Bücher und Buchkapitel
Mielke, S.; Gasperini, D. Plant–Insect Bioassay for Testing Arabidopsis Resistance to the Generalist Herbivore Spodoptera littoralis (Champion, A. & Laplaze, L., eds.). Methods Mol Biol 2085, 69-78, (2020) ISBN: 978-1-0716-0142-6 DOI: 10.1007/978-1-0716-0142-6_5
Jasmonates are essential engineers of plant
defense responses against many pests, including herbivorous insects.
Herbivory induces the production of jasmonic acid (JA) and its bioactive
conjugate jasmonoyl-l-isoleucine (JA-Ile), which then triggers a large
transcriptional reprogramming to promote plant acclimation. The
contribution of the JA pathway, including its components and regulators,
to defense responses against insect herbivory can be evaluated by
conducting bioassays with a wide range of host plants and insect pests.
Here, we describe a detailed and reproducible protocol for testing
feeding behavior of the generalist herbivore Spodoptera littoralis on
the model plant Arabidopsis thaliana and hence infer the contribution of
JA-mediated plant defense responses to a chewing insect.
Publikationen in Druck
Dallery, J.-F.; Zimmer, M.; Halder, V.; Suliman, M.; Pigné, S.; Le Goff, G.; Gianniou, D. D.; Trougakos, I. P.; Ouazzani, J.; Gasperini, D.; O’Connell, R. J. Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B bioRxiv (2019) DOI: 10.1101/651562
Infection of Arabidopsis thaliana by the
ascomycete fungus Colletotrichum higginsianum is characterised by an
early symptomless biotrophic phase followed by a destructive
necrotrophic phase. The fungal genome contains 77 secondary
metabolism-related biosynthetic gene clusters (BGCs), and their
expression during the infection process is tightly regulated. Deleting
CclA, a chromatin regulator involved in repression of some BGCs through
H3K4 trimethylation, allowed overproduction of 3 families of terpenoids
and isolation of 12 different molecules. These natural products were
tested in combination with methyl jasmonate (MeJA), an elicitor of
jasmonate responses, for their capacity to alter defence gene induction
in Arabidopsis. Higginsianin B inhibited MeJA-triggered expression of
the defence reporter VSP1p:GUS, suggesting it may block bioactive JA-Ile
synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS,
we found that higginsianin B, but not three other structurally-related
molecules, suppressed JA-Ile signalling by preventing degradation of JAZ
proteins, the repressors of JA responses. Higginsianin B likely blocks
the 26S proteasome-dependent degradation of JAZ proteins because it
inhibited chymotrypsin- and caspase-like protease activities. The
inhibition of target degradation by higginsianin B also extended to
auxin signalling, as higginsianin B treatment reduced IAA-dependent
expression of DR5p:GUS. Overall, our data indicate that specific fungal
secondary metabolites can act similarly to protein effectors to subvert
plant immune and developmental responses.
Publikation
Schulze, A.; Zimmer, M.; Mielke, S.; Stellmach, H.; Melnyk, C. W.; Hause, B.; Gasperini, D. Shoot-to-root translocation of the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) coordinates plant growth responses following tissue damage bioRxiv (2019) DOI: 10.1101/517193
Multicellular organisms rely upon the movement of signaling molecules across cells, tissues and organs to communicate among distal sites. In plants, herbivorous insects, necrotrophic pathogens and mechanical wounding stimulate the activation of the jasmonate (JA) pathway, which in turn triggers the transcriptional changes necessary to protect plants against those challenges, often at the expense of growth. Although previous evidence indicated that JA species can translocate from damaged into distal sites, the identity of the mobile compound(s), the tissues through which they translocate and the consequences of their relocation remain unknown. Here, we demonstrated that endogenous JA species generated after shoot injury translocate to unharmed roots via the phloem vascular tissue in Arabidopsis thaliana. By wounding wild-type shoots of chimeric plants and by quantifying the relocating compounds from their JA-deficient roots, we uncovered that the JA-Ile precursor 12-oxo-phytodienoic acid (OPDA) is a mobile JA species. Our data also showed that OPDA is a primary mobile compound relocating to roots where, upon conversion to the bioactive hormone, it induces JA-mediated gene expression and root growth inhibition. Collectively, our findings reveal the existence of long-distance transport of endogenous OPDA which serves as a communication molecule to coordinate shoot-to-root responses, and highlight the importance of a controlled distribution of JA species among organs during plant stress acclimation.
