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I would like to open with I was having a spirited discussion the other day with a member on another forum and they were making the claim that mycho additives were a waste of time and money for cannabis.
What I would like is for someone to confirm my opinion that this is incorrect and mychos can and will populate the cannabis rhizospere. So I went hunting to substantiate my belief. With that said it was tough to find anything due to lack of studies on the genus cannabis as we all know for obvious reasons. I was able to determine based on 1 study that mychos will inhabit the cannabis rhizopshere. Keeping in mind not ALL plant life has a symbiotic relationship with mychos. Legumes are one such species that utilize mycho as part of nitrogen fixation process. I will paste the paper and in review see page # 10 and it makes reference to the genus Cannabaceae.
Upon a wikipedia search I found this. http://en.wikipedia.org/wiki/Cannabaceae
Cannabaceae is a small family of flowering plants. As now circumscribed, the family includes about 170 species grouped in about 11 genera, including Cannabis (hemp),Humulus (hops) and Celtis (hackberries). Celtis is by far the largest genus, containing about 100 species.[1]
Other than a shared evolutionary origin (see Phylogeny below), members of the family have few common characteristics; some are trees (e.g. Celtis), others areherbaceous plants (e.g. Cannabis).
So here is the study paper titled Exploiting an Ancient Signalling Machinery
to Enjoy a Nitrogen Fixing Symbiosis
Here is the link.
Here is the paper, Based on my understanding I am correct and my sparring partner is wrong. Be sure to go review the entire paper as it is very interesting for the science lovers in the group. Peace
Abstract
Almost for a century now it is speculated that a transfer of the largely
legume-specific symbiosis with nitrogen fixing rhizobium would be
profitable in agriculture (Burrill and Hansen, 1917; Charpentier and
Oldroyd, 2010). Till now such step was not achieved, despite intensive
research in this era. Novel insights in the underlying signalling networks
leading to intracellular accommodation of rhizobium as well as
mycorrhizal fungi of the Glomeromycota order show extensive
commonalities between both interactions. As mycorrhizae symbiosis can
be established basically with most higher plant species it raises questions
why only in a few taxonomic lineages the underlying signalling network
could be hijacked by rhizobium. Unravelling this, will lead to insights
that are essential to achieve an old dream.
Introduction
Rhizobium bacteria and arbuscular mycorrhizal fungi of the
Glomeromycota phylum can both establish an endosymbiosis with
plants that facilitates growth in a nitrogen or phosphate deficient
environment, respectively. Mycorrhizal fungi are obligatory biotrophs.
Their hyphae penetrate the root intercellularly or intracellularly,
depending on the plant host, and subsequently form arbuscules in inner
cortical cells. These arbuscules are highly branched intracellular hyphae
that are surrounded by a membrane formed by the host. This periarbuscular
membrane functions as a symbiotic interface as transporters
are present that facilitate exchange of nutrients. These are mainly
phosphates, but also nitrates that are taken up by the mycelium outside
the plant root (the so called extraradical mycelium) (Smith et al., 2011;
Javot et al., 2011). In return the fungus obtains photosynthates of the
plant for which it has specific monosaccharide transporters in its
arbuscular membrane (Helber et al., 2011). In between the periarbuscular
membrane and the branched hyphae a structured plant cell
wall is practically absent to maximize reciprocal exchange between both
organisms.
Unlike mycorrhizal fungi, some soil bacteria of the Rhizobaceae
family -collectively called rhizobia- have a dual lifestyle. They can be
free living in soil, but in case the appropriate legume host is present
they can establish a biotrophic endosymbiosis. For this they carry a set
of symbiotic genes that are located on a large symbiotic (sym)
plasmid(s) or are present as symbiotic islands in the genome. These
symbiotic genes do include; (I) genes required for nitrogen fixation (the
nif and fix genes) and (II) genes essential to establish symbiosis (the nod,
nol and noe genes). This second set of genes encodes the machinery that
is essential for biosynthesis and secretion of lipo-chitooligosaccharides
(LCOs) that function as signal molecules and are named Nod factors.
Nod factors are perceived by the host plant and set in motion
symbiotic engagement.
page 10 Chapter 1
The nitrogen fixing rhizobium symbiosis is basically restricted to
legumes -the Fabaceae-, with one exception the genus Parasponia that
belongs to the Cannabaceae. Cannabaceae and Fabaceae diverged ~100
million years ago represented by the split of Fabales and the orders
Fagales, Cucurbitales and Rosales (Wang et al., 2009), in all probability
supporting the idea that emergence of rhizobium symbioses occurred in
parallel in both lineages (Fig. 1).
Figure 1: Phylogenetic relation of plant families in the orders Fabales, Fagales, Cucurbitales
and Rosales. The occurrence of rhizobium symbiosis is indicated with a red asterisk and
Frankia symbiosis with yellow circle. Within a family, symbiosis is occurring only in one or a few
genera, with the exception of legumes (Fabaceae) (Doyle et al., 2011). Drawing is based on
published phylogenetic trees (Wang et al., 2009; Zhang et al., 2011; Li et al., 2004; Zhang et
al., 2006).
