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Phosphorus is an important macronutrient required for plant growth and development. It is absorbed by the roots in the form of inorganic phosphate (Pi). Under Pi limiting conditions, plants activate the Phosphate Starvation Response (PSR) system to enhance Pi acquisition. The NITROGEN LIMITATION ADAPTION (NLA) gene is a component of the Arabidopsis PSR, and its expression is post-transcriptionally regulated by miR827. We show that loss-of-function of NLA and MIR827 overexpression increases Pi level and enhances resistance to infection by the fungal pathogen Plectosphaerella cucumerina in Arabidopsis. Upon pathogen infection, high Pi plants (e.g. nla plants and wild type plants grown under high Pi supply) showed enhanced callose deposition. High Pi plants also exhibited superinduction of camalexin biosynthesis genes which is consistent with increased levels of camalexin during pathogen infection. Pathogen infection and treatment with fungal elicitors, triggered up-regulation of MIR827 and down-regulation of NLA expression. Under non-infection conditions, the nla plants showed increased levels of SA and JA compared with wild type plants, their levels further increasing upon pathogen infection. Overall, the outcomes of this study suggest that NLA plays a role in Arabidopsis immunity, while supporting convergence between Pi signaling and immune signaling in Arabidopsis., This research was supported by Project RTI2018-101275-B-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” to BSS, and Plan de Promoción de la Investigación Universitat Jaume I (UJI-B2019-2) and “Servicios Centrales de Instrumentación Científica (SCIC), Universitat Jaume I to VF. MDM received the support of a fellowship from ”La Caixa” Foundation (ID 100010434), fellowship code LCF/BQ/DI20/11780039. We also acknowledge financial support from the MCIN/AEI through the “Severo Ochoa Programme for Centres of Excellence in R&D” SEV‐2015–0533 and CEX2019-000902-S, and the CERCA Programme / “Generalitat de Catalunya”. B.V-T was a recipient of a Ph. D. grant from the MINECO/AEI (BES-2016-076289)., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host’s roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) in Arabidopsis thaliana upon infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All GSH-deficient mutants exhibited impaired activation of defense marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant susceptibility to CN infection and the production of camalexin upon nematode infection. These results suggest that GSH levels affects plant susceptibility to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defense against infection.

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants., eLife digest In the fight against harmful fungi and bacteria, plants have an arsenal of chemicals at their disposal. For instance, species in the crucifer family – which includes mustard, cabbages and the model plant Arabidopsis thaliana – can defend themselves with camalexin, a compound produced soon after the plant receives signals from its attacker. What controls this precise timing, however, is still unclear. For the genes that rule the production of camalexin to be ‘read’, interpreted, and ultimately converted into proteins, their DNA sequences first need to be physically accessible to the cell. This availability is controlled, in part, by adding or removing chemical groups onto histones, the spool-like structures which DNA wraps around. These precisely controlled modifications ultimately help to activate or repress a gene. Sometimes, activating and inhibiting chemical groups can be present in the same location, creating what is known as a bivalent chromatin domain. Zhao et al. investigated whether histone modifications regulate when A. thaliana produces camalexin in response to an attack. A combination of bioinformatics and experimental approaches highlighted two chemical modifications (one repressive, the other activating) which were present on the same histone, forming a previously unknown bivalent chromatin domain. Mutant plants which did not carry these modifications could not produce camalexin at the right time. Further experiments showed that under normal conditions, both histone modifications were present. However, when the plant was under attack, the level of repressive and activating modifications respectively decreased and increased, leading to gene activation. Together, the results by Zhao et al. suggest that both histone modifications are required for camalexin genes to respond appropriately to signals from a harmful agent. A deeper understanding of this new mechanism could, in turn, allow scientists to engineer crops that are better at resisting disease.

