Commentary based on Paniagua Voirol et al. (2020) Plant responses
to insect eggs are not induced by egg-associated microbes, but by a
secretion attached to the eggs
A hungry caterpillar is making its way across a leaf, unsuspiciously
munching on the fresh plant tissue. Little does the insect know that its
nibbling has initiated avalanche-like effects underneath its tiny feet.
It is unaware that its food is not willing to resign to the fate of
being eaten without fighting back. And it is oblivious about the fact
that it is being meticulously observed and recorded - observed and
recorded by scientists who aim to reveal the very details of the complex
plant-herbivore interactions. The paper by Paniagua Voirol et al. in
this Issue focusses on the interface between plant and insect – an area
that is far less investigated than the plant’s internal defence
response.
One of the internal plant signals set off by chewing insects has
recently been demonstrated by Toyota et al. (2018). Using fluorescent
reporter plants, they visualized the rapid spread of the second
messenger Ca2+. The Ca2+ wave
originated from the site of attack, and travelled mainly through the
leaf vasculature to activate systemic responses throughout the entire
plant. This Ca2+-related signalling is triggered by a
release of the wound signal glutamate (Toyota et al., 2018), and is
further transduced and propagated by ion channels of the
GLUTAMATE-RECEPTOR-LIKE family (Mousavi et al., 2013). The systemic
defence responses include, for example, increased synthesis of jasmonic
acid, and accumulation of toxic and repellent compounds to limit the
amount of herbivore damage (Wu & Baldwin, 2010).
Reacting directly to larval herbivory is only one of the manifold
mechanisms that plants have developed in the course of the never-ending
evolutionary arms race with insect herbivores. Many species can
‘anticipate’ larval attacks and respond to the deposition of eggs before
the insects are hatching. Anticipatory defence mechanisms include the
release of volatile compounds to ward off adult insects or to attract
parasitoids (M. Hilker et al., 2002), production of harmful defence
compounds (Austel et al., 2016; Bandoly et al., 2016), mechanical egg
removal or destruction (Balbyshev & Lorenzen, 1997; Desurmont &
Weston, 2011), and locally restricted necrosis that increases egg
mortality (Geuss et al., 2017). The latter is a typical response to egg
deposition by Pieris butterflies. The processes involved
strikingly resemble the pathogen-induced hypersensitive response, in
which cells immediately surrounding the infection site are killed to
prevent the pathogen spread. The hypersensitive response is initiated by
membrane-bound receptors such as FLAGELLIN-SENSITIVE 2 (FLS2) and
BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) that perceive pathogen-associated
molecular patterns (Chinchilla et al., 2007), and induce complex
cellular signalling pathways, resulting in the accumulation of reactive
oxygen species and nitric oxide (Balint-Kurti, 2019; Clarke et al.,
2000). Similarly, egg deposition by Pieris butterflies and other
herbivorous insects causes accumulation of reactive oxygen species and
salicylic acid, callose deposition, up-regulation of
pathogenesis-related genes, and localized programmed cell death (Figure
1) (Bittner et al., 2017; Little et al., 2007).
With this in mind, Paniagua Voirol et al. hypothesized that the defence
responses to eggs of the large white butterfly Pierisbrassicae are in fact mediated and induced by egg-associated
microorganisms and not by the eggs themselves. The authors found that
butterfly larvae grew significantly less and developed more slowly on
egg-primed leaves than on un-primed ones. The egg-induced up-regulation
of defence mechanisms in the plant was reflected in increased expression
levels of defence marker genes such as PATHOGENESIS-RELATED GENE 1 (PR1)
and PATHOGENESIS-RELATED GENE 5 (PR5). Depositing surface-sterile eggs
from antibiotics-treated butterflies onto leaves, however, resulted in
significantly lower marker gene transcript levels, both inArabidopsis thaliana and in the natural butterfly hostBrassica nigra . Larvae feeding on these plants put on more weight
compared to plants that were treated with non-sterile eggs, and they
developed faster into pupae. Do these findings already solve the case
and prove a tripartite insect-microbe-plant relationship? As usual in
biology, the answer is more complex. Surprisingly, the authors found
that also ‘unsterile’ egg clutches of P. brassicae harboured only
negligible amounts of bacteria. Furthermore, treatment of
Arabidopsis leaves with egg-associated bacterial mixtures, cultivatedin situ , did not prime the plant anti-herbivore defence. Larvae
feeding on these leaves grew and developed similarly well as on
untreated leaves, making bacteria unlikely culprits for inducing the
hypersensitive response-like plant defence mechanisms.