To host rhizobium novel organs are formed, named nodules, which
provide an optimal niche for nitrogen fixing rhizobia. Legume nodules
have a unique ontology and originate from primordia formed in the root
cortex. Legume nodules contain a large central tissue of which its
infected cells harbour hundreds of rhizobia. In most legumes these
bacteria are hosted individually, or in small clusters, surrounded by a
plant-derived membrane; a unit that is called symbiosome. This
membrane compartment facilitates exchange of fixed ammonium for
other nutrients, including photosynthates. To reach the nodule
primordia, in general sophisticated intracellular infection threads are
formed. Starting at a root hair that curls around a (single) bacterium a
membrane bound tubular infection thread is formed that guides the
clonally propagating microsymbiont to the nodule primordium.
Subsequently, bacteria are released from the infection thread and
develop in their symbiotic form. Infection threads that enter nodule cells
are bound by a thick cell wall and to release rhizobia from such infection
thread cell wall-free patches are created, so-called unwalled droplets. At
such sites, bacteria are in close contact with the surrounding host
membrane enabling pinching off of symbiosomes.
In contrast to legumes, Parasponia nodules seem much more primitive.
Nodule ontology resembles that of a lateral root. Parasponia is infected
intercellularly by rhizobium and only in the nodule cortex rhizobium
triggers formation of intracellular infection threads. These are
invaginations of the plasmamembrane and are bound by a thick cell
wall. From these infection threads, fixation threads are formed.
Fixation threads are also bound by a plant membrane and a plant cell
wall, however this cell wall is markedly thinner than the infection
thread cell wall. This type of fixation threads also occurs in nodules
of some basal legumes. Rhizobium is not released as symbiosomes
from the fixation threads most likely due to the presence of this cell
wall.
Commonalities in Signalling
Since several years it is known that in legumes rhizobium and
mycorrhizal fungi signals activate different receptors, but in turn these 12 Chapter 1
activate a common signalling module that subsequently diverges in the
two symbiotic interactions (Radutoiu et al., 2003). This commonality in
symbiotic signalling has been characterized in two model legume species,
Lotus japonicus and Medicago truncatula, and is shown to occur in other
legumes and non-legumes that interact with mycorrhizal fungi (Gutjahr
et al., 2008; Kouchi et al., 2010). The common signalling module
stretches from a plamamembrane receptor kinase (named LjSYMRK in
L. japonicus and MtDMI2 in M. truncatula), a cation channel located in the
nuclear envelope (LjCASTOR, LjPOLLUX/MtDMI1), and a nuclear
localized protein complex of a calcium Calmodulin dependent kinase
(CCaMK) and a coiled-coil protein (LjCYCLOPS/MtIPD3).
Furthermore, several subunits of the nuclear pore have been found to be
essential for rhizobium and mycorrhizae induced signalling (For recent
reviews see: Kouchi et al., 2010; Oldroyd et al., 2011). An essential step in
rhizobium symbiosis is the recognition of Nod factors, which holds for
(almost all) legumes as well as for Parasponia species. In legumes, Nod
factors are perceived by two distinct transmembrane LysM-type receptor
kinases (LjNFR1, LjNFR5 / MtLYK3, MtNFP). Studies in heterologous
systems indicate that these receptors can form a heterodimeric complex
(Madsen et al., 2011), whereas in legumes itself the subcellular regulation
is highly dynamic and affected upon Nod factor signalling (Haney et al.,
2011). Upon Nod factor perception these LysM-type receptor kinases,
together with the common signalling module, set in motion bacterial
entry as well as root nodule organogenesis.
Legume LysM-type Nod factor receptors are not essential for
mycorrhization. However, two complementary approaches strongly
support the idea that mycorrhizal fungi activate LysM-type receptor
kinases. Recently it was shown that the model mycorrhizal fungus
Glomus intraradices also produces LCOs, molecules that are named Myc
factors (Maillet et al., 2011). Application of these Myc factors to plant
roots increases mycorrhization at least twofold, an effect that seems
generic in higher plants as it can be triggered in legumes as well as nonlegumes
(Maillet et al., 2011).Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 13
Myc factors and Nod factors are structurally very similar (Maillet et al.,
2011). As the rhizobium symbiosis is relatively young in comparison to
mycorrhizal symbiosis it suggests that Nod factor perception evolved
from the mycorrhizal fungal symbiosis. Strong support for this came
from studies on Parasponia andersonii. Parasponia-rhizobium symbiosis is
relatively young based on the close phylogenetic relation of Parasponia
species with non-nodulating sister species in the Trema genus (Sytsma et
al., 2002; Yesson et al., 2004). Therefore co-evolution of rhizobium and
Parasponia has been relatively limited when compared to legumes. In line
with this it is anticipated that in Parasponia the LysM-type receptor kinase
family is less diverged, similar as seen in other non-legumes species
(Zhang et al., 2007; Zhang et al., 2009). In comparison L. japonicus and M.
