Differential tetraspanin genes expression and subcellular localization during mutualistic interactions in Phaseolus vulgaris
Differential tetraspanin genes expression and subcellular localization during mutualistic interactions in Phaseolus vulgaris
Saul Jimenez-JimenezID 0 3
Olivia Santana 0 3
Fernando Lara-Rojas 0 3
Manoj- Kumar Arthikala 3
Elisabeth Armada 0 3
Kenji Hashimoto 1 3
Kazuyuki Kuchitsu 1 3
Sandra Salgado 0 3
Jes u?s Aguirre 2 3
Carmen Quinto 0 3
Luis C a?rdenasID 0 3
0 Departamento de Biolog ??a Molecular de Plantas, Instituto de Biotecnolog ??a, Universidad Nacional Aut o ?noma de M e ?xico, Cuernavaca, Morelos, M e ?xico, 2 Ciencias Agrogeno ?micas, Escuela Nacional de Estudios Superiores Unidad Leo ? n-Universidad Nacional Aut o ?noma de M e ?xico , Le o ?n, Guanajuato, M e ?xico
1 Department of Applied Biological Science, Tokyo University of Science , Yamazaki, Noda, Chiba , Japan
2 Instituto de Fisiolog ??a Celular, Universidad Nacional Auto ? noma de M e ?xico, Ciudad de M e ?xico , M e ?xico
3 Editor: Ricardo Aroca, Estacion Experimental del Zaidin , SPAIN
Arbuscular mycorrhizal fungi and rhizobia association with plants are two of the most successful plant-microbe associations that allow the assimilation of P and N by plants, respectively. These mutualistic interactions require a molecular dialogue, i.e., legume roots exude flavonoids or strigolactones which induce the Nod factors or Myc factors synthesis and secretion from the rhizobia or fungi, respectively. These Nod or Myc factors trigger several responses in the plant root, including calcium oscillations, and reactive oxygen species (ROS). Furthermore, superoxide and H2O2 have emerged as key components that regulate the transitions from proliferation to differentiation in the plant meristems. Similar to the root meristem, the nodule meristem accumulates superoxide and H2O2. Tetraspanins are transmembrane proteins that organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking and ROS generation. Plant tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, and hormonal regulation and recently have been reported as a specific marker of exosomes in animal and plant cells and key players at the site of plant fungal infection. In this study, we conducted transcriptional profiling of the tetraspanin family in common bean (Phaseolus vulgaris L. var. Negro Jamapa) to determine the specific expression patterns and subcellular localization of tetraspanins during nodulation or under mycorrhizal association. Our results demonstrate that the tetraspanins are transcriptionally modulated during the mycorrhizal association, but are also expressed in the infection thread and nodule meristem development. Subcellular localization indicates that tetraspanins have a key role in vesicular trafficking, cell division,
and root hair polar growth.
role in study design, data collection and analysis,
decision to publish, or preparation of the
The symbiotic interaction between rhizobia and legumes requires a molecular dialogue that
involves the exchange of specific signaling molecules. Legumes secrete particular flavonoids or
strigolactones that are specifically recognized by rhizobia or mycorrhizal fungi [
compounds induce the expression of specific genes, which encode proteins involved in the synthesis
and secretion of Nod factors (NFs) from rhizobia, which are lipochitin-oligosaccharides or the
Myc factor from the mycorrhizal fungi [
]. Thereafter, NFs or Myc factors are specifically
recognized by the plant root and induce several responses, including ionic changes, membrane
depolarization, cytoskeleton rearrangements, reactive oxygen species (ROS) generation, and
gene expression [
]. Soon after the rhizobia reach the root hair, microfilament structures and
Ca2+ gradients undergo dramatic changes in the tip region of root hair cells in response to NFs
]. In Arabidopsis thaliana root hairs, ROS-mediated Ca2+ channel activity supports polar
]. Therefore, ionic responses, ROS production, and cytoskeletal rearrangements
have been suggested as key players in reprogramming root hair growth, which allows the root
hair tip curling response during the establishment of symbiotic interactions in legume plants.
Then, a tunnel-like infection thread (IT) forms through invagination of the plasma membrane
and the cell wall . While the bacteria travel inside the IT through the root hair, the cortical
cells divide in a NF-dependent manner to form the nodule primordia that the rhizobia colonize
in structures named symbiosome. Once mature, the nodule is able to fix atmospheric nitrogen
]. On the other hand, the arbuscular mycorrhizal association induces the hypopodium and
the further invasion of the cortical cells, which end up with the arbuscule formation. Both
processes, the bacterial colonization trough the infection thread and arbuscule formation, require
an active vesicular trafficking, endocytosis and exocytosis in order to increase the membranal
surface required for symbiosome and arbuscule formation [
Given their wide distribution in mammals, insects, fungi, mosses, and plants, tetraspanins
likely coemerged in multicellular organisms during evolution [
]. In animal cells,
tetraspanins are typically localized at the cell?cell interface in tetraspanin-enriched microdomains
(TEMs), where they associate with each other and with other membrane-bound molecules and
build important molecular platforms or cell?cell interactions [
]. Tetraspanins get their
name from their four transmembrane domains. The N and C tails of tetraspanins are localized
on the cytoplasmic side of the membrane, and four transmembrane domains allow for the
formation of two extracellular loops, one small and one large. The large loop has highly conserved
cysteine residues, which could act as redox and pH sensors or promote protein?protein
interactions. The small loop in plant tetraspanins contains a cysteine residue that is absent in
animal tetraspanins . Although tetraspanins in animal cells have been involved in various
biological functions, such as cell motility, morphology, signaling, plasma membrane dynamics,
and protein trafficking, how tetraspanins engage in plant cells functions at the cellular level is
largely unknown [
]. Furthermore, tetraspanin have been described in animal and recently in
plant cells as key components and specific markers of the exosomes, which are vesicles derived
from the exocytic multivesicular bodies (MVB) that carry important molecules such as lipids,
proteins, messenger RNA, and microRNAs, that play important roles in cell-to-cell
communication in animal [
]. In plant cells it has been recently reported that exosomes go beyond
organism boundaries and inhibit a pathogenic interaction in plants [
]. There is also an
emerging idea that tetraspanins are part of a mechanism that generates ROS [
]. In animal
cells, H2O2 has been described as an important component of axonal regeneration after acute
injury . However, the injured neurons do not express the NADPH oxidase required for
ROS generation. Instead, macrophages recruited to the vicinity secrete exosomes carrying
NOX enzymes, which are taken up by the injured neuron via endocytosis and promote axonal
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growth in a ROS-dependent manner with a key participation of exosomes [
]. On the
other hand, the cuticle exoskeleton of the nematode Caenorhabditis elegans, in a similar way to
the cell wall in plant cells, requires the ROS-dependent cross-linking of tyrosine residues, in a
process assisted by BLI-3, which is a DUOX NADPH oxidase, and tetraspanin TSP-15 [
]. When plants are infected with the pathogenic fungus Magnaporthe grisea, the fungal
tetraspanin Pls1 and an NADPH oxidase are localized at the infection sites or appressorium
structure to generate a ROS accumulation response that is required to reestablish the
appressorium polarity [
24, 25, 31?33
]. Therefore, accumulating evidence links tetraspanin with ROS
and polarity. Furthermore, tetraspanins have been identified at the tip of growing pollen tubes,
a region that requires NADPH oxidase activity for the generation of ROS, a key player
regulating polar growth . However, we do not know if tetraspanins also function in pollen tube or
even root hair polar growth [
Tetraspanins also accumulate in the female gametophyte, suggesting that they have an
active role during gametophyte development or fertilization, a well-described ROS-dependent
process. Furthermore, tetraspanins function during the transition from the floral meristem to
the gynoecium as well as during the somatic-to-reproductive cell fate transition during
megasporogenesis, suggesting that these proteins are regulators of cell fate determination [
tetraspanins are also expressed in specialized tissues, such as the the quiescent center or the early
initial cells that give rise to lateral roots meristems, these proteins may function in specific tissues
or contribute to cell fate determination [
18, 35, 38
]. The meristematic distribution of some
tetraspanins suggests that these proteins might be involved in regulating meristematic activity, which
is highly dependent on ROS accumulation generated by NADPH oxidase activity, with
superoxide-promoting meristematic activity and H2O2-promoting cell differentiation [
On the other hand, NADPH-oxidase-derived ROS is a key component during nodule
development and mycorhizal formation [
]. Since superoxide and H2O2 are produced
during nodulation [
], it is possible that the molecular mechanism that maintains
meristematic activity in the root is conserved during nodule meristem development . Therefore, it
is plausible that the ROS requirement for meristematic activity in the plant root is conserved
during primordium development during nodule organogenesis and that tetraspanins have
similar functions during the early stages of nodule development, as described in lateral root
formation in Arabidopsis .
Here, we report the differential expression profile of the tetraspanin family in P. vulgaris in
response to rhizobia or NF inoculation in several specific plant cells, such as root primordia,
root hairs, and nodules at several developmental stages. PvTET8, PvTET4 and PvTET3 were
highly expressed in the root meristematic region and during the early stages of primordium
nodule development. Since PvTET8 was highly induced during nodulation, but not during
mycorrhizal association, we suggest that this tetraspanin plays a particular role during
nodulation, including the infection thread formation. Furthermore, the subcellular localization of
some tetraspanins at the apical plasma membrane of P. vulgaris root hairs and in cytoplasmic
vesicles suggests that these proteins function in polar growth, a central process during
infection thread formation and migration. Our findings suggest that tetraspanins may also
contribute to the vesicular trafficking required for localized exocytosis during the infection process or
contribute to the exosome biogenesis as described during the pathogenic responses.
Materials and methods
Using the methodology described by Huang et al. (2010), tetraspanin sequences containing four
transmembrane domains were database screened, including a small and large extracellular loop,
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using bioinformatics tools. The sequences were aligned in ClustalW. To avoid subjective bias,
manual alignment editing was minimized. A phylogenetic tree was built using MEGA version
6.0.6 with 1000 bootstrap tests and pairwise deletion.
Vector construction for analyzing the activity promoter and subcellular
To evaluate promoter activity, the pPvTET1A::GFP-GUS, pPvTET8::GFP-GUS and pPvTET3::
GFP-GUS construct, which includes the 1000-bp fragment upstream of the initiation codon of
TET1A, TET8 and TET3 respectively, was created by PCR using P. vulgaris cv Negro Jamapa
genomic DNA as template and gene-specific primers (S1 Fig). Each product of PCR was
cloned into the pENTR/D-TOPO vector (Invitrogen) and recombined into the destination
binary vector pBGWFS7.0 using Gateway LR Clonase II Enzyme Mix (Invitrogen) [
each step, the presence of the insert was confirmed by Sanger sequencing and PCR. All
constructed plasmids were introduced by electroporation into Agrobacterium rhizogenes strain
K599. To design constructs for overexpressing PvTET10, PvTET6 and PvTET3, the open
reading frame of each gene was isolated from P. vulgaris cDNA and inserted into the pH7WG2D.1
binary vector under the control of the constitutive 35S promoter [
]. Empty pH7WG2D.1
vector, which constitutively expresses GFP, was used as the control in the overexpression
system. All constructed plasmids were introduced by electroporation into A. rhizogenes strain
K599 and used for further generation of transgenic roots or Agrobacterium tumefaciens strain
AGL1 for transient expression in N. benthamiana leaves.
Bean hairy root transformation
Common bean seeds (Phaseolus vulgaris cv Negro Jamapa) were used for A. rhizogenes
K599-mediated transformation to generate hairy roots harboring a construct of interest in
composite plants using a previously described method [
Treatment with Nod factors by nebulization
Common bean seedlings were incubated with Nod factors at 48 h post germination (hpg) by
nebulization with the Omron ComAir Nebulizer System Model NE-C801 according to the
manufacturer?s instructions. The Nod factors were purified according to our reported method
]. A kinetic of transcript accumulation was conducted in roots incubated with Nod factors
at 12, 24, and 36 h post inoculation for each tetraspanin. Nebulization with 1% CHAPS (w/v)
was used as a control.
