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Microbial symbioses Root-nodule symbiosis Nitrogen availability • Nitrogen is required in large amounts as an essential component of proteins, nucleic acids and other cellular constituents. • Abundant supply of nitrogen in the earth's atmosphere -nearly 79% in the form of N2 gas • N2 is unavailable for use by most organisms -triple bond makes N2 molecule almost inert • N2 must be fixed to ammonium (NH4+) or nitrate (NO3-) ions to make it available for growth and metabolism Sources of Available Nitrogen - weathering of rocks releases negligible amounts of ammonium (NH4+) or nitrate (NO3-) ions - small amount of ammonia/nitrate is produced by lightning - ammonia/nitrate is also produced industrially Terrestrial ecosystems, 80% to 90% of nitrogen available to plants originates from biological nitrogen fixation 80% of the total comes from symbiotic associations Three Major Types of Nitrogen Fixing symbioses 1) Rhizobia – Gram negative bacteria that form nodules mainly on roots. legume host plants: peanut, soybean, lentil, bean, pea, clover and alfalfa. non-legume host: Parasponia 2) Actinomycetes - Gram positive bacteria (genus Frankia) diverse hosts: many trees and woody shrubs Important role in nitrogen economies of forests and other natural Ecosystems. 3) Cyanobacteria – Gram negative photosynthetic bacteria diverse hosts: cycads, ferns, liverworts and hornworts Anabaena associates with the water fern Azolla and is used as a co-crop in rice paddies allowing sustainable rice cultivation Morphological variations in the rhizobium–legume symbiosis. (a) Cupriavidus taiwanensis – Mimosa pudica and occasionally on the stems of legumes (b) Azorhizobium caulinodans – Sesbania rostrata (c) Bradyrhizobium sp. ORS322 – Aeschynomene afraspera (d) Bradyrhizobium sp. ORS278 – Aeschynomene sensitiva Nodules display various shapes: -round (e) Sinorhizobium fredii – soybean -coralloid (f) Methylobacterium nodulans – Crotalaria perrottetii - elongated (g) Sinorhizobium meliloti – Medicago sativa Cortical tissue infection proceeds via transcellular infection threads (h) S. meliloti (tagged with red and green fluorescent proteins)– M. sativa or crack-entry at emergence of lateral roots (i) Bradyrhizobium sp. ORS285 (green tagged) – Aeschynomene indica Invasion of plant roots Barrel medic Rhizobium Flavonoids (2-phenyl-1,4-benzopyrone derivatives) Flavonoids bind bacterial NodD proteins, which are members of the LysR family of transcriptional regulators, and activate these proteins to induce the transcription of rhizobial genes, namely the nod genes. Flavonoids from non-host plants inhibit nod gene transcription. Structure of Nod factors Nod factors (or lipochitooligosaccharides) have: -a backbone of β 1–4-linked N-acetylglucosamine residues (black) with N-linked acyl groups (green) and other hostspecific decorations ( red ). -Each species produces multiple Nod factors; e.g., the number of glucosamine residues can be four or five, the acyl chains of the Nod factors from S. meliloti and R. leguminosarum bv. viciae can be C18:1 instead of C16:2 and C18:4 as shown, and not all Nod factors necessarily carry all the host-specific decorations -The decoration groups can be fucosyl, sulphuryl, acetyl, methyl, carbamoyl and arabinosyl residues Nod factors responses: - Increase in the intracellular levels of calcium in root hairs, followed by strong calcium oscillations (spiking) and alterations to root hair cytoskeleton - Curling of the root hairs, which traps rhizobial bacteria within what is known as a tight colonized curled root hair (CCRH) - Simultaneously, Nod factors stimulate root cortex cells to reinitiate mitosis and these cells will form the nodule primordium, and give rise to the cells that will receive the invading bacteria Root hair curling and cortical cell divisions Nod factor signal transduction system Infection thread development Bacterial mutants unable to produce cyclic b-glucans cannot attach to the root hairs Bacteria have to produce a simbiotically active exopolysaccharide Reorganization of cellular polarity causes the inversion of the tip growth and the formation of the infection thread Only bacteria at the tip of the infection thread are actively dividing S. meliloti produces the exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II) Succinoglycan is more efficient than galactoglucan in mediating infection thread formation in Medicago sativa and the only one in M. truncatula Infection thread failure can be caused by plant or bacterial defects A- wild-type S. meliloti B- S. meliloti exoY mutant C- M. truncatula lin mutant D- S. meliloti nodF nodL mutant E- M. trucatula partially depleted of MtNFP F- S. meliloti nodF nodL mutant on M. truncatula partially depleted for MtLYK3 Reactive oxygen species (ROS) are generated in the infection thread but their importance on the progression of the symbiosis is unknown Endocytosis of bacteria and bacteroid differentiation Nitrogen fixation Overall stoichiometry of dinitrogen reduction: N2 + 8H+ + 8e- + 16ATP 2NH4+ + 16ADP + 16Pi + H2 N2 is reduced to NH4+ by the enzymatic complex nitrogenase: - dinitrogenase (a2b2 tetramer, 8Fe-7S, MoFe7S9.homocitrate) - dinitrogenase reductase (homodimer, 4Fe-4S) It has a slow activity with 3 molecules of N2 per second Structure of the nitrogenase complex Genes involved in nitrogenase biosynthesis and nitrogen fixation regulation Turnover cycle of the nitrogenase 8 electrons: 6 to reduce N2 and 2 for H2 The electron source is ferredoxin or flavodoxin One electron is transferred in each cycle of oxidation/reduction 2 ATP are used per electron transferred In the presence of oxygen, the dinitrogenase reductase has a halflife of 30 s, and dinitrogenase has 10 min Distribution of oxygen in symbiotic nitrogen-fixing nodules Nif gene expression O2 N2 + 8e- + 10H+ nitrogenase 16 ATP 2NH4+ + H2 16 Pi + 16 ADP Nitrogenase is regulated by oxygen and ammonia availability and also by the cell energetic status Regulatory cascade controlling nif genes transcription FixL/FixJ- two component regulatory system NifA- is the master regulator of nitrogen fixation Metabolic reactions within the symbiosome Nodulation strategies in rhizobia Rhizobia induce the formation of nodules on legumes using either a NF-dependent (a) or a NF-independent (b) process. (b) The need for one initial plant signal remains to be demonstrated. The bacteria enter in the plant via cracks in the epidermis which result from the emergence of lateral roots. Accumulation in these infection zones of cytokinin-like compounds synthesized by the bacteria might directly bypass the early NF signaling pathway and trigger nodule organogenesis.