truncatula have at least 2 members of the LjNFR5/MtNFP-type receptor
kinases, of which one is a Nod factor receptor, whereas the second Nod
factor receptor, LjNFR1/MtLYK3, underwent even series of
duplications (Zhang et al., 2007; Limpens et al., 2003; Arrighi et al., 2006;
Lohmann et al., 2010). In contrast P. andersonii only has a single
NFR5/NFP-type receptor, PaNFP. Functional analysis revealed that
PaNFP has a dual symbiotic function. It controls the formation of the
symbiotic interface of rhizobium as well as mycorrhizal fungi (Op den
Camp et al., 2011). Together with the observation that mycorrhizal fungi
secrete LCOs this leads to the hypothesis that not only the common
signalling module, but also the rhizobium Nod factor perception
mechanism is recruited from endomycorrhizae.
The duplications of Nod factor receptors in M. truncatula and L. japonicus
do not stand on their own. Like most plant lineages also the legume
lineage experienced whole genome duplications (WGDs) (Cannon et al.,
2006). One such duplication event occurred early in evolution of the
Papilionoideae about 58 million years ago. This legume subfamily
represents most nodulating legumes, including all prominent crop
species. Hundreds of paralogous gene pairs that originate from this
duplication showed to be maintained in M. truncatula, L. japonicus and
soybean (Glycine max), and for many of these one or even both genes 14 Chapter 1
showed to be expressed in root nodules. As these legumes diverged
more than 55 million years ago, it indicates that this WGD provided a
genetic redundancy that contributed to the evolution of the rhizobium
nodule symbiosis in the Papilionoid subfamily (Camp et al., 2011; Young
et al., 2011).
Downstream of the Common Signalling Module
Formation of a symbiotic interface that facilitates exchange of nutrients
is a crucial step in endosymbiosis. Studies in Parasponia indicate that this
process is tightly controlled by LCO signalling in case of fixation thread
formation by rhizobium as well as arbuscule formation by mycorrhizal
fungi. Also in legumes Nod factor signalling plays a prominent role in
the formation of a symbiotic interface. Knock down (or loss of function)
of several genes of the common signalling module results in nodules
with numerous intracellular infection threads, but symbiosomes are not
formed (Ivanov et al., 2012; Ovchinnikova et al., 2011; Horvath et al.,
2011; Limpens et al., 2005).Taken together, it suggests that rhizobium
and mycorrhizal fungi trigger similar cellular responses.
Recently it was shown that two GRAS-type transcription factors, NSP1
and NSP2, both essential for basically all Nod factor induced responses
in legumes, also have a function in the absence of the microsymbionts.
These transcription factors were shown to control expression of
DWARF27, a gene essential for strigolactone biosynthesis (Liu et al.,
2011; Lin et al., 2009). As a consequence, functional nsp1 and nsp2
mutants are significantly hampered in strigolactone biosynthesis. This
holds for M. truncatula as well as rice (Oryza sativa), suggesting that the
transcriptional regulation of a key enzyme in strigolactone biosynthesis is
largely conserved in higher plants (Liu et al., 2011). Strigolactones are
important secondary metabolites in plants that can act as hormones as
well as ex planta attractants for mycorrhizal fungi. Plant roots secrete
strigolactones in response to phosphate starvation, which, in M.
truncatula, correlates with a NSP1-NSP2 dependent transcriptional Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 15
activation of MtDWARF27 (Liu et al., 2011). This supports that NSP1
and NSP2 have a function in mycorrhizal symbiosis and suggests that
this function might have been recruited during evolution of rhizobium
symbiosis. However, to date only very little evidence supports a role of
strigolactone signalling in rhizobium symbiosis (Soto et al., 2010; Foo
and Davies, 2011). Further, all NSP mediated responses in the
rhizobium symbiosis depend on Nod factor perception and the common
signalling module. In contrast, strigolactone biosynthesis does not
depend on the common signalling module. Therefore it is very well
possible that not primarily the strigolactone function of NSP1-NSP2 has
been recruited in rhizobium symbiosis, but rather that both transcription
factors gained novel primary targets in case of legume nodulation.