Significance To address mechanisms by which plants control their associated microorganisms, we designed a screen using a bacterial activity in soil. A detailed analysis of a candidate gene from the screen, CYP71A27, identified an additional root-specific component of synthesis of camalexin, a natural product involved in plant defense against pathogens. Loss of function of CYP71A27 affected not only the microbiota in soil but also interactions with single plant growth-promoting bacteria. The loss of the promoting effect in the mutants could be complemented chemically by adding camalexin to the bacteria. Thus, our study identified an additional role for camalexin in facilitating interaction with microbes in the rhizosphere and an additional gene for its synthesis., Plants in their natural ecosystems interact with numerous microorganisms, but how they influence their microbiota is still elusive. We observed that sulfatase activity in soil, which can be used as a measure of rhizosphere microbial activity, is differently affected by Arabidopsis accessions. Following a genome-wide association analysis of the variation in sulfatase activity we identified a candidate gene encoding an uncharacterized cytochrome P450, CYP71A27. Loss of this gene resulted in 2 different and independent microbiota-specific phenotypes: A lower sulfatase activity in the rhizosphere and a loss of plant growth-promoting effect by Pseudomonas sp. CH267. On the other hand, tolerance to leaf pathogens was not affected, which agreed with prevalent expression of CYP71A27 in the root vasculature. The phenotypes of cyp71A27 mutant were similar to those of cyp71A12 and cyp71A13, known mutants in synthesis of camalexin, a sulfur-containing indolic defense compound. Indeed, the cyp71A27 mutant accumulated less camalexin in the roots upon elicitation with silver nitrate or flagellin. Importantly, addition of camalexin complemented both the sulfatase activity and the loss of plant growth promotion by Pseudomonas sp. CH267. Two alleles of CYP71A27 were identified among Arabidopsis accessions, differing by a substitution of Glu373 by Gln, which correlated with the ability to induce camalexin synthesis and to gain fresh weight in response to Pseudomonas sp. CH267. Thus, CYP71A27 is an additional component in the camalexin synthesis pathway, contributing specifically to the control of plant microbe interactions in the root.

Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

Indole alkaloid camalexin has potential medicinal properties such as suppressing the viability of leukemic but not normal cells. Camalexin is not produced in plants and an external factor is required to activate its biosynthesis. In this work, we stimulated camalexin biosynthesis in Arabidopsis calli by blocking one of repressors of the jasmonate pathway, the jasmonate ZIM-domain protein 1 (JAZ1) by using amiRNA targeting JAZ1 gene transcripts. Inhibition of the JAZ1 gene led to an increase in camalexin content from trace amounts in control culture to 9 µg/g DW in the jaz1 line without affecting growth. In addition, JAZ1 silencing enhanced tolerance to cold stress with simultaneous increasing camalexin content up to 30 µg/g DW. Real-time quantitative PCR determination of marker gene expression showed that effects caused by the JAZ1 silencing might be realized through crosslinking JA, ROS, and abscisic acid signaling pathways. Thus, targeting the distal components of signaling pathways can be suggested as a tool for bioengineering of secondary metabolism, along with standard techniques for targeting biosynthetic genes or genes encoding transcription factors.

Plants are able to detect insect eggs deposited on leaves. In Arabidopsis, eggs of the butterfly species Pieris brassicae (common name large white) induce plant defenses and activate the salicylic acid (SA) pathway. We previously discovered that oviposition triggers a systemic acquired resistance (SAR) against the bacterial hemibiotroph pathogen Pseudomonas syringae. Here, we show that insect eggs or treatment with egg extract (EE) induce SAR against the fungal necrotroph Botrytis cinerea BMM and the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. This response is abolished in ics1, ald1 and fmo1, indicating that the SA pathway and the N-hydroxypipecolic acid (NHP) pathway are involved. Establishment of EE-induced SAR in distal leaves potentially involves tryptophan-derived metabolites, including camalexin. Indeed, SAR is abolished in the biosynthesis mutants cyp79B2 cyp79B3, cyp71a12 cyp71a13 and pad3-1, and camalexin is toxic to B. cinerea in vitro. This study reveals an interesting mechanism by which lepidopteran eggs interfere with plant–pathogen interactions.

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions., Zarattini et al. confirm the capacity of Lytic Polysaccharide Monooxygenases (LPMO) active on cellulose to trigger immune responses in Arabidopsis. These results bring insight to the field of cell wall modifying enzymes and their roles in plant defense mechanisms.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific phosphatases of the type 2C Ser/Thr protein phosphatase (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. Our work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant and ap2c1 mkp1 double mutant displayed enhanced wound-induced activation of MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants show an autoimmune-like response, associated with elevated levels the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. Interestingly, this phenotype is reduced in ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MPK6 misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MPK6 activity, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.HighlightDouble MAPK phosphatase mutant plants ap2c1 mkp1 exhibit constitutive, autoimmune-like stress responses, dependent on their substrate MAPK MPK6.

