1. Indian Agricultural Research Institute, New Delhi
WELCOME

2. Biochemical and physiological basis of Arbuscular mycorrhiza fungi
Indian Agricultural Research Institute, New Delhi
Application of Molecular Markers for Testing the Genetic Purity of Pearl Millet Hybrids and Parental Lines
Rahul Kumar
Roll no
Division of Horticulture
Indian Agricultural Research Institute
New Delhi
Biochemical and physiological basis of Arbuscular mycorrhiza fungi
(AMF)-Host Association in Horticultural crops
Biochemical and physiological basis of AMF-Host Association in Horticultural crops

3. Arbuscular mycorrhiza fungi
Indian Agricultural Research Institute, New Delhi
WHAT IS AMF ?
Arbuscular mycorrhiza fungi
Arbuscular mycorrhizas (AMs) are characterized by the formation of unique structures, arbuscules and vesicles by fungi of the phylum Glomeromycota (AM fungi). AM fungi (AMF) help plants to capture nutrients such as phosphorus, sulfur, nitrogen and micronutrients from the soil. It is believed that the development of the arbuscular mycorrhizal symbiosis played a crucial role in the initial colonisation of land by plants and in the evolution of the vascular plants

4. Archaeospora Gerdemannii
Indian Agricultural Research Institute, New Delhi
Classification
Order
Sub order
Family
Genus
GLOMALES
GIGASPORINAE
Gigasporaceae
Gigaspora
Scutellospora
GLOMINEAE
Glomaceae
Glomus
Sclerocystis
Acaulosporaceae
Acaulospora
Entrophospora
Archaeosporaceae
Archaeospora Gerdemannii
Paraglomaceae
Paraglomus

5. Introduction
Arbuscular mycorrhizae fungi are restricted to the order Glomales with three families having six genera, namely Glomus, Acaulospora, Gigaspora, Sclerocystis, Scutellospora and Entrophospora which colonize the root cortex (Morton and Redecker 2001)
Brassicaceae Caryophyllaceae, Chenopodiaceae, or Urticaceae, do not engage in AM interactions (Smith and Read 1997; Vierheilig et al. 1996).
Chemical mutagenesis has revealed that AM symbiosis is established through a multistep process consisting of a “cascade of recognition events” leading to a complete morphological an physiological interaction of two partners. (Azcon-Aguilar and Barea 1996).

6. Continued…
The formation of appressoria is the first signs of recognition (Bonfante and Perotto 1992).
Seedlings, which are colonized by these fungi, perform better during transplantation and plants are also more tolerant towards heavy metal toxicity (Samantry et al 1998).
Mycorrhizal plants show higher tolerance to high soil temperature and various soil and root borne pathogens (Azcon-Aguilar and Barea 1996).

7. Event undergoing AM spore Hyphal growth germination Host plant
Growth stops
Recognition events
Mycorrhizal root
Fungal cell cycle activation
non-aggressive lytic enzyme
Plant cell cycle modification
Changes in root morphology
Changes in cell structure
Changes in gene expression
Interface cellular and functional compatibility

8. 2 . Growth and morphogenesis of the presymbiotic mycelium
Indian Agricultural Research Institute, New Delhi
Steps of symbiosis
Presymbiosis
1 . Spore germination
2 . Growth and morphogenesis of the presymbiotic mycelium
3. Formation of appressoria
4. Penetration of the root epidermis and exodermis
5. Colonization of the root cortx
6. Groth of extraradical ,symbiotic mycelium
7 . Production of viable and infective spores
Symbiosis
Nutrient uptake and exchange

9. Compatible reaction between host and AMF
Indian Agricultural Research Institute, New Delhi
Compatible reaction between host and AMF
Step A: A germinated spore show a linear growth pattern, consisting of branches extending in all directions, functional for soil exploration and for an efficient exploitation of resources.
Step B: 24 h after the perception of host derived signals, a different hyphal growth pattern is expressed,36 h after the beginning of symbionts interaction, dramatic morphogenetical changes occur in hyphal tips, leading to the formation of appressoria.
Step C: After root penetration, intercellular hyphae colonize the root, producing intracellular branched structures, the arbuscules, h after the beginning of the interaction