The question remains how mycorrhizae and rhizobium LCO induced
signalling on one hand can trigger similar cellular responses and on the
other hand can control symbiosis specific responses of which nodule
formation in case of rhizobium is most prominent. Both, mycorrhizae
and rhizobium LCOs trigger Ca2+ oscillations in the perinuclear region
within minutes after application. It was hypothesized that the amplitude
and oscillation frequencies were different and that CCaMK, possibly in
conjunction with interacting proteins like LjCYCLOPS/MtIPD3, can
translate these different calcium signatures in specific responses (Kosuta
et al., 2008). However, studies using a nuclear-targeted version of the
Ca2+ sensor cameleon reveals that the Ca2+ oscillation responses
triggered by rhizobium and a mycorrhizal fungus are indistinguishable
(Sieberer et al., 2012). This makes it unlikely that a signature in Ca2+
oscillation is a discriminating factor between both symbionts. Instead a
difference is observed between (cortical) cells that perceive LCOs and
cells that become actually intracellularly infected by either rhizobium or
mycorrhizal fungus. In latter case, the cells display and enhanced
amplitude in Ca2+ oscillations (Sieberer et al., 2012). Still, CCaMK might
be a component that can discriminate differences in input signal; e.g. in
strength of the signal. A recent study shows that rhizobium and
mycorrhzal symbioses have different requirements for binding of 16 Chapter 1
Calmodulin (CaM) to CCaMK (Shimoda et al., 2012). CaM is a Ca2+
binding protein that functions as messenger to transduce signals and in
such could be a determinant of CCaMK specificity. However, the CaM
binding domain in CCaMK is highly conserved and transcomplementation
experiments demonstrated that non-legume CCaMK
could functionally complement a corresponding mutation in legumes.
This makes it unlikely that this domain obtained different properties in
legumes to serve the rhizobium symbiosis. However, it does illustrate
that there are more stringent demands to CCaMK functioning in
rhizobium symbiosis when compared to mycorrhizae.
In trying to understand the difference in rhizobium and mycorrhizal
induced responses it is important to realise that our knowledge on ‘when,
where, which and how much’ LCOs are needed in the different steps to
achieve a symbiosis with rhizobium or a mycorrhizal fungus is still
scanty. The differences in demands for Nod factor receptor is clearly
illustrated by a M. truncatula Mtlyk3 splicing mutant (hcl-4) that produces
markedly reduced levels of functional receptor protein (90% reduction
of correctly spliced mRNA) (Smit et al., 2007). In this mutant root hair
curling is not affected, whereas infection thread formation is almost
completely blocked. Nevertheless both processes depend on the
activation of the same common signalling pathway, including CCaMK.
As mycorrhizal fungi seem to produce extremely low quantities of these
LCOs when compared to rhizobium (Maillet et al., 2011), it is possible
that the difference in response is also a matter of amounts of ligand.
Evolutionary Constraints and Multiple Events
As discussed above, it seems very probable that the evolution of
rhizobium nodule symbiosis in legumes as well as in Parasponia involved
the recruitment of the mycorrhizal LCO perception mechanism as well
as the common signalling module. This suggests that genetic constraints
rather than invention of novelties determined the evolution of the
rhizobium endosymbiosis. As the essential genes are likely present in all Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 17
plant species that are able to establish an endomycorrhizal symbiosis, it
in theory provides a red-carpet welcome for microbes to evolve a
(symbiotic) biotrophic relation. This raises the question whether the
common signalling pathway has been recruited in evolution more than
twice. Some observations indicate that this is indeed the case. Current
knowledge about legume phylogeny in relation to occurrence of
rhizobium symbiosis suggests that this character could have evolved up
to 6 times within the Fabaceae (Doyle, 2011). However, no evidence yet
has been provided that in all these cases the same set of genes has been
co-opted. First such evidence has been obtained in a different plantnitrogen
fixing endosymbiosis; namely between the gram-positive
bacteria of the genus Frankia and species collectively known as
actinorhizal plants. Actinorhizal plants make lateral root-like nodules
similar as found on Parasponia roots. Filamentous Frankia hyphae infect
actinorhizal roots intercellularly, but once inside the nodule cortical cells
are infected intracellularly to form fixiation thread-like structures.
Actinorhyzal plants do not form a single phylogenetic lineage and most
probably the symbiosis with Frankia species evolved several times in
parallel (Fig. 1) (Doyle, 2011). Studies in two unrelated actinorhizal
species, Datisca glomerata and Casuarina glauca revealed that gene homologs
of the legume Nod factor signalling pathway are expressed in young root
nodules (Hocher et al., 2011). RNA interference knockdown experiments
revealed that the LRR-type receptor kinase SYMRK/DMI2 is essential
for nodule formation as well as intracellular infection (Gherbi et al., 2008;
Markmann et al., 2008). This makes it probable that also in actinorhizal
species the common signalling module has been recruited. These
findings suggest that parallel evolutionary events of nitrogen fixing
nodular endosymbioses with either rhizobium or Frankia at least in part
leaned on the ancient and widespread mycorrhizal symbiosis.
Taken into account these recent insights in the genetic constraints
underlying nitrogen fixing endosymbioses, it raises questions why not
more plant species have gained such -at first sight- profitable interaction.
Current advances in genomics and metabolomics provide unprecedented 18 Chapter 1
new tools to tackle this question. Thereby it should be the ambition to
provide a proof of concept, and demonstrate that a transfer of nitrogen
fixing symbiosis is achievable.
