogen of cereals and grasses. Survives in the infected residues of one crop, then invades the roots of g crop. p Exceptional p the following cases kills whole crop; hence the name "take-all". G. graminis also causes a patch disease of turf grasses, especially of Agrostis species (the "bent grasses") which are used in high-quality turf such as on bowling greens and golfcourse greens. Fluorescent pseudomonds produce phenazine antibiotics which kill Gg - these antibioticproducing bacteria might be applied commercially as seed coatings, to prevent or reduce take-all or other root diseases.

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Biotechnological applications of PlantMicrobe Interactions

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Agrobacterium A b t i radiobacter di b t strain t i K84 control of Agrobacterium tumefaciens Agrobacterium tumefaciens causes crown gall disease on dicotyledonous (broad)p plants, , - apple, pp , p pear, ,p peach, , cherry, y, leaved) almond, raspberry, roses, grapevine. Bacterium transfers part of its DNA to the plant, - DNA integrates into the plants genome, causing the production of tumours and associated changes in plant metabolism. Agrobacterium radiobacter strain K84 produces a bacteriocin -agrocin 84 - enters the pathogen cells and blocks DNA synthesis. Unique mode of action of A. tumefaciens enabled this bacterium to be used as a tool in plant breeding Plants have been engineered to express the Bt gene - insects attempting to eat these plants are killed. Several crops also engineered for resistance to herbicides e.g. glyphosate - herbicide can be used for weed control without damaging the crop. E.g. "Roundup Ready" Monsanto crops soybean, canola, cotton

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F t Future advances d i in PMI

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New advances molecular biology unlocking secrets of black box of unknown soil organisms Identifying new organisms/enzymes Biotechnological applications Biocontrol Genetic engineering of plants - pesticide and herbicide resistance Biocatalysis next decade 90% processes Bioremediation eg. Wood decay fungal enzymes detoxify pollutants, delignify agricultural wastes leave cellulose as cheap commercial substrate Functional Genomics microarrays poised to revolutionise our understanding and exploitation p of PMI E.g. Projects now being funded to sequence genes in Phytophthora involved in infection process Microarrays y which g genes are turned on in plants and pathogens during infection
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PLANT-MICROBE PLANT MICROBE INTERACTION

Mechanism of interaction

Nitrogen fixing organisms: representative i of f symbiotic bi i bacteria b i

N legume Non l

Alder is a N-fixing plant that forms a symbiotic association with bacteria (more specifically an actinomycete) of the genus Frankia. There are about 21 genera of non-legumes non legumes that fix N, called actinorhizal plants .

Gunnera sp., an unusual angiosperm that contains nitrogen-fixing cyanobacteria in pockets at the base of petioles

Legume
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The legume family (Leguminosae or Fabaceae) includes many important crop species such as pea, alfalfa, clover, common peanut, and lentil bean, p

soybean

pea

R.leguminosarum biovar viciae colonizes pea ( Pisum spp.) ) R.l. R l biovar trifolii colonizes clover ( Trifolium spp) R l biovar phaseoli colonizes bean ( Phaseolus spp.) Bradyrhizobium japonicum colonizes soybean ) Rhizobium NGR 234 colonizes Parasponia and tropical plants; very broad host range

Nodule development process

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1. Bacteria encounter root; th are chemotactically they h t ti ll attracted tt t d toward t d specific ifi plant chemicals (flavonoids) exuding from root tissue especially in response to nitrogen limitation tissue,

2. Bacteria attracted to the root, attach themselves to the root hair surface producing lectins and secrete specific oligosaccharide signal molecules (nod factors). )

3. I 3 In response t to oligosaccharide li h id signals, i l th the root hair becomes deformed and curls at the tip; bacteria become enclosed in small pocket. z Cortical cell division is induced within the root.

4. Bacteria then invade the root hair cell and move along an internal plant-derived internal, plant derived infection thread, z multiplying, and secreting polysaccharides that fill the channel. channel

infection thread

Rhizobium cells expressing GFP (green fluorescent protein) invade a host root hair

5. Infection thread penetrates through several layers of cortical cells and then ramifies within the cortex. Cells in advance of the thread divide and organize themselves into a nodule primordium. 6. The 6 Th branched b h d infection i f i thread h d enters the h nodule d l primordium zone and penetrates individual primordium cells. 7. Bacteria are released from the infection thread into the cytoplasm of the host cells, but remain surrounded by the peribacteroid ib t id membrane b . Failure F il to t f form th the PBM results lt in i the activation of host defenses and/or the formation of ineffective nodules. 8. Infected root cells swell and cease dividing. Bacteria within the swollen cells change form to become endosymbiotic bacteroids, which begin to fix nitrogen.