Publikation
Schulze, A.; Zimmer, M.; Mielke, S.; Stellmach, H.; Melnyk, C. W.; Hause, B.; Gasperini, D. Wound-Induced Shoot-to-Root Relocation of JA-Ile Precursors Coordinates Arabidopsis Growth Mol Plant 12, 1383-1394, (2019) DOI: 10.1016/j.molp.2019.05.013
Multicellular organisms rely on the movement of
signaling molecules across cells, tissues, and organs to communicate
among distal sites. In plants, localized leaf damage activates jasmonic
acid (JA)-dependent transcriptional reprogramming in both harmed and
unharmed tissues. Although it has been indicated that JA species can
translocate from damaged into distal sites, the identity of the mobile
compound(s), the tissues through which they translocate, and the effect
of their relocation remain unknown. Here, we found that following shoot
wounding, the relocation of endogenous jasmonates through the phloem is
essential to initiate JA signaling and stunt growth in unharmed roots of
Arabidopsis thaliana. By employing grafting experiments and hormone
profiling, we uncovered that the hormone precursor
cis-12-oxo-phytodienoic acid (OPDA) and its derivatives, but not the
bioactive JA-Ile conjugate, translocate from wounded shoots into
undamaged roots. Upon root relocation, the mobile precursors
cooperatively regulated JA responses through their conversion into
JA-Ile and JA signaling activation. Collectively, our findings
demonstrate the existence of long-distance translocation of endogenous
OPDA and its derivatives, which serve as mobile molecules to coordinate
shoot-to-root responses, and highlight the importance of a controlled
redistribution of hormone precursors among organs during plant stress
acclimation.
Publikationen in Druck
Mielke, S.; Gasperini, D. Interplay between Plant Cell Walls and Jasmonate Production Plant Cell Physiol (2019) DOI: 10.1093/pcp/pcz119
Plant cell walls are sophisticated
carbohydrate-rich structures representing the immediate contact surface
with the extracellular environment, often serving as the first barrier
against biotic and abiotic stresses. Notably, a variety of perturbations
in plant cell walls result in upregulated jasmonate (JA) production, a
phytohormone with essential roles in defense and growth responses.
Hence, cell wall-derived signals can initiate intracellular JA-mediated
responses and the elucidation of the underlying signaling pathways could
provide novel insights into cell wall maintenance and remodeling, as
well as advance our understanding on how is JA biosynthesis initiated.
This Mini Review will describe current knowledge about cell wall-derived
damage signals and their effects on JA biosynthesis, as well as provide
future perspectives.
Publikation
Kowalski, A. M.; Gooding, M.; Ferrante, A.; Slafer, G. A.; Orford, S.; Gasperini, D.; Griffiths, S. Agronomic assessment of the wheat semi-dwarfing gene Rht8 in contrasting nitrogen treatments and water regimes Field Crops Res 191, 150-160, (2016) DOI: 10.1016/j.fcr.2016.02.026
Reduced height 8 (Rht8) is the main alternative
to the GA-insensitive Rht alleles in hot and dry environments where it
reduces plant height without yield penalty. The potential of Rht8 in
northern-European wheat breeding remains unclear, since the close
linkage with the photoperiod-insensitive allele Ppd-D1a is unfavourable
in the relatively cool summers. In the present study, two near-isogenic
lines (NILs) contrasting for the Rht8/tall allele from Mara in a
UK-adapted and photoperiod-sensitive wheat variety were evaluated in
trials with varying nitrogen fertiliser (N) treatments and water regimes
across sites in the UK and Spain.The Rht8 introgression was
associated with a robust height reduction of 11% regardless of N
treatment and water regime and the Rht8 NIL was more resistant to
root-lodging at agronomically-relevant N levels than the tall NIL. In
the UK with reduced solar radiation over the growing season than the
site in Spain, the Rht8 NIL showed a 10% yield penalty at standard
agronomic N levels due to concomitant reduction in grain number and
spike number whereas grain weight and harvest index were not
significantly different to the tall NIL. The yield penalty associated
with the Rht8 introgression was overcome at low N and in irrigated
conditions in the UK, and in the high-temperature site in Spain.
Decreased spike length and constant spikelet number in the Rht8 NIL
resulted in spike compaction of 15%, independent of N and water regime.
The genetic interval of Rht8 overlaps with the compactum gene on 2DS,
raising the possibility of the same causative gene. Further genetic
dissection of these loci is required.
Publikation
Gasperini, D.; Acosta, I. F.; Farmer, E. E. Cotyledon Wounding of Arabidopsis Seedlings Bio Protoc 6, e1712, (2016) DOI: 10.21769/BioProtoc.1712
Damage to plant organs through both biotic and
abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old
(5-do) seedlings represent an excellent system in which to study plant
responses to mechanical wounding, both at the site of the damage and in
distal unharmed tissues. Seedlings of wild type, transgenic or mutant
lines subjected to single or repetitive cotyledon wounding can be used
to quantify morphological alterations (e.g., root length, Gasperini et
al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et
al., 2015; Gasperini et al., 2015), follow transcriptional changes by
quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or
examine additional aspects of the wound response with a plethora of
downstream procedures. Here we illustrate how to rapidly and reliably
wound cotyledons of young seedlings, and show the behavior of two
promoters driving the expression of β-glucuronidase (GUS) in entire
seedlings and in the primary root meristem, following single or
repetitive cotyledon wounding respectively. We describe two procedures
that can be easily adapted to specific experimental needs.