What I would like is for someone to confirm my opinion that this is incorrect and mychos can and will populate the cannabis rhizospere. So I went hunting to substantiate my belief. With that said it was tough to find anything due to lack of studies on the genus cannabis as we all know for obvious reasons. I was able to determine based on 1 study that mychos will inhabit the cannabis rhizopshere. Keeping in mind not ALL plant life has a symbiotic relationship with mychos. Legumes are one such species that utilize mycho as part of nitrogen fixation process. I will paste the paper and in review see page # 10 and it makes reference to the genus Cannabaceae.
Upon a wikipedia search I found this. http://en.wikipedia.org/wiki/Cannabaceae
Cannabaceae is a small family of flowering plants. As now circumscribed, the family includes about 170 species grouped in about 11 genera, including Cannabis (hemp),Humulus (hops) and Celtis (hackberries). Celtis is by far the largest genus, containing about 100 species.[1]
Other than a shared evolutionary origin (see Phylogeny below), members of the family have few common characteristics; some are trees (e.g. Celtis), others areherbaceous plants (e.g. Cannabis).
So here is the study paper titled Exploiting an Ancient Signalling Machinery
to Enjoy a Nitrogen Fixing Symbiosis
Here is the link.
Here is the paper, Based on my understanding I am correct and my sparring partner is wrong. Be sure to go review the entire paper as it is very interesting for the science lovers in the group. Peace
Abstract
Almost for a century now it is speculated that a transfer of the largely
legume-specific symbiosis with nitrogen fixing rhizobium would be
profitable in agriculture (Burrill and Hansen, 1917; Charpentier and
Oldroyd, 2010). Till now such step was not achieved, despite intensive
research in this era. Novel insights in the underlying signalling networks
leading to intracellular accommodation of rhizobium as well as
mycorrhizal fungi of the Glomeromycota order show extensive
commonalities between both interactions. As mycorrhizae symbiosis can
be established basically with most higher plant species it raises questions
why only in a few taxonomic lineages the underlying signalling network
could be hijacked by rhizobium. Unravelling this, will lead to insights
that are essential to achieve an old dream.
Introduction
Rhizobium bacteria and arbuscular mycorrhizal fungi of the
Glomeromycota phylum can both establish an endosymbiosis with
plants that facilitates growth in a nitrogen or phosphate deficient
environment, respectively. Mycorrhizal fungi are obligatory biotrophs.
Their hyphae penetrate the root intercellularly or intracellularly,
depending on the plant host, and subsequently form arbuscules in inner
cortical cells. These arbuscules are highly branched intracellular hyphae
that are surrounded by a membrane formed by the host. This periarbuscular
membrane functions as a symbiotic interface as transporters
are present that facilitate exchange of nutrients. These are mainly
phosphates, but also nitrates that are taken up by the mycelium outside
the plant root (the so called extraradical mycelium) (Smith et al., 2011;
Javot et al., 2011). In return the fungus obtains photosynthates of the
plant for which it has specific monosaccharide transporters in its
arbuscular membrane (Helber et al., 2011). In between the periarbuscular
membrane and the branched hyphae a structured plant cell
wall is practically absent to maximize reciprocal exchange between both
organisms.
Unlike mycorrhizal fungi, some soil bacteria of the Rhizobaceae
family -collectively called rhizobia- have a dual lifestyle. They can be
free living in soil, but in case the appropriate legume host is present
they can establish a biotrophic endosymbiosis. For this they carry a set
of symbiotic genes that are located on a large symbiotic (sym)
plasmid(s) or are present as symbiotic islands in the genome. These
symbiotic genes do include; (I) genes required for nitrogen fixation (the
nif and fix genes) and (II) genes essential to establish symbiosis (the nod,
nol and noe genes). This second set of genes encodes the machinery that
is essential for biosynthesis and secretion of lipo-chitooligosaccharides
(LCOs) that function as signal molecules and are named Nod factors.
Nod factors are perceived by the host plant and set in motion
symbiotic engagement.
page 10 Chapter 1
The nitrogen fixing rhizobium symbiosis is basically restricted to
legumes -the Fabaceae-, with one exception the genus Parasponia that
belongs to the Cannabaceae. Cannabaceae and Fabaceae diverged ~100
million years ago represented by the split of Fabales and the orders
Fagales, Cucurbitales and Rosales (Wang et al., 2009), in all probability
supporting the idea that emergence of rhizobium symbioses occurred in
parallel in both lineages (Fig. 1).
Figure 1: Phylogenetic relation of plant families in the orders Fabales, Fagales, Cucurbitales
and Rosales. The occurrence of rhizobium symbiosis is indicated with a red asterisk and
Frankia symbiosis with yellow circle. Within a family, symbiosis is occurring only in one or a few
genera, with the exception of legumes (Fabaceae) (Doyle et al., 2011). Drawing is based on
published phylogenetic trees (Wang et al., 2009; Zhang et al., 2011; Li et al., 2004; Zhang et
al., 2006).
