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Abstract BackgroundIdentifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agentPhytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. ResultsMining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestanslife cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families withinP. infestansoften discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genesin vivo. ConclusionsWe established a large dataset of binding specificities forP. infestansTFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms. 

CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about ten-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R), which had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. 

Phytophthora is a long-established, well known and globally important genus of plant pathogens. Phylogenetic evidence has shown that the biologically distinct, obligate biotrophic downy mildews evolved from Phytophthora at least twice. Since, cladistically, this renders Phytophthora ‘paraphyletic’, it has been proposed that Phytophthora evolutionary clades be split into multiple genera (Runge et al. 2011; Crous et al. 2021; Thines et al. 2023; Thines 2024). In this letter, we review arguments for the retention of the generic name Phytophthora with a broad circumscription made by Brasier et al. (2022) and by many delegates at an open workshop organized by the American Phytopathological Society. We present our well-considered responses to this proposal in general terms and to the specific proposals for new genera; together with new information regarding the biological properties and mode of origin of the Phytophthora clades. We consider that the proposals for new genera are mostly non-rigorous and not supported by the scientific evidence. Further, given (1) the apparent lack of any distinguishing biological characteristics (synapomorphies) between the Phytophthora clades; (2) the fundamental monophyly of Phytophthora in the original Haeckelian sense; (3) the fact that paraphyly is not a justification for taxonomic splitting; and (4) the considerable likely damage to effective scientific communication and disease management from an unnecessary break-up of the genus, we report that Workshop delegates voted unanimously in favour of preserving the current generic concept and for seeking endorsement of this view by a working group of the International Commission on the Taxonomy of Fungi. 

ABSTRACT The oomycete Phytophthora capsici is a destructive pathogen of a wide range of vegetable hosts, especially peppers and cucurbits. A 94.17-Mb genome assembly was constructed using PacBio and Illumina data and annotated with support from transcriptome sequencing (RNA-Seq) reads. 

Abstract MotivationDe novo genome assembly is a challenging computational problem due to the high repetitive content of eukaryotic genomes and the imperfections of sequencing technologies (i.e. sequencing errors, uneven sequencing coverage and chimeric reads). Several assembly tools are currently available, each of which has strengths and weaknesses in dealing with the trade-off between maximizing contiguity and minimizing assembly errors (e.g. mis-joins). To obtain the best possible assembly, it is common practice to generate multiple assemblies from several assemblers and/or parameter settings and try to identify the highest quality assembly. Unfortunately, often there is no assembly that both maximizes contiguity and minimizes assembly errors, so one has to compromise one for the other. ResultsThe concept of assembly reconciliation has been proposed as a way to obtain a higher quality assembly by merging or reconciling all the available assemblies. While several reconciliation methods have been introduced in the literature, we have shown in one of our recent papers that none of them can consistently produce assemblies that are better than the assemblies provided in input. Here we introduce Novo&Stitch, a novel method that takes advantage of optical maps to accurately carry out assembly reconciliation (assuming that the assembled contigs are sufficiently long to be reliably aligned to the optical maps, e.g. 50 Kbp or longer). Experimental results demonstrate that Novo&Stitch can double the contiguity (N50) of the input assemblies without introducing mis-joins or reducing genome completeness. Availability and implementationNovo&Stitch can be obtained from https://github.com/ucrbioinfo/Novo_Stitch.