Publikation
Gasperini, D.; Chételat, A.; Acosta, I. F.; Goossens, J.; Pauwels, L.; Goossens, A.; Dreos, R.; Alfonso, E.; Farmer, E. E. Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth PLOS Genet 11, e1005300, (2015) DOI: 10.1371/journal.pgen.1005300
Physical damage can strongly affect plant
growth, reducing the biomass of developing organs situated at a distance
from wounds. These effects, previously studied in leaves, require the
activation of jasmonate (JA) signalling. Using a novel assay involving
repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a
function of JA in suppressing cell division and elongation in roots.
Regulatory JA signalling components were then manipulated to delineate
their relative impacts on root growth. The new transcription factor
mutant myc2-322B was isolated. In vitro transcription assays and
whole-plant approaches revealed that myc2-322B is a dosage-dependent
gain-of-function mutant that can amplify JA growth responses. Moreover,
myc2-322B displayed extreme hypersensitivity to JA that totally
suppressed root elongation. The mutation weakly reduced root growth in
undamaged plants but, when the upstream negative regulator NINJA was
genetically removed, myc2-322B powerfully repressed root growth through
its effects on cell division and cell elongation. Furthermore, in a
JA-deficient mutant background, ninja1 myc2-322B still repressed root
elongation, indicating that it is possible to generate JA-responses in
the absence of JA. We show that NINJA forms a broadly expressed
regulatory layer that is required to inhibit JA signalling in the apex
of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4
displayed cell layer-specific localisations and MYC3 and MYC4 were
expressed in mutually exclusive regions. In nature, growing roots are
likely subjected to constant mechanical stress during soil penetration
that could lead to JA production and subsequent detrimental effects on
growth. Our data reveal how distinct negative regulatory layers,
including both NINJA-dependent and -independent mechanisms, restrain JA
responses to allow normal root growth. Mechanistic insights from this
work underline the importance of mapping JA signalling components to
specific cell types in order to understand and potentially engineer the
growth reduction that follows physical damage.
Publikation
Gasperini, D.; Chauvin, A.; Acosta, I. F.; Kurenda, A.; Stolz, S.; Chételat, A.; Wolfender, J.-L.; Farmer, E. E. Axial and Radial Oxylipin Transport Plant Physiol 169, 2244-2254, (2015) DOI: 10.1104/pp.15.01104
Jasmonates are oxygenated lipids (oxylipins)
that control defense gene expression in response to cell damage in
plants. How mobile are these potent mediators within tissues? Exploiting
a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis
(Arabidopsis thaliana) that displays impaired jasmonic acid (JA)
synthesis in specific cell types and using JA-inducible reporters, we
mapped the extent of the transport of endogenous jasmonates across the
plant vegetative growth phase. In seedlings, we found that jasmonate (or
JA precursors) could translocate axially from wounded shoots to
unwounded roots in a LOX2-dependent manner. Grafting experiments with
the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin
transport. Next, we used rosettes to investigate radial cell-to-cell
transport of jasmonates. After finding that the LOX6 protein localized
to xylem contact cells was not wound inducible, we used the lox234
triple mutant to genetically isolate LOX6 as the only JA
precursor-producing LOX in the plant. When a leaf of this mutant was
wounded, the JA reporter gene was expressed in distal leaves. Leaf
sectioning showed that JA reporter expression extended from contact
cells throughout the vascular bundle and into extravascular cells,
revealing a radial movement of jasmonates. Our results add a crucial
element to a growing picture of how the distal wound response is
regulated in rosettes, showing that both axial (shoot-to-root) and
radial (cell-to-cell) transport of oxylipins plays a major role in the
wound response. The strategies developed herein provide unique tools
with which to identify intercellular jasmonate transport routes.
Publikation
Farmer, E. E.; Gasperini, D.; Acosta, I. F. The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding New Phytol 204, 282-288, (2014) DOI: 10.1111/nph.12897
Jasmonates are lipid mediators that control
defence gene expression in response to wounding and other environmental
stresses. These small molecules can accumulate at distances up to
several cm from sites of damage and this is likely to involve
cell‐to‐cell jasmonate transport. Also, and independently of jasmonate
synthesis, transport and perception, different long‐distance wound
signals that stimulate distal jasmonate synthesis are propagated at
apparent speeds of several cm min–1 to tissues distal to wounds in a
mechanism that involves clade 3 GLUTAMATE RECEPTOR‐LIKE (GLR) genes. A
search for jasmonate synthesis enzymes that might decode these signals
revealed LOX6, a lipoxygenase that is necessary for much of the rapid
accumulation of jasmonic acid at sites distal to wounds. Intriguingly,
the LOX6 promoter is expressed in a distinct niche of cells that are
adjacent to mature xylem vessels, a location that would make these
contact cells sensitive to the release of xylem water column tension
upon wounding. We propose a model in which rapid axial changes in xylem
hydrostatic pressure caused by wounding travel through the vasculature
and lead to slower, radially dispersed pressure changes that act in a
clade 3 GLR‐dependent mechanism to promote distal jasmonate synthesis.