10. Spores (S) Soil Hyphae Hyphal Proliferation in the Cortex
Indian Agricultural Research Institute, New Delhi
Soil Hyphae
Spores (S)
Hyphal Proliferation in the Cortex
Root Contact and Penetration
1.Mycorrhizal root system washed carefully from coarse sand to reveal the intact network with external hyphae (arrow) with spores (S) produced by Glomus mosseae.
2.Darkly pigmented soil hyphae (H) of a Scutellospora species with auxiliary cells (AV).
3. Soil hyphae have produced 2 appressoria between epidermal cells (arrows). These are seen here in a surface view of a root with attached hyphae.
4. Hyphae at an entry point (E) penetrating cortex cells (arrows) approximately 1 day after contact with the root
5. Alternating long (L) and short (S) cells in the dimorphic exodermis of a root. Hyphae of VAM fungi have penetrated unsuberised short cells (arrows). 6.
Erythronium americanum------coiling type
Glomus versiforme-----linear type
Coiling (Paris)
Arbuscular Mycorrhizas
(ii) Linear (Arum)
Arbuscular Mycorrhizas

11. Arbuscules External Hyphae Vesicles
Indian Agricultural Research Institute, New Delhi
Arbuscules
External Hyphae
Vesicles
1.Developing arbuscule of Glomus mosseae in a root cell with fine branch hyphae (arrows). The trunk (T) of this arbuscule branched from an intercellular hyphae.
2. Vesicles (V) produced by a Glomus species in a leek root. This root also contains many intercellular hyphae.

12. Physiological and Biochemical changes during symbiosis
In the presence of root exudates growth and branching of hyphae is strongly increased (Tamasloukht et al. 2003).
This presymbiotic reaction leads to activation of specific genes followed by physiological and morphological changes.
In return, germinating spores produce diffusible factors which are perceived by plant roots leading to the expression of specific genes (Kosuta et al. 1998).
The arbuscules or dead end are responsible for nutrient exchange ,finally senesce and collapse after 4–10 days of symbiosis ((Bonfante et al.,).

13. COLONIZATION CONSEQUENCE OF ENZYMATICAND MECHANICAL PROCESS
Plant cell wall is degraded by enzymes of microbial origin like pectinase, cellulase and lyase and polygalactouronases are important determinants of pathogenecity. (Varma and Bonfante 1994).
Host allow the fungus to colonize the host tissues and to obtain nutrients from the degradation of pectic substrates (Varma 1998).
AM fungi do not penetrate the endodermis or any other walls, contain suberin and lignin because low rate of production of cell wall hydrolytic enzymes.
Appressoria run within huge air channels, producing penetration peg and causing only limited and subtle changes in the structure of the host wall (Brundrett and Kendrick 1990).

14. CREATION OF APOPLASTIC COMPARTMENT
Indian Agricultural Research Institute, New Delhi
CREATION OF APOPLASTIC COMPARTMENT
Structurally complex
Zone of high molecular complexity
PERI ARBUSCULAR MEMBRANE
b-1,4glucans,
Nonesterfied polygalactouronase,
Hemicellulose such as xyloglucans,
Protein rich in hydroxyproline (HRGPs) Arabinogalactan proteins
GOLGI
1.Apoplastic material is laid down between the invaginated plasma membrane and fungal cell surface, creating a new compartment.
2.since it is composed of the host membrane, the interfacial material, the fungal wall and the membrane (Gollotte et al. 1996a).
3. Current model assumes that plant cell wall consist of three interwoven domains:
1.A network of cellulose and hemicellulose,
2.Heterogeneous pectin’s
3.Proteins.
The presence of these molecules typical of the primary plant cell wall indicates that the newly synthesized membrane, termed the perifungal membrane, retains the enzymatic machinery involved in the synthesis (cellulose) and secretion (pectins, hemicellulose, HRGPs) of cell wall material.
In mycorrhizal association, because of its position around the fungus, the term perifungal membrane is suggested, which has a wider meaning
High amount of H+-ATPase activity
Phosphate transporters
(Gollotte et al. 1996a).
Bonfante-Fasolo et al.

15. Modification in fungus and host cell architecture
Indian Agricultural Research Institute, New Delhi
Modification in fungus and host cell architecture
Modifications in the fungus
Cell wall-progressively thinner.
Cytoplasm changes its organization.
Changes also occur in the storage components from lipid to glycogen
Nuclear reorganization change the physico-chemical properties of the fungal wall, resulting in alternation of its permeability and resistance to turgor pressure
Specific fungal enzyme activities i.e vacuolar alkaline phosphatase
(Bonfante and Scannerini 1992 and Tisserant et al.1993)).

