The May 2020 Editor’s pick for MPMI
is “Arabidopsis
Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease
Resistance.”
The first author is Laura Bacete, a graduate student in the lab of Antonio Molina at the Universidad
Politécnica de Madrid. To read
more about Laura, you can find her bio here.
Laura is now a postdoc at the Institute for Biology at the Norwegian University
of Science and Technology. Antonio recently presented this work in a What’s
New in MPMI? Seminar. You can find a recording of his seminar here.
Plant Cell Wall Composition and Disease Resistance:
A Journey across Novel Mechanisms of Plant Immunity
Submitted by Laura Bacete and Antonio
Molina
Traditionally, the plant cell wall has been
considered simply a physical defensive barrier against pathogens. However, this
outdated view has evolved to a novel concept that considers the plant cell wall
as a dynamic structure regulating different processes of plant immunity and development
(Figure 1) (Bacete et al. 2018). Recently, we have published
in Molecular Plant-Microbe Interactions (MPMI) our last findings about
the impact of the alteration of the cytokinin-responsive Arabidopsis Response
Regulator 6 (ARR6) gene expression
on the modulation of plant cell wall composition and disease resistance responses
(Bacete et al. 2020). Here, we describe the
story of how we reached this fascinating discovery, and how our research group,
initially focused on A. thaliana resistance
to necrotrophic fungi, started a journey that led us to identify a novel mechanism
of plant immunity and to determine the relevance of plant cell wall composition
in disease resistance. This journey led us to the conviction that plant cell wall-mediated
immunity is a key and dynamic component of plant disease resistance against necrotrophic
fungi—our initial pathogens of interest—but also against all the plant pathogens
we have studied.
The complexity of the plant immune system
The complexity of the plant immunity
system, comprising different mechanisms of resistance, was well known at the beginning
of this century. These mechanisms include diverse molecular monitoring systems that
perceive stresses-derived signals, as well as microbe-associated molecular patterns
(MAMPs) and effectors (avirulent proteins) from pathogens, which trigger specific
resistance responses upon perception by specific plant receptors (Jones and Dangl 2006). The evolution of such
monitoring systems has enabled plants to fine-tune their defensive responses
and to adapt their physiological response to environmental condition changes. Also,
it is well known that plant defensive responses are mediated by phytohormones, like
salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), which were initially
described as mainly required for plant resistance to biotrophic (SA) and nectrotrophic
(ET and JA) pathogens, respectively (Robert-Seilaniantz
et al. 2011). In recent years, other phytohormones have been added to
this list of “defensive hormones.” These include abscisic acid (ABA),
brassinosteroids, gibberellins, auxins, and more recently cytokinin, as shown in
recent articles and in our MPMI paper (Bacete
et al. 2020; Argueso et al. 2012; Gupta et al. 2020).
Two decades ago, the plant cell wall was considered
in the plant immunity field to be simply a structure displaying a physical defensive
role—a sort of passive barrier with no essential function in a complex plant
immune system. Nevertheless, it had been demonstrated by several groups that the
plant cell wall is a dynamic and highly regulated structure with several
important functions for plant growth and development. All plant cells have a primary
plant cell wall that is mainly composed of cellulose—which is the principal load-bearing
component—pectins, hemicelluloses, and structural
glycoproteins. In addition, cells that have completed their cellular expansion and
need to strengthen their structure for functional reasons (e.g., to form vessel
or fiber cells) generate a secondary cell wall that also includes lignin.
The plant cell wall is a prominent structure to manage mechanical stresses
caused by either internal (e.g., due to osmotic pressure) or external (e.g.,
caused by pathogen attack) physiological/environmental changes. Therefore, an
important question arose several years ago: how do plants perceive these
changes in their cell walls? In recent years, the status of the plant cell wall
has been shown to be constantly monitored through a series of cell wall integrity
(CWI) surveillance mechanisms (Bacete and Hamann
2020), and the wall has been found to be a source of damage-associated molecular
patterns (DAMPs), mainly of carbohydrate-based compositions, that trigger immune
responses (Bacete et al. 2018, 2020).
Exploring Arabidopsis
thaliana disease resistance to necrotrophic fungi: The initials
Early in the foundation
of our lab at the Technical University of Madrid (UPM, Spain), we performed several
screenings of A. thaliana mutant collections and quantitative trait loci
(QTL) analyses of ecotypes to identify novel genetic components of plant resistance
to necrotrophic fungi. The reason for this initial objective was that the genetic
determinants of plant resistance to this type of fungi were understudied,
despite the fact that necrotrophic fungi cause important yield loses in
agriculture. We selected for these initial screenings several strains from different
necrotrophic fungi species, but we particularly focused on one strain that had been
serendipitously isolated by Brigitte Mauch-Mani (Neuchatel University,
Switzerland) from Arabidopsis plants growing under her lab conditions (Ton and Mauch-Mani 2004). This necrotrophic fungal strain
was an ascomycete from Plectosphaerella cucumerina, which was very easy to
handle in the lab and, more importantly, gave very reproducible necrotrophic symptoms
in different A. thaliana genotypes. We named this isolate PcBMM to
recognize the contribution of Brigitte Mauch-Mani to its discovery. PcBMM
transformed our scientific goals, changed our view of the genetic determinants of
plants resistance to necrotrophic fungi, and revealed an unexpected and relevant
contribution of the plant cell wall to immunity. This exciting journey with Plectospherella
has recently reached an important milestone with the publication in MPMI
of the first sequence and annotation of the genomes and transcriptomes of three
Plectospherella strains (including PcBMM) with different lifestyles
on A. thaliana genotypes (Muñoz-Barrios
et al. 2020).
In our early screenings
with PcBMM we identified several A. thaliana cell wall mutants, like
ern1/irx1/lew2 (impaired in AtCESA8 required for secondary cell wall
cellulose synthesis), displaying broad-spectrum resistance to PcBMM and other
necrotrophic and biotrophic pathogens and enhanced resistance to abiotic
stresses. This initial finding was shocking, but exciting, since it was not in accordance
with the classical view of plant disease resistance to necrotrophic pathogens. Intriguingly,
the molecular bases of irx1 resistance did not seem to be dependent on canonical
defensive pathways (e.g., the expected ET and JA for necrotrophic fungi), but instead
it relied on novel mechanisms of immunity involving ABA signaling and antimicrobial
compounds like tryptophan-derived metabolites and peptides (Hernandez-Blanco et al. 2007). Moreover, in additional
screening aimed at deciphering PcBMM genetic resistance, we frequently found
A. thaliana mutants with enhanced susceptibility to PcBMM and additional
pathogens, which showed alterations in their plant cell wall composition. Among
these mutants were erecta (er), impaired in a receptor-like protein
kinase, and agb1, defective in the beta-subunit of Arabidopsis heterotrimeric
G protein, that display different biochemical alterations in their cell wall composition
compared with that of wild-type plants (Delgado-Cerezo
et al. 2012; Llorente et al., 2005; Sánchez-Rodríguez et al. 2009; Torres et al. 2013). These and additional exciting results suggested that ER and heterotrimeric
G proteins play roles in regulating novel mechanisms of disease resistance mediated
by the cell wall in addition to their function in plant development (Sánchez-Rodríguez
et al. 2009). The function of ER-mediated
pathway in immunity was further corroborated by the characterization of the role
in plant immunity of YODA, a mitogen-activated protein kinase kinase kinase (MAPK3)
functioning downstream of ER in plant development (Bergmann 2004). YODA has been found to regulate broad-spectrum disease resistance through
noncanonical defensive mechanisms involving cell wall-mediated resistance and the
up-regulation of the expression of specific protein receptors and peptidic DAMPs
(Sopeña-Torres et al. 2018; Téllez et al. 2020).
Exploring the
contribution of the plant cell wall to A. thaliana immunity: The ARR6
example
The findings described
above led us to the conviction that plant cell wall composition and integrity were
essential components of
A. thaliana immunity. To explore this regulatory
effect of the plant cell wall on
A. thaliana immunity and resistance to different
type of pathogens, we decided to follow a biased mutant screening approach and to
perform a detailed analysis of the resistance to different pathogens of a collection
of selected
Arabidopsis mutants impaired in either the primary or secondary
cell wall (Molina et al. 2020). In this biased
screening (
Figure 2),
an astonishingly high number of cell wall mutants showed altered susceptibility/resistance
to one or more of the pathogens tested compared with wild-type plants, further supporting
the key contribution of the plant cell wall to disease resistance (for further details are provided in Molina et al. 2020).
One of the cell wall
mutants with disease resistance alterations was impaired in the ARR6 gene (arr6), and it is
characterized in our MPMI paper (Bacete et al. 2020). Our first observations
on two mutant alleles (arr6-3 and arr6-2) of ARR6 indicated
that they both had alterations in their cell wall composition and in their resistance
to different pathogens with different colonization styles. ARR proteins have been
described as components of the cytokinin signaling pathway, which has previously
been involved in the modulation of some disease-resistance responses (Argueso et al. 2012; Gupta et al. 2020). In
our work recently published in MPMI (Bacete
et al. 2020), we describe a previously unknown function of ARR6 by
showing that ARR6 is actually a regulator of cell wall composition and of disease
resistance responses against different pathogens causing important diseases,
like the necrotrophic fungus PcBMM and the vascular bacterium Ralstonia
solanacearum. arr6 mutants, which do not have functional versions of the ARR6
gene, are more resistant to PcBMM fungus but more rapidly and intensely develop
the disease symptoms caused by the vascular bacterium R. solanacearum. In
contrast, plants that display higher levels of ARR6 expression (by
transgenic overexpression) than wild-type plants or arr6-3 (e.g.,
overexpressor and complementation lines, respectively) are more resistant to the
bacteria but more susceptible to the fungus. Transcriptomic and metabolomic analyses
revealed that, in arr6 plants, canonical
disease-resistance pathways, like those activated by defensive phytohormones, were
not altered, whereas immune responses triggered by microbe-associated molecular
patterns were slightly enhanced. As in previous research approaches performed
in the lab, our findings of the bases of the resistance were again original and
out of the canons, which is something that always triggers researchers’
curiosity, making our work even more intriguing and exciting, but also risky
for publication. Moreover, the characterization of ARR6-mediated resistance reinforced
our view of plant cell wall relevance in the modulation of specific immune responses
and confirmed the opportunities provided by plant cell wall mutants for the identification
of novel and uncharacterized mechanisms of plant immunity.
We next
hypothesized that some cell wall component could be released from arr6 walls due to their observed alteration in composition and that
this compound might function as DAMP that will be recognized by a plant
receptor, triggering immunity. However, cell walls are very complex, so we had
to obtain simpler cell wall fractions enriched in main biochemical components. Remarkably,
pectin-enriched cell wall fractions from arr6 plants activated more intense
immune responses than similar wall fractions from wild-type plants, suggesting that
the arr6 pectin fraction is enriched in wall-related DAMPs. The next step
we performed in this research area was the purification of these putative DAMP molecules
from arr6 pectin fractions. Actually, we have recently described the characterization
of the immune-active pectin fractions of arr6 by further fractionation
of it by chromatographic means (Mélida et al. 2020). These analyses pointed to the
role of pentose-based oligosaccharides in triggering plant immune responses in arr6.
Specifically, we have identified pentose-based oligosaccharide structures, such
as beta-1,4-xylooligosaccharides, with specific degrees of polymerization carrying
arabinose decorations. Remarkably, these novel DAMPs, which trigger immune responses
in Arabidopsis, also activate immune responses in crops and confer enhanced disease
resistance to pathogens, including necrotrophic fungi (Mélida et al. 2020). The characterization of these new cell wall-derived
plant DAMPs represents the culmination of a long journey across novel mechanisms
of plant immunity in our lab that led us to determine the significant and specific
contribution of plant cell wall composition in disease resistance. This has been
a journey that we initiated with the necrotrophic fungus PcBMM and that has
taken us to the identification of novel, noncanonical, cell wall-mediated mechanisms
of immunity of relevance for different sets of pathogens. We sincerely guess
that our research can contribute to the development of innovative crop protection
technologies to reach the desire goal of more sustainable agriculture that will
feed the growing human population.