The nodule provides an oxygen-controlled environment (leghemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen metabolites from the bacteroids to the plant vascular system, and of photosynthate from the host plant to the bacteroids.

Patriarca et al. 2002. Microbiology and Mol. Biol. Rev. 66:202-223

SUMMARY : 1. NodD p protein recognition g of different compounds p in root exudates (flavonoids) contributes to host specificity. 2. The nod genes encode functions that are involved in the regulation of gene expression (e.g., NodD), producing the core Nod factor (NodABC) and d d decorating ti nod d f factors t (h (hsn,e.g., N NodH, dH N NoDE). DE) 3. Differences in Nod factor decoration (side chain modifications) contribute to specificity. Specificity is a due to a combination of factors: 1. Types of flavonoids made by host plants 2. Nod D sensor proteins in the bacterial symbionts 3. Types of Nod factors produced by the bacteria 4. Amounts of Nod factors p produced by y the bacteria 5. Exopolysaccharide - for indeterminant nodules (role unclear) 6. Plant hydrolases (e.g. chitinases that may degrade Nod factors) 7 Plant lectins - carbohydrate binding proteins (possible role in attachment of 7. bacteria to plant cells) 8. Plant receptors for Nod factors
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z z z z

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nodC gene encodes NodC protein - an enzyme with N-acetylglucosaminyltransferase activity. Synthesis of the N-acetylglucosamine backbone. backbone nodB gene encodes the NodB protein - a deacetylase enzyme that removes an acetyl group from the Nod factor. nodAgene encodes the NodA protein - an acyltransferase that adds a fatty acid chain to the site deacetylated by NodB Sulfation ( nodH, noeE). Sulfotransferases that modify Nod factors (e.g., in R. meliloti). Fatty acid addition ( nodEF). Different NodEF proteins from different Rhizobium species can put different fatty acids onto Nod factors and g specificity. p y change Arabinosylation ( noeC). The NoeC protein adds arabinose to Nod factors - can influence number of nodules that are formed on host plants. F Fucosylation l (e.g., ( nodZ). d ) F Fucose addition dd changes h specificity f of f nodulation for some symbionts such as Bradyrhizobium japonicum. Acetylation (e.g., nodL). Acetlyation can influence Nod factor activity for R. R leguminosarumand le uminosarumand B. B japonicumNGR234 Methylation and Carbamoylation (e.g., nodS). Mutation changes host range for R. tropici, R. loti, R. frediiand B. japonicum.

Factors Affecting Nitrogen Fixation Soil pH -- as soil pH decreases (i (i.e., e soil becomes acidic) - infection and nodulation decreases Soil potassium level -- as soil potassium level decreases - number and size of nodules decrease and nitrogenase activity decreases Soil nitrate level -- as soil nitrate level increases, infection, nodulation, nodule growth, and amount nitrogen fixation decreases

Gene roc s roa

hab and hac inf noi bar bad nif cof nop

multiplication of rhizobia at or near the root surface adhesion dh of f rhizobia h b to root hair h surface f root hair branching and root curling formation of an infection thread nodule initiation: formation of nodule meristem, nodule development and differentiation bacteroid release from infection thread bacterial differentiation onset of nitrogen fixation biochemical and physiological functions associated with nitrogen fixation maintenance (persistence) of nodule function

Mycorhizae y

Soil and Site Factors Influencing Mycorrhizas 1. Mycorrhizal Inoculum 2 S 2. Soil il Disturbance Di t b mycorrhizal fungi is present in most soils. mycorrhizal fungi may be absent from soils where severe soil disturbance has resulted in topsoil loss, or where host plants are limited by adverse soil or site factors such as y, aridity, y, waterlogging, gg g, or climatic extremes salinity, High rates of P and N fertilizers suppress ectomycorrhiza development in the field Excessive NaCl in soil inhibit mycorrhizal formation and restrict the activity of most mycorrhizal fungi, but some can tolerant these conditions ECM fungi can be highly sensitive to waterlogging of soils, while VAM fungi may be less sensitive

3. Soil Fertility

4. Adverse Soil Conditions

Infection process (VA Mycorrhizae)

1. root grows nearby 2. spore germinates p 3. penetration 4. arbuscules form, then vesicles 5. produce external spores

