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The identification of several fructan exohydrolases (FEHs, EC 3.2.1.80) in non‐fructan accumulating plants raised the question of their roles. FEHs may be defense‐related proteins involved in the interactions with fructan‐accumulating microorganisms. Since known defense‐related proteins are upregulated by defense‐related phytohormones, we tested the hypothesis that FEHs of non‐fructan accumulating plants are upregulated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) using the model plant Arabidopsis thaliana and the agronomically relevant and genetically related species Brassica napus. By sequence homologies with the two known FEH genes of A. thaliana, At6‐FEH, and At6&1‐FEH, the genes coding for the putative B. napus FEHs, Bn6‐FEH and Bn6&1‐FEH, were identified. Plants were treated at root level with SA, methyl jasmonate (MeJA) or 1‐aminocyclopropane‐1‐carboxylic acid (ACC). The transcript levels of defense‐related and FEH genes were measured after treatments. MeJA and ACC did not upregulate FEHs, while HEL (HEVEIN‐LIKE PREPROTEIN) expression was enhanced by both phytohormones. In both species, the expression of AOS, encoding a JA biosynthesis enzyme, was enhanced by MeJA and that of the defensine PDF1.2 and the ET signaling transcription factor ERF1/2 by ACC. In contrast, SA not only increased the expression of genes encoding antimicrobial proteins (PR1 and HEL) and the defense‐related transcription factor WRKY70 but also that of FEH genes, in particular 6&1‐FEH genes. This result supports the putative role of FEHs as defense‐related proteins. Genotypic variability of SA‐mediated FEH regulation (transcript level and activities) was observed among five varieties of B. napus, suggesting different susceptibilities toward fructan‐accumulating pathogens. [ABSTRACT FROM AUTHOR]

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Nitrogen fertilization has been reported to influence the development of clubroot, a root disease of Brassicaceae species, caused by the obligate protist Plasmodiophora brassicae. Our previous works highlighted that low-nitrogen fertilization induced a strong reduction of clubroot symptoms in some oilseed rape genotypes. To further understand the underlying mechanisms, the response to P. brassicae infection was investigated in two genotypes "Yudal" and HD018 harboring sharply contrasted nitrogen-driven modulation of resistance toward P. brassicae. Targeted hormone and metabolic profiling, as well as RNA-seq analysis, were performed in inoculated and non-inoculated roots at 14 and 27 days post-inoculation, under high and low-nitrogen conditions. Clubroot infection triggered a large increase of SA concentration and an induction of the SA gene markers expression whatever the genotype and nitrogen conditions. Overall, metabolic profiles suggested that N-driven induction of resistance was independent of SA signaling, soluble carbohydrate and amino acid concentrations. Low-nitrogen-driven resistance in "Yudal" was associated with the transcriptional regulation of a small set of genes, among which the induction of NRT2 - and NR -encoding genes. Altogether, our results indicate a possible role of nitrate transporters and auxin signaling in the crosstalk between plant nutrition and partial resistance to pathogens. [ABSTRACT FROM AUTHOR]

Nitrogen fertilization can affect the susceptibility of Brassica napus to the telluric pathogen Plasmodiophora brassicae. Our previous works highlighted that the influence of nitrogen can strongly vary regarding plant cultivar/pathogen strain combinations, but the underlying mechanisms are unknown. The present work aims to explore how nitrogen supply can affect the molecular physiology of P. brassicae through its life epidemiological cycle. A time-course transcriptome experiment was conducted to study the interaction, under two conditions of nitrogen supply, between isolate eH and two B. napus genotypes (Yudal and HD-018), harboring (or not harboring) low nitrogen-conditional resistance toward this isolate (respectively). P. brassicae transcriptional patterns were modulated by nitrogen supply, these modulations being dependent on both host-plant genotype and kinetic time. Functional analysis allowed the identification of P. brassicae genes expressed during the secondary phase of infection, which may play a role in the reduction of Yudal disease symptoms in low-nitrogen conditions. Candidate genes included pathogenicity-related genes ("NUDIX," "carboxypeptidase," and "NEP-proteins") and genes associated to obligate biotrophic functions of P. brassicae. This work illustrates the importance of considering pathogen's physiological responses to get a better understanding of the influence of abiotic factors on clubroot resistance/susceptibility. [ABSTRACT FROM AUTHOR]

