Shared infection strategy of a fungal pathogen across diverse lineages of land plants, the Fusarium example.

Plants engage with a wide variety of microorganisms either in parasitic or mutualistic relationships, which have helped them to adapt to terrestrial ecosystems. Microbial interactions have driven plant evolution and led to the emergence of complex interaction outcomes via suppression of host defenses by evolving pathogens. The evolution of plant-microbe interactions is shaped by conserved host and pathogen gene modules and fast-paced lineage-specific adaptability which determines the interaction outcome. Recent findings from different microbes ranging from bacteria, oomycetes, and fungi suggest recurrent concepts in establishing interactions with evolutionarily distant plant hosts, but also clade-specific adaptation that ultimately contributes to pathogenicity. Here, we revisit some of the latest features that illustrate shared colonization strategies of the fungal pathogen Fusarium oxysporum on distant plant lineages and lineage-specific adaptability of mini-chromosomal units encoding effectors, for shaping host-specific pathogenicity in angiosperms.

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.

Conserved secreted effectors contribute to endophytic growth and multihost plant compatibility in a vascular wilt fungus.

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum.

Root-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they can also infect nonvascular plants, which diverged from vascular plants over 450 million years ago. We established a pathosystem between the nonvascular plant Marchantia polymorpha (Mp) and the root-infecting vascular wilt fungus Fusarium oxysporum (Fo). On angiosperms, Fo exhibits exquisite adaptation to the plant xylem niche as well as host-specific pathogenicity, both of which are conferred by effectors encoded on lineage-specific chromosomes. Fo isolates displaying contrasting lifestyles on angiosperms - pathogenic vs endophytic - are able to infect Mp and cause tissue maceration and host cell killing. Using isogenic fungal mutants we define a set of conserved fungal pathogenicity factors, including mitogen activated protein kinases, transcriptional regulators and cell wall remodelling enzymes, that are required for infection of both vascular and nonvascular plants. Markedly, two host-specific effectors and a morphogenetic regulator, which contribute to vascular colonisation and virulence on tomato plants are dispensable on Mp. Collectively, these findings suggest that vascular wilt fungi employ conserved infection strategies on nonvascular and vascular plant lineages but also have specific mechanisms to access the vascular niche of angiosperms.

Immunity onset alters plant chromatin and utilizes EDA16 to regulate oxidative homeostasis.

Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation.

Omega hydroxylated JA-Ile is an endogenous bioactive jasmonate that signals through the canonical jasmonate signaling pathway.

Jasmonates are fatty acid derivatives that control several plant processes including growth, development and defense. Despite the chemical diversity of jasmonates, only jasmonoyl-L-isoleucine (JA-Ile) has been clearly characterized as the endogenous ligand of the jasmonate co-receptors (COI1-JAZs) in higher plants. Currently, it is accepted that ω-hydroxylation of JA-Ile leads to inactivation of the molecule. This study shows that ω-hydroxylated JA-Ile (12-OH-JA-Ile) retains bioactivity and signals through the canonical JA-pathway. The results suggest that 12-OH-JA-Ile differentially activates a subset of JA-Ile co-receptors that may control and/or modulate particular jasmonate dependent responses. It is proposed that after a strong immune response mediated by JA-Ile, the ω-hydroxylated form modulates JA-Ile activated processes thereby improving plant resilience.

An Evolutionarily Ancient Immune System Governs the Interactions between Pseudomonas syringae and an Early-Diverging Land Plant Lineage.

Evolutionary molecular plant-microbe interactions (EvoMPMI) is an emerging field bridging the gap between molecular phytopathology and evolutionary studies. EvoMPMI research is currently challenging due to the scarcity of pathogenic model systems in early-diverging land plants. Liverworts are among the earliest diverging land-plant lineages, and Marchantia polymorpha has emerged as a liverwort model for evolutionary studies. However, bacterial pathogens of Marchantia have not yet been discovered, and the molecular mechanisms controlling plant-pathogen interactions in this early-diverging land plant remain unknown. Here, we describe a robust experimental plant-bacterial pathosystem for EvoMPMI studies and discover that an ancient immune system governs plant-microbe interactions between M. polymorpha and the hemi-biotrophic pathogenic bacteria Pseudomonas syringae. We show that P. syringae pv tomato (Pto) DC3000, causal agent of tomato bacterial speck disease, colonizes M. polymorpha and activates typical hallmarks of plant innate immunity. Virulence of Pto DC3000 on M. polymorpha relies on effector activities inside liverwort cells, including conserved AvrPto and AvrPtoB functions. Host specificity analyses uncovered pathogenic differences among P. syringae strains, suggesting that M. polymorpha-P. syringae interactions are controlled by the genetic backgrounds of both host and pathogen. Finally, we show that ancient phytohormone defensive networks govern M. polymorpha-P. syringae interactions. Altogether, our results demonstrate that the basic structure of the plant immune system of extant angiosperms is evolutionarily ancient and conserved in early-diverging land plants. This basic immune system may have been instrumental for land colonization by the common ancestor of land plants.

Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2.

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade-off is a major challenge for obtaining broad-spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co-receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co-receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C-terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade-off in a crop, by spatially uncoupling the SA-JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas-based strategy for crop protection that could be readily implemented in the field.

Differential Suppression of Nicotiana benthamiana Innate Immune Responses by Transiently Expressed Pseudomonas syringae Type III Effectors.

The plant pathogen Pseudomonas syringae injects about 30 different virulence proteins, so-called effectors, via a type III secretion system into plant cells to promote disease. Although some of these effectors are known to suppress either pattern-triggered immunity (PTI) or effector-triggered immunity (ETI), the mode of action of most of them remains unknown. Here, we used transient expression in Nicotiana benthamiana, to test the abilities of type III effectors of Pseudomonas syringae pv. tomato (Pto) DC3000 and Pseudomonas syringae pv. tabaci (Pta) 11528 to interfere with plant immunity. We monitored the sequential and rapid bursts of cytoplasmic Ca2+ and reactive oxygen species (ROS), the subsequent induction of defense gene expression, and promotion of cell death. We found that several effector proteins caused cell death, but independently of the known plant immune regulator NbSGT1, a gene essential for ETI. Furthermore, many effectors delayed or blocked the cell death-promoting activity of other effectors, thereby potentially contributing to pathogenesis. Secondly, a large number of effectors were able to suppress PAMP-induced defense responses. In the majority of cases, this resulted in suppression of all studied PAMP responses, suggesting that these effectors target common elements of PTI. However, effectors also targeted different steps within defense pathways and could be divided into three major groups based on their suppressive activities. Finally, the abilities of effectors of both Pto DC3000 and Pta 11528 to suppress plant immunity was conserved in most but not all cases. Overall, our data present a comprehensive picture of the mode of action of these effectors and indicate that most of them suppress plant defenses in various ways.

JAZ2 controls stomata dynamics during bacterial invasion.

Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants. Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Δjas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs. Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs.

The Proteasome Acts as a Hub for Plant Immunity and Is Targeted by Pseudomonas Type III Effectors.

Recent evidence suggests that the ubiquitin-proteasome system is involved in several aspects of plant immunity and that a range of plant pathogens subvert the ubiquitin-proteasome system to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis (Arabidopsis thaliana). Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv tomato DC3000 (Pst) and Pseudomonas syringae pv maculicola ES4326. Both proteasome subunits are required for pathogen-associated molecular pattern-triggered immunity responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR In addition, we show that Pst inhibits proteasome activity in a type III secretion-dependent manner. A screen for type III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1, and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome, leading to its inhibition. Thus, the proteasome is an essential component of pathogen-associated molecular pattern-triggered immunity and SAR, which is targeted by multiple bacterial effectors.

Redundancy and specificity in jasmonate signalling.

Jasmonates (JAs) are essential phytohormones regulating plant development and environmental adaptation. Many components of the JA-signalling pathway have been identified. However, our insight into the mechanisms by which a single bioactive JA hormone can regulate a myriad of physiological processes and provide specificity in the response remains limited. Recent findings on molecular components suggest that, despite apparent redundancy, specificity is achieved by (1) distinct protein-protein interactions forming unique JAZ/transcription factor complexes, (2) discrete spatiotemporal expression of specific components, (3) variable hormone thresholds for the formation of multiple JA receptor complexes and (4) integration of several signals by JA-pathway components. The molecular modularity that is thereby created enables a single bioactive hormone to specifically modulate multiple JA-outputs in response to different environmental and developmental cues.

How Microbes Twist Jasmonate Signaling around Their Little Fingers.

Plant immunity relies on a complex network of hormone signaling pathways in which jasmonic acid (JA) plays a central role. Successful microbial pathogens or symbionts have developed strategies to manipulate plant hormone signaling pathways to cause hormonal imbalances for their own benefit. These strategies include the production of plant hormones, phytohormone mimics, or effector proteins that target host components to disrupt hormonal signaling pathways and enhance virulence. Here, we describe the molecular details of the most recent and best-characterized examples of specific JA hormonal manipulation by microbes, which exemplify the ingenious ways by which pathogens can take control over the plant's hormone signaling network to suppress host immunity.

FILAMENTOUS FLOWER Is a Direct Target of JAZ3 and Modulates Responses to Jasmonate.

