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

How co-evolution has shaped the interaction between plants andtheir associated microbes remains a central question in organis-mic interactions (Bonfante & Genre, 2010; Delaux & Schor-nack, 2021). Plants have evolved a sophisticated and multilayeredimmune system to ward off potential microbial invaders (Jones& Dangl, 2006; Boller & Felix, 2009). In addition, pathogenshave developed mechanisms allowing them to enter living plants,colonise their tissues and overcome their defence responses.Pathogenicity factors can be either broadly conserved or speciesspecific and include regulators of cell signalling, gene expressionor development, as well as secreted effector molecules that modu-late the host environment (Jongeet al., 2011; Turr aet al., 2014;Weiberget al., 2014; Prestiet al., 2015; Ryder & Talbot, 2015;van der Does & Rep, 2017).A particularly destructive group of plant pathogens are thosecausing vascular wilt diseases, which infect the roots and colonisethe highly protected and nutrient poor niche of the xylem(Yadeta & Thomma, 2013). The ascomycete fungusFusariumoxysporum(Fo) represents a species complex with worldwidedistribution that provokes devastating losses in more than 150different crops (Deanet al., 2012). Fo exhibits a hemibiotrophlifestyle with an initial biotrophic phase characterised by intercel-lular growth in the root cortex, followed by invasion of the vascu-lature and transition to the necrotrophic phase resulting inmaceration and death of the colonised host (Redkaret al., 2021).In the soil, Fo is able to locate roots by sensing secreted plant per-oxidases via its sex pheromone receptors and the cell wallintegrity mitogen activated protein kinase (MAPK) pathway(Turr aet al., 2015). Once inside the root, the fungus secretes asmall regulatory peptide that mimics plant Rapid ALkalinisationFactor (RALF) to induce host alkalisation, which in turn activatesa conserved MAPK cascade that promotes plant invasive growth(Masachiset al., 2016). Additional pathogenicity determinantsinclude transcriptional regulators, fungus/plant cell wall remod-elling components or secondary metabolites, among others(Michielse & Rep, 2009).Individual Fo isolates exhibit host-specific pathogenicity,which is determined by lineage-specific (LS) chromosomes thatencode distinct repertoires of effectors known as Secreted inXylem (Six) (Maet al., 2010; van Damet al., 2016). Some Six proteins appear to primarily target plant defence responses, butcan also be recognised as avirulence factors by specific host recep-tors (Houtermanet al., 2009; Tintoret al., 2020). In addition tothe pathogenic forms, the Fo species complex (FOSC) alsoincludes endophytic isolates such as Fo47, which was isolatedfrom a natural disease suppressive soil (Alabouvette, 1986; Wanget al., 2020). Fo47 colonises plant roots without causing wiltsymptoms and functions as a biological control agent againstpathogenic Fo strains. How vascular wilt fungi such as Fo gainedthe ability to associate with plant hosts and evolved endophyticand pathogenic lifestyles remains poorly understood.The bryophyteMarchantia polymorpha(Mp) belongs to theancient lineage of liverworts and has emerged as the primenonvascular plant model for studying the evolution of molecularplant–microbe interactions (Evo-MPMI), due to its low geneticredundancy, the simplicity of its gene families and an accessiblemolecular genetic toolbox (Ishizakiet al., 2008; Lockhart, 2015;Bowmanet al., 2017; Upsonet al., 2018; Gimenez-Ibanezet al.,2019). Importantly, Mp possesses receptor-like kinases (RLKs),nucleotide binding, leucine-rich repeat receptors (NLRs) and sal-icylic acid (SA) pathway genes similar to those mediatingimmune signalling in angiosperms (Xueet al., 2012; Bowmanet al., 2017), therefore allowing the study of plant–microbe inter-actions across evolutionarily distant land plant lineages such asliverworts and eudicots, which diverged>450 million years ago(Ma) (Carellaet al., 2018). A current shortcoming of Mp is thatonly few pathogen infection models have been developed forin vitropathogenicity assays. These include the fungiXylariacubensisandColletotrichum sp1, the oomycetePhytophthorapalmivoraand the Gram-negative bacteriumPseudomonassyringae(Nelsonet al., 2018; Carellaet al., 2019; Gimenez-Ibanezet al., 2019). A survey of the Mp microbiome identified anumber of fungal endophytes, some of which can also act aspathogens (Matsuiet al., 2019; Nelson & Shaw, 2019). Whetherroot-infecting vascular wilt fungi can colonise this land plant lin-eage, which is evolutionarily distant to eudicots and lacks bothtrue roots and xylem, is currently unknown.Here we established a new pathosystem between Fo and Mp.We find that Fo isolates that are either endophytic or pathogenicon different crops (tomato, banana, cotton) are all able tocolonise and macerate the thallus of this nonvascular plant. Infec-tion of Mp by Fo requires fungal core pathogenicity factors,whereas LS effectors are dispensable suggesting that this vascularwilt fungus employs conserved mechanisms during infection ofevolutionarily distant plant lineages. We further show that thefungal transition from biotrophic intercellular growth tonecrotrophic maceration and sporulation, which on angiospermsrelies on host-specific factors promoting xylem invasion, occursdirectly on the nonvascular plant Mp