To host rhizobium novel organs are formed, named nodules, which
provide an optimal niche for nitrogen fixing rhizobia. Legume nodules
have a unique ontology and originate from primordia formed in the root
cortex. Legume nodules contain a large central tissue of which its
infected cells harbour hundreds of rhizobia. In most legumes these
bacteria are hosted individually, or in small clusters, surrounded by a
plant-derived membrane; a unit that is called symbiosome. This
membrane compartment facilitates exchange of fixed ammonium for
other nutrients, including photosynthates. To reach the nodule
primordia, in general sophisticated intracellular infection threads are
formed. Starting at a root hair that curls around a (single) bacterium a
membrane bound tubular infection thread is formed that guides the
clonally propagating microsymbiont to the nodule primordium.
Subsequently, bacteria are released from the infection thread and
develop in their symbiotic form. Infection threads that enter nodule cells
are bound by a thick cell wall and to release rhizobia from such infection
thread cell wall-free patches are created, so-called unwalled droplets. At
such sites, bacteria are in close contact with the surrounding host
membrane enabling pinching off of symbiosomes.
In contrast to legumes, Parasponia nodules seem much more primitive.
Nodule ontology resembles that of a lateral root. Parasponia is infected
intercellularly by rhizobium and only in the nodule cortex rhizobium
triggers formation of intracellular infection threads. These are
invaginations of the plasmamembrane and are bound by a thick cell
wall. From these infection threads, fixation threads are formed.
Fixation threads are also bound by a plant membrane and a plant cell
wall, however this cell wall is markedly thinner than the infection
thread cell wall. This type of fixation threads also occurs in nodules
of some basal legumes. Rhizobium is not released as symbiosomes
from the fixation threads most likely due to the presence of this cell
wall.
Commonalities in Signalling
Since several years it is known that in legumes rhizobium and
mycorrhizal fungi signals activate different receptors, but in turn these 12 Chapter 1
activate a common signalling module that subsequently diverges in the
two symbiotic interactions (Radutoiu et al., 2003). This commonality in
symbiotic signalling has been characterized in two model legume species,
Lotus japonicus and Medicago truncatula, and is shown to occur in other
legumes and non-legumes that interact with mycorrhizal fungi (Gutjahr
et al., 2008; Kouchi et al., 2010). The common signalling module
stretches from a plamamembrane receptor kinase (named LjSYMRK in
L. japonicus and MtDMI2 in M. truncatula), a cation channel located in the
nuclear envelope (LjCASTOR, LjPOLLUX/MtDMI1), and a nuclear
localized protein complex of a calcium Calmodulin dependent kinase
(CCaMK) and a coiled-coil protein (LjCYCLOPS/MtIPD3).
Furthermore, several subunits of the nuclear pore have been found to be
essential for rhizobium and mycorrhizae induced signalling (For recent
reviews see: Kouchi et al., 2010; Oldroyd et al., 2011). An essential step in
rhizobium symbiosis is the recognition of Nod factors, which holds for
(almost all) legumes as well as for Parasponia species. In legumes, Nod
factors are perceived by two distinct transmembrane LysM-type receptor
kinases (LjNFR1, LjNFR5 / MtLYK3, MtNFP). Studies in heterologous
systems indicate that these receptors can form a heterodimeric complex
(Madsen et al., 2011), whereas in legumes itself the subcellular regulation
is highly dynamic and affected upon Nod factor signalling (Haney et al.,
2011). Upon Nod factor perception these LysM-type receptor kinases,
together with the common signalling module, set in motion bacterial
entry as well as root nodule organogenesis.
Legume LysM-type Nod factor receptors are not essential for
mycorrhization. However, two complementary approaches strongly
support the idea that mycorrhizal fungi activate LysM-type receptor
kinases. Recently it was shown that the model mycorrhizal fungus
Glomus intraradices also produces LCOs, molecules that are named Myc
factors (Maillet et al., 2011). Application of these Myc factors to plant
roots increases mycorrhization at least twofold, an effect that seems
generic in higher plants as it can be triggered in legumes as well as nonlegumes
(Maillet et al., 2011).Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 13
Myc factors and Nod factors are structurally very similar (Maillet et al.,
2011). As the rhizobium symbiosis is relatively young in comparison to
mycorrhizal symbiosis it suggests that Nod factor perception evolved
from the mycorrhizal fungal symbiosis. Strong support for this came
from studies on Parasponia andersonii. Parasponia-rhizobium symbiosis is
relatively young based on the close phylogenetic relation of Parasponia
species with non-nodulating sister species in the Trema genus (Sytsma et
al., 2002; Yesson et al., 2004). Therefore co-evolution of rhizobium and
Parasponia has been relatively limited when compared to legumes. In line
with this it is anticipated that in Parasponia the LysM-type receptor kinase
family is less diverged, similar as seen in other non-legumes species
(Zhang et al., 2007; Zhang et al., 2009). In comparison L. japonicus and M.
