The relationship between transgenerational acquired resistance and global DNA methylation in Arabidopsis.

Progeny of heavily diseased plants develop transgenerational acquired resistance (TAR). In Arabidopsis, TAR can be transmitted over one stress-free generation. Although DNA methylation has been implicated in the regulation of TAR, the relationship between TAR and global DNA methylation remains unknown. Here, we characterised the methylome of TAR-expressing Arabidopsis at different generations after disease exposure. Global clustering of cytosine methylation revealed TAR-related patterns in the F3 generation, but not in the F1 generation. The majority of differentially methylated positions (DMPs) occurred at CG context in gene bodies. TAR in F3 progeny after one initial generation of disease, followed by two stress-free generations, was lower than TAR in F3 progeny after three successive generations of disease. This difference in TAR effectiveness was proportional to the intensity of differential methylation at a sub-set of cytosine positions. Comparison of TAR-related DMPs with previously characterised cytosine methylation in mutation accumulation lines revealed that ancestral disease stress preferentially acts on methylation-labile cytosine positions, but also extends to methylation-stable positions. Thus, the TAR-related impact of ancestral disease extends beyond stochastic variation in DNA methylation. Our study has shown that the Arabidopsis epigenome responds globally to disease in previous generations and we discuss its contribution to TAR.

Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea.

Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction.IMPORTANCE Fungal fruiting bodies are formed by sexual reproduction. We studied the development of fruiting bodies ("apothecia") of the ubiquitous plant-pathogenic ascomycete Botrytis cinerea The role of mating type genes in apothecium development was investigated by targeted mutation. Two genes are essential for the initiation of sexual development; mutants in these genes are sterile. Two other genes were not essential for development of stipes; however, they were essential for stipes to develop a disk and produce sexual ascospores. We examined gene expression profiles during apothecium development, as well as in ascospores sampled from apothecia. We provide the first study ever of the transcriptome of pure ascospores in a filamentous fungus. The expression of numerous genes involved in plant infection was induced in the ascospores, implying that ascospores are developmentally primed for infection before their release from apothecia.

A gapless genome sequence of the fungus Botrytis cinerea.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

The role of DNA (de)methylation in immune responsiveness of Arabidopsis.

DNA methylation is antagonistically controlled by DNA methyltransferases and DNA demethylases. The level of DNA methylation controls plant gene expression on a global level. We have examined impacts of global changes in DNA methylation on the Arabidopsis immune system. A range of hypo-methylated mutants displayed enhanced resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), whereas two hyper-methylated mutants were more susceptible to this pathogen. Subsequent characterization of the hypo-methylated nrpe1 mutant, which is impaired in RNA-directed DNA methylation, and the hyper-methylated ros1 mutant, which is affected in DNA demethylation, revealed that their opposite resistance phenotypes are associated with changes in cell wall defence and salicylic acid (SA)-dependent gene expression. Against infection by the necrotrophic pathogen Plectosphaerella cucumerina, nrpe1 showed enhanced susceptibility, which was associated with repressed sensitivity of jasmonic acid (JA)-inducible gene expression. Conversely, ros1 displayed enhanced resistance to necrotrophic pathogens, which was not associated with increased responsiveness of JA-inducible gene expression. Although nrpe1 and ros1 were unaffected in systemic acquired resistance to Hpa, they failed to develop transgenerational acquired resistance against this pathogen. Global transcriptome analysis of nrpe1 and ros1 at multiple time-points after Hpa infection revealed that 49% of the pathogenesis-related transcriptome is influenced by NRPE1- and ROS1-controlled DNA methylation. Of the 166 defence-related genes displaying augmented induction in nrpe1 and repressed induction in ros1, only 25 genes were associated with a nearby transposable element and NRPE1- and/or ROS1-controlled DNA methylation. Accordingly, we propose that the majority of NRPE1- and ROS1-dependent defence genes are regulated in trans by DNA methylation.

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina.

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.

A novel Zn2 Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea.

D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2 Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative d-galacturonate transporters.

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.

Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Effector identification in the lettuce downy mildew Bremia lactucae by massively parallel transcriptome sequencing.

Lettuce downy mildew (Bremia lactucae) is a rapidly adapting oomycete pathogen affecting commercial lettuce cultivation. Oomycetes are known to use a diverse arsenal of secreted proteins (effectors) to manipulate their hosts. Two classes of effector are known to be translocated by the host: the RXLRs and Crinklers. To gain insight into the repertoire of effectors used by B. lactucae to manipulate its host, we performed massively parallel sequencing of cDNA derived from B. lactucae spores and infected lettuce (Lactuca sativa) seedlings. From over 2.3 million 454 GS FLX reads, 59 618 contigs were assembled representing both plant and pathogen transcripts. Of these, 19 663 contigs were determined to be of B. lactucae origin as they matched pathogen genome sequences (SOLiD) that were obtained from >270 million reads of spore-derived genomic DNA. After correction of cDNA sequencing errors with SOLiD data, translation into protein models and filtering, 16 372 protein models remained, 1023 of which were predicted to be secreted. This secretome included elicitins, necrosis and ethylene-inducing peptide 1-like proteins, glucanase inhibitors and lectins, and was enriched in cysteine-rich proteins. Candidate host-translocated effectors included 78 protein models with RXLR effector features. In addition, we found indications for an unknown number of Crinkler-like sequences. Similarity clustering of secreted proteins revealed additional effector candidates. We provide a first look at the transcriptome of B. lactucae and its encoded effector arsenal.

How do oomycete effectors interfere with plant life?

Oomycete genomes have yielded a large number of predicted effector proteins that collectively interfere with plant life in order to create a favourable environment for pathogen infection. Oomycetes secrete effectors that can be active in the host's extracellular environment, for example inhibiting host defence enzymes, or inside host cells where they can interfere with plant processes, in particular suppression of defence. Two classes of effectors are known to be host-translocated: the RXLRs and Crinklers. Many effectors show defence-suppressive activity that is important for pathogen virulence. A striking example is AVR3a of Phytophthora infestans that targets an ubiquitin ligase, the stabilisation of which may prevent host cell death. The quest for other effector targets and mechanisms is in full swing.

Identification of Hyaloperonospora arabidopsidis transcript sequences expressed during infection reveals isolate-specific effectors.

Biotrophic plant pathogens secrete effector proteins that are important for infection of the host. The aim of this study was to identify effectors of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) that are expressed during infection of its natural host Arabidopsis thaliana. Infection-related transcripts were identified from Expressed Sequence Tags (ESTs) derived from leaves of the susceptible Arabidopsis Ws eds1-1 mutant inoculated with the highly virulent Hpa isolate Waco9. Assembly of 6364 ESTs yielded 3729 unigenes, of which 2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted proteins were identified. Of these, 75 were found to be Hpa-specific and six isolate Waco9-specific. Among 42 putative effectors identified there were three Elicitin-like proteins, 16 Cysteine-rich proteins and 18 host-translocated RXLR effectors. Sequencing of alleles in different Hpa isolates revealed that five RXLR genes show signatures of diversifying selection. Thus, EST analysis of Hpa-infected Arabidopsis is proving to be a powerful method for identifying pathogen effector candidates expressed during infection. Delivery of the Waco9-specific protein RXLR29 in planta revealed that this effector can suppress PAMP-triggered immunity and enhance disease susceptibility. We propose that differences in host colonization can be conditioned by isolate-specific effectors.