Plant Root Endosymbioses

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Plant Root Endosymbioses

• An investigation of the genetic overlap between
  arbuscular mycorrhiza and root nodule symbioses

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The story of two intracellular symbioses
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First, some history The evolution of plant root
endosymbioses
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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)

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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.
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The basic plot AM

• 1m Pre-infection development (Pid)
• 2m Appressorium formation (Apf)
• 3m Intercellular mycelium growth (Img)
• 4m Arbuscule development (Ard)
• 5m Mycobiont persistence (Myp)

2m
1m
3m
3m
4m
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The basic plot RNS
2n/3n
1n
4n

• 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)

5n
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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)

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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
• 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)

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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.

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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

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Observation of AM/RNS-associated phenotypes in
Lotus SYMRK mutants

• Root hair responses to bacterical innoculatio
• Wild-type w/ M.lotiR7A
• Lotus SYMRK mutant cac41.5 w/ M.lotiR7A
• Wild-type w/ M.lotiR7AC2 (a nodCTn5 mutant)
• Lotus SYMRK mutant cac41.5 w/ M.lotiR7AC2

I NF-dependent signaling leading to root hair
deformation is independent of Lotus SYMRK
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Relative positioning in biological pathway

• Gene expression of LB in Wild-type and SYMRK
  mutant (cac41.5) roots analysed by RT-PCR over 48
  hours.

I NF-induced gene activation is Lotus
SYMRK-dependent
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Genetic Isolation/Identification

• Positional Cloning of Lotus SYMRK
• Genetic linkage map of Chromosome 2
• Physical map of TAC contig
• Intron-exon map of SYMRK gene
• SYMRK cDNA Analysis
• Constitutive expression of SYMRK in roots

I The SYMRK gene is identical with the predicted
RLK gene and is constitutively expressed in roots
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Theoretical Characterization

• Features of SYMRK that place it the LRR1
  class of RLKs
• Signal peptide
• Leucine-rich repeats
• Trans-membrane domain
• Protein kinase domain

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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.

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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
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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

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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.
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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.
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Genetic identification isolation

• Positional cloning of HAR1 gene
• Genetic linkage map of Chromosome 3
• Physical Map of BAC contig

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Genetic verification of cloning

• Complementary conformation of the cloning of the
  complete HAR1 gene

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Theoretical characterization comparison to
previously studied systems

• I Amino-acid characterization of HAR1 as a
  leucine-rich repeat receptor-like kinase (LRR-RLK)

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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 not have the same expression pattern
and is present as a single copy in the Lotus
genome. Therefore, there are genes in leguminous
plants that bear a close resemblance to
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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.
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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/ecologi
  cal implications of symbiosis?