Download Bi358 3/3/2005 1 Nitrogen-fixing Symbioses II BI358 I. Molecular

Bi358 3/3/2005
Nitrogen-fixing Symbioses II
BI358
I.
Molecular and Cellular events surrounding onset of symbiosis in Rhizobium legumes
A. So as have been saying there is now a lot of info on molecular crosstalk between
partners in this sym
1. Today we’re going to get through how this crosstalk has been dissected
B. Are going to go through the onset of symbiosis in legumes that form
indeterminate nodules and Rhizobium.
1. Plant Signaling
2. Root hair curling – bacteria signaling
3. Infection thread formation
4. Development of a nodule
C. Plant Signaling
1. Plant exudes compounds into the soil
a) Root exudate now known to contain a variety of flavenoids that can be
species-specific - show slide
(1) First spot where specificity can work
(a) Different plants exude slightly different flavenoids
2. Rhizobium migrates towards the root - presumably up a concentration
gradient - in response to the flavenoids show slide
a) Bacteria are flagellated and can move upto 2 cm/day
b) Bacteria begin to proliferate
c) These flavenoids move into Rhizobium and bind to protein nodD
(1) NodD coded for by one of the nodulin genes
(2) Nodulin genes are a group of genes – on an operon
(a) In Rhizobium these genes are on a symbiosis plasmid (recall
the same set up in Buchnera aphid symbiont)
(b) genes code for biosynthetic pathway for production of the nod
factors
(i) important in next signaling step
(c) In this group of up to 20 genes, only one, nodD, is
constitutively expressed in freeliving Rhizobium
(3) NodD is a trancription factor
(a) When nodD binds the flavenoid
(i) Changes shape
(ii) then it in turn binds to promotor regions or “nod box” on
the nod operon
(b) This turns on the other nodulin genes
(i) Nod genes start making nod factor
(c) Nod factors are small oligosaccharides
(d) Nod factors produced and released into the soil as the bacteria
approach and attach to the uninfected plant root
(4) This is another place where there are mechanisms for specificity
(a) There are several copies of the nodD gene that bind to slightly
different flavenoid compounds
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(b) This results in recognition and specificity events between the
partners
3. How was all of this demonstrated experimentally??
a) For work on induction of nod gene expression:
(1) Fractionated the root exudate into all of its separate compounds and purified them
(2) Had all of the nod operon hooked up to reporter genes
(a) Could not measure for presence of nod factor – didn’t even yet
know what they were
(3) Subjected the bacteria to the variety of different compounds from
the fractionation and looked for which ones resulted in expression
of the reporter gene
D. Root hair curling – bacterial signaling
1. Proliferating bacteria reach root
2. root hair curling around the site of bacteria binding
3. proliferation of the cortical cells at the base of the root hair cell - far from
the binding site
a) this is the ultimate site of the nodule
b) Commences creating a new meristem - in the case of indeterminate
nodules
c) Interesting that cortical cell proliferation happens remotely – clear that
remote signaling is taking place
4. Root hair curling caused by nod factors exuded from bacteria
a) As mentioned above; Nod factors are small oligosaccharides
(1) Nod factors produced and released into the soil as the bacteria
approach and attach to the uninfected plant root
b) This process was elucidated by creating mutants in the nod operon and
look at what happens to development of nodule
c) Can get root hair curling by adding nod factor alone – no bacteria
required
d) Also placed the nod genes in Agrobacterium - could get Agrobacterium
to cause nodulation
5. Specificity: These compounds bind to the surface of the plant root in a
specific fashion
a) They bind to lectins, carbohydrate-binding proteins extending from the
surface of the root – lock and key
b) Hence another spot for recognition and specificity between partners
c) The sugars are specific to a strain of bacterium - participate in host
range determination
d) These events were teased apart experimentally by substituting nod
operons in one strain for another and successfully getting infection in
plants that normally host the other strain
E. Infection
1. Infection thread
a) Formation
(1) At site of root hair curling hydrolysis of plant cell wall takes place
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(a) not known what causes this
(2) Plasma membrane starts to invaginate and the bacteria move into
this invagination - called an infection thread
(3) Infection thread grows through root moves down to developing
nodule
(4) Infection threads never form without the physical presence of
bacteria
2. Bacterial migration through infection thread and into nodule
a) Infection thread grows down to base of root hair cell and bacteria move
down thread
b) Once they reach the developing nodule, infection thread branches entering individual cells
c) Bacteria enter into cortical cells by blebbing bits of membrane off that
surround the bacteria
(1) Once in the cells - the bacteria differentiate into bacteroids
F. Maturation of nodule:
1. Infection threads continue to branch and grow into new cells throughout life
of an indeterminate nodule - so bacteria continue to divide in the infection
thread
2. Once cortical cells are infected - they are new symbiotic cells
a) Cells elongate and bacteria proliferate
3. These mature into central portion - zone of N-fixation
a) nif genes are not expressed in bacteria - only in bacteroids - so there is
another unexplored set of signals that initiate differentiation in the
bacteria
b) nif- mutants can do everything including forming normal nodules - just
not functional at fixing N
II. Actinorhizal Plants/Frankia symbiosis
A. Bacteria: Frankia
1. were not cultured successfully until 1978 and are difficult to work on
because they are slow growing
2. Phylogenetics: has been recently reported
a) 3 different clades with overlapping specificity
3. They have a fungus-like morphology and were thought for a long time to be
fungi - still named actinomycetes - confusing show slide
a) form chains of cells in lab even called hyphae
b) also vesicles - thick-walled cells that are site of nitrogen fixation
(1) presence of proteins that help exclude O2 from vesicle
4. Unlike Rhizobium can fix N in lab at near ambient O2 points to a different
kind of morph and function of symbiosis
5. Have not been found in free-living in soil - but likely that they are there as
trees can nodulate in soils with no growth for >100 yrs
B. Host plants:
1. Members of a disparate set of angiosperms - almost all woody
a) No taxonomic correlation - suggests that symbiosis arose several times
in different sets of orgs
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b) 8 families, 140 species
c) well-known ones include alder, Casuarina, Coenothus
2. Ecological contribution
a) Contribution to nitrogen availability in forest and other habitats
(1) alder - comprises significant part of Pacific NW forests
(2) Coenothus important plant in chaparral
b) in succession - have been shown to be important pioneers in very
nitrogen poor soil
(1) after volcanic eruptions some tropical species have been shown to
be first to recolonize
(2) in riparian soils that are water-logged - stabilize against stream
erosion
c) Management of forests
(1) has proved useful in forest management - after logging allow
actinorhizals to come in and provide nitrogen enriching - some of
this is currently done in Oregon with alder
(a) alder is a good pulp and timber tree - so has use for logging
industry
(2) Casuarina - looks like conifer but isn’t - has great promise in
developing world - mitigate some effects of deforestation by
planting this tree - good for lumber and enriches soil
(a) much better than continuing to cut down virgin forest
C. Nodules called actinorhizas
1. Morphology: very different from legumes show overhead
a) Essentially modified lateral root
(1) many form in cluster
(2) with intact meristem at tip of nodule
(3) vascular tissue runs up middle of symbiotic tissue - so it surrounds
it in a doughnut
b) “Hyphae” will invade developing, uninfected cortical cells near
meristem
c) Bacteria maintain their different cell types in the nodule - form densely
branched vesicles at tips of filaments within a host cell - this is site of
root
stele
Actinorhiza
nodule
meristem
Cortical cells becoming
infected
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N-fixation show slide
2.
can be long lived - upto to 4 years
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