Clubroot, caused by Plasmodiophora brassicae Woronin, is one of the most important diseases of oilseed rape (Brassica napus L.). The rapid erosion of monogenic resistance in clubroot-resistant (CR) varieties underscores the need to diversify resistance sources controlling disease severity and traits related to pathogen fitness, such as resting spore production. The genetic control of disease index (DI) and resting spores per plant (RSP) was evaluated in a doubled haploid (DH) population consisting of 114 winter oilseed rape lines, obtained from the cross 'Aviso' × 'Montego,' inoculated with P. brassicae isolate "eH." Linkage analysis allowed the identification of three quantitative trait loci (QTLs) controlling DI (PbBn_di_A02, PbBn_di_A04, and PbBn_di_C03). A significant decrease in DI was observed when combining effects of the three resistance alleles at these QTLs. Only one QTL, PbBn_rsp_C03, was found to control RSP, reducing resting spore production by 40%. PbBn_rsp_C03 partially overlapped with PbBn_di_C03 in a nucleotide-binding leucine-rich repeat (NLR) gene-containing region. Consideration of both DI and RSP in breeding for clubroot resistance is recommended for the long-term management of this disease. [ABSTRACT FROM AUTHOR]

Plant disease resistance is often under quantitative genetic control. Thus, in a given interaction, plant cellular responses to infection are influenced by resistance or susceptibility alleles at different loci. In this study, a genetic linkage analysis was used to address the complexity of the metabolic responses of Brassica napus roots to infection by Plasmodiophora brassicae. Metabolome profiling and pathogen quantification in a segregating progeny allowed a comparative mapping of quantitative trait loci (QTLs) involved in resistance and in metabolic adjustments. Distinct metabolic modules were associated with each resistance QTL, suggesting the involvement of different underlying cellular mechanisms. This approach highlighted the possible role of gluconasturtiin and two unknown metabolites in the resistance conferred by two QTLs on chromosomes C03 and C09, respectively. Only two susceptibility biomarkers (glycine and glutathione) were simultaneously linked to the three main resistance QTLs, suggesting the central role of these compounds in the interaction. By contrast, several genotype-specific metabolic responses to infection were genetically unconnected to resistance or susceptibility. Likewise, variations of root sugar profiles, which might have influenced pathogen nutrition, were not found to be related to resistance QTLs. This work illustrates how genetic metabolomics can help to understand plant stress responses and their possible links with disease. [ABSTRACT FROM AUTHOR]

Summary: Nitrogen (N) availability can impact plant resistance to pathogens by the regulation of plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered to be the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or nitrate‐deficient medium. N deficiency led to enhanced or reduced susceptibility depending on the plant genotype. Focusing on four genotypes displaying contrasting responses, we determined the impact of N deficiency on plant growth and shoot N concentration, and performed expression analyses on N‐ and defence‐related genes, as well as the quantification of soluble phenolics and different amino acids in roots. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation or to mechanisms previously identified to be involved in plant resistance. Furthermore, our studies highlight a role of glutamine in mediating the susceptibility to A. euteiches in M. truncatula. [ABSTRACT FROM AUTHOR]

