Transcript Plant Root Intracellular Symbioses

Plant Root Endosymbioses
An investigation of the genetic
overlap between arbuscular
mycorrhiza and root nodule
symbioses
The story of two intracellular symbioses
Biological
Setting
Arbuscular Mycorrhiza
(AM)
Root Nodules
Symbioses (RNS)
Major
Most land plant species (80Characters (Host 90%) and zygomycete fungi
and
of the order Glomales
microsymbiont)
Legumes and rhizobia
(Gram-negative
bacteria)
Tone
Promiscuity and plurality
Specificity
Theme
The exchange of hospitable
environs for organic
phosphate.
The exchange of
hospitable environs for
fixed nitrogen.
Historical
Context
Evolved with the colonization Evolved as needed to
of land by plants and fungi
meet the nitrogenous
needs of plants
First, some history: The evolution of plant
root endosymbioses
The thematic significance of symbioses
• Anton de Bary (1879):
“Symbiosis is a prolonged living together of different
organisms that is beneficial for at least one of them.”
• So what are the economics of these relationships?
• Plant root endosymbioses provide an ecological niche
for the microsymbionts, as well as a structural
background for metabolic/signal exchange between the
partners and for the control of symbionts by hosts.
• Plants tend to deal in a currency of photosynthates and
are “in it” for the acquisition of valued chemical
resources: phosphates (AM), nitrogenous compounds
(RNS)
Biological Setting
The mechanized actualization of these themes create two biological settings that have
striking structural similarities. The infection thread symbiosis represents a hypothetical
evolutionary intermediate between the two extant forms of plant root intracellular
symbioses.
The basic plot: AM
2m
1m
3m
4m
3m
• 1m: Pre-infection
development (Pid)
• 2m: Appressorium
formation (Apf)
• 3m: Intercellular
mycelium growth (Img)
• 4m: Arbuscule
development (Ard)
• 5m: Mycobiont
persistence (Myp)
The basic plot: RNS
2n/3n
1n
4n
5n
• 1n: non-deformed root hair
• 2n: hair curling and infection
thread (IT) initiation (Hac/Iti)
• 3n: IT growth in root hair (Ith)
• 4n: IT development in root
cortex (Itr)
• 5n: IT development in the
juvenile nodule tissue (Itn)
• 6n: mature nodule (with distinct
meristem, infection zone,
nitrogen fixation zone and zone
of degredation)
The cast of chemical characters and their
patterns of communication
• Molecular model
• Green: the SYM
pathway defined by
plant genes required
for both bacterial and
fungal symbioses (AM
& RNS)
• Orange: the
components of the
bacterial recognition
module (RNS)
Back to the biological big picture: Have we seen this
story before? Are we dealing with stock characters?
The interactions of the common
endosymbiotic characters are
suggestive of the mechanisms
involved in differential signal
perception in other biological systems
a)
b)
c)
d)
e)
SYMRK/NORK conceptual receptor
complex (AM and RNS)
LRR-RLK CLAVATA1 (a regulator of
meristem development in plants)
LRR-RLK BRI1 (a mediator of plant
steroid signaling)
Toll-like receptor (TLR: a mammalian
complex involved in the perception of
microbial patterns)
Toll-receptor (an insect complex
involved in the perception of
microbial patterns)
Case Studies
• An investigation of
Genetic Overlap:
– “A plant receptor-like
kinase required for
both bacterial and
fungal symbiosis”
Nature, Vol 417. 27
June 2002
• A demonstration of
Autoregulation
– “HAR1 mediates
systemic regulation of
symbiotic organ
development.” Nature.
Vol 420. 28 November
2002.
The cloning and characterization of SYMRK
• Observation of AM/RNS-associated phenotypes in
Lotus SYMRK mutants
• Relative positioning of participants in a biological
pathway
• Genetic isolation/identification of SYMRK
• Theoretical characterization of SYMRK gene
product
• Comparison of SYMRK form and function to
those of previously studied systems
Observation of AM/RNS-associated
phenotypes in Lotus SYMRK mutants
Root hair responses to
bacterical innoculatio
a) Wild-type w/ M.lotiR7A
b) Lotus SYMRK mutant
cac41.5 w/ M.lotiR7A
c) Wild-type w/
M.lotiR7AC2 (a
nodC::Tn5 mutant)
d) Lotus SYMRK mutant
cac41.5 w/ M.lotiR7AC2
I: NF-dependent signaling leading to root hair deformation is
independent of Lotus SYMRK
Relative positioning in biological pathway
Gene expression
of LB in Wildtype and
SYMRK mutant
(cac41.5) roots
analysed by RTPCR over 48
hours.
I: NF-induced gene activation is Lotus SYMRK-dependent
Genetic Isolation/Identification
Positional Cloning of
Lotus SYMRK
a) Genetic linkage map of
Chromosome 2
b) Physical map of TAC
contig
c) Intron-exon map of
SYMRK gene
d) SYMRK cDNA Analysis
e) Constitutive expression of
SYMRK in roots
I: The SYMRK gene is identical with
the predicted RLK gene and is
constitutively expressed in roots
Theoretical Characterization
Features of
SYMRK that place
it the LRR1 class
of RLKs:
• Signal peptide
• Leucine-rich
repeats
• Trans-membrane
domain
• Protein kinase
domain
Comparison to previously studied systems
Comparison of members of the protein family of receptors containing
extracellular LRRs. This tree of protein relatedness compares examples
from various subfamilies in animals and plants and indicates the breath of
species in which the receptors are found, and the variety of functions that
they have.
SYMRK has a lot in common with other
genes required for both AM and RNS
• Similar phenotype to pea SYM19 mutants
• SYM19 is also closely linked to the SHMT
marker
• Coding regions of cDNA sequence are
similar (85.7% on the nucleotide level and
82.8% on the peptide level)
• Sequentially similar mutant alleles
I: Pea SYM19 and Lotus SYMRK are orthologous genes
How are endosymbiotic systems regulated?
The cloning and characterization of HAR1
•
•
•
•
•
Observation of unregulated phenotype
Localization of responsible genotype
Genetic Isolation/Identification
Theoretical Characterization
Comparison to previously studied systems
Observation of unregulated mutant phenotype
• Grafting experiments,
Table 1
Split-root experiments. a, Split-root system using L. japonicus. b,
Autoregulation experiments with wild type and har1 mutants. Nodules
on root B were counted 5 weeks after the second inoculation. Bars in
the graph represent the mean and standard deviation of nodule
numbers. Thirteen to 18 plantlets were measured for each value.
Observation of unregulated mutant phenotype
I: The har1 mutant, like the soybean nts1 and pea hypernodulating
mutants is unable to produce an autoregulation signal from the roots.
Genetic identification & isolation
Positional cloning of HAR1 gene
a) Genetic linkage map of Chromosome 3
b) Physical Map of BAC contig
Genetic verification of cloning
• Complementary
conformation of the
cloning of the
complete HAR1 gene
Theoretical characterization & comparison to
previously studied systems
I: Amino-acid characterization of HAR1 as a leucine-rich
repeat receptor-like kinase (LRR-RLK)
Localization and expression of the
responsible genotype
RNA (a) and DNA (b) blot analysis of HAR1 gene
I: Though structurally similar to CLV1(Arabidopsis), HAR1 does
Theoretical model & comparison to previously
studied systems
I: Genes in leguminous plants bearing a close resemblance to CLV1
regulate nodule development systematically by means of organ-organ
communication.
The Sales Pitch: Why is the story
of symbiosis worth studying?
• Relation to SET (Serial Endosymbiosis Theory)endosymbiosis as
an agent of evolution.
• Is Margulis on to something with her notion of symbiogenesis?
Is there really evidence that hereditary symbiosis,
supplemented by the gradual accumulation of heritable
mutation, results in the origin of new species and
morphological novelty. Is endosymbiosis an agent of
evolution?
• Economic Implications:
• What would the manipulation of these systems do for
agricultural efficiency?
• What is the morale of the story? What engineering lessons can
we learn from the evolutionary/ecological implications of
symbiosis?