A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli.

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Assembly, annotation, and comparison of Macrophomina phaseolina isolates from strawberry and other hosts.

BACKGROUND: Macrophomina phaseolina is a fungal plant pathogen with a broad host range, but one genotype was shown to exhibit host preference/specificity on strawberry. This pathogen lacked a high-quality genome assembly and annotation, and little was known about genomic differences among isolates from different hosts. RESULTS: We used PacBio sequencing and Hi-C scaffolding to provide nearly complete genome assemblies for M. phaseolina isolates representing the strawberry-specific genotype and another genotype recovered from alfalfa. The strawberry isolate had 59 contigs/scaffolds with an N50 of 4.3 Mb. The isolate from alfalfa had an N50 of 5.0 Mb and 14 nuclear contigs with half including telomeres. Both genomes were annotated with MAKER using transcript evidence generated in this study with over 13,000 protein-coding genes predicted. Unique groups of genes for each isolate were identified when compared to closely related fungal species. Structural comparisons between the isolates reveal large-scale rearrangements including chromosomal inversions and translocations. To include isolates representing a range of pathogen genotypes, an additional 30 isolates were sequenced with Illumina, assembled, and compared to the strawberry genotype assembly. Within the limits of comparing Illumina and PacBio assemblies, no conserved structural rearrangements were identified among the isolates from the strawberry genotype compared to those from other hosts, but some candidate genes were identified that were largely present in isolates of the strawberry genotype and absent in other genotypes. CONCLUSIONS: High-quality reference genomes of M. phaseolina have allowed for the identification of structural changes associated with a genotype that has a host preference toward strawberry and will enable future comparative genomics studies. Having more complete assemblies allows for structural rearrangements to be more fully assessed and ensures a greater representation of all the genes. Work with Illumina data from additional isolates suggests that some genes are predominately present in isolates of the strawberry genotype, but additional work is needed to confirm the role of these genes in pathogenesis. Additional work is also needed to complete the scaffolding of smaller contigs identified in the strawberry genotype assembly and to determine if unique genes in the strawberry genotype play a role in pathogenicity.

Detection of Fusarium oxysporum f. sp. fragariae from Infected Strawberry Plants.

Isolates of the Fusarium oxysporum species complex have been characterized as plant pathogens that commonly cause vascular wilt, stunting, and yellowing of the leaves in a variety of hosts. F. oxysporum species complex isolates have been grouped into formae speciales based on their ability to cause disease on a specific host. F. oxysporum f. sp. fragariae is the causal agent of Fusarium wilt of strawberry and has become a threat to production as fumigation practices have changed in California. F. oxysporum f. sp. fragariae is polyphyletic and limited genetic markers are available for its detection. In this study, next-generation sequencing and comparative genomics were used to identify a unique genetic locus that can detect all of the somatic compatibility groups of F. oxysporum f. sp. fragariae identified in California. This locus was used to develop a TaqMan quantitative polymerase chain reaction assay and an isothermal recombinase polymerase amplification (RPA) assay that have very high sensitivity and specificity for more than 180 different isolates of the pathogen tested. RPA assay results from multiple field samples were validated with pathogenicity tests of recovered isolates.

Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and Soil.

Macrophomina phaseolina is a broad-host-range fungus that shows some degree of host preference on strawberry, and causes symptoms that include crown rot and root rot. Recently, this pathogen has affected strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan polymerase chain reaction assay which is able to quantify as little as 2 to 3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.

Transcriptome and Small RNAome Dynamics during a Resistant and Susceptible Interaction between Cucumber and Downy Mildew.

Cucumber ( L.) downy mildew, caused by the obligate oomycete pathogen (Berk. and Curt.) Rostov., is the primary factor limiting cucumber production. Although sources of resistance have been identified, such as plant introduction line PI 197088, the genes and processes involved in mediating resistance are still unknown. In the current study, we conducted a comprehensive transcriptome and small RNAome analysis of a resistant (PI 197088) and susceptible ('Vlaspik') cucumber during a time course of infection using Illumina sequencing. We identified significantly differentially expressed (DE) genes within and between resistant and susceptible cucumber leaves over a time course of infection. Weighted gene correlation network analyses (WGCNA) created coexpression modules containing genes with unique expression patterns between Vlaspik and PI 197088. Recurring data trends indicated that resistance to cucumber downy mildew is associated with earlier response to the pathogen, hormone signaling, and regulation of nutrient supply. Candidate resistance genes were identified from multiple transcriptome analyses and literature support. Additionally, parallel sequencing of small RNAs (sRNAs) from cucumber and during the infection time course was used to identify and quantify novel and existing microRNA (miRNA) in both species. Predicted miRNA targets of cucumber transcripts suggest a complex interconnectedness of gene expression regulation in this plant-pathogen system. This work bioinformatically uncovered gene expression patterns involved in the mediation of or response to resistance. Herein, we provide the foundation for future work to validate candidate resistance genes and miRNA-based regulation proposed in this study.

Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis.

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

Light and the circadian clock mediate time-specific changes in sensitivity to UV-B stress under light/dark cycles.

In Arabidopsis, the circadian clock regulates UV-B-mediated changes in gene expression. Here it is shown that circadian clock components are able to inhibit UV-B-induced gene expression in a gene-by-gene-specific manner and act downstream of the initial UV-B sensing by COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and UVR8 (UV RESISTANCE LOCUS 8). For example, the UV-B induction of ELIP1 (EARLY LIGHT INDUCIBLE PROTEIN 1) and PRR9 (PSEUDO-RESPONSE REGULATOR 9) is directly regulated by LUX (LUX ARRYTHMO), ELF4 (EARLY FLOWERING 4), and ELF3. Moreover, time-dependent changes in plant sensitivity to UV-B damage were observed. Wild-type Arabidopsis plants, but not circadian clock mutants, were more sensitive to UV-B treatment during the night periods than during the light periods under diel cycles. Experiments performed under short cycles of 6h light and 6h darkness showed that the increased stress sensitivity of plants to UV-B in the dark only occurred during the subjective night and not during the subjective day in wild-type seedlings. In contrast, the stress sensitivity of Arabidopsis mutants with a compromised circadian clock was still influenced by the light condition during the subjective day. Taken together, the results show that the clock and light modulate plant sensitivity to UV-B stress at different times of the day.

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif.

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.