truncatula have at least 2 members of the LjNFR5/MtNFP-type receptor
kinases, of which one is a Nod factor receptor, whereas the second Nod
factor receptor, LjNFR1/MtLYK3, underwent even series of
duplications (Zhang et al., 2007; Limpens et al., 2003; Arrighi et al., 2006;
Lohmann et al., 2010). In contrast P. andersonii only has a single
NFR5/NFP-type receptor, PaNFP. Functional analysis revealed that
PaNFP has a dual symbiotic function. It controls the formation of the
symbiotic interface of rhizobium as well as mycorrhizal fungi (Op den
Camp et al., 2011). Together with the observation that mycorrhizal fungi
secrete LCOs this leads to the hypothesis that not only the common
signalling module, but also the rhizobium Nod factor perception
mechanism is recruited from endomycorrhizae.
The duplications of Nod factor receptors in M. truncatula and L. japonicus
do not stand on their own. Like most plant lineages also the legume
lineage experienced whole genome duplications (WGDs) (Cannon et al.,
2006). One such duplication event occurred early in evolution of the
Papilionoideae about 58 million years ago. This legume subfamily
represents most nodulating legumes, including all prominent crop
species. Hundreds of paralogous gene pairs that originate from this
duplication showed to be maintained in M. truncatula, L. japonicus and
soybean (Glycine max), and for many of these one or even both genes 14 Chapter 1
showed to be expressed in root nodules. As these legumes diverged
more than 55 million years ago, it indicates that this WGD provided a
genetic redundancy that contributed to the evolution of the rhizobium
nodule symbiosis in the Papilionoid subfamily (Camp et al., 2011; Young
et al., 2011).
Downstream of the Common Signalling Module
Formation of a symbiotic interface that facilitates exchange of nutrients
is a crucial step in endosymbiosis. Studies in Parasponia indicate that this
process is tightly controlled by LCO signalling in case of fixation thread
formation by rhizobium as well as arbuscule formation by mycorrhizal
fungi. Also in legumes Nod factor signalling plays a prominent role in
the formation of a symbiotic interface. Knock down (or loss of function)
of several genes of the common signalling module results in nodules
with numerous intracellular infection threads, but symbiosomes are not
formed (Ivanov et al., 2012; Ovchinnikova et al., 2011; Horvath et al.,
2011; Limpens et al., 2005).Taken together, it suggests that rhizobium
and mycorrhizal fungi trigger similar cellular responses.
Recently it was shown that two GRAS-type transcription factors, NSP1
and NSP2, both essential for basically all Nod factor induced responses
in legumes, also have a function in the absence of the microsymbionts.
These transcription factors were shown to control expression of
DWARF27, a gene essential for strigolactone biosynthesis (Liu et al.,
2011; Lin et al., 2009). As a consequence, functional nsp1 and nsp2
mutants are significantly hampered in strigolactone biosynthesis. This
holds for M. truncatula as well as rice (Oryza sativa), suggesting that the
transcriptional regulation of a key enzyme in strigolactone biosynthesis is
largely conserved in higher plants (Liu et al., 2011). Strigolactones are
important secondary metabolites in plants that can act as hormones as
well as ex planta attractants for mycorrhizal fungi. Plant roots secrete
strigolactones in response to phosphate starvation, which, in M.
truncatula, correlates with a NSP1-NSP2 dependent transcriptional Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 15
activation of MtDWARF27 (Liu et al., 2011). This supports that NSP1
and NSP2 have a function in mycorrhizal symbiosis and suggests that
this function might have been recruited during evolution of rhizobium
symbiosis. However, to date only very little evidence supports a role of
strigolactone signalling in rhizobium symbiosis (Soto et al., 2010; Foo
and Davies, 2011). Further, all NSP mediated responses in the
rhizobium symbiosis depend on Nod factor perception and the common
signalling module. In contrast, strigolactone biosynthesis does not
depend on the common signalling module. Therefore it is very well
possible that not primarily the strigolactone function of NSP1-NSP2 has
been recruited in rhizobium symbiosis, but rather that both transcription
factors gained novel primary targets in case of legume nodulation.
