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 Characters (Host and microsymbiont) Most land plant species (80- 90%) and zygomycete fungi of the order Glomales Legumes and rhizobia (Gram-negative bacteria) TonePromiscuity and pluralitySpecificity ThemeThe exchange of hospitable environs for organic phosphate. The exchange of hospitable environs for fixed nitrogen. Historical Context Evolved with the colonization of land by plants and fungi Evolved as needed to 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 1m: Pre-infection development (Pid) 2m: Appressorium formation (Apf) 3m: Intercellular mycelium growth (Img) 4m: Arbuscule development (Ard) 5m: Mycobiont persistence (Myp) 1m 2m 3m 4m

The basic plot: RNS 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) 1n 2n/3n 4n 5n

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)SYMRK/NORK conceptual receptor complex (AM and RNS) b)LRR-RLK CLAVATA1 (a regulator of meristem development in plants) c)LRR-RLK BRI1 (a mediator of plant steroid signaling) d)Toll-like receptor (TLR: a mammalian complex involved in the perception of microbial patterns) e)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 June 2002 A demonstration of Autoregulation –“HAR1 mediates systemic regulation of symbiotic organ development.” Nature. Vol 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 Wild- type and SYMRK mutant (cac41.5) roots analysed by RT- PCR 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 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

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?