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

Figure 1. Schema of Arabidopsis thaliana cell wall composition and wall roles in disease resistance responses. Primary cell walls are composed of cellulose and different pectins and hemicelluloses. Secondary cell walls are reinforced with lignin. Cell wall function in disease resistance to pathogens as a first passive physical barrier that pathogens need to overcome for infection progression, but also as a constantly monitored dynamic structure whose integrity is perceived by different molecular systems. The plant cell wall is also a source of metabolites and proteins with direct activity against pathogens and of signaling molecules, such as damage-associated molecular patterns (DAMPs), that trigger immunity responses.

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 However, this outdated view has evolved to a novel concept that considers the plant cell wall as a dynamic structure regulating different processes of plant immunity and development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

Figure 2. Selection of cell wall mutants and biased resistance screening performed. Collection of cell wall mutants tested included well-known mutants and putative cell wall mutants impaired in either genes encoding proteins implied in the biosynthesis or remodeling of plant cell wall or genes expressed during cell wall biogenesis processes (Pesquet et al. 2005). The resistance of these mutants to four different type of pathogens with different colonization styles was determined. A significant number of mutants showed altered susceptibility/resistance to one or more pathogens compared with wild-type plants (additional details provided in Molina et al. 2020).

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.

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