The plant hormone jasmonate (JA) plays an important role in regulating growth, development, and immunity. Activation of the JA-signaling pathway is based on the hormone-triggered ubiquitination and removal of transcriptional repressors (JASMONATE-ZIM DOMAIN [JAZ] proteins) by an SCF receptor complex (SCF(COI1)/JAZ). This removal allows the rapid activation of transcription factors (TFs) triggering a multitude of downstream responses. Identification of TFs bound by the JAZ proteins is essential to better understand how the JA-signaling pathway modulates and integrates different responses. In this study, we found that the JAZ3 repressor physically interacts with the YABBY (YAB) family transcription factor FILAMENTOUS FLOWER (FIL)/YAB1. In Arabidopsis thaliana, FIL regulates developmental processes such as axial patterning and growth of lateral organs, shoot apical meristem activity, and inflorescence phyllotaxy. Phenotypic analysis of JA-regulated responses in loss- and gain-of-function FIL lines suggested that YABs function as transcriptional activators of JA-triggered responses. Moreover, we show that MYB75, a component of the WD-repeat/bHLH/MYB complex regulating anthocyanin production, is a direct transcriptional target of FIL. We propose that JAZ3 interacts with YABs to attenuate their transcriptional function. Upon perception of JA signal, degradation of JAZ3 by the SCF(COI1) complex releases YABs to activate a subset of JA-regulated genes in leaves leading to anthocyanin accumulation, chlorophyll loss, and reduced bacterial defense.

Novel players fine-tune plant trade-offs.

Jasmonates (JAs) are essential signalling molecules that co-ordinate the plant response to biotic and abiotic challenges, as well as co-ordinating several developmental processes. Huge progress has been made over the last decade in understanding the components and mechanisms that govern JA perception and signalling. The bioactive form of the hormone, (+)-7-iso-jasmonyl-L-isoleucine (JA-Ile), is perceived by the COI1-JAZ co-receptor complex. JASMONATE ZIM DOMAIN (JAZ) proteins also act as direct repressors of transcriptional activators such as MYC2. In the emerging picture of JA-Ile perception and signalling, COI1 operates as an E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S proteasome, thereby derepressing transcription factors such as MYC2, which in turn activate JA-Ile-dependent transcriptional reprogramming. It is noteworthy that MYCs and different spliced variants of the JAZ proteins are involved in a negative regulatory feedback loop, which suggests a model that rapidly turns the transcriptional JA-Ile responses on and off and thereby avoids a detrimental overactivation of the pathway. This chapter highlights the most recent advances in our understanding of JA-Ile signalling, focusing on the latest repertoire of new targets of JAZ proteins to control different sets of JA-Ile-mediated responses, novel mechanisms of negative regulation of JA-Ile signalling, and hormonal cross-talk at the molecular level that ultimately determines plant adaptability and survival.

Rational design of a ligand-based antagonist of jasmonate perception.

(+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile) regulates developmental and stress responses in plants. Its perception involves the formation of a ternary complex with the F-box COI1 and a member of the JAZ family of co-repressors and leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the COI1-JAZ co-receptor with higher affinity than JA-Ile. On the basis of the co-receptor structure, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists. One derivative, coronatine-O-methyloxime (COR-MO), has strong activity in preventing the COI1-JAZ interaction, JAZ degradation and the effects of JA-Ile or COR on several JA-mediated responses in Arabidopsis thaliana. Moreover, it potentiates plant resistance, preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for plant biology research, our results underscore its biotechnological potential for safer and sustainable agriculture.

The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis.

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

bHLH003, bHLH013 and bHLH017 are new targets of JAZ repressors negatively regulating JA responses.

Cell reprogramming in response to jasmonates requires a tight control of transcription that is achieved by the activity of JA-related transcription factors (TFs). Among them, MYC2, MYC3 and MYC4 have been described as activators of JA responses. Here we characterized the function of bHLH003, bHLH013 and bHLH017 that conform a phylogenetic clade closely related to MYC2, MYC3 and MYC4. We found that these bHLHs form homo- and heterodimers and also interact with JAZ repressors in vitro and in vivo. Phenotypic analysis of JA-regulated processes, including root and rosette growth, anthocyanin accumulation, chlorophyll loss and resistance to Pseudomonas syringae, on mutants and overexpression lines, suggested that these bHLHs are repressors of JA responses. bHLH003, bHLH013 and bHLH017 are mainly nuclear proteins and bind DNA with similar specificity to that of MYC2, MYC3 and MYC4, but lack a conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Moreover, expression of bHLH017 is induced by JA and depends on MYC2, suggesting a negative feed-back regulation of the activity of positive JA-related TFs. Our results suggest that the competition between positive and negative TFs determines the output of JA-dependent transcriptional activation.