Rhizobium tropici CIAT899 bacteria were grown in 250-mL flasks containing 100 mL of PY
broth supplemented with 7 mM CaCl2, 50 ?g/mL rifampicin, and 20 ?g/mL nalidixic acid, in a
shaking incubator (250 rpm) at 30?C until the suspension reached an OD600 of 0.8. For
nodulation assays, transgenic composite plants were transplanted under hydroponic conditions in
glass tubes containing Fahreus medium and inoculated with 1 mL of a R. tropici CIAT899
suspension diluted to an OD600 of 0.05 in 10 mM MgSO4 and grown in a controlled environment
chamber (16 h light/8 h darkness, at 26?C). At the indicated time points after inoculation,
inoculated roots were frozen in liquid nitrogen and stored at ?80?C. At 5 and 7 days post
infection (dpi), the root region close to the tail that is most susceptible to nodule formation
was selected, and at 10, 14, and 18 dpi, only nodules were selected. In all cases, the equivalent
root region of uninoculated roots was collected as a control.
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Mycorrhizal spore inoculation and mycorrhization
Common bean seedlings at 48 hpg were transferred into pots (20-cm diameter) with
vermiculite that was previously well inoculated in the pot with 1 gr of Rhizophagus irregularis substrate
containing on average 800 spores homogeneously distributed. Inoculated plants were irrigated
twice weekly with half-strength B&D solution containing a low concentration of potassium
phosphate (50 ?M; a 95% reduction compared with the control (500?M) to potentiate AM
]). As controls, two conditions without spores of R. irregularis were used: one set
of plants were irrigated with 50 ?M potassium phosphate (scarcity phosphate) and the other
with 500 ?M potassium phosphate (standard condition). AM fungal colonization status was
determined by light and confocal microscopy, as indicated. Root samples were collected at 1,
2, 3, and 6 wpi and frozen in liquid nitrogen. All samples were stored at ?80?C.
Quantification of transcript levels by RT-qPCR analysis
Total RNA was extracted from frozen tissues using TRIzol reagent (Life Technologies)
according to the manufacturer?s instructions. To eliminate contaminating genomic DNA,
total RNA samples (1 ?g in 20 ?L) were treated with 1 unit of DNaseI (RNase-free;
Invitrogen) at 37?C for 30 min and then at 65?C for 10 min. Two-step RT-qPCR was performed
using Maxima SYBR Green qPCR Master Mix (2X; Thermo Fisher Scientific), following the
manufacturer?s instructions. Each reaction was set up using 100 ng of cDNA as template in a
20-?L final volume. Gene-specific primers used in RT-qPCR reactions are listed in S1 Fig.
qPCRs were performed in a LightCycler 480 real-time PCR system (Roche). Relative
transcript abundance was calculated using the formulae reported by Schmitteng et al. [
vulgaris Elongation Factor 1? (Pv-EF1?) was used as a reference gene, as previously described.
RT-qPCR data are averages of three independent experiments or biological replicates with
two technical replicates.
Root hair and root isolation
The roots of P. vulgaris seedlings were divided and separated at 48 hpg. First, the region of the
primary root that contained root hairs was cut into three equal segments named zones I, II,
and III. Zone I contained the tip of the root that contained the initial or bulging out root hairs,
zone II contained the rapidly growing root hairs, and zone III contained the mature or
fullgrown root hairs. Each fraction was collected in different containers in liquid nitrogen.
Samples were stirred vigorously to separate the root hairs from the roots. Root hairs were isolated
by pouring the liquid nitrogen mixture through a metal strainer. At the end, the root hairs
were separated from the root (now shaved root) using a strainer. The shaved roots
corresponding to regions I, II, and III were collected for tetraspanin transcript analysis. Fractions were
stored at ?80?C until use. In each biological replicate, in order to confirm the different
developmental stage of root hairs, the level of RabA2 transcript was measured to assess the
differential expression of this gene in each enriched tissue fraction.
Subcellular localization of common bean tetraspanins
Transient expression assays were conducted in Nicotiana benthamiana leaves to determine the
subcellular localization of PvTET3 and PvTET6 proteins. The molecular construction carrying
35S:PvTET3-GFP and 35S:PvTET6-GFP was transferred to A. tumefaciens AGL1. For
transient assays, leaves from 4- to 6-week-old wild-type N. benthamiana plants were coinfiltrated
with the agrobacterium suspension harboring 35S:PvTETx-GFP. The infiltrated plants were
marked and kept in a growth room at 16 h light/8 h darkness at 25 ? 2?C. Plasmolysis was
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induced using a 1M NaCl hypertonic solution. Fluorescence was visualized 48?72 h after
infiltration using a spinning disk confocal microscope (Yokogawa, Japan) as described below.
Microscopy imaging and analysis
Transgenic roots were mounted in chambers adapted from large Petri dishes with a hole in the
center. The hole was covered with a large glass coverslip. The chamber contained a layer of
solid Fahreus medium (with Phytagel at 0.8%) and cellophane paper to prevent root
movement. Roots were visualized under the inverted microscope (Nikon Eclipse Ti-E, Japan) with a
40x/1.25 NA water immersion lens (Nikon). For confocal images, we used a spinning disk
confocal system (Intelligent Imaging Innovations /3i, USA) consisting of a CSU-W1 confocal
head (Yokogawa, Japan) and a modular solid-state laser stack; Slidebook software was used to
control the system and capture images (Intelligent Imaging Innovations /3i, USA). Images
were recorded with a digital camera (Andor-IXON 3; AndorTM Technology) for 1?2 min at
512-nm resolution and with frame rates of 100?300 ms. GFP fluorescence was obtained by
exciting with an argon/2 ion laser (488 nm), and emitted fluorescence was collected using an
emission filter (500 to 530 nm).
Data processing and statistical analysis were performed using GraphPad Prism version 6.00
for Windows (GraphPad Software). An unpaired two-tailed Student?s t-test was used to
determine whether data from two different groups were significantly different, Double, or triple
asterisks above the columns indicate differences that are statistically significant
(pvalue < 0.05).
Plant tetraspanins have a unique cysteine residue in the small extracellular
To determine the number of tetraspanin members in common bean (Phaseolus vulgaris L. var.
Negro Jamapa), we searched the database of nonredundant protein sequences in NCBI using
BLASTP (Protein-protein BLAST) using AtTET10 as query. AtTET10 and OsTET14 from O.
sativa can be considered the founders of the tetraspanin family in their respective species [
]. We identified 13 putative tetraspanin sequences in P. vulgaris, 25 in Glycine max, 9 in
Medicago truncatula, and 5 in Lotus japonicus (Fig 1 and S2 Fig). Seventeen tetraspanins have
been described in the model plant Arabidopsis and 15 in Oryza sativa [
]. PvTET10, and
other orthologs of AtTET10 and OsTET14, have between 10 and 12 introns in all reported
genomes, including Marchantia polymorpha and Physcomitrella patens.
The amino acid sequences and functional motifs previously described in Arabidopsis
allowed us to select tetraspanin members in P. vulgaris and compare them to other
tetraspanins in the legume genome database reported in Phytozome (https://phytozome.jgi.doe.gov/
pz/portal.html). Next, we conducted a bioinformatic screen based on several sequences,
including the transmembrane domains, the cysteine pattern in the large extracellular loop
(LEL), the single cysteine residue located exclusively in plant tetraspanins in the small
extracellular loop (SEL), and the GCC(K/R)P signature in the large extracellular loop. Multiple
sequence alignment and motif analyses revealed that common bean tetraspanins maintain the
general features described for plant tetraspanins, with high conservation in specific motifs. For
instance, in P. vulgaris, these proteins share an average of 38% identity and 42% similarity,
which is comparable to that observed for O. sativa and Arabidopsis.
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Fig 1. A rooted neighbor-joining phylogenetic tree of common bean tetraspanin. (A) On the left shown is the phylogenetic tree constructed using
MEGA6.06, with amino acidic sequences from phytozome.org. Numbers above branches indicate bootstrap percentage values. PvTET proteins were clustered
based on a significant bootstrap value of 50%. (B) On the right using an online tool, Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn/), was
used to draw the tetraspanin gene structure. Red boxes indicate exons, black lines depict introns, upstream/downstream sequences are shown as oranges
boxes. Intron phases are indicated at exon?intron junctions.
Based on these data, we generated an unrooted phylogenetic three using PvTET10 as a
query from Arabidopsis (S2 Fig and S3 Fig). In our analysis, PvTET10 clustered with AtTET10
and OsTET14, whereas PvTET1A and PvTET1B clustered with AtTET1 (TORNADO) and
AtTET2, and PvTET8 clustered with OsTET7-9 and AtTET8-9. We did not find a homolog of
AtTET13 in the P. vulgaris genome or in any other legume genome. This could suggest the
presence of a particular clade in Arabidopsis or in the Brassicaceae that does not exist in other
plants, at least not in P. vulgaris or in legumes. In our molecular tree, we identified 7 groups,
each of which contained at least two members, except the group that contained PvTET10
alone. Sequence identity and similarity analysis coupled with the number of introns supported
the idea that PvTET10 is a common ancestor of tetraspanin in common bean, as described for
its homologs in Arabidopsis (Fig 1). By contrast, PvTET12, which has no introns, is the most
recent tetraspanin member and arose by functional divergence and loss of introns [
GmTET12A (accession number XP_003551251.1) and GmTET12B (accession number
XP_003547633.1) are homologs of PvTET12 (accession number XM_007138229.1). In M.
truncatula, we identified one homologue of TET12 (accession number XP_013463988.1);
however, we did not find a corresponding homolog in L. japonicus. The cytoplasmic N and C
termini of plant tetraspanins each have 6?10 aa, making them shorter than animal tetraspanins,
which contain between 9 and 40 aa, and even shorter than fungal ones, which contain between
4 and 100 aa.
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In addition, we conducted an exhaustive in silico characterization of tetraspanins in
common bean to predict disulfide bond formation. We used a bioinformatic tool for disulfide
connectivity prediction named DiANNA (clavius.bc.edu/~clotelab/DiANNA/) and found that
disulfide bonds form between the single cysteine located in the SEL and the cysteine residues
found in the LEL of all common bean tetraspanins, as previously suggested for tetraspanins in
Tetraspanins PvTET3 and PvTET1A are highly expressed in root and root
To establish the transcript accumulation for the different tetraspanin genes in P. vulgaris roots,
we selected root tissues and sectioned different regions with different developmental stages
according to the experimental requirements (described in Materials and methods). In our
experiments with P. vulgaris, we considered the three developmental root hair zones, which
were evident by 48 h post germination (hpg). As depicted in Fig 2C we sectioned the three
different root zones and separated the root hair cells from the root tissue, generating the enriched
root hair fraction from the three developmental stages (zones I, II, and III) and the
corresponding shaved root (Fig 2C). Transcript accumulation for all tetraspanin genes, except
PvTET2B due to the lack of specific regions for primers design for amplification (accession
number XP 007152918.1), was determined in each one of the generated samples (root hairs
and shaved root); this analysis was conducted by selecting each tetraspanin family member
and determining its accumulation in root hairs at different developmental stages (Fig 2B).
The monomeric GTPase PvRabA2 is a good marker of the different developmental stages
of root hairs due to its key role in root hair tip growth (S4 Fig), although it is also expressed in
root tissue [
]. We observed that RabA2 transcript accumulated in root hairs; as expected,
the transcript accumulation was higher in zones II and III, where the cells are rapidly
expanding or reaching full length, compared with zone I, where root hairs are just bulging out (S4
Fig). As expected, the shaved root section that lacked root hairs also had increased PvRabA2
transcript accumulation in zones I, II, and III, again with higher accumulation in zones II and
III (S4 Fig). Therefore, these root sections were used to determine the transcript accumulation
for selected tetraspanin members in P. vulgaris. We found that PvTET3 and PvTET1A are
more abundant in shaved roots or root hairs from zones II and III than from zone I (Fig 2A
and 2B). However, PvTET1A present a very low expression in root hairs from zone II, but it is
expressed in those from zone III (Fig 2B). This differential expression contrasts with shaved
roots, where PvTET1A transcript accumulation increases gradually beginning from zone I to
zone II and from zone II to zone III (Fig 2A). Furthermore, an analysis of the reported
expression atlas of P. vulgaris  indicates that PvTET3 is ubiquitously expressed during the
different root developmental stages, while PvTET1A is constitutively expressed in all root tissue, but
with higher expression in some stages of root development (S5 Fig).