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before symbiosis takes place, the germinating spores need certain factors to promote hyphal p yp g growth. At this stage, g , the fungus g is still trophically p y dependent p on spore reserves, and it has been proposed that some root exudates regulate the ability of the fungus to use its endogenous reserves fl flavonoids id play l an iimportant t t role l iin VA mycorrhizal hi l symbioses. bi The flavanones hesperitin and naringenin and the flavone apigenin stimulated hyphal growth of Gigaspora margarita The two isoflavones formononetin and biochanin A and, to a lesser extent, the flavone chrysin y have been shown to stimulate formation of VA mycorrhizae y between a Glomus sp. and white clover. The flavonol glycoside quercetin-3-O-galactoside, 4',7-dihydroxy flavone, and 4' 7 dihydroxy flavanone 4',7-dihydroxy flavanone, identified in seed and root exudates of alfalfa alfalfa, enhanced spore germination of two Glomus species

Agrobacterium

Acetosyringone: Phenolic compound, alpha-Hydroxyacetosyringone,Catechol,Ferulic acid,Gallic acid,p-Hydroxybenzoic acid Protocatechuic acid,Pyrogallic acid,Resorcylic acid, sinapinic acid,Syringic acid,Vanillin

H How P Pathogens th Att Attack k Pl Plants t ?

Pathogens P th attack tt k plants l t because b they th have h evolved l d th the unique i ability to gain access to plant tissues and live off their contents In order to do cause disease, pathogens must be able to: 1. Recognize the host 2. Penetrate host barriers 3. Move (invasion) through host tissues 4 Utilize 4. Utili e host components (nutrition) (n trition) for gro growth th and reproduction 5 In addition 5. addition, they must be able to suppress host defense systems

Chemotaxis of Phytophthora zoospores Tactic response allows zoospores to reach i f i sites infection i i in roots of fh host plants l Positive chemotaxis towards plant compounds (ex: P. sojae to isoflavones) Electrotactic responses also noted (weak electric fields generated by roots)
Phytophthora and Pythium zoospores swim towards amino acids and sugars Pythium zoospores swimming to root caps

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B. Surface sensing (Thigmotropism and Chemical signals)

This p process is best chacterized for rust spores p ( Uromyces, y Puccinia) ) The spore reorients the direction of germ tube growth based on cues from the host plant surface. For example, Puccini graminis germlings grow perpendicular to the veins of the leaf surface. surface The spores appear to have a mechanism for sensing appropriate points of entry. Uromyces germlings will recognize topographical features of a surface (leaf stomate g guard cells, artificial surfaces with grooves g or ridges) g and differentiate i into an appressorium.The i Th signal i l for f differentiation diff i i i is a change h in i elevation l i of f 0.5 0 5 micrometres. Other signals can influence germling differentiation and these include: Sugars, Potassium and Calcium for rust fungi Cutin monomers or lipid monomers for Magnaporthe grisea and Colletotrichum species. p Secondary messenger molecules can trigger infection structure formation, e.g., (cAMP) can trigger gg appressorium pp formation in M. grisea.

z z z

A Adhesion A.Adhesion
a spore lands on the plant surface. adhesion : hydrophobic interactions with the plant cuticle. After the spore germinates and forms a germ tube, a film is secreted to surround the germ tube and presumably attach it to the surface. what ? Proteins or glycoproteins in the film (extracellular matrix) e.g. hydrophobins that are bound to the fungal germling cell surface. The sheath may also contain degradative enzymes (such as cutinase) that begin to break down the plant surface. surface Magnaporthe grisea (Rice Blast fungus) is an exception : The spore tips contain a packet of a spore tip mucilage (STM) that acts like a glue to hold the conidia onto the leaf surface (even in the presence of flowing water). This fungus also senses surface hardness, and surface hydrophobicity

a)Bacterial movement (swimming) on p plant surface. b)Bacterial multiplication p on surface/and invasion of the apoplast. c)Flagellin recognition g and subsequent response.

Quorum sensing
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Some bacteria use quorum sensing to monitor their local population density. They accomplish this by secreting and monitoring small diffusible signaling molecules. and when the concentration reaches a threshold, receptors signal a change in gene expression. N-acyl homoserine lactones are the most common types of signaling molecules that bacteria use for quorum sensing. Plant Pl t pathogenic th i b bacteria t i use quorum sensing i t to control t l pathogenesis th i and d colonization l i ti of the host.

Thus quorum sensing is used for : 1. Production of extracellular polysaccharides 2. Production of degradative enzymes p (low molecular weight g iron binding g factors) 3. Production of siderophores 4. Expression of type III secretion apparatus (hrp genes). 5. Transfer of the Ti plasmid between strains of Agrobacterium tumefaciens.
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General steps/components involved in a response (signal transduction) are conserved between insects, plants and animals. 1. Ligand binding to a Receptor 2. Signaling across the membrane 3. Protein kinase/protein 3 phosphorylation cascade. Often this involves mitogen activated ti t d protein t i ki kinases (MAPK (MAPKs) ) 4. Activation of transcription factors 5. Change in gene expression defense genes g