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Significance Most plants resist most plant pathogens. Barley resists wheat-infecting powdery mildew races (and vice versa), and both barley and wheat resist potato late blight. Such “nonhost” resistance could result because the pathogen fails to suppress defense or triggers innate immunity due to failure to evade detection. Albugo candida causes white rust on most Brassicaceae, and we investigated Arabidopsis NHR to Brassica-infecting races. Transgressive segregation for resistance in Arabidopsis recombinant inbred lines revealed genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. Some of these NLR-encoding genes confer resistance to white rust in Brassica sp. This genetic method thus provides a route to reveal resistance genes for crops, widening the pool from which such genes might be obtained., Arabidopsis thaliana accessions are universally resistant at the adult leaf stage to white rust (Albugo candida) races that infect the crop species Brassica juncea and Brassica oleracea. We used transgressive segregation in recombinant inbred lines to test if this apparent species-wide (nonhost) resistance in A. thaliana is due to natural pyramiding of multiple Resistance (R) genes. We screened 593 inbred lines from an Arabidopsis multiparent advanced generation intercross (MAGIC) mapping population, derived from 19 resistant parental accessions, and identified two transgressive segregants that are susceptible to the pathogen. These were crossed to each MAGIC parent, and analysis of resulting F2 progeny followed by positional cloning showed that resistance to an isolate of A. candida race 2 (Ac2V) can be explained in each accession by at least one of four genes encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. An additional gene was identified that confers resistance to an isolate of A. candida race 9 (AcBoT) that infects B. oleracea. Thus, effector-triggered immunity conferred by distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to these crop pathogens.

To understand the population genetics of structural variants (SVs), and their effects on phenotypes, we developed an approach to mapping SVs, particularly transpositions, segregating in a sequenced population, and which avoids calling SVs directly. The evidence for a potential SV at a locus is indicated by variation in the counts of short-reads that map anomalously to the locus. These SV traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between an SV trait at one locus and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3x) population sequence data from 488 recombinant inbredArabidopsisgenomes, we identified 6,502 segregating SVs. Remarkably, 25% of these were transpositions. Whilst many SVs cannot be delineated precisely, PCR validated 83% of 44 predicted transposition breakpoints. We show that specific SVs may be causative for quantitative trait loci for germination, fungal disease resistance and other phenotypes. Further we show that the phenotypic heritability attributable to sequence anomalies differs from, and in the case of time to germination and bolting, exceeds that due to standard genetic variation. Gene expression within SVs is also more likely to be silenced or dysregulated. This approach is generally applicable to large populations sequenced at low-coverage, and complements the prevalent strategy of SV discovery in fewer individuals sequenced at high coverage.

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.

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, Darwin's 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, eLife digest Many microorganisms live as parasites inside another living organism, and gain nutrients at their host's expense. Plants and animals have immune systems that serve to protect against this kind of exploitation, but successful parasites have evolved ways to avoid detection by their hosts' immune systems, and/or to suppress hosts' defence mechanisms. Parasites often avoid detection by releasing molecules that interfere with specific aspects of a host's immune system. The same molecules, however, can be recognised by the immune systems of other species and trigger defence responses that eradicate the parasites; this explains why most parasites can colonise only a limited number of host species. It is less clear how parasites evolve to become ‘generalists’ that can infect many host species. However, some generalist parasites have several distinct subgroups—each of which is specialised to infect a limited number of host species. Albugo candida is a generalist parasite that infects over 200 plant species, including mustard greens, oilseed and vegetable crops. Even though its looks and its lifestyle resemble those of a fungus, A. candida is actually an oomycete: a group of organisms that are more closely related to golden-brown algae than they are to fungi. About 24 subgroups (or ‘races’) of this generalist parasite have been identified to date, but it remains unclear how these subgroups have evolved. McMullan, Gardiner et al. tested different isolates of A. candida—four from southeast England and one from western Canada—which had been collected from different plant species and confirmed that each could only infect a narrow range of plant hosts. Next, the genome sequences of these five A. candida strains were assembled and compared. This analysis revealed that the five strains represented three distinct subgroups (or races) of A. candida. Moreover, some parts of one subgroup's genome were most similar to those found in a second subgroup; and other parts were more like sections of the third subgroup's genome. McMullan, Gardiner et al. point out that such ‘mosaic-like genomes’ indicate crossbreeding between the different subgroups. But as A. candida must infect a plant in order to reproduce, and different subgroups infect different host plants, how can different subgroups meet in order to mate and reproduce? In answer to this question, McMullan, Gardiner et al. showed that a plant that is infected with one subgroup of A. candida becomes susceptible to co-infection with other subgroups, including those that couldn't normally infect this plant species on their own. These findings reveal that generalist parasites can therefore evolve new subgroups via a mechanism that is similar to the way that crossbreeding (or hybridization) between existing species can lead to the evolution of new species. DOI: http://dx.doi.org/10.7554/eLife.04550.002

International audience; 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.

The HopPtoF locus in Pseudomonas syringae pv. tomato DC3000 harbors two genes, ShcF and HopF2 (previously named ShcFPto and HopFPto), that encode a type III chaperone and a cognate effector protein, respectively. The HopF2 gene has a rare initiation codon, ATA that was reported to be functional only in mitochondrial genes. Here, we report that the native HopPtoF locus of DC3000 confers an avirulence function in tobacco W38 plants, indicating that the ATA start codon directs the synthesis of a functional effector. However, disruption of HopF2 in DC3000 genome did not alter the bacterial virulence in tomato plants. The HopPtoF locus displayed a measurable virulence activity in two strains of P. syringae pv. tomato when the ATA start codon was changed to ATG, and this change also elevated the avirulence function in W38 plants. HopF2 contains a putative myristoylation site. Mutational analysis indicated that this site is required for plasma membrane localization and virulence and avirulence activities of HopF2.

Background Next 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. Results We 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. Conclusions EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-341) contains supplementary material, which is available to authorized users.

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., Author Summary Plant pathogens that cannot grow except on their hosts are called obligate biotrophs. How such biotrophy evolves is poorly understood. In this study, we sequenced the genome of the obligate biotroph white rust pathogen (Albugo laibachii, Oomycota) of Arabidopsis. From comparisons with other oomycete plant pathogens, diatoms, and the human pathogen Plasmodium falciparum, we reveal a loss of important metabolic enzymes. We also reveal the appearance of defence-suppressing “effectors”, some carrying motifs known from other oomycete effectors, and discover and experimentally verify a novel class of effectors that share a CHXC motif within 50 amino acids of the signal peptide cleavage site. Obligate biotrophy involves an intimate association within host cells at the haustorial interface (where the parasite penetrates the host cell's cell wall), where nutrients are acquired from the host and effectors are delivered to the host. We found that A. laibachii, like Hyaloperonospora arabidopsidis and Plasmodium falciparum, lacks molybdopterin-requiring biosynthetic pathways, suggesting relaxed selection for retention of, or even selection against, this pathway. We propose that when defence suppression becomes sufficiently effective, hosts become such a reliable source of nutrients that a free-living phase can be lost. These mechanisms leading to obligate biotrophy and host specificity are relevant not only to plant pathogenic oomycetes but also to human pathogens.