PvTET1A, PvTET8, PvTET3 and PvTET4 are induced in response to NFs or
We then evaluated the transcript accumulation of some tetraspanin genes in response to
nanomolar concentration of NFs (10?9 M), which can reprogram polar growth and nodule
primordia development [
]. In agreement with previous observations [
], treatment with 10?9 M NFs
caused root hairs to swell and undergo morphological changes (S6B Fig). As expected, no
morphological responses were observed when chitosan was used as a negative control (S6A Fig).
We initially considered to evaluate the transcript accumulation in response to NFs or
rhizobia for PvTET1A and PvTET3 since these genes were the most highly expressed in shaved root
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Fig 2. Tetraspanin gene expression profile in P. vulgaris root or root hairs from different developmental stages.
The relative expression of each PvTET gene was evaluated by qRT?PCR in three different sections of root at 48 h post
germination (hpg). Mature zone or Zone III (blue), elongation zone or Zone II (red), and meristematic, elongating and
differentiating region or Zone I (green). Transcript accumulation was normalized to the expression of EF1a, which was
used as a reference gene. Bars represent means ? SEM from at least three independent biological replicates with three
technical repeats. (A) Tetraspanin (TET) gene expression in shaved root and (B) root hairs at different development
stages at 48 hpg. Tissues enriched with emerging or bulging root hairs from Zone I (green bar), tissues enriched with
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growing root hairs (red bar) and tissues enriched with mature root hairs (blue bar). Transcript accumulation was
normalized to the expression of EF1a, which was used as a reference gene. Bars represent means ? SEM from at least
three independent biological replicates with three technical repeats. P-values <0.05 are marked with two asterisks
(Student?s t-test). (C) Cartoon depicting the different root and root hairs zones analyzed.
and root hairs under normal conditions, other tetraspanin genes were also tested, under NFs
or Rhizobia inoculation, but the one that did not respond were not considered for further
analysis (S7 Fig). PvTET1A was downregulated in P. vulgaris roots in the first 36 h days after
inoculation with NFs, but we detected a response at 5?10 days post inoculation with rhizobia (Fig
3A). By contrast, PvTET3 expression did not change within the first 24 h following inoculation
with NFs, but a significative decrease was observed at 36 h after treatment. On the other hand,
when inoculated with R. tropici, a further decrease was observed at 5 and 14?18 dpi (Fig 3B).
Thus, some tetraspanin transcript levels respond to NF treatment or bacterial inoculation
depending of the developmental stage, i.e at 5 dai, PvTET1A increases while PvTET3 decreases.
To confirm that P. vulgaris roots respond to rhizobia inoculation, we assessed the transcript
accumulation of PvENOD40, an early nodulin gene induced during nodule development.
PvENOD40 is clearly induced after rhizobia inoculation, indicating that the nodule program is
induced under our experimental conditions (Fig 3C).
We then further analyzed the transcript accumulation of PvTET4 and PvTET8 in roots
treated with NFs and rhizobia. PvTET4 was upregulated after R. tropici inoculation (Fig 3D),
whereas PvTET8 did not increase in response to NFs within the first 6 days, but it was
specifically induced in response to rhizobia inoculation after 7 days (Fig 3E). Therefore, we selected
PvTET8 to examine whether transcript accumulation was correlated with promoter activity.
We cloned the promoter and generated the pPvTET8::GUS-GFP construct, which revealed that
pPvTET8 was induced during nodule primordium development as well as in the meristematic
region of the apical root, including the lateral root primordium (Fig 3F and 3G). We also
observed clear promoter activity during the emergence of lateral root primordium, which
originates from the pericycle (Fig 4B?4E), and this promoter activity remained in the root
primordium (Fig 4F). Whereas the nodule primordium arises in the outer cells of the cortex (Figs 3G
and 5D), the lateral root originates from the internal pericycle cells (Fig 3F). We examined
promoter activity by assessing GUS activity (Fig 4B?4E) and fluorescence from the pPvTET8::
GUS-GFP fusion. Both approaches yielded the same results (Fig 4G) including the pPvTET10::
GUS-GFP (Fig 4I).
Next, we assessed PvTET8 promoter activity during nodule development, from infection
thread formation to fully grown nodules, and we found that the pPvTET8::GUS-GFP promoter
is highly active during the early stages of nodule development, which includes infection thread
formation in the root hairs (Fig 5A and 5B). Thereafter, when the cortical cells started to
divide, forming cells that will give rise to the nodule primordium, clear promoter activity was
observed in the outer cells that form the primordium (Fig 5C?5E). The fully developed nodule
also depicts a clear and specific promoter activity in the infected zone of the nodule (Fig 5E).
We also evaluated the promotor activity for pPvTET1A::GUS-GFP (Fig 5F and 5G) and
pPvTET3::GUS-GFP (Fig 5H) the two most highly expressed gene in P. vulgaris root. Although
these genes do not seem to have a strong increase during nodulation, when the promotor
activity was assayed, there is a clear evidence that apart from the vascular bundle, the
expression in the nodule is very clear.
Since pPvTET8 was specifically induced in response to rhizobia inoculation, we analyzed
the promoter sequence from PvTET8 up to 1.0 kb upstream of the translation start site of
PvTET8 using PlantCARE (database) to identify putative cis-acting regulatory elements. In
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Fig 3. Expression of PvTET1A, PvTET3, PvTET4, and PvTET8 in P. vulgaris during nodule development. Transcript accumulation is observed during
nodule development under rhizobia colonization. (A) PvTET1A, (B) PvTET3 and (C) PvENOD40 transcript accumulation after Nod factor (NF) treatment or
inoculation with R. Tropici CIAT 899 are depicted. PvTET4 (D) and PvTET8 (E) specifically respond to rhizobia inoculation. These results are compared with
uninoculated roots harvested at the same time (green bars). Expression values were normalized with those of EF1a. Bars represent means ? SEM of at least three
independent biological replicates with three technical repeats. P-values <0.05 are marked with two asterisks (Student?s t-test). Promotor activity of PvTET8 in
lateral root primordia (F) and nodule primordia development (G).
this analysis, we identified at least 14 different regulatory elements with a length of between 4
and 11 bp. Some cis-elements are represented in Fig 4A. The cis-regulatory elements TATA
and CAAT were highly repeated in the promoter sequences. In addition, several putative
ciselements involved in the light, heat, and drought stress response, circadian control, defense,
and anaerobic induction were present, as was one box associated with the YABBY
transcription factor. Thus, cis-elements associated with defense and stress responsiveness and cell fate
were well represented in this promoter region, as represented in Fig 4A.
Since pPvTET3::GUS-GFP was highly expressed in the root (Fig 5H), we prompted to
explore if there is promotor activity during different stages of nodule development. We found
that pPvTET3::GUS-GFP is expressed in the root meristematic region and vascular bundles
(Fig 6A and 6B), including a low promotor activity during the early cell divisions in the nodule
primordium (Fig 6C and 6D) and during the early nodule development (Fig 6E), again with
higher activity in the vascular bundles, however, a clear localization in the nodule was found,
although at latter stages of development in fully developed nodules (Fig 6F and 6G).
Furthermore, the pPvTET3 seems to be more localized in the cortex of the nodule, as compared to the
pPvTET8, which seems more localized in the central region (Fig 5E) and most related to the
infected region. These results suggest that both, pPvTET8 and pPvTET3 could be differentially
expressed in the same organ, but in different region.
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Fig 4. PvTET8 transcript accumulation and promotor activity during lateral root and nodule primordia
development. (A) Analysis of putative cis-regulatory elements in the promoter region of PvTET8. (B-F), pPvTET8::
GUS-GFP promotor activity in P. vulgaris root during lateral root emergence. (G and H) show the promoter activity
during the onset of lateral root development and the meristematic region of the emerging lateral root as depicted by
fluorescence. (I), Subcellular localization of 35S:PvTET10-GFP during lateral root formation. Transgenic composite
plants from P. vulgaris were generated by the A. rhizogenes method.
Mycorrhizal association downregulates PvTET3, while phosphate scarcity
upregulates PvTET12 and downregulates PvTET3
Since the nodulation process recruited many genes from the mycorrhizal association, we also
explored if the tetraspanin genes could be modulated in response and mycorrhizal association
and low phosphate. Therefore, we determined the effect of mycorrhizal interaction with P.
vulgaris on the accumulation of different TET transcripts. We inoculated P. vulgaris seedlings
with R. irregularis and included two controls, one in which plants were uninoculated and
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Fig 5. Promotor activity of PvTET8 during nodule development and subcelular localization of PvTET6 in P.
vulgaris during the infection process with rhizobia. (A-C) pPvTET8::GUS-GFP promotor activity at the early stages
of infection thread formation in root hairs. (C and D) Promotor activity of pPvTET8::GUS-GFP during the early stages
of cell division during primordia development and (E) in fully developed mature nodule, as depicted the promotor is
highly expressed in the infection zone of the nodule. (F-G) Promotor activity for pPvTET1A::GUS-GFP and (H)
promotor activity for pPvTET3::GUS-GFP. Transgenic composite plants were generated with A. rhizogenes and
promotor expression analyzed by GUS activity. Bars represent 20 ?m in all images.
irrigated with a standard concentration of phosphate (500 ?M) and another in which plants
were uninoculated and irrigated with medium deficient in phosphate (phosphate scarcity at
50 ?M), which is expected to induce the plant root response to phosphate scarcity, such as
lateral root formation and root hair proliferation. Both controls were included to show the
specific effect of mycorrhization on different tetraspanin transcript accumulation as a result of
root hair proliferation and lateral root formation. While PvTET8, PvTET4, and PvTET3
expression was modulated during nodule formation (Fig 3A and 3B and Fig 4A), inoculation
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Fig 6. Promotor activity of PvTET3 in the root and during nodule development in P. vulgaris during symbiotic conditions. (A and B) pPvTET3::GUS-GFP
promotor activity in the apical root and vascular bundles. (C and D) PvTET3 promotor activity at the early stages of nodule primordium formation. (E- G) Promotor
activity during the nodule development. Transgenic composite plants were generated with A. rhizogenes and promotor expression analyzed by GUS activity.
with R. irregularis only affected PvTET3 expression during the mycorrhizal association (see Fig
7A). As expected, the expression of the phosphate transporter PT4 which is induced during the
mycorrhizal association, was found to be induced under our experimental condition (Fig 7C).
Fig 7. Tetraspanin transcript accumulation profile in P. vulgaris under R. irregularis colonization. (A) Tetraspanin transcript accumulation in P. vulgaris roots
colonized with R. irregularis in the symbiotic stage at 6 wpi as compared with uninoculated plants under phosphate scarcity with potassium phosphate at 50 ?M.
Transcript accumulation was normalized to the expression of Ef1a, which was used as a reference gene. (B) Expression of tetraspanin PvTET12 in P. vulgaris roots
during abiotic stress induced by NaCl conditions at 100 mM at 24 hpi. (C) Phosphate transporter PT4 transcript accumulation under mycorrhizal condition. Data
are the means ? SEM of two biological experiments (three roots collected from each biological experiment and for each period were used).
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Fig 8. PvTET3 and PvRbohB expression during mycorrhiza formation. (A) Quantitative RT-PCR analysis of relative expression levels of PvTET3 in roots
(wild type) inoculated with R. irregularis and under un-inoculated condition at the indicated number of weeks post-inoculation (wpi). Transcript
accumulation was normalized to the expression of Ef1a, which was used as a reference gene. Data are the means ? SEM of two biological experiments (three
roots collected from each biological experiment and for each period were used). (B) Expression of PvRbohB in P. vulgaris roots colonized by R. irregularis.
Quantitative RT-PCR analysis of relative expression levels of PvRbohB in roots (wild type) inoculated with R. irregularis compared with expression in
uninoculated roots of P. vulgaris at different weeks post-inoculation (wpi). Transcript accumulation was normalized to the expression of Ef1a, as a reference
gene. The data are the means ? SEM of two biological experiments (three roots collected from each biological experiment and for each period).
A temporal analysis of PvTET3 transcript accumulation under mycorrhization confirmed
that PvTET3 transcript levels were lower at 2, 3, and 6 weeks after mycorrhization compared
with the control (Fig 8A). This suggests that PvTET3 was downregulated during
mycorrhization (Fig 7A and Fig 8A). It has been reported that transcript accumulation of PvRbOHB
decreases during mycorrhizal association [
]. Therefore, we evaluated the accumulation of
PvRbOHB transcript at 1, 2, 3, and 6 weeks post inoculation (wpi) to define its temporal
response and correlate it with that of PvTET3 (Fig 8B). PvRbOHB was not induced during the
first week post inoculation, during which its expression was comparable to that of the control
condition (Fig 8B). However, after 2?3 weeks, a clear increase in PvRbOHB transcript
accumulation was observed, but a decreased PvTET3 expression was found. However, at 6 weeks, a
clear decrease in both PvRbOHB and PvTET3 was observed, and thus confirming the
previously reported data for PvRbOHB under mycorrhizal association [
We found no significant differences at the transcriptional level for the other tetraspanins
under our experimental conditions (S7 Fig). However, we identified a clear PvTET12
upregulation under phosphate scarcity (Fig 7A). It is interesting that under normal growth
conditions, we did not observe significant PvTET12 expression in root hairs at different
developmental stages or even in the shaved roots (Fig 2A and 2B), even though its homolog,
AtTET12, has been identified as a transcriptional signature in root hairs and pollen tubes [
To determine if another different stress, such as osmotic or saline treatment, could modify the
PvTET12 transcript level, we treated the P. vulgaris root with 100 mM NaCl; which is known
to induce a saline response [
]. Under saline stress conditions, we found that PvTET12
expression was also upregulated (Fig 7B).
P. vulgaris tetraspanins localize to the apical plasma membrane,
intracellular vesicles, and meristematic regions
Tetraspanin proteins in general have been described to be plasma membrane proteins and
component of cytoplasmic vesicles. However, TET3 in Arabidopsis has been found in
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Fig 9. Subcellular localization of PvTET6 and PvTET3 in Nicotiana benthamiana leaves and growing root hairs of P. vulgaris. (A) Confocal analysis of GFP
expression in the leaves of transgenic N. benthamiana plants. (B) 35S:PvTET3-GFP. (C) 35S:PvTET6-GFP. (D) 35S:PvTET6-GFP (close-up of region indicated in
C). (E-H) 35S:PvTET6-GFP subcellular localization in transgenic living P. vulgaris root hairs. Images in bright field (E), Merge (G), GFP signal (F), Z-projection
(H). Bars = 20 ?m.
proteomic analysis of plasmodesmata. Therefore, we determined the subcellular localization of
PvTET3 and PvTET6 by generating GFP fusions of their encoding genes and transiently
expressing these constructs in N. benthamiana leaves (Fig 9A?9D). An analysis of leaves
expressing the 35S:PvTET3-GFP and 35S:PvTET6-GFP constructs under the control of a
constitutive promoter (CaMV35S) showed that these proteins examined in this study accumulated
at the periphery of N. benthamiana epidermal cells, indicating a membrane localization (Fig
9B and S8 Fig), and clearly differentiated from the cytoplasmic localization of GFP in control
agroinfiltrated cells (Fig 9A). Furthermore, some of the tetraspanins localized to fluorescent
spots in the plasma membrane, suggesting plasmodesmata localization (Fig 9C, inset). We also
generated transgenic P. vulgaris composite plants expressing 35S:PvTET6-GFP using A.
rhizogenes and found that some tetraspanins are expressed in vesicular structures that are swept
along by cytoplasmic flux in growing root hairs, but are also localized in the apical plasma
membrane of these cells (Fig 9E?9H, S1 Movie and S2 Movie). In order to determine the
plasma membrane localization, we generated agroinfiltrated N. benthamiana epidermal cells
expressing the tetraspanin and subjected to NaCl treatment for plasmolysis. These results
suggest that the signal remains associated to the plasma membrane (S8 Fig).
We then evaluated 35S:PvTET10-GFP expression in composite plants. TET10 exhibited
similar localization to TET6, namely, apical localization in the plasma membrane and dynamic
vesicles in the cytoplasm of growing root hair cells in a pattern that sometimes follows the
cytoplasmic streaming with higher ambulation at the tip dome (Fig 10A?10C). Furthermore,
in P. vulgaris composite plants expressing 35S:PvTET10-GFP under nodulation conditions,
the cortical cells that enter the division process that will generate the nodule primordium are
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Fig 10. Subcellular localization of PVTET10 in growing root hairs from P. vulgaris. (A, B and C) Apical membrane
localization of 35S:PvTET10-GFP at different developmental stages of the growing root hair. (D, E and F) Cytoplasmic
vesicle localization for PvTET10, and its accumulation in the infection site where the infection thread and nodule
primordia are induced. P. vulgaris plants were transformed by A. rhizogenes in order to generate the composite plants.
Bacterial colonization is in red and the subcellular localization of PvTET10 is in green.
enriched in these cytoplasmic vesicles (Fig 10D?10F). Since TET3, TET6, and TET10 localized
to moving vesicles, we further examined the dynamics and behavior of these vesicles.
We selected PvTET3, which we know localizes to vesicular structures in N. benthamiana
(Fig 11A and 11B) and P. vulgaris composite plants (Fig 11C?11J), to determine the dynamics
and shapes of these vesicles. In P. vulgaris epidermal cells, these vesicles were tracked in a time
lapse of 1.6 seconds for 3 min in transgenic roots overexpressing 35S:PvTET3-GFP. We
observed fusion events that resulted in larger vesicles, with amorphous shapes that resemble
protrusions (Fig 11C?11J).
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Fig 11. Subcellular localization of PvTET3 in N. benthamiana leaves and P. vulgaris roots. (A and B) 35S:PvTET3-GFP localization in cytoplasmic vesicles in
agroinfiltrated N. benthamiana leaves. (C-J) Subcellular localization of 35S:PvTET3:GFP in P. vulgaris composite epidermal root cells. Cytoplasmic vesicles were
tracked over time in order to show the fusion and morphological changes resembling protrusions during the cytoplasmic streaming. Vesicles were tracked in a
time lapse of 1.6 seconds for 3 min in transgenic roots expressing 35S:PvTET3-GFP.
We found that PvTET1A, PvTET3, PvTET4 and PvTET8 are induced and differentially
expressed during early nodule development, as depicted by transcript accumulation and
promoter activity and in the fully grown and mature nodule, but with different timing and
strength. Furthermore, these expression patterns resemble those found in primordia during
lateral root formation which also requires a new program that involves cell division and ROS
production. The finding that some tetraspanin presents a clear membrane localization at the
tip of the root hairs and some others in what appears to be associated with plasmodesmata
(PD), and cytoplasmic vesicles, suggest a role in symplastic communication trough regulation
of PD or in cell trafficking as described in animal cells [
We identified the tetraspanin family in P. vulgaris and found PvTET10 to be a conserved
tetraspanin with at least one representative in each legume species examined, except for
soybean, which contained more members due to a genome duplication event [
clusters with AtTET10 and OsTET14, which contain similar structural features of introns/exons,
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suggesting that PvTET10 is the ancestral tetraspanin gene, as is AtTET10 in the Brassicaceae
and OsTET14 in O. sativa [
]. These structural intron/exon patterns indicate a similar
origin as suggested for deuterostomes, in which tetraspanins are the product of a divergent
evolutionary process and originated from at least one common ancestor [
]. In our unrooted
phylogenetic tree, PvTET1A and PvTET1B grouped with AtTET1 (TORNADO) and AtTET2,
while PvTET8, OsTET7-9, and AtTET8-9 clustered together (S2 Fig and S3 Fig).
We found that PvTET3 and PvTET1A transcripts are more abundant and ubiquitously
expressed during root and root hair development (Fig 2A and 2B). In angiosperms plants such
as Arabidopsis and P. vulgaris, primary root formation takes place during embryogenesis,
whereas lateral roots form post-embryonically from pericycle founder cells, and both of these
processes are mediated by changes in hormone levels [
]. PvTET1A is expressed
postembryonically, between 2 and 4 dpg, in the seedling when root growth begins and before
lateral roots emerge . According with our phylogenetic data, PvTET1A is the homolog of
TORNADO in Arabidopsis (AtTET1 or AtTRN1), which regulates cell specification in the root
epidermis during radial pattern formation and thus in agreement with our transcriptional
profile for PvTET1A with a high expression in root . However, our transcriptional profile does
not correspond with the reported higher TRN1 promoter activity in the meristematic zone in
the root tip. This discrepancy with higher expression in root zone II and III and lower in zone
I that correspond to the meristematic region could be due to the fact that the promoter region
of PvTET1A does not contain a putative cis-regulatory element, GCCACT, that exists in
Arabidopsis TRN1, and associated with meristematic expression and auxin dependency [
Therefore, PvTET1A could have a different regulation, or its expression pattern could differ
from that reported in Arabidopsis.
The finding that PvTET3 transcript accumulation was downregulated under nodulation
and mycorrhizal conditions in P. vulgaris roots and that the encoded protein localizes to the
PM and PD, suggests that PD could be a regulated structure during the mutualistic
interactions. We also found that PvTET6 is specifically targeted to the PM and PD, in agreement with
the subcellular localization of some homologous tetraspanins identified in Arabidopsis, such as
AtTET3 and AtTET5 [
]. PD are connections between cells that mediate symplastic
communication from single cells to tissue domains, these structures have essential roles in
cell-to-cell communication . Both the density and aperture size of PD are developmentally
regulated through the deposition or solubilization of callose by callose synthases or glucanases,
allowing the formation of spatial symplastic domains that establish tissue-specific
developmental programs [
]. Numerous non-cell-autonomous proteins (NCAPs) and small RNAs travel
through the PD and play crucial roles in cell fate determination and organ patterning during
plant development. In vascular plants, it has been suggested that PD networks are associated
with shoot apical meristem (SAM) organization in Arabidopsis and maize (Zea mays) [
Typical PD proteins, such as AtPDLP1, have a specific signal peptide, LVL, located in a
transmembrane domain [
], which was also found in PvTET6 and PvTET3. Transmembrane
domains in tetraspanin have turned to play important roles. For instance, a single point
mutation, L31S substitution in the third amino acid of TM1 has been associated with field-evolved
resistance of cotton bollworm to transgenic Bt cotton [
In rice, blast disease caused by the hemibiotrophic fungus Magnaporthe oryzae grow from
one cell to the next through PD. This response is coordinated by chitin perception that
requires a receptor kinase (CERK1) and a chitin elicitor binding protein (CEBiP), which sense
the chitin and induce a reduction in the cell-to-cell connectivity via PD [
]. During the
nodulation process it has been recently described that PD connection between the
phloemearly primordium-epidermal cell forming the infection thread is a key step for the infection
]. Again, it seems that PD are key components of the photogenic and
19 / 31
mutualistic response and PD localized tetraspanins could be important modulators of the
intercellular flux regulating the symplastic continuity and molecular flux between cells. It
could involve ROS to regulate PD permeability by facilitating the cross-linking process of
]. Therefore, the observed downregulation or upregulation of PvTET3 could affect PD
composition by modulating the capability of tetraspanin to recruit some additional molecular
components required to modify the callose composition in PD during mycorrhizal or rhizobia
association. This may also facilitate the hypha or infection thread to move cell-to-cell through
the PD connections or facilitate the diffusion of some proteins, such as transcription factors or
metabolites, required to fine-tune the regulation of cortical and cortex responses.
Nodulation involves substantial crosstalk between NFs and auxin signaling in Medicago
truncatula, with a high accumulation of auxin at the site of nodule meristem formation [
Indeed, there is a large overlap between genes induced in response to NFs and auxin, including
two tetraspanin genes with homology to PvTET3 (Medtr4g061010) and PvTET1A
]. In addition, a previous report showed that PIN1 expression was
reduced in the Arabidopsis trn2-1 mutant, which has compromised auxin transport activity
during the transition from floral meristem termination to gynoecium development, which
suggests a link between tetraspanin and auxin homeostasis [
]. Therefore, in addition to
altering auxin transport, Nod factors could modulate the expression of specific tetraspanins that
influence hormone levels, which could regulate cell division, a well-described process during
]. If PvTET1A and PvTET3 are downregulated during the interaction with NFs,
this response could be associated with the disruption of auxin transport or the stimulation of
auxin biosynthesis [
]. We suggest that NFs signaling has a profound impact on PvTET1A
and PvTET3 expression by affecting auxin levels and thereby coordinates nodule primordium
development. Indeed, it is well known that NFs interfere with auxin transport, biosynthesis,
and homeostasis [
The finding that PvTET4 and PvTET8 are induced during early nodule development with
different timing and localization, as depicted by transcript accumulation and promoter activity
and in the fully grown and mature nodule. Our results indicate that PvTET8 could be involved
at different developmental stages of nodule primordia formation and in the infected thread
region, and PvTET3 although we did not find a high transcript accumulation, the promotor
activity indicates that is expressed in root and mature nodule, suggesting a role in the mature
nodule or later on during senescence. Furthermore, these expression patterns resemble those
found during lateral root formation. Both lateral root primordium and nodule primordium
formation require differentiated cells to become dedifferentiated and then to enter a new
program that involves cell division. Therefore, this tetraspanin could be involved in the molecular
mechanism that maintains meristem activity.
Furthermore, the roles of tetraspanin have been expanded beyond intercellular boundaries.
It has been reported recently that Arabidopsis cells secrete exosome-like extracellular vesicles
that are derived from the multivesicular bodies (MVBs), which contain cytoplasmic material,
such as proteins, miRNA, and lipids [
]. Under certain circumstances, these MVBs fuse to
the plasma membrane and release the internal vesicles into the extracellular space. It has been
demonstrated recently that tetraspanins are key components and specific markers for animal
(CD63) and plant exosomes (AtTET8 and AtTET9) . In Arabidopsis, exosomes transport
key miRNAs that induce silencing of fungal genes critical for pathogenicity in Botrytis cinerea,
defining a role for tetraspanin and exosome biogenesis in intercellular and inter-kingdom
]. This could explain why AtTET8 expression is upregulated upon
treatment with pathogen elicitors  and why, during our experimental conditions, PvTET8 is
induced when plants are infected with rhizobia. It is possible that exosomes could play an
important role during the mutualistic interaction. Furthermore, tetraspanin genes are among
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the induced genes in the reported legume transcriptome of wild peanut (Arachis spp) roots
infected with nematodes, suggesting that the pathogenic response in legumes also involves
The upregulation of PvTET8 during the defense response also can be explained by the
enrichment of defense response cis-regulatory elements in its promoter region. Indeed, it has
been widely suggested that the molecular mechanism underlying mutualism was derived from
that underlying pathogenic interactions, but suppression of the immune response allows the
symbiont to colonize the plant host [
]. It will be interesting to determine the specific role of
PvTET8 during nodulation and mycorrhization and explore the role that vesicles and
exosomes could play in recruiting proteins or membrane-associated proteins that are required in
the plasma membrane during mutualistic interactions.
Our data showing that PvTET12 is upregulated under phosphate scarcity, but not under
mycorrhizal association, suggest that PvTET12 expression is highly dependent on
environmental nutrition conditions. In Arabidopsis, AtTET12 has been identified in root hairs and in
the pollen tube [
]. Furthermore, the microarray data of Glycine max reported on the
Soybean Efb Browser website shows a differential expression of GmTET12 in roots and root hairs
at 24 h after inoculation with bacteria [
35, 57, 93
]. These data suggest a role for PvTET12 in the
massive proliferation of root hairs as a response to phosphate acquisition [
In animal cells, tetraspanins have multiple antagonistic effects. For example, the tetraspanin
CD82 is downregulated during metastases, while the tetraspanin CD151 and tetraspanin 8 are
induced and able to support tumor progression [
]. It has been reported that the CD63
tetraspanin in animal cells recruits a H+-ATPase beta-subunit in parietal cells that affects its trafficking.
Also, saline stress has been used to induce changes in the H+-ATPase localization mediated by
]. Here, we also found that NaCl could upregulate PvTET12. Furthermore, in
Arabidopsis, a mutant of one isoform of PM H+-ATPase, AHA7, exhibited reduced root hair density
and lower H+ density efflux in the root hair zone, while the transcript was upregulated under
lowphosphate conditions [
]. Since phosphate scarcity affects PvTET12 expression, it could affect
the recruitment of protein related to the change in H+ efflux, and it is tempting to think that
tetraspanins are somehow related to the regulation or trafficking of H+-ATPase. Furthermore, a PT4/
PT11 H+-ATPase has been found to be important for arbuscule maintenance and AM-mediated
phosphate uptake [
]. Plants have developed several strategies to increase phosphate
acquisition, including changes to root architecture and the formation of root hairs [
the finding that other TETs, such as tetraspanin-1, are also related to changes in root hair
regulation suggests the existence of a more complex regulation [
There is an emerging association between tetraspanins and the mechanisms of ROS
]. In the nematode C. elegans, the exoskeleton, the cuticle composed of collagen, is
tyrosine cross-linked in a ROS-dependent manner, in a process assisted by BLI-3, a DUOX
NADPH oxidase [
]. This process also requires the participation of tetraspanin TSP-15,
which allows recruitment of the NADPH oxidase [
24, 25, 30
]. Inactivation of this tetraspanin
or BLI-3 produces similar phenotypes. Furthermore, during plant infection with the
pathogenic fungus M. grisea or B. cinerea, the tetraspanin PLS1 is required for infection site
formation. Therefore, the tetraspanin and ROS generated by a NADPH oxidase is needed to
coordinate ROS production at the infection site. In pathogenic C. lindemuthianum, a
tetraspanin is also required to reestablish appressorium polarity [
24, 25, 31?33
]. Furthermore, in
Claviceps purpurea, Nox2 and Pls1 are important for a balanced host?pathogen interaction, while in
HeLa cells, the cotransfection of tetraspanin CD82 and GTPase Cdc42 induces apoptosis by
generating ROS [
]. These data add weight to the strong connection between
tetraspanins and the ROS-generating machinery, both in animal and plant cells, to coordinate
localized ROS production [
24, 25, 30, 105
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During the mycorrhizal association of P. vulgaris with R. irregularis, only PvTET3 was
downregulated. This response coincides with previous reports in which a decrease in ROS
production induced by RNAi of PvRbOHB enhanced colonization by R. irregularis [
]. Thus, a
coordinated decrease in ROS production and PvTET3 expression could be required for
mycorrhizal colonization, as previously suggested [
56, 78, 79
This spatial ROS requirement is also observed during the Casparian strip lignification that
occurs in plants, where CASP, a protein recruiting the NADPH oxidase, plays a role similar to
tetraspanin, bringing together NADPH oxidase and peroxidase and ensuring localized
activation of the oxidase [
]. Tetraspanins have also been localized to the tip of growing pollen
tubes and root hairs (this work), and it is well known that the tip regions require NADPH
oxidase activity for the generation of ROS, key players in the regulation of polar growth [
The subcellular localization of some tetraspanins at the apical plasma membrane suggests that
tetraspanins may play a key role during polar growth. Furthermore, the particular expression
of PvTET8 in root hairs in which an infection thread forms during early cell division in cortical
cells could be related to the NADPH-oxidase-mediated ROS generation that is required for
meristematic activity. Therefore, it is tempting to speculate that tetraspanins also play a role in
recruiting the ROS-generating machinery to a specific cellular location. Moreover, tetraspanin
also accumulates at the site of female gametophyte differentiation, a well-described
ROSdependent process .
Finally, the site-specific localization of PvTET8 in root and nodule meristematic regions
points to a role in meristem maintenance. The well-described role of O2.- accumulation in the
root apical meristem (RAM) and shoot apical meristem (SAM) in inhibiting the transition
from proliferation to differentiation suggests a key role for tetraspanins in undifferentiated
cells to inhibit differentiation in plant. Indeed, the balance of O2./H2O2 levels in
undifferentiated and differentiated cells is crucial for WUSCHEL (WUS) activation to promote stem cell
differentiation. For instance, ROS can activate WUS and thereby repress expression of the TF
YABBY, which regulates the tetraspanin TORNADO at the transcriptional level. Therefore, the
tetraspanin TORNADO could be involved in processes that regulate ROS production, both at
the temporal and spatial level. It is interesting that the WUS-RELATED HOMEOBOX (WOX)
family transcription factor WOX5 like PvTET8, PvTET3 (this work), are also highly expressed
during nodule organogenesis, suggesting that WOX genes are common regulators of cell
proliferation in different systems, such as the SAM, RAM, and nodule primordium, including in
36, 40, 86, 107
Calcium and ROS are important cellular messengers and key players during mutualistic
]. ROS, and therefore the enzymes that generate ROS (e.g., NADPH
oxidases), play a key role in root hair tip growth, both in the presence and absence of pathogenic
or mutualistic interactions, and in the growth of other tip-growing cells such as pollen tubes
. The observation that FNs from rhizobia have a different effect on ROS accumulation
than do pathogenic signals, such as elicitors, suggests that plant cells differentiate symbiotic
from pathogenic signals [
6, 14, 109
]. We previously reported that P. vulgaris produces 9
NADPH oxidases (Rboh), some of which are mainly expressed in roots, root hairs, or nodules
]. The overexpression of one, RbohB, results in increased nodulation, but with a reduced
mycorrhizal association [
], suggesting that ROS are important players in mutualistic
42, 43, 56
]. We suggest that the co-occurrence of tetraspanin and NADPH oxidase in
the apical root hair cells or the early infection thread, and nodule primordia, could be related
to the ROS-generating machinery. The role of tetraspanins in regulating the plasma membrane
by recruiting the required proteins to specific membrane microdomains enriched in
tetraspanins (tetraspanin web) or by affecting its vesicular trafficking have been well described. It is
important to bear in mind that the release of the rhizobia from the infection thread or the
22 / 31
arbuscule formation requires a complete coordination with the secretory system from the host
plant cells for symbiont accommodation. This involves a high rate of exocytosis of specific
components required for cell wall remodeling, including the extra membrane required to form
the peribacteroideal membrane. In this scenery, tetraspanins could also play a central role
organizing those membrane domains, but also facilitating the required vesicular trafficking to
specific places as those reported for photogenic interactions that involves the specific exosomes
S1 Fig. Oligonucleotides used in this study.
S2 Fig. Phylogenetic tree of TET family proteins from Arabidopsis and P. vulgaris. The
phylogenetic tree was generated from the alignment of tetraspanin proteins with n = 1000
bootstrap replicates. The TET proteins were classified into clades based on phylogenetic
analysis using the neighbor-joining (NJ) method. We used as query all tetraspanins reported by
Boavida et al., 2013.
S3 Fig. An unrooted neighbor-joining three phylogenetic tree was constructed based on
the amino acid sequences alignment of some legumes tetraspanins using the
neighborjoining method (NJ)(Saitou and Nei, 1987, Takata et al., 2013). We selected amino acid
sequences from Medicago truncatula, Phaseolus vulgaris, Glycine max, and Lotus japonicus. In
this phylogenetic tree we schematize seven groups formed with legumes tetraspanin and are
represented by different color branch. We selected a bootstrapping method to build the
phylogenetic tree with 1000 replicates using MEGA Version 6.0.6 (Tamura et al., 2013)
S4 Fig. Rab2A transcript levels during root hair development. Transcript levels were
quantified by reverse transcription and real-time PCR (RT-qPCR) and calculated using the
expression levels of Elongation Factor 1? as reference. Measures were performed in each enriched
tissues and different zones in root of common bean at 48 hpg. The number of biological
replicates (n = 3) is indicated. Error bars indicate mean and SEM (?SEM).
S5 Fig. Expression level reported in transcriptomic atlas of common bean. FY- Young
flowers, collected prior to floral emergence; LF- Leaf tissue from fertilized plants collected at the
same time of LE and LI; L5- Leaf tissue collected 5 days after plants were inoculated with
effective rhizobium; LE- Leaf tissue collected 21 days after plants were inoculated with effective
rhizobium; LI- Leaf tissue collected 21 days after plants were inoculated with ineffective
rhizobium; N5- Pre-fixing (effective) nodules collected 5 days after inoculation; NE-
Effectively fixing nodules collected 21 days after inoculation; NI- Ineffectively fixing nodules
collected 21 days after inoculation; P1- Pods between 10 and 11 cm long, associated with stage 1
seeds (pod only); P2- Pods between 12 and 13 cm long associated with stage 2 seeds (pod
only); PH- Pods approximately 9cm long, associated with seeds at heart stage (pod only);
PYYoung pods, collected 1 to 4 days after floral senescence. Samples contain developing embryos
at globular stage; R- Whole roots from fertilized plants collected at the same time as RE and
RI; R5- Whole roots separated from 5 day old pre-fixing nodules; RE- Whole roots separated
from fix+ nodules collected 21 days after inoculation; RI- Whole roots separated from
fixnodules collected 21 days after inoculation; RT- Root tips, 0.5 cm of tissue, collected from
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fertilized plants at 2nd trifoliate stage of development.; S1- Stage 1 seeds, between 6 and 7 mm
across and approximately 50 mg; S2- Stage 2 seeds, between 8 and 10 mm across and between
140 and 150 mg; SH- Heart stage seeds, between 3 and 4 mm across and approximately 7 mg;
ST- Shoot tip, including the apical meristem, collected at the 2nd trifoliate stage; YL- Fully
expanded 2nd trifoliate leaf tissue from plants provided with fertilizer; YR- Whole roots,
including root tips, collected at the 2nd trifoliate stage of development; YS- All stem
internodes above the cotyledon collected at the 2nd trifoliate stage. Common bean atlas source
S6 Fig. Common bean root responses to NFs treatment. (A) Representative image of root
hairs of roots of common bean under control condition treated with chitosan 10?9 M and (B)
root hair subjected to a treatment with 10?9 M of NFs for 4 h (Scale = 100 ?m).
S7 Fig. Expression of the other of tetraspanin members in P. vulgaris during nodule
formation. Different transcript abundance under two treatments: incubated with Nod Factor or
inoculated with R. Tropici CIAT 899 (separated with a dotted line). The experiment included
uninoculated roots harvested as control at the same time (green bars). Expression values were
normalized with EF1a. Bars represent means ? SEM from at least three independent biological
replicates with three technical repeats. P-values <0.05 are marked with two asterisks,
respectively (Student?s t-test).
S8 Fig. Subcellular localization of 35S:PvTET3-GFP, 35S:PvTET6-GFP after plasmolysis.
Agroinfiltrated cells from N. benthamiana leaves under plasmolysis induced by NaCl. A, B
and C, 35S:GFP, 35S:PvTET3-GFP, 35S:PvTET6-GFP respectively, showing the regular
cytoplasmic protein localization under control condition (left panel) and under plasmolysis (right
small panels). Arrows in B and C indicates the 35S:PvTET3-GFP and 35S:PvTET6-GFP
fluorescence associated with the retracted plasma membrane, while the 35S:GFP remains in the
cytoplasm in A.
S1 Movie. Composite plants from P. vulgaris expressing the fusion protein 35S:
PvTET6-GFP. Images were acquired with a spinning disk confocal system (Intelligent
Imaging Innovations /3i, USA) consisting of a CSU-W1 confocal head (Yokogawa, Japan) and a
modular solid-state laser stack; Slidebook software was used to control the system and capture
images (Intelligent Imaging Innovations /3i, USA). Movie represent 40 images taken 15
seconds apart. Note the apical membrane localization and the cytoplasmic localization in
vesicular structures that follows the cytoplasmic streaming.
S2 Movie. Composite plants from P. vulgaris expressing the fusion protein 35S:
PvTET6-GFP in epidermal cells. Images were acquired with a spinning disk confocal system
(Intelligent Imaging Innovations /3i, USA) consisting of a CSU-W1 confocal head (Yokogawa,
Japan) and a modular solid-state laser stack; Slidebook software was used to control the system
and capture images (Intelligent Imaging Innovations /3i, USA). Movie represent 40 images
taken 15 seconds apart. Note the apical membrane localization and the cytoplasmic
localization in vesicular structures that follows the cytoplasmic streaming.
24 / 31
We acknowledge the technical support from Noreide Nava, Juan E. Olivares Grajales, Rosana
Sa?nchez, Georgina Estrada, the facility for Oligonucleotides synthesis, and the National
Advanced Microscopy Laboratory (LNMA).
Conceptualization: Luis Ca?rdenas.
Funding acquisition: Kazuyuki Kuchitsu, Jesu?s Aguirre, Carmen Quinto, Luis Ca?rdenas.
Investigation: Saul Jimenez-Jimenez, Olivia Santana, Fernando Lara-Rojas, Manoj-Kumar
Arthikala, Elisabeth Armada, Kenji Hashimoto, Sandra Salgado, Luis Ca?rdenas.
Methodology: Saul Jimenez-Jimenez, Kenji Hashimoto, Kazuyuki Kuchitsu, Jesu?s Aguirre,
Supervision: Luis Ca?rdenas.
Writing ? original draft: Saul Jimenez-Jimenez, Luis Ca?rdenas.
Writing ? review & editing: Kenji Hashimoto, Kazuyuki Kuchitsu.
25 / 31
26 / 31
grisea. Proceedings of the National Academy of Sciences of the United States of America. 2001; 98
(12):6963?8. https://doi.org/10.1073/pnas.111132998 PMID: 11391010; Central PMCID: PMC34461.
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1. Fisher RF , Long SR . Rhizobium-plant signal exchange . Nature . 1992 ; 357 ( 6380 ): 655 - 60 . https://doi. org/10.1038/357655a0 PMID: 1614514 .
2. Oldroyd GE . Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants . Nature reviews Microbiology . 2013 ; 11 ( 4 ): 252 - 63 . https://doi.org/10.1038/nrmicro2990 PMID: 23493145 .
3. Ferguson BJ , Mens C , Hastwell AH , Zhang M , Su H , Jones CH , et al. Legume nodulation: The host controls the party . Plant , cell & environment. 2019 ; 42 ( 1 ): 41 - 51 . https://doi.org/10.1111/pce.13348 PMID: 29808564 .
4. Zepeda I , Sanchez-Lopez R , Kunkel JG , Banuelos LA , Hernandez-Barrera A , Sanchez F , et al. Visualization of highly dynamic F-actin plus ends in growing phaseolus vulgaris root hair cells and their responses to Rhizobium etli nod factors . Plant & cell physiology . 2014 ; 55 ( 3 ): 580 - 92 . https://doi.org/ 10.1093/pcp/pct202 PMID: 24399235 .
5. Denarie J , Debelle F , Prome JC . Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis . Annual review of biochemistry . 1996 ; 65 : 503 - 35 . https://doi.org/10.1146/annurev.bi. 65 .070196.002443 PMID: 8811188 .
6. Ca?rdenas L , Quinto C . Reactive oxygen species (ROS) as early signals in root hair cells responding to rhizobial nodulation factors . Plant Signaling & Behavior . 2008 ; 3 : 1 - 3 .
7. Cardenas L , Holdaway-Clarke TL , Sanchez F , Quinto C , Feijo JA , Kunkel JG , et al. Ion changes in legume root hairs responding to Nod factors . Plant physiology . 2000 ; 123 ( 2 ): 443 - 52 . https://doi.org/ 10.1104/pp. 123 . 2 .443 PMID: 10859175; Central PMCID : PMC1539257 .
8. Ca?rdenas L , Feijo? JA, Kunkel JG , Sa?nchez F , Holdaway-Clarke T , Hepler PK , et al. Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs . Plant J . 1999 ; 19 ( 12657 ): 347 - 52 .
9. Shaw SL , Long SR . Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells . Plant physiology . 2003 ; 131 ( 3 ): 976 - 84 . https://doi.org/10.1104/pp.005546 PMID : 12644650; Central PMCID : PMC166863 .
10. Miller DD , de Ruijter NCA , Bisseling T , Emons AM . The role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug . Plant J . 1999 ; 17 ( 12336 ): 141 - 54 .
11. Yokota K , Fukai E , Madsen LH , Jurkiewicz A , Rueda P , Radutoiu S , et al. Rearrangement of Actin Cytoskeleton Mediates Invasion of Lotus japonicus Roots by Mesorhizobium loti . The Plant cell . 2009 ; 21 ( 1 ): 267 - 84 . https://doi.org/10.1105/tpc.108.063693 PMID: 19136645 .
12. Foreman J , Demidchik V , Bothwell JH , Mylona P , Miedema H , Torres MA , et al. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth . Nature . 2003 ; 422 ( 6930 ): 442 - 6 . https:// doi.org/10.1038/nature01485 PMID: 12660786 .
13. Takeda S , Gapper C , Kaya H , Bell E , Kuchitsu K , Dolan L. Local positive feedback regulation determines cell shape in root hair cells . Science . 2008 ; 319 ( 5867 ): 1241 - 4 . https://doi.org/10.1126/science. 1152505 PMID: 18309082 .
14. Cardenas L. New findings in the mechanisms regulating polar growth in root hair cells . Plant signaling & behavior . 2009 ; 4 ( 1 ): 4 - 8 . https://doi.org/10.4161/psb.4.1.7341 PMID: 19568333; Central PMCID : PMC2634060 .
15. Oldroyd GE , Murray JD , Poole PS , Downie JA . The rules of engagement in the legume-rhizobial symbiosis . Annual review of genetics . 2011 ; 45 : 119 - 44 . https://doi.org/10.1146/annurev-genet- 110410 - 132549 PMID: 21838550 .
16. Russo G , Carotenuto G , Fiorilli V , Volpe V , Chiapello M , Van Damme D , et al. Ectopic activation of cortical cell division during the accommodation of arbuscular mycorrhizal fungi . The New phytologist . 2019 ; 221 ( 2 ): 1036 - 48 . https://doi.org/10.1111/nph.15398 PMID: 30152051 .
17. Heck C , Kuhn H , Heidt S , Walter S , Rieger N , Requena N. Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription Factor MIG1 . Current biology: CB . 2016 ; 26 ( 20 ): 2770 - 8 . https://doi.org/10.1016/j.cub. 2016 . 07 .059 PMID: 27641773 .
18. Reimann R , Kost B , Dettmer J. TETRASPANINs in Plants . Frontiers in plant science. 2017 ; 8 : 545 . https://doi.org/10.3389/fpls. 2017 .00545 PMID: 28458676
19. Wang F , Vandepoele K , Van Lijsebettens M. Tetraspanin genes in plants. Plant science: an international journal of experimental plant biology . 2012 ; 190 : 9 - 15 . https://doi.org/10.1016/j.plantsci. 2012 . 03 .005 PMID: 22608515 .
20. Huang C , Fu C , Wren JD , Wang X , Zhang F , Zhang YH , et al. Tetraspanin-enriched microdomains regulate digitation junctions . Cellular and molecular life sciences: CMLS . 2018 . https://doi.org/10. 1007/s00018-018-2803-2 PMID: 29589089 .
21. Zo?ller M. Tetraspanins: push and pull in suppressing and promoting metastasis . Nature Reviews Cancer . 2009 ; 9 ( 1 ): 40 . https://doi.org/10.1038/nrc2543 PMID: 19078974
22. Couto N , Caja S , Maia J , Strano Moraes MC , Costa-Silva B . Exosomes as emerging players in cancer biology . Biochimie . 2018 . https://doi.org/10.1016/j.biochi. 2018 . 03 .006 PMID: 29555374 .
23. Cai Q , Qiao L , Wang M , He B , Lin F-M , Palmquist J , et al. Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes . Science . 2018 ; 360 ( 6393 ): 1126 - 9 . https://doi.org/ 10.1126/science.aar4142 PMID: 29773668
24. Moribe H , Mekada E. Co-occurrence of tetraspanin and ROS generators: Conservation in protein cross-linking and other developmental processes . Worm . 2013 ; 2 ( 2 ):e23415. https://doi.org/10.4161/ worm.23415 PMID: 24058871; Central PMCID : PMC3704445 .
25. Moribe H , Konakawa R , Koga D , Ushiki T , Nakamura K , Mekada E . Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans . PLoS genetics . 2012 ; 8 ( 9 ):e1002957. https://doi.org/10.1371/journal.pgen.1002957 PMID: 23028364; Central PMCID : PMC3447965 .
26. Hervera A , De Virgiliis F , Palmisano I , Zhou L , Tantardini E , Kong G , et al. Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons . Nature cell biology . 2018 ; 20 ( 3 ): 307 - 19 . https://doi.org/10.1038/s41556-018-0039 -x PMID : 29434374 .
27. Krishnamoorthy L , Chang CJ . Exosomal NADPH Oxidase: Delivering Redox Signaling for Healing . Biochemistry . 2018 . https://doi.org/10.1021/acs. biochem.8b00429 29889508. PMID: 29889508
28. Morelli AE , Larregina AT , Shufesky WJ , Sullivan ML , Stolz DB , Papworth GD , et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells . Blood . 2004 ; 104 ( 10 ): 3257 - 66 . https:// doi.org/10.1182/blood-2004 -03-0824 PMID: 15284116.
29. Edens WA , Sharling L , Cheng G , Shapira R , Kinkade JM , Lee T , et al. Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox . The Journal of cell biology . 2001 ; 154 ( 4 ): 879 - 91 . https://doi.org/10. 1083/jcb.200103132 PMID: 11514595 .
30. Moribe H , Yochem J , Yamada H , Tabuse Y , Fujimoto T , Mekada E . Tetraspanin protein (TSP-15) is required for epidermal integrity in Caenorhabditis elegans . Journal of cell science . 2004; 117(Pt 22 ): 5209 - 20 . https://doi.org/10.1242/jcs.01403 PMID: 15454573 .
31. Clergeot PH , Gourgues M , Cots J , Laurans F , Latorse MP , Pepin R , et al. PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe
32. Veneault-Fourrey C , Parisot D , Gourgues M , Lauge R , Lebrun MH , Langin T. The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum . Fungal genetics and biology: FG & B . 2005 ; 42 ( 4 ): 306 - 18 . https://doi.org/10.1016/j. fgb. 2005 . 01 .009 PMID: 15749050 .
33. Siegmund U , Heller J , van Kan JA , Tudzynski P. The NADPH oxidase complexes in Botrytis cinerea: evidence for a close association with the ER and the tetraspanin Pls1 . PloS one . 2013 ; 8 ( 2 ):e55879. https://doi.org/10.1371/journal.pone.0055879 PMID: 23418468; Central PMCID : PMC3572182 .
34. Damiani I , Pauly N , Puppo A , Brouquisse R , Boscari A . Reactive Oxygen Species and Nitric Oxide Control Early Steps of the Legume-Rhizobium Symbiotic Interaction. Frontiers in plant science . 2016 ; 7 : 454 . https://doi.org/10.3389/fpls. 2016 .00454 PMID: 27092165; Central PMCID : PMC4824774 .
35. Boavida LC , Qin P , Broz M , Becker JD , McCormick S . Arabidopsis tetraspanins are confined to discrete expression domains and cell types in reproductive tissues and form homo- and heterodimers when expressed in yeast . Plant physiology . 2013 ; 163 ( 2 ): 696 - 712 . https://doi.org/10.1104/pp. 113 . 216598 PMID: 23946353; Central PMCID : PMC3793051 .
36. Lieber D , Lora J , Schrempp S , Lenhard M , Laux T. Arabidopsis WIH1 and WIH2 genes act in the transition from somatic to reproductive cell fate . Current biology: CB . 2011 ; 21 ( 12 ): 1009 - 17 . https://doi. org/10.1016/j.cub. 2011 . 05 .015 PMID: 21658947 .
37. Yamaguchi H , Kawaguchi K , Sagawa T. Dynamical crossover in a stochastic model of cell fate decision . Physical review E . 2017 ; 96 ( 1-1 ): 012401 . https://doi.org/10.1103/PhysRevE.96.012401 PMID: 29347197 .
Wang F , Muto A , Van de Velde J , Neyt P , Himanen K , Vandepoele K , et al. Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development . Plant physiology. 2015 ; 169 ( 3 ): 2200 - 14 . https://doi.org/10.1104/pp. 15 .01310 PMID: 26417009; Central PMCID : PMC4634101 .
39. Tsukagoshi H , Busch W , Benfey PN . Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root . Cell . 2010 ; 143 ( 4 ): 606 - 16 . https://doi.org/10.1016/j.cell. 2010 . 10 . 020 PMID: 21074051 .
40. Zeng J , Dong Z , Wu H , Tian Z , Zhao Z . Redox regulation of plant stem cell fate . The EMBO journal . 2017 ; 36 ( 19 ): 2844 - 55 . https://doi.org/10.15252/embj.201695955 PMID: 28838936; Central PMCID : PMC5623875 .
41. Montiel J , Arthikala MK , Cardenas L , Quinto C . Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation . International journal of molecular sciences . 2016 ; 17 ( 5 ). https://doi.org/ 10.3390/ijms17050680 PMID: 27213330; Central PMCID : PMC4881506 .
42. Arthikala MK , Sanchez-Lopez R , Nava N , Santana O , Cardenas L , Quinto C . RbohB, a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization . The New phytologist . 2014 ; 202 ( 3 ): 886 - 900 . https://doi. org/10.1111/nph.12714 PMID: 24571730 .
43. Montiel J , Nava N , Cardenas L , Sanchez-Lopez R , Arthikala MK , Santana O , et al. A Phaseolus vulgaris NADPH oxidase gene is required for root infection by Rhizobia . Plant & cell physiology . 2012 ; 53 ( 10 ): 1751 - 67 . https://doi.org/10.1093/pcp/pcs120 PMID: 22942250 .
44. Karimi M , Inze D , Depicker A . GATEWAY vectors for Agrobacterium-mediated plant transformation . Trends in plant science . 2002 ; 7 ( 5 ): 193 - 5 . PMID: 11992820 .
45. Rezaie J , Ajezi S , Avci CB , Karimipour M , Geranmayeh MH , Nourazarian A , et al. Exosomes and their Application in Biomedical Field: Difficulties and Advantages . Molecular neurobiology . 2018 ; 55 ( 4 ): 3372 - 93 . https://doi.org/10.1007/s12035-017-0582-7 PMID: 28497202 .
46. Estrada-Navarrete G , Alvarado-Affantranger X , Olivares JE , Guillen G , Diaz-Camino C , Campos F , et al. Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes . Nature protocols . 2007 ; 2 ( 7 ): 1819 - 24 . https://doi.org/10.1038/nprot. 2007 .259 PMID: 17641650 .
47. Cardenas L , Dominguez J , Quinto C , Lopez-Lara IM , Lugtenberg BJ , Spaink HP , et al. Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli . Plant molecular biology . 1995 ; 29 ( 3 ): 453 - 64 . PMID: 8534845 .
48. Smith SE , Smith FA , Jakobsen I. Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses . Plant physiology . 2003 ; 133 ( 1 ): 16 - 20 . https://doi.org/10.1104/pp. 103 . 024380 PMID: 12970469; Central PMCID : PMC1540331 .
49. Schmittgen TD , Livak KJ . Analyzing real-time PCR data by the comparative C(T) method . Nature protocols. 2008 ; 3 ( 6 ): 1101 - 8 . PMID: 18546601 .
50. Mani B , Agarwal M , Katiyar-Agarwal S. Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress. Frontiers in plant science . 2015 ; 6 : 1088 . https://doi.org/10.3389/fpls. 2015 .01088 PMID: 26697042; Central PMCID : PMC4675852 .
51. Garcia-Espana A , Chung PJ , Sarkar IN , Stiner E , Sun TT , Desalle R . Appearance of new tetraspanin genes during vertebrate evolution . Genomics . 2008 ; 91 ( 4 ): 326 - 34 . https://doi.org/10.1016/j.ygeno. 2007 . 12 .005 PMID: 18291621 .
52. Garcia-Espa?a A , Mares R , Mung-Tien S , DeSalle R. Intron Evolution: Testing Hypotheses of Intron Evolution Using the Phylogenomics of Tetraspanins . PloS one . 2009 ; 4 ( 3 ): 1 - 12 .
53. Blanco FA , Meschini EP , Zanetti ME , Aguilar OM . A small GTPase of the Rab family is required for root hair formation and preinfection stages of the common bean-Rhizobium symbiotic association . The Plant cell . 2009 ; 21 ( 9 ): 2797 - 810 . https://doi.org/10.1105/tpc.108.063420 PMID: 19749154; Central PMCID : PMC2768941 .
54. Dalla Via V , Traubenik S , Rivero C , Aguilar OM , Zanetti ME , Blanco FA . The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis . Plant molecular biology . 2017 ; 93 ( 6 ): 549 - 62 . https://doi.org/10.1007/s11103- 016-0581-5 PMID: 28074430 .
55. O 'Rourke JA , Iniguez LP , Fu F , Bucciarelli B , Miller SS , Jackson SA , et al. An RNA-Seq based gene expression atlas of the common bean . BMC genomics . 2014 ; 15 : 866 . https://doi.org/10.1186/ 1471 - 2164-15-866 PMID: 25283805; Central PMCID : PMC4195886 .
56. Arthikala MK , Montiel J , Nava N , Santana O , Sanchez-Lopez R , Cardenas L , et al. PvRbohB negatively regulates Rhizophagus irregularis colonization in Phaseolus vulgaris . Plant & cell physiology . 2013 ; 54 ( 8 ): 1391 - 402 . https://doi.org/10.1093/pcp/pct089 PMID: 23788647 .
57. Becker JD , Takeda S , Borges F , Dolan L , Feijo JA . Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature . BMC plant biology . 2014 ; 14 : 197 . https://doi.org/10. 1186/s12870-014-0197-3 PMID: 25080170; Central PMCID : PMC4236730 .
58. Borges A , Tsai SM , Caldas DG . Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses . Plant cell reports . 2012 ; 31 ( 5 ): 827 - 38 . https://doi.org/10.1007/ s00299-011-1204 -x PMID : 22193338 .
59. Delgado MJ , Ligero F , Lluch C. Effects of salt stress on growth and nitrogen fixation by pea, fababean, common bean and soybean plants . Soil Biology and Biochemistry . 1994 ; ( 26(3 )): 371 - 6 .
60. Kaymakanova M , N. Stoeva N. Physiological hystological reaction of bean plants (Phaseolus Vulg . L.) to salt stress . Gen Appl Plant Physiology . 2008 ; 34 ( 3-4 ): 177 - 88 .
61. Schmutz J , Cannon SB , Schlueter J , Ma J , Mitros T , Nelson W , et al. Genome sequence of the palaeopolyploid soybean . Nature . 2010 ; 463 ( 7278 ): 178 - 83 . https://doi.org/10.1038/nature08670 PMID: 20075913 .
62. Garcia-Espana A , Chung PJ , Zhao X , Lee A , Pellicer A , Yu J , et al. Origin of the tetraspanin uroplakins and their co-evolution with associated proteins: implications for uroplakin structure and function . Molecular phylogenetics and evolution . 2006 ; 41 ( 2 ): 355 - 67 . https://doi.org/10.1016/j.ympev. 2006 . 04 . 023 PMID: 16814572 .
63. Malamy JE , Benfey PN . Organization and cell differentiation in lateral roots of Arabidopsis thaliana . Development . 1997 ; 124 ( 1 ): 33 - 44 . PMID: 9006065 .
64. Dubrovsky JG , Sauer M , Napsucialy-Mendivil S , Ivanchenko MG , Friml J , Shishkova S , et al. Auxin acts as a local morphogenetic trigger to specify lateral root founder cells . Proceedings of the National Academy of Sciences of the United States of America . 2008 ; 105 ( 25 ): 8790 - 4 . https://doi.org/10.1073/ pnas.0712307105 PMID: 18559858; Central PMCID : PMC2438385 .
65. Cnops G , Wang X , Linstead P , Van Montagu M , Van Lijsebettens M , Dolan L. Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root . Development . 2000 ; 127 ( 15 ): 3385 - 94 . PMID: 10887093 .
66. Chaubet N , Flenet M , Clement B , Brignon P , Gigot C . Identification of cis-elements regulating the expression of an Arabidopsis histone H4 gene . The Plant journal: for cell and molecular biology . 1996 ; 10 ( 3 ): 425 - 35 . PMID: 8811858 .
67. Thomas CL , Bayer EM , Ritzenthaler C , Fernandez-Calvino L , Maule AJ . Specific targeting of a plasmodesmal protein affecting cell-to-cell communication . PLoS biology . 2008 ; 6 ( 1 ):e7. https://doi.org/ 10.1371/journal.pbio.0060007 PMID: 18215111; Central PMCID : PMC2211546 .
68. Fernandez-Calvino L , Faulkner C , Walshaw J , Saalbach G , Bayer E , Benitez-Alfonso Y , et al. Arabidopsis plasmodesmal proteome . PloS one . 2011 ; 6 ( 4 ):e18880. https://doi.org/10.1371/journal.pone. 0018880 PMID: 21533090; Central PMCID : PMC3080382 .
69. Ehlers K , Kollmann R . Primary and secondary plasmodesmata: structure, origin, and functioning . Protoplasma . 2001 ; 216 ( 1-2 ): 1 - 30 . PMID: 11732191 .