The authors concluded that the eliciting factor had to be an attribute
of the eggs themselves. Pieris brassicae deposits its eggs in
clutches that get ‘glued’ onto the leaf surface by secretory substances
produced by the butterfly female’s accessory reproductive gland (ARG).
Application of various amounts of the ARG secretion product onto leaves,
and following larvae feeding experiments revealed a dose-dependent
relationship with the plant defence response and larvae performance.
But how then explain the initial findings with surface-sterile eggs?
Further analysis revealed that antibiotics treatment of the female
butterflies significantly decreased the biomass of the butterflies’
accessory reproductive gland, and consequently reduced the secretion
quantity. Less ARG secretion resulted in poor plant defence response and
consequently prosperous larvae growth (Figure 2). The causes for the
reduced ARG biomass in the treated butterflies remain unknown, as do(es)
the specific elicitor(s) contained in the secretion mix and the
mechanism for its perception on the plant epidermis.
This study by Paniagua Voirol et al. shows that plants seem to have
gained the upper hand in this specific insect-plant relationship. High
amounts of ARG secretion are necessary for proper attachment of
gregariously laid butterfly egg clutches, and therefore indispensable
for successful insect recreation. This type of egg deposition contrasts
with single laid eggs, which in turn are more prone to being killed by
the hypersensitive response-like leaf necrosis. What appears to be an
advantageous adaptation conferring a fitness benefit to the butterflies
has been turned into a payoff by the evolutionary co-adaptation of the
plant. Such an imbalance in “armaments” between interacting species
pairs can occur if the strength of selection imposed by the species on
one another is not perfectly equal. Natural selection is frequently
acting more strongly on species with more at stake in terms of
evolutionary performance, i.e. reproductive success, leading to a faster
evolution of fitness-enhancing adaptations for species under stronger
selective pressure (Anderson et al., 2010; Humphreys & Ruxton, 2020).
These latest findings by the Hilker group come at a significant time for
research on plant biotic interactions. The year 2020 has been declared
as “The International Year of Plant Health” by the General Assembly of
the United Nations to raise global awareness for the devastating damage
caused by plant pests and diseases. Not only crops but whole ecosystems
are more threatened than ever by biotic stressors. International trade
and travel, monocultural farming systems, and cultivation of non-endemic
species have accelerated the fast spread of transboundary pests and
diseases with dramatic consequences. The Food and Agricultural
Organization of the United Nations estimates that the global annual crop
loss caused by phytopathogens and herbivores to be up to 40% leading to
trading losses of over $220 billion (FAO, 2019). The herbivorous
caterpillars of the large white cabbage butterfly Pieris
brassicae investigated by Paniagua Voirol et al. are considered a
serious and highly destructive pest that can cause significant economic
losses in brassicaceous crops (CABI, 2020). The caterpillars are
specialist herbivores feeding only on plants containing glucosinolates.Pieris brassicae is mainly distributed in Europe, Asia, and
Northern Africa, but can also be found in America and Australia due to
the migratory nature of the adult insect. Many of the host plants such
as cabbage, rape seed, and cauliflower are of economic importance in
these regions, and caterpillar infestations can cause severe yield
losses (Hasan & Ansari, 2011) with financial and social consequences on
all levels of food production.
The United Nations emphasize that protecting crops against
phytopathogens and herbivores holds the potential to reduce poverty and
hunger, and they urge researchers to develop innovative practices and
technologies for environmentally friendly methods such as integrated
pest management. The adverse environmental impacts of conventional
synthetic broadband pesticides have been flagged up in recent years
because of the concerning decline in the global bee population.
Integrated pest management aims to avoid poisonous substances when
dealing with pests. Biological control practices are promoting
insectivores, or usage of ‘biological insecticides’ derived from
microorganisms or plants that have less severe side effects. The
findings by Paniagua Voirol et al. therefore have practical value,
because the application of the secretory substances from female
butterflies have potential to strengthen the plant’s natural defence,
and consequently reduce the herbivore damage. Identifying the active
component of the ARG secretion is the obvious next challenge, but this
study already represents a fundamental stepping-stone that paves the way
for an environmentally friendly crop pest control in the future.