Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete is highly variable and rapidly overcomes resistance genes and fungicides. The use of multiple read types results in a high-quality, near-chromosome-scale, consensus assembly. Flow cytometry plus resequencing of 30 field isolates, 37 sexual offspring, and 19 asexual derivatives from single multinucleate sporangia demonstrates a high incidence of heterokaryosis in B . lactucae . Heterokaryosis has phenotypic consequences on fitness that may include an increased sporulation rate and qualitative differences in virulence. Therefore, selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.
Pathogens that infect plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen Bremia lactucae by searching the predicted proteome for the WY domain, a structural fold found in effectors that has been implicated in immune suppression as well as effector recognition by host resistance proteins. We predicted 55 WY domain containing proteins in the genome of B. lactucae and found substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis of 21 effectors containing WY domains revealed 11 that elicited cell death on wild accessions and domesticated lettuce lines containing resistance genes, indicative of recognition of these effectors by the host immune system. Only two of the 11 recognized effectors contained the canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.
An RNAseq-based analysis of the cassava plants inoculated with Xam allowed the identification of transcriptional upregulation of genes involved in jasmonate metabolism, phenylpropanoid biosynthesis and putative targets for a TALE. Cassava bacterial blight, a disease caused by the gram-negative bacterium Xanthomonas axonopodis pv. manihotis (Xam), is a major limitation to cassava production worldwide and especially in developing countries. The molecular mechanisms underlying cassava susceptibility to Xam are currently unknown. To identify host genes and pathways leading to plant susceptibility, we analyzed the transcriptomic responses occurring in cassava plants challenged with either the non-pathogenic Xam strain ORST4, or strain ORST4(TALE1 Xam ) which is pathogenic due to the major virulence transcription activator like effector TALE1 Xam . Both strains triggered similar responses, i.e., induction of genes related to photosynthesis and phenylpropanoid biosynthesis, and repression of genes related to jasmonic acid signaling. Finally, to search for TALE1 Xam virulence targets, we scanned the list of cassava genes induced upon inoculation of ORST4(TALE1 Xam ) for candidates harboring a predicted TALE1 Xam effector binding element in their promoter. Among the six genes identified as potential candidate targets of TALE1 Xam a gene coding for a heat shock transcription factor stands out as the best candidate based on their induction in presence of TALE1 Xam and contain a sequence putatively recognized by TALE1 Xam .
Many plant-pathogenic bacteria suppress pathogen-associated molecular pattern (PAMP)-triggered immunity by injecting effector proteins into the host cytoplasm during infection through the type III secretion system (TTSS). This type III secretome plays an important role in bacterial pathogenicity in susceptible hosts. Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight, injects several effector proteins into the host cell, including TALE1(Xam) . This protein is a member of the Transcriptional Activator-Like effector (TALE) protein family, formerly known as the AvrBs3/PthA family. TALE1(Xam) has 13.5 tandem repeats of 34 amino acids each, as well as two nuclear localization signals and an acidic activation domain at the C-terminus. In this work, we demonstrate the importance of TALE1(Xam) in the pathogenicity of Xam. We use versions of the gene that lack different domains in the protein in structure-function studies to show that the eukaryotic domains at the 3' end are critical for pathogenicity. In addition, we demonstrate that, similar to the characterized TALE proteins from other Xanthomonas species, TALE1(Xam) acts as a transcriptional activator in plant cells. This is the first report of the identification of a TALE in Xam, and contributes to our understanding of the pathogenicity mechanisms employed by this bacterium to colonize and cause disease in cassava.
Pathogens infecting plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen, Bremia lactucae, using comparative genomics and bioinformatics to search for the WY domain. This conserved structural element is found in Phytophthora effectors and some other oomycete pathogens; it has been implicated in the immune-suppressing function of these effectors as well as their recognition by host resistance proteins. We predicted 54 WY domain containing proteins in isolate SF5 of B. lactucae that have substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis effectors containing WY domains revealed eleven out of 21 that triggered necrosis, which is characteristic of the immune response on wild accessions and domesticated lettuce lines containing resistance genes. Only two of the eleven recognized effectors contained a canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.Author SummaryThere is a microscopic battle that takes place at the molecular level during infection of plants and animals by pathogens. Some of the weapons that pathogens battle with are known as “effectors,” which are secreted proteins that enter host cells to alter physiology and suppress the immune system. Effectors can also be a liability for plant pathogens because plants have evolved ways to recognize these effectors, triggering a defense response leading to localized cell death, which prevents the spread of the pathogen. Here we used computer models to predict effectors from the genome of Bremia lactucae, the causal agent of lettuce downy mildew. Three effectors were demonstrated to suppress the basal immune system of lettuce. Eleven effectors were recognized by one or more resistant lines of lettuce. In addition to contributing to our understanding of the mechanisms of pathogenesis, this study of effectors is useful for breeding disease resistant lettuce, decreasing agricultural reliance on fungicides.
Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete pathogen is highly variable and has rapidly overcome resistance genes and fungicides deployed in attempts to control it. The described high-quality genome assembly of B. lactucae provides the foundation for detailed understanding of this economically important pathogen. The biotrophic nature of B. lactucae coupled with high levels of heterozygosity and the recently expanded repeat content made genome assembly challenging. The combined use of multiple read types, including synthetic long reads, single molecule sequences, and Hi-C, resulted in a high-quality, chromosome-scale, consensus assembly of this diploid organism. Phylogenetic analysis supports polyphyly in the downy mildews consistent with the biotrophic mode of pathogenesis evolving more than once in the Peronosporaceae. Flow cytometry plus resequencing of 30 field isolates as well as sexual offspring and asexual derivatives from multinucleate single sporangia demonstrated a high incidence of heterokaryosis in B. lactucae. Heterokaryons have phenotypic differences and increased fitness compared to homokaryotic derivatives. Consequently, B. lactucae exhibits somatic hybrid vigor and selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.
BackgroundGenetic maps are an important resource for validation of genome assemblies, trait discovery, and breeding. Next generation sequencing has led to the production of high-density genetic maps constructed with 10,000s of markers. Most current approaches require a genome assembly of the organism under study, or a close relative, to identify markers. Our Assembly Free Linkage Analysis Pipeline (AFLAP) removes this requirement by using uniquely segregating k-mers as markers. This removes potential biases including preferential read alignment and variant calling.ResultsWe tested AFLAP using 100 F2 individuals of Arabidopsis thaliana, sequenced to low coverage. Genetic maps generated using k-mers specific for each parent contained over 130,000 markers that were concordant with the genomic assembly of the five chromosomes. AFLAP was then applied to 83 F1 individuals, sequenced to >5x coverage, generated from two crosses of the oomycete Bremia lactucae. The genetic map contained over 90,000 markers ordered in 19 large linkage groups. The genetic map was used to fragment, order, orient, and scaffold the reference assembly of B. lactucae, resulting in a much improved, genetically consistent genome assembly.ConclusionsAFLAP can therefore be used to generate high density linkage maps and improve draft genome assemblies of non-model organisms when a mapping population sequenced to adequate coverage is available. The genetic maps produced for B. lactucae were accurately aligned to the reference assembly and resulted in significant improvements of the assembly.
Our assembly-free linkage analysis pipeline (AFLAP) identifies segregating markers as k-mers in the raw reads without using a reference genome assembly for calling variants and provides genotype tables for the construction of unbiased, high-density genetic maps without a genome assembly. AFLAP is validated and contrasted to a conventional workflow using simulated data. AFLAP is applied to whole genome sequencing and genotype-by-sequencing data of F1, F2, and recombinant inbred populations of two different plant species, producing genetic maps that are concordant with genome assemblies. The AFLAP-based genetic map for Bremia lactucae enables the production of a chromosome-scale genome assembly.
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