Highlight Modifications in glutamine synthetase OsGS1-2 expression and fungal pathogenicity underlie nitrogen-induced susceptibility to rice blast. Understanding why nitrogen fertilization increase the impact of many plant diseases is of major importance. The interaction between Magnaporthe oryzae and rice was used as a model for analyzing the molecular mechanisms underlying Nitrogen-Induced Susceptibility (NIS). We show that our experimental system in which nitrogen supply strongly affects rice blast susceptibility only slightly affects plant growth. In order to get insights into the mechanisms of NIS, we conducted a dual RNA-seq experiment on rice infected tissues under two nitrogen fertilization regimes. On the one hand, we show that enhanced susceptibility was visible despite an over-induction of defense gene expression by infection under high nitrogen regime. On the other hand, the fungus expressed to high levels effectors and pathogenicity-related genes in plants under high nitrogen regime. We propose that in plants supplied with elevated nitrogen fertilization, the observed enhanced induction of plant defense is over-passed by an increase in the expression of the fungal pathogenicity program, thus leading to enhanced susceptibility. Moreover, some rice genes implicated in nitrogen recycling were highly induced during NIS. We further demonstrate that the OsGS1-2 glutamine synthetase gene enhances plant resistance to M. oryzae and abolishes NIS and pinpoint glutamine as a potential key nutrient during NIS. [ABSTRACT FROM AUTHOR]

Background: The induction of alcohol fermentation in roots is a plant adaptive response to flooding stress and oxygen deprivation. Available transcriptomic data suggest that fermentation-related genes are also frequently induced in roots infected with gall forming pathogens, but the biological significance of this induction is unclear. In this study, we addressed the role of hypoxia responses in Arabidopsis roots during infection by the clubroot agent Plasmodiophora brassicae. Results: The hypoxia-related gene markers PYRUVATE DECARBOXYLASE 1 (PDC1), PYRUVATE DECARBOXYLASE 2 (PDC2) and ALCOHOL DEHYDROGENASE 1 (ADH1) were induced during secondary infection by two isolates of P. brassicae, eH and e2. PDC2 was highly induced as soon as 7 days post inoculation (dpi), i.e., before the development of gall symptoms, and GUS staining revealed that ADH1 induction was localised in infected cortical cells of root galls at 21 dpi. Clubroot symptoms were significantly milder in the pdc1 and pdc2 mutants compared with Col-0, but a null T-DNA insertional mutation of ADH1 did not affect clubroot susceptibility. The Arg/N-end rule pathway of ubiquitin-mediated proteolysis controls oxygen sensing in plants. Mutants of components of this pathway, ate1 ate2 and prt6, that both exhibit constitutive hypoxia responses, showed enhanced clubroot symptoms. In contrast, gall development was reduced in quintuple and sextuple mutants where the activity of all oxygen-sensing Group VII Ethylene Response Factor transcription factors (ERFVIIs) is absent (erfVII and prt6 erfVII). Conclusions: Our data demonstrate that the induction of PDC1 and PDC2 during the secondary infection of roots by P. brassicae contributes positively to clubroot development, and that this is controlled by oxygen-sensing through ERFVIIs. The absence of any major role of ADH1 in symptom development may also suggest that PDC activity could contribute to the formation of galls through the activation of a PDH bypass. [ABSTRACT FROM AUTHOR]

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site. [ABSTRACT FROM AUTHOR]

The role of salicylic acid (SA) and jasmonic acid (JA) signaling in resistance to root pathogens has been poorly documented. We assessed the contribution of SA and JA to basal and partial resistance of Arabidopsis to the biotrophic clubroot agent Plasmodiophora brassicae. SA and JA levels as well as the expression of the SA-responsive genes PR2 and PR5 and the JA-responsive genes ARGAH2 and THI2.1 were monitored in infected roots of the accessions Col-0 (susceptible) and Bur-0 (partially resistant). SA signaling was activated in Bur-0 but not in Col-0. The JA pathway was weakly activated in Bur-0 but was strongly induced in Col-0. The contribution of both pathways to clubroot resistance was then assessed using exogenous phytohormone application and mutants affected in SA or JA signaling. Exogenous SA treatment decreased clubroot symptoms in the two Arabidopsis accessions, whereas JA treatment reduced clubroot symptoms only in Col-0. The cpr5-2 mutant, in which SA responses are constitutively induced, was more resistant to clubroot than the corresponding wild type, and the JA signalingdeficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(d)-acetylornithine biosynthesis in infected Col-0 roots. The 35S::NATA1 and nata1 lines displayed reduced or enhanced clubroot symptoms, respectively, thus suggesting that in Col-0 this pathway was involved in the JA-mediated basal clubroot resistance. Overall, our data support the idea that, depending on the Arabidopsis accession, both SA and JA signaling can play a role in partial inhibition of clubroot development in compatible interactions with P. brassicae. [ABSTRACT FROM AUTHOR]

Camalexin has been reported to play defensive functions against several pathogens in Arabidopsis. In this study, we investigated the possible role of camalexin accumulation in two Arabidopsis genotypes with different levels of basal resistance to the compatible eH strain of the clubroot agent Plasmodiophora brassicae. Camalexin biosynthesis was induced in infected roots of both Col-0 (susceptible) and Bur-0 (partially resistant) accessions during the secondary phase of infection. However, the level of accumulation was four-to-seven times higher in Bur-0 than Col-0. This was associated with the enhanced transcription of a set of camalexin biosynthetic P450 genes in Bur-0: CYP71A13, CYP71A12, and CYP79B2. This induction correlated with slower P. brassicae growth in Bur-0 compared to Col-0, thus suggesting a relationship between the levels of camalexin biosynthesis and the different levels of resistance. Clubroot-triggered biosynthesis of camalexin may also participate in basal defense in Col-0, as gall symptoms and pathogen development were enhanced in the pad3 mutant (Col-0 genetic background), which is defective in camalexin biosynthesis. Clubroot and camalexin responses were then studied in Heterogeneous Inbred Families (HIF) lines derived from a cross between Bur-0 and Col-0. The Bur/Col allelic substitution in the region of the previously identified clubroot resistance QTL PbAt5.2 (Chromosome 5) was associated with both the enhanced clubroot-triggered induction of camalexin biosynthesis and the reduced P. brassicae development. Altogether, our results suggest that high levels of clubroot-triggered camalexin biosynthesis play a role in the quantitative control of partial resistance of Arabidopsis to clubroot. [ABSTRACT FROM AUTHOR]

The model brown alga Ectocarpus siliculosus undergoes extensive transcriptomic changes in response to abiotic stress, many of them related to primary metabolism and particularly to amino acid biosynthesis and degradation. In this study we seek to improve our knowledge of the mechanisms underlying the stress tolerance of this alga, in particular with regard to compatible osmolytes, by examining the effects of these changes on metabolite concentrations. We performed extensive metabolic profiling (urea, amino acids, sugars, polyols, organic acids, fatty acids) of Ectocarpus samples subjected to short-term hyposaline, hypersaline and oxidative stress, and integrated the results with previously published transcriptomic data. The most pronounced changes in metabolite concentrations occurred under hypersaline stress: both mannitol and proline were accumulated, but their low final concentrations indicate that, in this stress condition, both compounds are not likely to significantly contribute to osmoregulation at the level of the entire cell. Urea and trehalose were not detected in any of our samples. We also observed a shift in fatty acid composition from n-3 to n-6 fatty acids under high salinities, and demonstrated the salt stress-induced accumulation of small amounts of γ-aminobutyric acid (GABA). GABA could be synthesized in E. siliculosus through a salt stress-induced putrescine-degradation pathway. [ABSTRACT FROM AUTHOR]