Temporal dynamics of gene expression underpins the responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at the target genes, and recruitment of the transcriptional machinery that includes transcription factors. As one of the most potent specialized metabolites inArabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, it remains unknown how the rapid activation of genes in this pathway upon a pathogen signal happens. By combining publicly available epigenomic data within vivochromatin mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, H3K27me3 (repression) and H3K18ac (activation), to form a bivalent chromatin. Mutants with reduced H3K27m3 or H3K18ac showed that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that this type of bivalent chromatin, which we name a kairostat, plays an important role controlling the timely induction of gene expression upon stimuli in plants.

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile (4OH-ICN), and indole glucosinolate (IGS), show broad-spectrum antifungal activity. However, the upstream regulators of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed a positive role of WRKY33 in host resistance to Alternaria brassicicola by directly regulating the transcription of genes involved in the biosynthesis and atypical hydrolysis of IGS both in Arabidopsis and Chinese kale. Indole-3-yl-methylglucosinolate (I3G) and 4-methoxyindole-3-yl-methylglucosinolate (4MI3G) are the main components of IGS. WRKY33 induces the expression of MYB51 and CYP83B1 which promotes the biosynthesis of I3G, the precursor of 4MI3G. Moreover, it also directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side chain modification of I3G to produce 4MI3G, which is in turn hydrolyzed by PEN2. However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp-metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.One-sentence SummaryPathogen-responsive WRKY33 directly regulates indole glucosinolates biosynthesis and atypical hydrolysis, conferring to host resistance to Alternaria brassicicola in Arabidopsis and Brassica crops.

Plants are exposed to regular diurnal rhythms of light and dark. Changes in the photoperiod by the prolongation of the light period cause photoperiod stress in short day-adapted Arabidopsis thaliana. Here we report on the transcriptional response to photoperiod stress of wild-type A. thaliana and photoperiod stress-sensitive cytokinin signalling and clock mutants. Transcriptomic changes induced by photoperiod stress included numerous changes in reactive oxygen (ROS)-related transcripts and showed a strong overlap with changes occurring in response to ozone stress and pathogen attack, which have in common the induction of an apoplastic oxidative burst. A core set of photoperiod stress-responsive genes has been identified, including salicylic acid (SA) biosynthesis and signalling genes. Genetic analysis revealed a central role for NPR1 in the photoperiod stress response as npr1-1 mutants were stress-insensitive. Photoperiod stress treatment led to a strong increase in camalexin levels which is consistent with shared photoperiod stress and pathogen response pathways. Photoperiod stress induced resistance of Arabidopsis plants to a subsequent infection by Pseudomonas syringae cv. tomato DC3000 indicating priming of the defence response. Together, photoperiod stress causes transcriptional reprogramming resembling plant pathogen defence responses and induces systemic acquired resistance in the absence of a pathogen.One sentence summaryPhotoperiod stress results in significant dynamic transcriptomic changes related to oxidative stress similar to those caused by pathogen attack and primes the defence response against a subsequent pathogen infection.

The metabolites produced in elicited leaves of the wild crucifer Neslia paniculata (L.) Desv. were investigated in abiotically stressed plants. Herein the phytoalexins camalexin, 7-methoxycamalexin and 6,7-dimethoxycamalexin together with the signalling metabolites arabidopsides A and D are reported. This is the first report of occurrence of 7-methoxy and 6,7-dimethoxycamalexins in planta and the third example of synchronized biosynthesis of phytoalexins and galacto-oxylipins. It is suggested that arabidopsides and structurally similar metabolites functioning as signalling molecules are likely to occur in a much larger number of plant species than originally hypothesized. N. paniculata is potentially useful to generate hybrid plants resistant to blackleg disease caused by Leptosphaeria maculans.

Ethylene response factor (ERF) Group VII members generally function in regulating plant growth and development, abiotic stress responses, and plant immunity in Arabidopsis; however, the details of the regulatory mechanism by which Group VII ERFs mediate plant immune responses remain elusive. Here, we characterized one such member, ERF72, as a positive regulator that mediates resistance to the necrotrophic pathogen Botrytis cinerea. Compared with the wild-type (WT), the erf72 mutant showed lower camalexin concentration and was more susceptible to B. cinerea, while complementation of ERF72 in erf72 rescued the susceptibility phenotype. Moreover, overexpression of ERF72 in the WT promoted camalexin biosynthesis and increased resistance to B. cinerea. We identified the camalexin-biosynthesis genes PAD3 and CYP71A13 and the transcription factor WRKY33 as target genes of ERF72. We also determined that MPK3 and MPK6 phosphorylated ERF72 at Ser151 and improved its transactivation activity, resulting in increased camalexin concentration and increased resistance to B. cinerea. Thus, ERF72 acts in plant immunity to coordinate camalexin biosynthesis both directly by regulating the expression of biosynthetic genes and indirectly by targeting WRKK33.