16. Modifications in the plant
Indian Agricultural Research Institute, New Delhi
Continued…
Modifications in the plant
Elicitor degradation,
Nutritional and hormonal plant defense regulation,
Activation of regulatory symbiotic gene expression.
Modifications in the cell’s architecture
-Invagination of the plant plasmalema,
-Fragmentation of the vacuoles,
-Disappearance of amyloplast
-Increase in the number of organelles, such as golgi
-Position of the nucleus is also affected (peripheral position to central position ).
(Bonfante and Perotto 1992).

17. Signal exchange between the plant root and the hyphae of AM fungi
Indian Agricultural Research Institute, New Delhi
Signal exchange between the plant root and the hyphae of AM fungi
1.Receptor kinase
2.Predicted ion channel
3.Calmodulin-dependent protein
SYMRK/DMI2 receptor kinase may be the earliest to act in the AM signalling pathway
SYMRK-Collective gene
DMI-Does not make infection
Medigo truncatula
Ion channel
Inter cellular kinase
Rhizobium-made Nod factors induce rapid changes in both Ca2þ
and gene expression. Mutations and
inhibitors that abolish Nod-factor-induced Ca2þ
spiking block gene induction, indicating a specific role for Ca2þ
spiking in signal transduction. We used transgenic Medicago truncatula expressing a ‘cameleon’ Ca2þ
sensor
to assess the relationship between Nod-factor-induced Ca2þ
spiking and the activation of downstream gene
expression. In contrast to ENOD11 induction, Ca2þ
spiking is activated in all root-hair cells and in epidermal or
pre-emergent root hairs cells in the root tip region. Furthermore, cortical cells immediately below the
epidermal layer also show slow Ca2þ
spiking and these cells lack Nod-factor-inducedENOD11 expression. This
indicates a specialization in nodulation gene induction downstream of Nod-factor perception and signal
transduction. There was a gradient in the frequency of Ca2þ
spiking along the root, with younger root-hair cells
having a longer period between spikes than older root hairs. Using a Ca2þ
-pump inhibitor to block Ca2þ
spiking
at various times after addition of Nod factor, we conclude that about 36 consecutive Ca2þ
spikes are sufficient
to induce ENOD11–GUS expression in root hairs. To determine if the length of time of Ca2þ
spiking or the
number of Ca2þ
spikes is more critical for Nod-factor-induced ENOD11 expression, jasmonic acid (JA) was
added to reduce the rate of Nod-factor-induced Ca2þ
spiking. This revealed that even when the period between
Ca2þ
spikes was extended, an equivalent number of Ca2þ
spikes were required for the induction of ENOD11.
However, this JA treatment did not affect the spatial patterning of ENOD11–GUS expression suggesting that
although a minimal number of Ca2þ
spikes are required for Nod-factor-induced gene expression, other factors
restrict the expression of ENOD11 to a subset of responding cells.
Ion channel
Radutoiu et al. 2003; Parniske 2004

18. Epidermal opening: Continued…
Indian Agricultural Research Institute, New Delhi
Continued…
Epidermal opening:
-LjSYM15-Pectinolytic enzyme
CLEFT
Intracellular passage to the inner cortex
-LJSYMRK, LjSYM4-serine carboxy peptidase 2 promoter ::green fluorescent protein
Arbuscule development:
(Dissection of component thus transcriptone of arbuscule)
-LjSYM4, LjSYM15-Phosphate transporter
CEL1=Beta -1 4 glucanase
HYPERNODULATION AND ABERRANT ROOT FORMATION
-(HAR1) receptor kinase

19. Chemical messenger at pre-infection stage
Indian Agricultural Research Institute, New Delhi
Mycorrhiza is regulated by water-soluble sugar, amino acids, organic acids, flavonoids, and volatile exudates . (Rovira 1996)
Flavonoids from host
Pre-infection growth (Pif), and hyphal branching (Pab).
Appressoria(Apr)
1.Mycorrhiza is regulated by water-soluble mono and disaccharide’s, amino acids, organic acids, flavonoids, nucleotide and enzymes) and volatile exudates (alcohols, ketones, esters, phenols, terpenoids, organic acid) and by surface-bound recognition molecules. (Rovira 1996)
2. Volatile compounds are more favored candidate
Hyphae penetrate cortical cells
(Pen)
Arbuscules (Arb).

