Alexandre Robert-Seilaniantz

Hormone crosstalk in plant disease and defense : more than just JASMONATE-SALICYLATE antagonism

Until recently, most studies on the role of hormones in plant-pathogen interactions focused on salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). It is now clear that pathogen-induced modulation of signaling via other hormones contributes to virulence. A picture is emerging of complex crosstalk and induced hormonal changes that modulate disease and resistance, with outcomes dependent on pathogen lifestyles and the genetic constitution of the host. Recent progress has revealed intriguing similarities between hormone signaling mechanisms, with gene induction responses often achieved by derepression. Here, we report on recent advances, updating current knowledge on classical defense hormones SA, JA, and ET, and the roles of auxin, abscisic acid (ABA), cytokinins (CKs), and brassinosteroids in molding plant-pathogen interactions. We highlight an emerging theme that positive and negative regulators of these disparate hormone signaling pathways are crucial regulatory targets of hormonal crosstalk in disease and defense.

Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the “CHXCs”, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.

The microRNA miR393 re‐directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates

flg22 treatment increases levels of miR393, a microRNA that targets auxin receptors. Over-expression of miR393 renders plants more resistant to biotroph pathogens and more susceptible to necrotroph pathogens. In contrast, over-expression of AFB1, an auxin receptor whose mRNA is partially resistant to miR393 degradation, renders the plant more susceptible to biotroph pathogens. Here we investigate the mechanism by which auxin signalling and miR393 influence plant defence. We show that auxin signalling represses SA levels and signalling. We also show that miR393 represses auxin signalling, preventing it from antagonizing SA signalling. In addition, over-expression of miR393 increases glucosinolate levels and decreases the levels of camalexin. Further studies on pathogen interactions in auxin signalling mutants revealed that ARF1 and ARF9 negatively regulate glucosinolate accumulation, and that ARF9 positively regulates camalexin accumulation. We propose that the action of miR393 on auxin signalling triggers two complementary responses. First, it prevents suppression of SA levels by auxin. Second, it stabilizes ARF1 and ARF9 in inactive complexes. As a result, the plant is able to mount a full SA response and to re-direct metabolic flow toward the most effective anti-microbial compounds for biotroph resistance. We propose that miR393 levels can fine-tune plant defences and prioritize resources.

Evidence for suppression of immunity as a driver for genomic introgressions and host range expansion in races of Albugo candida, a generalist parasite

How generalist parasites with wide host ranges can evolve is a central question in parasite evolution. Albugo candida is an obligate biotrophic parasite that consists of many physiological races that each specialize on distinct Brassicaceae host species. By analyzing genome sequence assemblies of five isolates, we show they represent three races that are genetically diverged by ∼1%. Despite this divergence, their genomes are mosaic-like, with ∼25% being introgressed from other races. Sequential infection experiments show that infection by adapted races enables subsequent infection of hosts by normally non-infecting races. This facilitates introgression and the exchange of effector repertoires, and may enable the evolution of novel races that can undergo clonal population expansion on new hosts. We discuss recent studies on hybridization in other eukaryotes such as yeast, Heliconius butterflies, Darwins finches, sunflowers and cichlid fishes, and the implications of introgression for pathogen evolution in an agro-ecological environment. DOI: http://dx.doi.org/10.7554/eLife.04550.001

Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad-spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH-1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co-expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.

Both the Jasmonic Acid and the Salicylic Acid Pathways Contribute to Resistance to the Biotrophic Clubroot Agent Plasmodiophora brassicae in Arabidopsis

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 signaling-deficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(δ)-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.

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics

BackgroundNext Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10–20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5’ ends of mRNA or use of RNA ligations.ResultsWe have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3′ end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing.ConclusionsEXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.

Camalexin contributes to the partial resistance of Arabidopsis thaliana to the biotrophic soilborne protist Plasmodiophora brassicae

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.

A Biotic or Abiotic Stress

Throughout their lifespan plants are exposed to numerous biotic and abiotic stresses that may affect their normal growth, development and reproduction. In order to survive, they have evolved elaborate mechanisms to perceive and respond to each type of stress. A complex and still obscure network of interactions between hormones and genes expression allows the plant to fine tune the appropriate response. Each type of stress has received a great deal of attention and many important discoveries allow researchers to begin to unravel each signaling pathway. However, more and more evidence suggest that studying each response in isolation is an oversimplification. Indeed, plants are able to integrate multiple signals and respond to different stresses in a specific manner. Moreover, evidence indicates that each stress/pathway interacts with each other. Recent progress in transcriptome analysis and the construction of large databases centralizing microarray data from different laboratories, allows researchers to carry out comparative approaches. These types of approaches, revealed interesting and important networks. These give us the opportunity to understand the plant responses in a more comprehensive, integrative manner. In this chapter, we describe the role of different phyto-hormones in mediating various biotic and abiotic stress responses and also discuss the possible mechanisms by which they can provide tolerance to those stresses.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits

Structural Rearrangements can have unexpected effects on quantitative phenotypes. Surprisingly, these rearrangements can also be considered as... To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii, isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions.