10.6092/issn.2531-7342/11867
application/pdf
2531-7342
11867
en
Valentina  Fiorilli
https://creativecommons.org/licenses/by/4.0
Italian Journal of Mycology
orchid symbiosis
Laser microdissection as a tool to study fungal gene expression in mycorrhizal endosymbioses
Text.Serial.Journal
https://italianmycology.unibo.it/
50
Articles
2021-02-08
2020-11-30
2021-02-08
2021-06-25
1-9
Ital J Mycol
Valentina  Fiorilli
2021/02/08
10.6092/issn.2531-7342/11867
1
Department of Life Sciences and Systems Biology, University of Turin
2531-7342
orchid symbiosis
en
9
Zeng T, Holmer R, Hontelez J, te Lintel‐Hekkert B, Marufu L, de Zeeuw T, Wu F, Schijlen, E., Bisseling T, Limpens E. (2018) Host‐ and stage‐dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis. Plant Journal 94:411-425. https://doi.org/10.1111/tpj.13908
Laser microdissection as a tool to study fungal gene expression in mycorrhizal endosymbioses
https://italianmycology.unibo.it/article/download/11867/12313
50
                 IJM - Italian Journal of Mycology
                 ISSN 2531-7342 - Vol. 50 (2021): 1-9
                 Journal homepage: https://italianmycology.unibo.it/

Review

   Laser microdissection as a tool to study fungal gene expression
                  in mycorrhizal endosymbioses

                     Raffaella Balestrini1*, Silvia Perotto2, Valentina Fiorilli2

1 Institute for Sustainable Plant Protection - CNR, Turin, Italy
2 Department of Life Sciences and Systems Biology, University of Turin

Corresponding author e-mail: raffaella.balestrini@ipsp.cnr.it


Article info
Received 2/12/2020; accepted 28/1/2021
https://doi.org/10.6092/issn.2531-7342/11867

Abstract
Laser microdissection (LMD) is a microscopy technique that, through the collection of specific cell-type populations from
sections of heterogeneous tissues, allows the subsequent extraction of nucleic acids as well as primary and secondary
metabolites. In plants, LMD was widely used to study cell-specific gene expression during symbiotic interactions with
other organisms, including mycorrhizal fungi. In particular, LMD was extensively used to study cell-specificity in gene
expression profiles in arbuscular mycorrhizal (AM) and orchid mycorrhizal (ORM) interactions. These earlier studies
were mainly focused on the identification of functional markers in plant cells containing intracellular fungal structures,
i.e. arbuscules, the typical structures in AM, and coils, typical of ORM. Several plant and fungal genes coding for nutrient
transporters were identified in these cells thanks to LMD, suggesting that symbiotic nutrient exchange is cell specific. In
the absence of a stable transformation protocol for the expression of tagged genes in the mycorrhizal fungal partner, LMD
protocols represent a useful tool to study fungal gene expression in specific cell-type populations inside symbiotic plant
tissues.

Keywords
AM symbiosis, cell-specificity, LMD, gene expression, nutrient exchange, orchid symbiosis


Introduction
Mycorrhizal fungi are symbiotic soil fungi that assist plants in nutrient uptake since land colonization.
Endosymbiotic mycorrhizal fungi, such as arbuscular mycorrhizal (AM) or orchid mycorrhizal (ORM)
fungi, form specialized structures inside the host cells of colonized roots, which represent a heterogenous
landscape of different cell-type populations, expected to play different functions in the symbiosis. Since
several years, researchers have tried to characterize the different responses in these different cell-types
(Fiorilli et al., 2019) as well as to identify molecular markers related to the development of the typical
symbiotic fungal structures, such as arbuscules in the AM and coils in the ORM symbiosis. Most
attention has been focused on the AM symbiosis because AM fungi establish mycorrhizal interactions
with the roots of most crop species and play an important role as bio-fertilizers. As reported by Rouphael
et al. (2015), the development of more sustainable horticultural practices would greatly benefit from
the biostimulant functions displayed by AM fungi. The success of the AM symbiosis is mainly due to
the nutritional benefits to the host plant: AM fungi absorb inorganic phosphate (Pi) and water from the
soil, as well as other macro- and micronutrients, delivering them to the host plant. In turn, the fungus
receives photosynthesis-derived carbon compounds, such as carbohydrates and lipids (Balestrini and


Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
Balestrini, Perotto, Fiorilli                                        Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



Lumini, 2018). The use of AM fungi has been largely explored as an environmentally friendly strategy to
improve plant nutrition and growth and to enhance plant tolerance and resilience to different abiotic and
biotic stresses, although the impact and the reliability of these strategies across diverse environmental
conditions and production systems must be evaluated before practical recommendations can be
provided. Additionally, the exploitation of AM fungi in agricultural programs requires the identification
of the events that lead to the establishment and functioning of the symbiotic interaction, including
the mechanisms involved in nutrient exchange and water transport. Root colonization by AM fungi
occurs through the formation of inter- and intracellular hyphae and culminates, in the inner root cortical
layers, with complex and highly branched intracellular fungal structures called arbuscules. Arbuscules
are surrounded by a plant derived membrane and, because of the huge increase in the contact surface
between the fungus and the plant, are considered to be key structures in the AM symbiosis, where most
of the nutrient exchange between the two partners is likely to occur (Balestrini and Bonfante, 2005).
Once the first arbuscules are formed in the root cortex, all developmental stages of the symbiosis can
be found in a mycorrhizal root. Therefore, a mycorrhizal root is a heterogeneous assortment of different
cell types, including colonized and non-colonized epidermal and cortical plant cells. The presence of
multiple cell types, some of which involved in the different stages of the symbiotic interaction, makes
the root gene expression profiling very complicated.
       A similar situation is also found in other endomycorrhizal symbioses, such as orchid mycorrhiza
(Balestrini and Lumini, 2018). In nature, members of the large plant family Orchidaceae establish a
characteristic symbiotic interaction with some soil fungi that induce seed germination and development
of adult plants. The minute orchid seeds, called “dust seeds”, lack the endosperm and contain little
stored food reserves. Colonization by a compatible fungus is essential for their germination and for the
provision of major nutrients such as carbon and nitrogen (Balestrini and Lumini, 2018). Germinated
orchid seeds develop into tuber-like structures named protocorms that finally produce leaves and roots.
All stages of the orchid life cycle are mycorrhizal, and orchid mycorrhizal protocorms and roots are
heterogeneous structures made of different cell-types, where plant cells colonized by intracellular fungal
coils (the pelotons) are close to non-colonized plant cells. Moreover, the fungal coils undergo rapid
turnover inside the colonized cells, so that plant cells containing coils at different developmental stages
can be observed in the same tissue section. Thus, a serious drawback in the investigation of the molecular
aspects of mycorrhizal interactions in both AM and ORM is that the development of the symbiosis is
not a synchronous process and that colonized and non-colonized cells occur in the same tissues. As
a consequence, the transcriptional profile of whole mycorrhizal organs (e.g. roots and/or protocorms
for orchids) represents the simultaneous gene expression of multiple cell types and fungal structures
involved in the interaction (Balestrini and Fiorilli, 2020) and could mask cell type-specific differences.
In the last fifteen years, protocols based on the use of laser microdissection (LMD) were widely
applied to plant tissues and provided novel information on the role of different cell-type populations in
pathogenic (Tang et al., 2006; Chandran et al., 2010; Hacquard et al., 2010) and symbiotic plant-fungal
interactions that included mycorrhizal associations such as ectomycorrhizal, ORM and AM symbioses
(Fochi et al., 2017a, 2017b; Hacquard et al., 2013; Fiorilli et al., 2019; Balestrini and Fiorilli, 2020). In
particular, LMD technology was successfully applied to investigate the gene expression profiles of both
plant and fungal partners during different colonization stages in the AM symbiosis, but also to study
proteomic and metabolomic profiles in arbuscule-containing cells (Gaude et al., 2012, 2015). The main
objective of this review is to highlight the instrumental role of LMD to study fungal gene expression in
mycorrhizal endosymbioses.




                                                     2
Balestrini, Perotto, Fiorilli                                      Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



Application of LMD to study AM fungal functions in symbiosis
Since the first report by Balestrini et al. (2007), where LMD was applied to investigate gene expression
in mycorrhizal roots, several studies have been using this technology in conjunction with RT-PCR and
RT-qPCR. Many of these papers have described the results of targeted gene expression experiments
on RNA extracted from different cell-types isolated by LMD from mycorrhizal and non-mycorrhizal
roots, in particular cortical cells from non-mycorrhizal roots, cortical cells containing arbuscules and
non-colonized cells from mycorrhizal roots (reviewed in Fiorilli et al., 2019; Limpens, 2019; Balestrini
and Fiorilli 2020). Many LMD protocols have been successfully applied on different combinations
of plant and fungal species, leading to the identification of plant and fungal functional marker genes
associated with arbusculated cells in different plants (e.g. tomato, Lotus, Medicago). Concerning the
host plants, new information was obtained mainly on genes involved in fungal accommodation and
functioning of arbusculated cortical cells. Several genes coding for nutrient (and water) transporters
were found to be expressed in arbusculated cells, suggesting that functions such as nutrient exchange
are cell-specific and confirming the hypothesis that arbuscule-containing cells represent the core of a
functional AM symbiosis (reviewed in Fiorilli et al., 2019, Limpens 2019, Balestrini and Fiorilli 2020).
Interestingly, plant and fungal genes coding for proteins with the same function (i.e. phosphate and
ammonium transporters) have been found to be both expressed in arbusculated cells, suggesting that
plant and fungus may compete for the nutrient resources localized in the interfacial apoplast. A LMD-
based technique was also used to evaluate, in mycorrhizal roots of bean plants, the local impact of
water stress on gene expression (Recchia et al., 2018). In detail, gene expression data obtained for
root cortical cells, containing or not fungal arbuscules and collected from water deficit treated plants,
provided evidence that a cell type-specific modulation of the plant transcriptome occurs during
symbiosis under stress. The use of LMD to collect several cell-type populations from mycorrhizal
plants subjected to stressful conditions represents an additional advancement to understand the fine-
tune regulation of plant responses to adverse conditions mediated by the AM fungus. The use of LMD
also allowed an estimation of the percentage of fungal and plant transcripts in arbuscule-containing
cells. Using specific primers for both fungal and plant housekeeping genes, it was estimated that, in this
cell population, the plant transcripts represented about 80 % of the total transcripts, whereas the fungal
transcripts accounted for about 20% (Fiorilli et al., 2013).
       Most reports on the use of LMD to investigate the AM symbiosis have been focused on the
expression of plant genes, whereas few reports tried to unravel specific changes in gene expression
in the different fungal structures. The first experiments on an AM fungus consisted on studying the
expression of fungal genes coding for nutrient transporters in arbuscules (Balestrini and Fiorilli, 2020).
Tisserant et al. (2012) performed a non-targeted transcriptomic analysis on LMD-microdissected
arbuscule-containing root cells to investigate the fungal transcriptome in the arbuscules. These data
were validated by means of one-step RT-PCR on RNA extracted from AM fungal spores, extraradical
mycelium and arbusculated LMD-microdissected cells, and represented a starting point for further
studies on the expression of fungal genes in arbusculated cells (e.g., Belmondo et al., 2014). Unlike
plant genes, which were investigated by LMD in several AM host plant species, most results derived
by the use of LMD to study the fungal genes were obtained, with few exceptions, on Rhizophagus
irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler, which represents a model
system for studies on AM fungi. A list of the fungal genes reported to be expressed in arbusculated cells
can be found in Table 1.
       Additionally, Zeng et al. (2018) recently combined LMD of mycorrhizal root sections with
RNAseq analysis in order to gain novel insights into stage-specific fungal gene expression, focusing
the analysis on fungal genes coding for secreted proteins (SPs). It is well known that AM fungi secrete
an array of (small) proteins that may act as effector proteins (Kloppholz et al., 2011; Tisserant et al.,


                                                    3
Balestrini, Perotto, Fiorilli                                               Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867




      Table 1 - List of fungal genes identified in mycorrhizal roots as being preferentially or exclusively
expressed in arbuscule-containing cells by means of LMD technology (modified by Fiorilli et al., 2019).
     Fungal
                     Gene name                 Putative function                    Reference
     species
 Funneliformis             GmosPT                  Phosphate transporter                Balestrini et al., 2007
   mosseae                      GmHA                    H+ATPase                        Balestrini et al., 2007


                                RiARF             ADP-ribosylation factor                Gomez et al., 2009
                                RiAP            Probable autophagy protein               Gomez et al., 2009
                            Rip24p                 Related to p24 protein                Gomez et al., 2009
                                RIGS               Glutamine synthetase                  Gomez et al., 2009
                            RiARG                        Arginase                        Gomez et al., 2009
                                                    Probabile ornithine
                            RiOATR                                                       Gomez et al., 2009
                                                     aminotrasferase
                                RiASS           Argininocusscinate syntase               Gomez et al., 2009
                         Riacyl-CoA-
                                          Probable acyl-CoA dehydrogenase                Gomez et al., 2009
                            dehy
                                          Phospholipid-transporting ATPase
                            RiDSR2                                                       Gomez et al., 2009
                                                       DRS2
                                RiFAD           Delta-9 fatty acid denatuase             Gomez et al., 2009
  Rhizophagus              RiPEIP1        Preferentially Expressed in Planta             Fiorilli et al., 2016
   irregularis
                            GintPT                 Phosphate transporter                 Fiorilli et al., 2013
                          GintMST2              Monosaccaride transporter                Fiorilli et al., 2013
                            RiAMT1                Ammonium transporter                Perez-Tienda et al., 2011
                            RiAMT2                Ammonium transporter                Perez-Tienda et al., 2011
                      EXTB106676_b0                       No hit                        Tisserant et al., 2012
                       EXTA123933.b0                      No hit                        Tisserant et al., 2012
                            RiABCT                   ABC transporter                    Tisserant et al., 2012
                           RiATPase                       ATPase                       Hogekamp et al., 2011
                                RiRB               RNA binding protein                 Hogekamp et al., 2011
                                               C2H2 zinc finger transcription
                                RiZTF                                                  Hogekamp et al., 2011
                                                          factor
                            RiPTR2                 Dipeptide transporter               Belmondo et al., 2014
                                  -              Several secreted proteins*               Zeng et al., 2018

       *Due to the large dataset, specific proteins have been not reported here.




                                                          4
Balestrini, Perotto, Fiorilli                                       Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



2012; Lin et al., 2014). Through an LMD approach, Zeng et al., (2018) revealed that different SP sets
were expressed in the extraradical mycelium, the intraradical hyphae and the arbuscules. Interestingly,
these authors also observed that the gene expression levels of 42 candidate effectors varied from host to
host, suggesting that expression of secreted AM fungal proteins is stage and host dependent.
       In addition to functional genes, LMD has been applied, in combination with sequence-based
taxa identification, to study the AM fungal community inside mycorrhizal roots (Berruti et al., 2013).
Comparison between the AM fungal taxa identified inside arbusculated cells by LMD and those
identified in the DNA extracted from the whole root showed that the two AM fungal communities
differed remarkably. In fact, whereas five AM fungal taxa were found to be involved in the production
of arbuscules, two taxa were retrieved from the root DNA but were not found in the arbusculated
cells. Thus, the LMD technique can be used to obtain more precise data on the symbiotically active
intraradical AM fungal community. By focusing on the functional genes with a role in symbiosis, this
technique could be also used to evaluate the efficiency of different AM fungal species/isolates.

Application of LMD to study ORM fungal functions in symbiosis
All orchids depend on symbiotic mycorrhizal fungi for seed germination and produce a tuber-like
structure, i.e. the protocorm, that will eventually give rise to the true seedling. Basidiomycetes in the
genera Tulasnella, Ceratobasidium and Serendipita are the most common mycorrhizal symbionts in
orchid species that develop into fully photosynthetic adult plants. As described above, ORM protocorms
and roots are heterogeneous structures that comprise different plant cell-types, where cells colonized by
intracellular fungal coils (the pelotons) at different developmental stages, due to the rapid coil turnover
inside the colonized cells, are close to non-colonized plant cells in the same tissue section (Perotto et
al., 2014). A protocol based on LMD was used to localize specific plant and fungal gene transcripts in
different cell-type populations collected from mycorrhizal protocorms and roots of the Mediterranean
orchid Serapias vomeracea (Burm.f.) Briq. colonized by Tulasnella calospora (Boud.) Juel (Fochi et
al., 2017a, b; Balestrini et al., 2014). Total RNA was extracted from the different cell-type populations
(i.e. cells fully colonized by large fungal coils, cells containing condensed coils and non-colonized
plant cells). Transcripts corresponding to genes potentially involved in plant-fungus interactions were
then investigated by RT-PCR, as well as genes related to nitrogen (N) uptake and transport previously
identified as being up-regulated in symbiotic protocorms by transcriptomic experiments. The results
showed that SvNod1, coding for a S. vomeracea nodulin-like protein containing a plastocyanin-like
domain, is expressed only in protocorm cells containing intracellular fungal hyphae. Additionally,
fungal and plant genes involved in the transport and metabolism of nitrogen compounds were found
to be differentially expressed in cells containing fungal coils at different developmental stages, as well
as in non-colonized cells. A list of the fungal genes identified in coil-containing cells can be found in
Table 2.
       Overall, the data derived from the S. vomeracea - T. calospora ORM model system support
the hypothesis that ammonium is available in the apoplastic interface surrounding the pelotons and
is actively taken up by the fungus, as suggested by the strong induction in colonized cells of a low-
affinity ammonium transporter (TcAMT2). The induction, in the same colonized protocorm cells, of
the ammonium scavenging enzyme glutamine synthetase (TcGS1) suggests that ammonium is rapidly
assimilated once taken up by the fungal peloton (Fochi et al., 2017a). RNAs extracted from the same
cell-type populations were used also to verify the expression of plant genes potentially involved in
N uptake and transport, previously identified as being induced in mycorrhizal protocorms. A plant
ammonium transporter gene (SvAMT1) was found to be expressed in the colonized protocorm cells,
although it expression was not specific to this cell type (Fochi et al., 2017a, b). As hypothesized for
the AM symbiosis (Guether et al., 2009; Calabrese et al., 2016), the presence of both fungal and plant


                                                    5
Balestrini, Perotto, Fiorilli                                             Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



AMTs in the same colonized orchid cells may lead to a competition between the plant and the fungus
for nitrogen sources present in the interfacial apoplast. The LMD approach revealed that some plant
amino acid transporters were differentially expressed in protocorm cells containing fungal coils at
different developmental stages, adding an additional piece on the comprehension of the N metabolism
in orchid symbiosis (Fochi et al., 2017b) and leading to the identification of new functional marker
genes associated to coil-containing cells in ORM.

      Table 2 - Tulasnella calospora genes found to be expressed in Serapias vomeracea protocorm cells
containing fungal coils by means of LMD technology. Fungal transcripts code for transporters and enzymes
involved in N uptake and metabolism (Fochi et al., 2017a).
 Fungal species        Gene name                   Putative function                         References
                         TcAMT1                 Ammonium transporter
                         TcAMT2                 Ammonium transporter
                          TcGS1                  Glutamine synthetase
                          TcGS1                  Glutamine synthetase
                          TcCAR                        Arginase
                          TcURE                         Urease
    Tulasnella                                                                            Fochi et al., 2017a
                          TcOAT                Ornithine aminotransferase
    calospora
                         TcODC                  Ornithine decarboxylase
                         TcAAT1           Amino acid transporter/permease
                         TcAAT2           Amino acid transporter/permease
                         TcAAT3           Amino acid transporter/permease
                         TcAAT4           Amino acid transporter/permease
                         TcAAT5           Amino acid transporter/permease



Conclusions
LMD has proved to be a useful tool to study cell-type specific gene expression in symbiotic and pathogenic
interactions. Overall, the results obtained so far in symbiotic mycorrhizal interactions demonstrate that
LMD is a powerful technology to investigate the cellular complexity of both AM and ORM and can be
applied to investigate both plant and fungal gene expression. The possibility to successfully apply the
LMD technology to evaluate the expression level of both fungal and plant functional genes involved in
the AM and ORM symbioses should help to dissect the functional diversity in different fungal - plant
combinations and conditions. This knowledge may provide future guidelines to identify the best plant-
fungus combinations to improve plant fitness under a changing ecological scenario.

Authors’ contribution
RB, SP and VF equally contributed to writing this review.




                                                         6
Balestrini, Perotto, Fiorilli                                     Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



References
Balestrini R., Gomez-Ariza J, Lanfranco L, Bonfante P (2007) Laser microdissection reveals that
      transcripts for five plant and one fungal phosphate transporter genes are contemporaneously
      present in arbusculated cells. Molecular Plant Microbe Interaction 20:1055-106210. https://doi.
      org/10.1094/MPMI-20-9-1055
Balestrini R, Nerva L, Sillo F, Girlanda M, Perotto S (2014) Plant and fungal gene expression in
      mycorrhizal protocorms of the orchid Serapias vomeracea colonized by Tulasnella calospora.
      Plant Signaling & Behavior 9:11. https://doi.org/10.4161/15592324.2014.977707
Balestrini R, Bonfante P (2005). The interface compartment in arbuscular mycorrhizae: a special type
      of plant cell wall? Plant Biosystems 139:8-15. https://doi.org/10.1080/11263500500056799
Balestrini R, Lumini E (2018) Focus on mycorrhizal symbioses. Applied Soil Ecology 123:299-304.
      https://doi.org/10.1016/j.apsoil.2017.09.001
Balestrini R, Fiorilli V (2020) Laser microdissection as a useful tool to study gene expression in
      plant and fungal partners in AM symbiosis. In: Arbuscular Mycorrhizal Fungi: Methods and
      Protocols, Series Methods in Molecular Biology (N Ferrol, L Lanfranco, eds), Protocol Lab.
      Heidelberg, Germany, Springer. https://doi.org/10.1007/978-1-0716-0603-2_13
Belmondo S, Fiorilli V, Pérez-Tienda J, Ferrol N, Marmeisse R, Lanfranco L (2014) A dipeptide
      transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in
      the intraradical phase. Frontiers in Plant Science 5:436. https://doi.org/10.3389/fpls.2014.00436
Berruti A, Borriello R, Lumini E, Scariot V, Bianciotto V, Balestrini R (2013) Application of laser
       microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells.
       Frontiers in Plant Science 4:135. https://doi.org/10.3389/fpls.2013.00135
Calabrese S, Pérez-Tienda J., Ellerbeck, M., Arnould C, Chatagnier O, Boller T, Schüssler A,
       Brachmann A, Wipf D, Ferrol N, Courty PE (2016) GintAMT3 – a Low-Affinity Ammonium
       Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis. Frontiers in Plant Science
       7:679. https://doi.org/10.3389/fpls.2016.00679
Chandran D, Inada N, Hather G, Kleindt CK, Wildermuth MC (2010) Laser microdissection of
       Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and
       regulators. Proceedings of the National Academy of Sciences of the United States of America
       107:460-465. https://doi.org/10.1073/pnas.0912492107
Fiorilli V, Lanfranco L, Bonfante P (2013) The expression of GintPT, the phosphate transporter of
       Rhizophagus irregularis, depends on the symbiotic status and phosphate availability. Planta
       237: 1267-1277. https://doi.org/10.1007/s00425-013-1842-z
Fiorilli V, Belmondo S, Khouja HR, Abbà S, Faccio A, Daghino S, Lanfranco L (2016) RiPEIP1,
       a gene from the arbuscular mycorrhizal fungus Rhizophagus irregularis, is preferentially
       expressed in planta and may be involved in root colonization. Mycorrhiza 26:609-621. https://
       doi.org/10.1007/s00572-016-0697-0
Fiorilli V, Volpe V, Balestrini R (2019) Microscopic techniques coupled to molecular and genetic
       approaches to highlight cell-type specific differences in mycorrhizal symbiosis. In: Methods
       in Rhizosphere Biology Research (Reinhardt D, Sharma A.K., eds), Rhizosphere Biology.
       Singapore, Springer, pp 197-225. https://doi.org/10.1007/978-981-13-5767-1_11
Fochi V, Chitarra W, Kohler A, Voyron S, Singan VR, Lindquist EA, Barry KW, Girlanda M, Grigoriev
       IV, Martin F, et al. (2017a) Fungal and plant gene expression in the Tulasnella calospora–
       Serapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas.
       New Phytologist 213:365-379. https://doi.org/10.1111/nph.14279
Fochi V, Falla N, Girlanda M, Perotto S, Balestrini R (2017b) Cell-specific expression of plant nutrient
       transporter genes in orchid mycorrhizae. Plant Science 263:39-45. https://doi.org/10.1016/j.
       plantsci.2017.06.015

                                                   7
Balestrini, Perotto, Fiorilli                                    Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



Gaude N, Schulze WX, Franken P, Krajinski F (2012) Cell type-specific protein and transcription
      profiles implicate periarbuscular membrane synthesis as an important carbon sink in the
      mycorrhizal symbiosis. Plant Signal Behavior 7:461-4. https://doi.org/10.4161/psb.19650
Gaude N, Bortfeld S, Erban A, Kopka J, Krajinski F (2015) Symbiosis dependent accumulation of
      primary metabolites in arbuscule-containing cells. BMC Plant Biology 15:234. https://doi.
      org/10.1186/s12870-015-0601-7
Gomez SK, Javot H, Deewatthanawong P, Torres-Jerez I, Tang Y, Blancaflor EB, Udvardi MK,
      Harrison MJ (2009) Medicago truncatula and Glomus intraradices gene expression in cortical
      cells harboring arbuscules in the arbuscular mycorrhizal symbiosis. BMC Plant Biology 9:10.
      https://doi.org/10.1186/1471-2229-9-10
Guether M, Neuhäuser B, Balestrini R, Dynowski M, Ludewig U, Bonfante P (2009) A Mycorrhizal-
      Specific Ammonium Transporter from Lotus japonicus Acquires Nitrogen Released by
      Arbuscular Mycorrhizal Fungi.          Plant Physiology 150:73-83. https://doi.org/10.1104/
      pp.109.136390
Hacquard S, Delaruelle C, Legué V, Tisserant E, Kohler A, Frey P, Martin F, Duplessis S (2010)
      Laser Capture Microdissection of Uredinia Formed by Melampsora larici-populina Revealed
      a Transcriptional Switch Between Biotrophy and Sporulation. Molecular Plant-Microbe
      Interactions 23:1275-1286. https://doi.org/10.1094/MPMI-05-10-0111
Hacquard S, Tisserant E, Brun A, Legué V, Martin F, Kohler A (2013) Functional heterogeneity within
      ectomycorrhizas. Environmental Microbiology 15:1853-1869. https://doi.org/10.1111/1462-
      2920.12080
Hogekamp C, Arndt D, Pereira PA, Becker JD, Hohnjec N, Küster H (2011) Laser microdissection
      unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box
      transcription factor gene expression correlating with fungal contact and spread. Plant Physiology
      157:2023-2043. https://doi.org/10.1104/pp.111.186635
Kloppholz S, Kuhn H, Requena N (2011) A secreted fungal effector of Glomus intraradices promotes
      symbiotic biotrophy. Current Biology 21:1204-1209. https://doi.org/10.1016/j.cub.2011.06.044
Lin K, Limpens E, Zhang Z, et al. (2014) Single Nucleus Genome Sequencing Reveals High
      Similarity among Nuclei of an Endomycorrhizal Fungus. PLoS Genetics 10:e1004078. https://
      doi.org/10.1371/journal.pgen.1004078
Pérez-Tienda J, Testillano PS, Balestrini R, Fiorilli V, Azcón-Aguilar C, Ferrol N (2011) GintAMT2,
      a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus
      Glomus intraradices. Fungal Genetics & Biology 48:1044-1055. https://doi.org/10.1016/j.
      fgb.2011.08.003
Perotto S, Rodda M, Benetti A, Sillo, F, Ercole E, Rodda M, Girlanda M, Murat C, Balestrini R
      (2014) Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus
      relationship. Planta 239:1337–1349. https://doi.org/10.1007/s00425-014-2062-x
Recchia GH, Konzen ER, Cassieri F, Caldas DGG, Tsai SM (2018) Arbuscular mycorrhizal symbiosis
      leads to differential regulation of drought-responsive genes in tissue-specific root cells of
      common bean. Frontiers in Microbiology 9:1339. https://doi.org/10.3389/fmicb.2018.01339
Rouphael Y, Franken P, Schneider C, Schwarz D, Giovannetti M, Agnolucci M, De Pascale S, Bonini
      P, Colla G (2015) Arbuscular mycorrhizal fungi act as biostimulants in horticultural crops.
      Scientia Horticulturae 196:91-108. https://doi.org/10.1016/j.scienta.2015.09.002
Tang W, Coughlan S, Crane E, Beatty M, Duvick J (2006) The application of laser microdissection
      to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum
      graminicola. Molecular Plant Microbe Interaction 19:1240-1250. https://doi.org/10.1094/
      MPMI-19-1240


                                                  8
Balestrini, Perotto, Fiorilli                                    Italian Journal of Mycology 50 (2021): 1-9

https://doi.org/10.6092/issn.2531-7342/11867



Tisserant E, Kohler A, Dozolme-Seddas P, et al. (2012) The transcriptome of the arbuscular mycorrhizal
      fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate
      symbiont. New Phytologist 193:755-769. https://doi.org/10.1111/j.1469-8137.2011.03948.x
Zeng T, Holmer R, Hontelez J, te Lintel-Hekkert B, Marufu L, de Zeeuw T, Wu F, Schijlen, E., Bisseling
      T, Limpens E. (2018) Host- and stage-dependent secretome of the arbuscular mycorrhizal
      fungus Rhizophagus irregularis. Plant Journal 94:411-425. https://doi.org/10.1111/tpj.13908




                                                  9