20. Defense reactions of plant roots during colonization by AM fungi
Molecules
Modification
Phytoalexins
Late increase in some flavonoids PAL, CHS, and CHI during root colonization.
Localization of PAL and CHS transcripts in arbuscular containing cells.
No increase in IFR
Callose
β-1, 3 glucans in host wall at the base of arbuscule trunks
Peroxidase
Increase in total and wall bound activity in early stage of colonization. No localization in arbuscular containing cells
Chitinase
Early increase in transcripts and activity, suppression in later stage of colonization.
β-1,3 glucanase
No detectable quantitative changes initialy and decrease in later stage of colonization
PR-1 protein
Slight increase in transcripts. Localization around living arbuscules
Morandi et al.,2001; Gollotte et al.1995; Mc Arthur and Knowles 1992; Dumas et al.1989; Gianinazzi-Pearson et al respectively
PAL-l phenylalanine ammonia lyases
CHI-chalcone isomerase
CHS-chalcone synthetase
IFR-isoflavone oxidoreductase

21. Continued…
Alterations in anti-oxidative enzymes, such as catalase, peroxidase and superoxide dismutase, indicate corresponding genes might be expressed specifically during the colonization process ( Lambais 2000).
The expression of genes coding for PR proteins is strongly reduced, later stage PR proteins accumulate throughout infected root tissues (Tahiri-Alaoui et al. 1993).
In AM roots, a structured wall material hydroxyproline-rich glycoprotein is transiently deposited around hyphae specifically in cortex cells containing developing arbuscules (Gianinazzi-Pearson et al. 1992).
Lignification, considered as an important mechanism for disease resistance , may contribute to reducing pathogen proliferation in mycorrhizal roots.

22. Case study 1
Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi

23. Method of labeling and analysis of metabolism and transport in carrot roots colonized by Glomus intraradices.
13C-labeled glucose and fructose –fungus carbohydrates and lipids
Extraradical mycelium - not found
(c) Fungus converted sugars – lipids ERM
(d) Hexose undergoes PPP Pathway
NMR-Nuclear magnetic resonance spectography IPA=isopropyl alcohol
MeOH=Methonol
. Dacus carrota

24. Biochemical pathways of carbon metabolism active in symbiotic intraradical and extraradical AM fungi.
INTRA RADICAL
EXTRA RADICAL

25. Updated general overview of the translocation events implicating the three major nutrients (C, P and N) which may take place along the AM fungal colony.
RUNNER HYPHAE

26. In vivo multiphoton microscopy of fungal nuclei along an extraradical hypha of Glomus intraradices (upper pannel) and interpretation
of the micrograph (lower pannel), showing the main features of nuclear distribution along ERM in this fungus.

27. Case study 2
Arbuscular mycorrhizal symbiosis influences strigolactone production under salinity and alleviates salt stress in lettuce plants

28. (B) Effect on the stomatal conductance after 7 weeks.
(A) Effect on the efficiency of photosystem II after 7 weeks.
Phenotypic comparison of non-mycorrhizal (Control) and mycorrhizal (Glomus intraradices) lettuce plants growing for 7 weeks under increasing salinity levels ranging from 0 to 80 mM NaCl.
(B) Effect on the stomatal conductance after 7 weeks.

29. A.ABA content in lettuce roots after 7 weeks.
@ Influence of salinity and mycorrhizal colonization on strigolactone production.
@ Germination of Phelipanche ramosa seeds induced by root extracts of lettuce plants after 7 weeks.
@ GR24 (10−10 and 10−11 M) and demineralised water were used as positive and negative controls, respectively.
B.Gene expression analysis by real time PCR for the ABAbiosynthesis gene LsNCED2 after 7 weeks.

30. Conclusion
Approaches like RNAi ,radio active study ,transcriptone offers strategies for studying the expression and regulation of those fungal genes involved in the establishment, maintenance and functioning of the symbiosis.
The degree of colonization depend up on the kind of AMF species, which further varies with rhizospheric and root morphology
Root exudates have been shown to stimulate growth and branching of the AM fungal hyphae i.e. flavonoid/isoflavonoid compounds
The identification of mycorrhizal mutants is accelerating and the continued cloning of genes induced during the symbiosis will contribute to a molecular picture of the events accompanying development of the association.

31. FUTURE PERSPECTIVES
1. Optimizing physiological conditions to understand the mechanism of interaction
-The complete root colonization pattern
-The preferential colonization sites
-Specific infection structure
2. Identification of the plant proteins involved in the recognition events and the phytopromotion
-identification of component of the protein complexes
3. Identification and the specific function of the autofluorescent compound
-chemical nature of flurescent compound
-Purpose and physiological significance of the flurescence compound
4. Characterization of the signals involved with inhibition and promotion of mychorrhiza