70. AJ vB. Interaction between sieve element and companion cell and the consequences for photoassimilate distribution. Two structural hardware frames with associated physiological software packages in dicotyledons . Journal of experimental botany . 1996 ; 47 ( 1129 ): 1129 - 40 .
71. Salmon MS , Bayer EM . Dissecting plasmodesmata molecular composition by mass spectrometrybased proteomics . Frontiers in plant science . 2012 ; 3 : 307 . https://doi.org/10.3389/fpls. 2012 .00307 PMID: 23335932; Central PMCID : PMC3542633 .
72. Maule A , Faulkner C , Benitez-Alfonso Y. Plasmodesmata "in Communicado" . Frontiers in plant science . 2012 ; 3 : 30 . https://doi.org/10.3389/fpls. 2012 .00030 PMID: 22645579; Central PMCID : PMC3355775 .
73. Sevilem I , Yadav SR , Helariutta Y. Plasmodesmata : channels for intercellular signaling during plant growth and development . Methods in molecular biology . 2015 ; 1217 : 3 - 24 . https://doi.org/10.1007/ 978-1- 4939 -1523- 1 _1 PMID: 25287193 .
74. Juniper BE , Barlow PW . The distribution of plasmodesmata in the root tip of maize . Planta . 1969 ; 89 ( 4 ): 352 - 60 . https://doi.org/10.1007/BF00387235 PMID: 24504513 .
75. Imaichi R , Hiratsuka R . Evolution of shoot apical meristem structures in vascular plants with respect to plasmodesmatal network . American journal of botany . 2007 ; 94 ( 12 ): 1911 - 21 . https://doi.org/10.3732/ ajb.94.12. 1911 PMID: 21636385 .
76. Zhu T , Lucas WJ , Rost TL . Directional cell-to-cell communication in theArabidopsis root apical meristem I. An ultrastructural and functional analysis . Protoplasma . 1998 ; 203 : 35 - 47 .
77. Jin L , Wang J , Guan F , Zhang J , Yu S , Liu S , et al. Dominant point mutation in a tetraspanin gene associated with field-evolved resistance of cotton bollworm to transgenic Bt cotton . Proceedings of the National Academy of Sciences of the United States of America . 2018 ; 115 ( 46 ): 11760 - 5 . https://doi. org/10.1073/pnas.1812138115 PMID: 30381456; Central PMCID : PMC6243265 .
78. Genre A , Chabaud M , Faccio A , Barker DG , Bonfante P . Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota . The Plant cell . 2008 ; 20 ( 5 ): 1407 - 20 . https://doi.org/10. 1105/tpc.108.059014 PMID: 18515499; Central PMCID : PMC2438458 .
79. Murakami E , Cheng J , Gysel K , Bozsoki Z , Kawaharada Y , Hjuler CT , et al. Epidermal LysM receptor ensures robust symbiotic signalling in Lotus japonicus . eLife. 2018 ; 7 . https://doi.org/10.7554/eLife. 33506 PMID: 29957177; Central PMCID : PMC6025957 .
80. Faulkner C , Petutschnig E , Benitez-Alfonso Y , Beck M , Robatzek S , Lipka V , et al. LYM2 -dependent chitin perception limits molecular flux via plasmodesmata . Proceedings of the National Academy of Sciences of the United States of America . 2013 ; 110 ( 22 ): 9166 - 70 . https://doi.org/10.1073/pnas. 1203458110 PMID: 23674687; Central PMCID : PMC3670346 .
81. Kankanala P , Czymmek K , Valent B . Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus . The Plant cell . 2007 ; 19 ( 2 ): 706 - 24 . https://doi.org/10.1105/ tpc.106.046300 PMID: 17322409; Central PMCID : PMC1867340 .
82. Complainville A , Brocard L , Roberts I , Dax E , Sever N , Sauer N , et al. Nodule initiation involves the creation of a new symplasmic field in specific root cells of medicago species . The Plant cell . 2003 ; 15 ( 12 ): 2778 - 91 . https://doi.org/10.1105/tpc.017020 PMID: 14615602; Central PMCID : PMC282798 .
83. Gaudioso-Pedraza R , Beck M , Frances L , Kirk P , Ripodas C , Niebel A , et al. Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development . Current biology: CB . 2018 ; 28 ( 22 ): 3562 - 77 e6. https://doi.org/10.1016/j.cub. 2018 . 09 .031 PMID: 30416059 .
84. Benitez-Alfonso Y , Cilia M , San Roman A, Thomas C , Maule A , Hearn S , et al. Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport . Proceedings of the National Academy of Sciences of the United States of America . 2009 ; 106 ( 9 ): 3615 - 20 . https://doi. org/10.1073/pnas.0808717106 PMID: 19218459; Central PMCID : PMC2651306 .
85. Herrbach V , Chirinos X , Rengel D , Agbevenou K , Vincent R , Pateyron S , et al. Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula . Journal of experimental botany . 2017 ; 68 ( 3 ): 569 - 83 . https://doi.org/10.1093/jxb/erw474 PMID: 28073951; Central PMCID : PMC6055581 .
86. Yamaguchi N , Huang J , Xu Y , Tanoi K , Ito T . Fine-tuning of auxin homeostasis governs the transition from floral stem cell maintenance to gynoecium formation . Nature communications . 2017 ; 8 ( 1 ): 1125 . https://doi.org/10.1038/s41467-017-01252-6 PMID: 29066759; Central PMCID : PMC5654772 .
87. Larrainzar E , Riely BK , Kim SC , Carrasquilla-Garcia N , Yu HJ , Hwang HJ , et al. Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals . Plant physiology . 2015 ; 169 ( 1 ): 233 - 65 . https://doi.org/10.1104/ pp. 15 .00350 PMID: 26175514; Central PMCID : PMC4577383 .
88. Finet C , Jaillais Y. Auxology : when auxin meets plant evo-devo . Developmental biology . 2012 ; 369 ( 1 ): 19 - 31 . https://doi.org/10.1016/j.ydbio. 2012 . 05 .039 PMID: 22687750 .
89. Mathesius U , Schlaman HR , Spaink HP , Of Sautter C , Rolfe BG , Djordjevic MA . Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides . The Plant journal: for cell and molecular biology . 1998 ; 14 ( 1 ): 23 - 34 . https:// doi.org/10.1046/j. 1365 - 313X . 1998 . 00090 . x PMID : 15494052 .
90. Rutter BD , Innes RW . Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins . Plant physiology . 2017 ; 173 ( 1 ): 728 - 41 . https://doi.org/10.1104/pp. 16 .01253 PMID: 27837092; Central PMCID : PMC5210723 .
91. Guimaraes PM , Guimaraes LA , Morgante CV , Silva OB Jr., Araujo AC , Martins AC , et al. Root Transcriptome Analysis of Wild Peanut Reveals Candidate Genes for Nematode Resistance . PloS one . 2015 ; 10 ( 10 ):e0140937. https://doi.org/10.1371/journal.pone.0140937 PMID: 26488731; Central PMCID : PMC4619257 .
92. Chen T , Duan L , Zhou B , Yu H , Zhu H , Cao Y , et al. Interplay of Pathogen-Induced Defense Responses and Symbiotic Establishment in Medicago truncatula . Frontiers in microbiology. 2017 ; 8 : 973 . https://doi.org/10.3389/fmicb. 2017 .00973 PMID: 28611764; Central PMCID : PMC5447765 .
93. Libault M , Farmer A , Brechenmacher L , Drnevich J , Langley RJ , Bilgin DD , et al. Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection . Plant physiology . 2010 ; 152 ( 2 ): 541 - 52 . https://doi.org/10.1104/pp. 109 .148379 PMID: 19933387; Central PMCID : PMC2815892 .
94. Foehse D , Jungk A . Influence of phosphate and nitrate supply on root hair formation of rape, spinach and tomato plants . Plant and soil . 1983 ; 74 ( 3 ): 359 - 68 .
95. Bates TR , Lynch JP . Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability . Plant , cell & environment. 1996 ; 19 ( 5 ): 529 - 38 .
96. Bhosale R , Giri J , Pandey BK , Giehl RFH , Hartmann A , Traini R , et al. A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate . Nature communications . 2018 ; 9 ( 1 ): 1409 . https://doi.org/10.1038/s41467-018-03851-3 PMID: 29651114; Central PMCID : PMC5897496 .
97. Duffield A , Kamsteeg EJ , Brown AN , Pagel P , Caplan MJ . The tetraspanin CD63 enhances the internalization of the H,K-ATPase beta-subunit . Proceedings of the National Academy of Sciences of the United States of America . 2003 ; 100 ( 26 ): 15560 - 5 . https://doi.org/10.1073/pnas.2536699100 PMID: 14660791; Central PMCID : PMC307607 .
98. Hager A. Role of the plasma membrane H+ -ATPase in auxin-induced elongation growth: historical and new aspects . Journal of plant research . 2003 ; 116 ( 6 ): 483 - 505 . https://doi.org/10.1007/s10265- 003-0110 -x PMID : 12937999 .
99. Yuan W , Zhang D , Song T , Xu F , Lin S , Xu W , et al. Arabidopsis plasma membrane H+ -ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress . Journal of experimental botany . 2017 ; 68 ( 7 ): 1731 - 41 . https://doi. org/10.1093/jxb/erx040 PMID: 28369625; Central PMCID : PMC5441905 .
100. Krajinski F , Courty PE , Sieh D , Franken P , Zhang H , Bucher M , et al. The H+ -ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis. The Plant cell . 2014 ; 26 ( 4 ): 1808 - 17 . https://doi.org/10.1105/tpc.113.120436 PMID: 24781114; Central PMCID : PMC4036587 .
101. Wang E , Yu N , Bano SA , Liu C , Miller AJ , Cousins D , et al. A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula . The Plant cell . 2014 ; 26 ( 4 ): 1818 - 30 . https://doi.org/10.1105/tpc.113.120527 PMID: 24781115; Central PMCID : PMC4036588 .
102. Lopez-Bucio J , Hernandez-Abreu E , Sanchez-Calderon L , Nieto-Jacobo MF , Simpson J , HerreraEstrella L. Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system . Plant physiology . 2002 ; 129 ( 1 ): 244 - 56 . https://doi.org/10.1104/pp.010934 PMID : 12011355; Central PMCID : PMC155888 .
103. Schurmann J , Buttermann D , Herrmann A , Giesbert S , Tudzynski P . Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host-pathogen interaction . Molecular plant-microbe interactions: MPMI . 2013 ; 26 ( 10 ): 1151 - 64 . https://doi.org/10.1094/MPMI-03-13-0064 -R PMID : 23777432 .
104. Schoenfeld N , Bauer MK , Grimm S. The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates . FASEB journal: official publication of the Federation of American Societies for Experimental Biology . 2004 ; 18 ( 1 ): 158 - 60 . https://doi.org/10.1096/fj.03- 0420fje PMID: 14597553 .
105. Charrin S , Jouannet S , Boucheix C , Rubinstein E. Tetraspanins at a glance . Journal of cell science . 2014; 127(Pt 17 ): 3641 - 8 . https://doi.org/10.1242/jcs.154906 PMID: 25128561 .
106. Lee Y , Rubio MC , Alassimone J , Geldner N. A mechanism for localized lignin deposition in the endodermis . Cell . 2013 ; 153 ( 2 ): 402 - 12 . https://doi.org/10.1016/j.cell. 2013 . 02 .045 PMID: 23541512 .
107. Osipova MA , Mortier V , Demchenko KN , Tsyganov VE , Tikhonovich IA , Lutova LA , et al. Wuschelrelated homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation . Plant physiology . 2012 ; 158 ( 3 ): 1329 - 41 . https://doi.org/10.1104/pp. 111 .188078 PMID: 22232385; Central PMCID : PMC3291250 .
108. Cardenas L , Feijo JA , Kunkel JG , Sanchez F , Holdaway-Clarke T , Hepler PK , et al. Rhizobium nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs . The Plant journal: for cell and molecular biology . 1999 ; 19 ( 3 ): 347 - 52 . PMID: 10476081 .
109. Cardenas L , Martinez A , Sanchez F , Quinto C . Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs) . The Plant journal: for cell and molecular biology . 2008 ; 56 ( 5 ): 802 - 13 . https://doi.org/10.1111/j. 1365 - 313X . 2008 . 03644 . x PMID : 18680562 .