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.S.G-I was supported by a ‘‘Juan de la Cierva’’ fellowship from the Spanish Ministry for Science and Innovation. This work was funded by the Spanish Ministry for Science and Innovation grants BIO2010-21739, CSD2007-00057 and EUI2008- 03666 to R.S. J.P.R is an Australian Research Council Future Fellow (FT0992129)

The Receptor-Like Kinase SERK3/BAK1 Is Required for Basal Resistance against the Late Blight Pathogen Phytophthora infestans in Nicotiana benthamiana

BACKGROUND The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. PRINCIPAL FINDINGS We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.This work was supported by the Gatsby Charitable Foundation, BBSRC, Nuffield Foundation and the German Research Foundation (DFG). SS was supported by a personal research fellowship (SCHO1347/1-1). JPR is an Australian Research Council Future Fellow (FT0992129). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas syringae </it>is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. Pathogenicity of <it>P. syringae </it>strains is dependent on the type III secretion system, which secretes a suite of up to about thirty virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences. <it>P. syringae </it>pathovar <it>tabaci </it>naturally causes disease on wild tobacco, the model member of the Solanaceae, a family that includes many crop species as well as on soybean.</p> <p>Results</p> <p>We used the 'next-generation' Illumina sequencing platform and the Velvet short-read assembly program to generate a 145X deep 6,077,921 nucleotide draft genome sequence for <it>P. syringae </it>pathovar <it>tabaci </it>strain 11528. From our draft assembly, we predicted 5,300 potential genes encoding proteins of at least 100 amino acids long, of which 303 (5.72%) had no significant sequence similarity to those encoded by the three previously fully sequenced <it>P. syringae </it>genomes. Of the core set of Hrp Outer Proteins that are conserved in three previously fully sequenced <it>P. syringae </it>strains, most were also conserved in strain 11528, including AvrE1, HopAH2, HopAJ2, HopAK1, HopAN1, HopI, HopJ1, HopX1, HrpK1 and HrpW1. However, the <it>hrpZ1 </it>gene is partially deleted and <it>hopAF1 </it>is completely absent in 11528. The draft genome of strain 11528 also encodes close homologues of HopO1, HopT1, HopAH1, HopR1, HopV1, HopAG1, HopAS1, HopAE1, HopAR1, HopF1, and HopW1 and a degenerate HopM1'. Using a functional screen, we confirmed that <it>hopO1, hopT1, hopAH1</it>, <it>hopM1'</it>, <it>hopAE1</it>, <it>hopAR1</it>, and <it>hopAI1' </it>are part of the virulence-associated HrpL regulon, though the <it>hopAI1' </it>and <it>hopM1' </it>sequences were degenerate with premature stop codons. We also discovered two additional HrpL-regulated effector candidates and an HrpL-regulated distant homologue of <it>avrPto1</it>.</p> <p>Conclusion</p> <p>The draft genome sequence facilitates the continued development of <it>P. syringae </it>pathovar <it>tabaci </it>on wild tobacco as an attractive model system for studying bacterial disease on plants. The catalogue of effectors sheds further light on the evolution of pathogenicity and host-specificity as well as providing a set of molecular tools for the study of plant defence mechanisms. We also discovered several large genomic regions in <it>Pta </it>11528 that do not share detectable nucleotide sequence similarity with previously sequenced <it>Pseudomonas </it>genomes. These regions may include horizontally acquired islands that possibly contribute to pathogenicity or epiphytic fitness of <it>Pta </it>11528.</p

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 Receptor-Like Kinase SERK3/BAK1 Is Required for Basal Resistance against the Late Blight Pathogen Phytophthora infestans in Nicotiana benthamiana

Background: The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. Principal findings: We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. Conclusions/significance: We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1

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

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

BACKGROUND: The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. PRINCIPAL FINDINGS: We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE: We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1

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.Peer reviewe