The clubroot of crucifers caused by P. brassicae is a very serious devastating disease in more than 88 countries of the world with average loss of 10–15% yield and 2–6% in oil content. The pathogen is obligate, non-axenic, microscopic, single celled, soil borne with dormant, well protected resting spores which can survive for more than 20 years. Its genome is small in size (24.2–25.5 mb) with high gene density. It has two stages of infections as primary root hair and secondary cortical cell infection. The pathogen is highly variable and exists as mixture of pathotypes in the soil and even in single clubbed roots. It produces morphological structures like resting spores, zoospores, and plasmodia. Its life cycle is very complex consisting of different zoosporic stages, formation of primary and secondary plasmodia and resting spores inside the host cells. Its ultra-structures have been revealed through electron and scanning electron microscopy of infected host galls. The occurrence, distribution, and pattern of P. brassicae pathotypes/ virulence have been characterized from more than 20 countries of the world. The pathotypes have been designated as serial numbers, triplicates, and letters. Molecular markers have been developed for rapid identification and detection of pathotypes infecting Brassica species. CR811 gene has been identified in pathotypes 3 and 5 which plays role in host resistance and pathogenicity. Various physical, chemical and biological components of the soil environment have effect on the survival, growth, and reproduction of this pathogen being solely soil borne in nature. Infection process by the pathogen is initiated after germination of resting spores triggered by host exudates at 20–25 °C temperature. The clubroot development and severity of pathogenesis is regulated by metabolites, growth regulators, sugars, hormones, enzymes, proteins, lipids, and glucosinolates. Auxin and cytokinin causes increased cell division and cell elongation. Genes Pb-Bripa and Pb PGA are expressed at early stages of infection. The pathogen successfully completes its disease cycle through three stages of life cycle. The disease development is affected by temperature, soil pH, soil type, rainfall, drainage, boron, calcium, nitrogen, inoculum potential, host resistance, weeds, cultural practices and interactions of soil environ. The change in status and metabolism of hormones and compounds like auxins, cytokinins, glucosinolates, camalexin, brassinosteroids, gibberellin, JA, ethylene, flavonoids, and sugars have effects on club root phenotypes. Host resistance is government by qualitative and quantitative genes. Several QTL’s have been identified and mapped on chromosomes of Brassica species conferring resistance. Club root resistance in Brassica species is inherited by many dominant, recessive, additive and complementary genes. Club root resistance have been transferred in Brassica species through marker assisted selection, interspecific hybridization, distant hybridization and mutational approaches. Histological and systemic resistance has been identified in Brassica species. Molecular mechanisms of pathogenesis, plasmodial formation, and resting spores germination have been revealed through expression of genes Y10, trehalose-6- phosphate synthase and PbSTKL1. The genes PbPP2A and PbHMG are expressed at the early phases of infection. Multiomics approaches have revealed expression of several genes for infection, pathogenesis, metabolic pathways and regulation of molecular mechanisms of host resistance. Clubroot control has been aimed to target all the three stages of disease cycle. Clubroot management has been advocated through integration of approaches like cultural, chemical, biological, use of judicious nutrition and host resistance. Several standardized procedures, protocols, method and techniques have been developed to get better insight in Plasmodiophora-crucifer’s host pathosystem. However, there is always scope of improvement in our technology to get desired results for breeding high yielding disease resistant Brassica varieties for wider cultivation.

Genome-wide analysis of plant-growth-promoting Pseudomonas fluorescens strain SS101 (PfSS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, we investigated the effects of sulfur metabolism in PfSS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with PfSS101 or with a mutant disrupted in the adenylsulfate reductase cysH, a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type PfSS101 or its cysH mutant revealed that sulfur assimilation in PfSS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargeted metabolomics revealed that cysH-mediated sulfur assimilation in PfSS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study showed the metabolic interconnectedness of plants and their root-associated microbiota.