The question remains how mycorrhizae and rhizobium LCO induced
signalling on one hand can trigger similar cellular responses and on the
other hand can control symbiosis specific responses of which nodule
formation in case of rhizobium is most prominent. Both, mycorrhizae
and rhizobium LCOs trigger Ca2+ oscillations in the perinuclear region
within minutes after application. It was hypothesized that the amplitude
and oscillation frequencies were different and that CCaMK, possibly in
conjunction with interacting proteins like LjCYCLOPS/MtIPD3, can
translate these different calcium signatures in specific responses (Kosuta
et al., 2008). However, studies using a nuclear-targeted version of the
Ca2+ sensor cameleon reveals that the Ca2+ oscillation responses
triggered by rhizobium and a mycorrhizal fungus are indistinguishable
(Sieberer et al., 2012). This makes it unlikely that a signature in Ca2+
oscillation is a discriminating factor between both symbionts. Instead a
difference is observed between (cortical) cells that perceive LCOs and
cells that become actually intracellularly infected by either rhizobium or
mycorrhizal fungus. In latter case, the cells display and enhanced
amplitude in Ca2+ oscillations (Sieberer et al., 2012). Still, CCaMK might
be a component that can discriminate differences in input signal; e.g. in
strength of the signal. A recent study shows that rhizobium and
mycorrhzal symbioses have different requirements for binding of 16 Chapter 1
Calmodulin (CaM) to CCaMK (Shimoda et al., 2012). CaM is a Ca2+
binding protein that functions as messenger to transduce signals and in
such could be a determinant of CCaMK specificity. However, the CaM
binding domain in CCaMK is highly conserved and transcomplementation
experiments demonstrated that non-legume CCaMK
could functionally complement a corresponding mutation in legumes.
This makes it unlikely that this domain obtained different properties in
legumes to serve the rhizobium symbiosis. However, it does illustrate
that there are more stringent demands to CCaMK functioning in
rhizobium symbiosis when compared to mycorrhizae.
In trying to understand the difference in rhizobium and mycorrhizal
induced responses it is important to realise that our knowledge on ‘when,
where, which and how much’ LCOs are needed in the different steps to
achieve a symbiosis with rhizobium or a mycorrhizal fungus is still
scanty. The differences in demands for Nod factor receptor is clearly
illustrated by a M. truncatula Mtlyk3 splicing mutant (hcl-4) that produces
markedly reduced levels of functional receptor protein (90% reduction
of correctly spliced mRNA) (Smit et al., 2007). In this mutant root hair
curling is not affected, whereas infection thread formation is almost
completely blocked. Nevertheless both processes depend on the
activation of the same common signalling pathway, including CCaMK.
As mycorrhizal fungi seem to produce extremely low quantities of these
LCOs when compared to rhizobium (Maillet et al., 2011), it is possible
that the difference in response is also a matter of amounts of ligand.
Evolutionary Constraints and Multiple Events
As discussed above, it seems very probable that the evolution of
rhizobium nodule symbiosis in legumes as well as in Parasponia involved
the recruitment of the mycorrhizal LCO perception mechanism as well
as the common signalling module. This suggests that genetic constraints
rather than invention of novelties determined the evolution of the
rhizobium endosymbiosis. As the essential genes are likely present in all Exploiting an Ancient Signalling Machinery to Enjoy Symbiosis 17
plant species that are able to establish an endomycorrhizal symbiosis, it
in theory provides a red-carpet welcome for microbes to evolve a
(symbiotic) biotrophic relation. This raises the question whether the
common signalling pathway has been recruited in evolution more than
twice. Some observations indicate that this is indeed the case. Current
knowledge about legume phylogeny in relation to occurrence of
rhizobium symbiosis suggests that this character could have evolved up
to 6 times within the Fabaceae (Doyle, 2011). However, no evidence yet
has been provided that in all these cases the same set of genes has been
co-opted. First such evidence has been obtained in a different plantnitrogen
fixing endosymbiosis; namely between the gram-positive
bacteria of the genus Frankia and species collectively known as
actinorhizal plants. Actinorhizal plants make lateral root-like nodules
similar as found on Parasponia roots. Filamentous Frankia hyphae infect
actinorhizal roots intercellularly, but once inside the nodule cortical cells
are infected intracellularly to form fixiation thread-like structures.
Actinorhyzal plants do not form a single phylogenetic lineage and most
probably the symbiosis with Frankia species evolved several times in
parallel (Fig. 1) (Doyle, 2011). Studies in two unrelated actinorhizal
species, Datisca glomerata and Casuarina glauca revealed that gene homologs
of the legume Nod factor signalling pathway are expressed in young root
nodules (Hocher et al., 2011). RNA interference knockdown experiments
revealed that the LRR-type receptor kinase SYMRK/DMI2 is essential
for nodule formation as well as intracellular infection (Gherbi et al., 2008;
Markmann et al., 2008). This makes it probable that also in actinorhizal
species the common signalling module has been recruited. These
findings suggest that parallel evolutionary events of nitrogen fixing
nodular endosymbioses with either rhizobium or Frankia at least in part
leaned on the ancient and widespread mycorrhizal symbiosis.
Taken into account these recent insights in the genetic constraints
underlying nitrogen fixing endosymbioses, it raises questions why not
more plant species have gained such -at first sight- profitable interaction.
Current advances in genomics and metabolomics provide unprecedented 18 Chapter 1
new tools to tackle this question. Thereby it should be the ambition to
provide a proof of concept, and demonstrate that a transfer of nitrogen
fixing symbiosis is achievable.