Subterranean environments are characterized by the quasi absence of thermal variations (±1°C within a year), and organisms living in these biotopes for several millions of years, such as hypogean crustaceans, can be expected to have adapted to this very stable habitat. As hypogean organisms experience minimal thermal variation in their native biotopes, they should not be able to develop any particular cold adaptations to cope with thermal fluctuations. Indeed, physiological responses of organisms to an environmental stress are proportional to the amplitude of the stress they endure in their habitats. Surprisingly, previous studies have shown that a population of an aquatic hypogean crustacean, Niphargus rhenorhodanensis, exhibited a high level of cold hardiness. Subterranean environments thus appeared not to be following the classical above-mentioned theory. To confirm this counter-example, we studied seven karstic populations of N. rhenorhodanensis living in aquifers at approximately 10°C all year round and we analysed their behavioural, metabolic and biochemical responses during cold exposure (3°C). These seven populations showed reduced activities, and some cryoprotective molecules were accumulated. More surprisingly, the amplitude of the response varied greatly among the seven populations, despite their exposure to similar thermal conditions. Thus, the overall relationship that can be established between the amplitude of thermal variations and cold-hardiness abilities of ectotherm species may be more complex in subterranean crustaceans than in other arthropods. [ABSTRACT FROM AUTHOR]

Abstract: The Arabidopsis thaliana AtHMA4 is a P1B-type ATPase that clusters with the Zn/Cd/Pb/Co subgroup. It has been previously shown, by heterologous expression and the study of AtHMA4 knockout or overexpressing lines in Arabidopsis [1–3], that AtHMA4 is implicated in zinc homeostasis and cadmium tolerance. Here, we report the study of the heterologous expression of AtHMA4 in the yeast Saccharomyces cerevisiae. AtHMA4 expression resulted in an increased tolerance to Zn, Cd and Pb and to a phenotypic complementation of hypersensitive mutants. In contrast, an increased sensitivity towards Co was observed. An AtHMA4::GFP fusion protein was observed in endocytic vesicles and at the yeast plasma membrane. Mutagenesis of the cysteine and glutamate residues from the N-ter degenerated heavy metal binding domain impaired the function of AtHMA4. It was also the case when the C-ter His11 stretch was deleted, giving evidence that these amino acids are essential for the AtHMA4 binding/translocation of metals. [Copyright &y& Elsevier]

AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem. [Copyright &y& Elsevier]

The Arabidopsis thaliana AtHMA3 protein belongs to the P1B-adenosine triphosphatase (ATPase) transporter family, involved in heavy metal transport. Functional expression of AtHMA3 phenotypically complements the Cd/Pb-hypersensitive yeast strain Δycf1, but not the Zn-hypersensitive mutant Δzrc1. AtHMA3-complemented Δycf1 cells accumulate the same amount of cadmium as YCF1-complemented Δycf1 cells or wild-type cells, suggesting that AtHMA3 carries out an intracellular sequestration of Cd. A mutant of AtHMA3 altered in the P-ATPase phosphorylation domain did not complement Δycf1, suggesting that metal transport rather than chelation is involved. The fusion protein AtHMA3::green fluorescent protein (GFP) is localized at the vacuole, consistent with a role in the influx of cadmium into the vacuolar compartment. In A. thaliana, the mRNA of AtHMA3 was detected mainly in roots, old rosette leaves and cauline leaves. The expression levels were not affected by cadmium or zinc treatments. [Copyright &y& Elsevier]

Plant metabolomics is a set of fast-moving, analytical and chemometric tools and methods for plant functional genomics, phenotyping and systems biology. This multidisciplinary "omics" science can deliver qualitative and quantitative data that provide a detailed description of biochemical systems that are influenced by environmental changes, such as plant-pathogen interactions. This chapter examines key insights and recent outputs in the field of phytopathological metabolomics with a specific focus on the perturbations of primary compounds involved in central metabolism and infection-related metabolites including phytohormones. Priming of plant immune responses is also considered through the angle of metabolomics. Furthermore, the chapter highlights the recurring challenges in the distinction of host and pathogen metabolomes and the hurdles of metabolome annotation. The chapter concludes with perspectives indicating key avenues in ongoing efforts to decode metabolic landscapes of plants under biotic stress conditions. [ABSTRACT FROM AUTHOR]