In the last decade the importance of microbiota for plant performance has been overwhelmingly demonstrated. Plant microbiomes, both in roots and shoots, fulfill a plethora of functions that can strongly influence various plant traits. Metabolites are the main tools that plants use to actively shape their microbiome. Mechanistically, plants exude a complex mix of primary and secondary metabolites from roots, which serve as growth substrates for certain microbial strains, exert toxic effects on others, or act as signals in mediating the plant microbe interactions. Flavonoids and strigolactones have long been known to play important roles in enabling microbial symbiosis, even though their full complexity may not yet be fully elucidated. Recently, several other plant metabolites have also been implicated as important players mediating the interaction with the root microbiome, including molecules such as coumarins, camalexin and triterpenes. Because plants use such a wide range of chemical classes to shape their microbiome, mechanistic studies of plant-microbe interactions are increasingly applying high-throughput metabolomics techniques. In this review, we describe how metabolomics approaches have profiled the chemical complexity of plant rhizodeposits, and discuss how metabolomics can be utilized to functionally characterize specific metabolites that influence plant-microbe interactions. We will also discuss the advanced metabolite analyses that are needed to disentangle the complex metabolic interactions between plant hosts and their associated microbial communities.

Plants exude secondary metabolites from the roots to shape the composition and function of their microbiome. Many of these compounds are known for their anti-microbial activity and are part of the plant immunity, such as the indole-derived phytoalexin camalexin. Here we studied the dynamics of camalexin synthesis and exudation upon induction of Arabidopsis thaliana with a plant growth promotion bacteria Pseudomonas sp. CH267 or a bacterial pathogen Burkholderia glumae PG1. We show that while the camalexin accumulation and exudation is more rapidly but transiently induced upon interaction with the growth promoting strain, the pathogen induces a higher and more stable camalexin levels. The concentration of camalexin in shoots, roots and exudates is well correlated, triggering a question on the origin of the exuded camalexin. By combination of experiments with cut shoots and roots and grafting of wild type plant with mutants in camalexin synthesis we showed that while camalexin can be produced and released by both organs, in intact plant the exuded camalexin originates in the shoots. We show that camalexin synthesis in response to B. glumae PG1 is dependent on cooperation of four CYP71 genes and a loss of function of any of them reduces camalexin synthesis. In conclusion, camalexin synthesis seems to be controlled on a whole plant level and coordinated between shoots and roots.

The host range of parasites is an important factor in assessing the dynamics of disease epidemics. The evolution of pathogens to accommodate new hosts may lead to host range expansion, a process the molecular bases of which are largely enigmatic. The fungus Sclerotinia sclerotiorum parasitizes more than 400 plant species from diverse eudicot families while its close relative, S. trifoliorum, is restricted to plants from the Fabaceae family. We analyzed S. sclerotiorum global transcriptome reprogramming on hosts from six botanical families and reveal a flexible, host-specific transcriptional program driven by core and host-response co-expression (SPREx) gene clusters. We generated a chromosome-level genome assembly for S. trifoliorum and found near-complete gene space conservation in broad and narrow host range Sclerotinia species. However, S. trifoliorum showed increased sensitivity to the Brassicaceae defense compound camalexin. Inter-specific transcriptome analyses revealed a lack of transcriptional response to camalexin in S. trifoliorum and provide evidence that cis-regulatory variation associates with the genetic accommodation of Brassicaceae in the Sclerotinia host range. Our work demonstrates adaptive plasticity of a broad host range pathogen with specific responses to different host plants and demonstrates the co-existence of signatures for generalist and polyspecialist life styles in the genome of a plant pathogen. We reason that this mechanism enables the emergence of new disease with no or limited gene flow between strains and species, and could underlie the emergence of new epidemics originating from wild plants in agricultural settings.

Like animals, plants accommodate a rich diversity of microbes, typically without discernible disease symptoms. How their pathogenesis is prevented in the host remains obscure. Here, we show that the root-infecting fungus Colletotrichum fructicola of the C. gloeosporioides clade (CgE), isolated from field-grown healthy Brassicaceae plants, inhibits growth of pathogenic fungi in Arabidopsis thaliana, in a phosphate status-dependent manner. Loss of host ethylene signaling or phytoalexins, camalexin or indole glucosinolates, however, allows CgE to display pathogenesis, suggesting host contributions to endophytic CgE colonization and benefit. Compared to a closely-related C. gloeosporioides pathogen (CgP), CgE is characterized by genome expansion and >700 fungal genes (4.34%) specifically induced in the host roots when co-inoculated with CgP, including genes related to fungal secondary metabolism. This may underlie antimicrobial tolerance of CgE and its dominance over pathogenic fungi within the host, pointing to a role for fungus-fungus competition in asymptomatic fungal colonization in plants.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea. Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

International audience; The plant cell wall plays an important role in damage-associated molecular pattern-induced resistance to pathogens and herbivorous insects. Our current understanding of cell wall-mediated resistance is largely based on the degree of pectin methylesterification. However, little is known about the role of pectin acetylesterification in plant immunity. This study describes how one pectin-modifying enzyme, PECTIN ACETYLESTERASE 9 (PAE9), affects the Arabidopsis (Arabidopsis thaliana) transcriptome, secondary metabolome, and aphid performance. Electro-penetration graphs showed that Myzus persicae aphids established phloem feeding earlier on pae9 mutants. Whole-genome transcriptome analysis revealed a set of 56 differentially expressed genes (DEGs) between uninfested pae9-2 mutants and wild-type plants. The majority of the DEGs were enriched for biotic stress responses and down-regulated in the pae9-2 mutant, including PAD3 and IGMT2, involved in camalexin and indole glucosinolate biosynthesis, respectively. Relative quantification of more than 100 secondary metabolites revealed decreased levels of several compounds, including camalexin and oxylipins, in two independent pae9 mutants. In addition, absolute quantification of phytohormones showed that jasmonic acid (JA), jasmonoyl-Ile, salicylic acid, abscisic acid, and indole-3-acetic acid were compromised due to PAE9 loss of function. After aphid infestation, however, pae9 mutants increased their levels of camalexin, glucosinolates, and JA, and no long-term effects were observed on aphid fitness. Overall, these data show that PAE9 is required for constitutive up-regulation of defense-related compounds, but that it is not required for aphid-induced defenses. The signatures of phenolic antioxidants, phytoprostanes, and oxidative stress-related transcripts indicate that the processes underlying PAE9 activity involve oxidation-reduction reactions.

Robust nonhost resistance of Arabidopsis thaliana against the nonadapted hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive and CYP71A12/CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. Here we report that CYP71A12 and CYP71A13 are critical for Arabidopsis’ postinvasive resistance toward both the necrotrophic Alternaria brassicicola and the adapted hemibiotrophic C. higginsianum fungi. Metabolite analyses suggest that the production of indole-3-carboxylic acid derivatives (ICAs) and camalexin is induced upon pathogen invasion, while phenotypic comparison of cyp79B2 cyp79B3 and pen2 cyp71A12 cyp71A13 plants indicates that the contribution of ICAs to postinvasive resistance is dose-dependent. We also found that the disruption of intact pattern recognition receptor complex caused by bak1–5 mutation significantly reduced postinvasive resistance against C. tropicale and A. brassicicola, indicating that pattern recognition commonly contributes to this second defense-layer against pathogens with distinct infection strategies. However, the bak1–5 mutation had no detectable effects on Trp-metabolite accumulation triggered by pathogen invasion. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (i) bak1–5 insensitive Trp-metabolism that leads to antimicrobial secondary metabolites, and (ii) a bak1–5 sensitive immune pathway that activates the expression of antimicrobial proteins.

Phytoalexins are small antimicrobial molecules synthesized and accumulated by plants upon exposure to pathogens. Camalexin is an indole-derived phytoalexin, which is accumulated in plants including Arabidopsis thaliana, and other Brassicaceae, which plays a major role in disease resistance against fungal pathogens. The productivity of Brassica crops is adversely affected by Alternaria blight disease, which is caused by Alternaria brassicae. In Arabidopsis thaliana, MAP kinase signalling cascade is known to be involved in synthesis of camalexin, which contributes to disease resistance against a necrtrophic fungal pathogen, Botrytis cinerea. In the present study, MAPK signalling cascade leading to biosynthesis of camalexin that triggers defense responses in B. rapa upon exposure to the most devastating nectrophic fungus, Alternaria brassicae has been elucidated with the help of previously reported MAPK cascade in Arabidopsis thaliana, Molecular modelling, docking, and protein-protein interaction analysis of MAP kinases retrieved from Brassica rapa genome have been carried out to reveal the above cascade. The tertiary structure prediction of MAPKs obtained through molecular modelling revealed that all the protein models fulfil the criteria of being the stable structures. The molecular docking of predicted models for elucidating potential partners of MAPKs revealed strong interactions between MKK1, MKK4, MKK5, MAPK3 and MAPK6 with MKK9. The MAPK signalling cascade also shows different genes that express and play major role in camalexin biosynthesis in B. rapa during defense response to A. brassicae. The understanding of MAPK defense signaling pathway in B. rapa against devastating fungal pathogen Alternaria brassicae would help in devising strategies to develop disease resistance in Brassica crops.