A targeted sequencing approach for viability assessment and management of invasive Phytophthoras

Members of the Phytophthora genus are among the most serious invasive pathogens of forests and cause devastating ecological and economic impacts. Epidemics caused by the sudden oak death pathogen, Phytophthora ramorum, and root rot of Christmas trees caused by multiple Phytophthora species are among the many causing loss of millions of dollars to the nursery industry. In addition, a large volume of manufactured products is traded in packages made of wood and wood products globally raising phytosanitary concerns for global trade. Rigorous inspections and biosurveillance involving the monitoring of pathogens before and after applying phytosanitary measures are essential to keep these organisms under control. Despite technological advances in diagnostic assays based on genomics, assessment of treatment efficacy remains challenging for these pathogens. DNA remains stable during heat treatments and can remain as a relic in environments long after the pathogen is dead. Culture methods used for viability testing are time consuming and often report false negatives. We propose a solution to the problem using a transcriptomics approach applying the heat lability of RNA for the prediction of viability and sequence variability for taxonomic identity. We measure levels of RNA degradation by simulating phytosanitary treatment and show that RNA degradation by heat can be assessed from the length of the RNA transcript transcribed and amplified by RT-PCR. Phytophthora transcript sequences identified from an RNAseq study and reference genomes of eight clades of Phytophthora are used to create a panel of oligonucleotides that can target multiple regions along the length of several transcripts commonly expressed during host infection. Significant reduction (60-99%) in mean coverage within transcripts is observed after heat treatments for the 18 genes targeted by the panel. We present the design and testing of a rapid and high-throughput transcriptomics assay for viability assessment of Phytophthoras using the power of targeted next generation sequencing. The assay can be applied for implementation of trade regulations and management of Phytophthora infections in nurseries by regulatory agencies.Forestry, Faculty ofGraduat

Transgenic expression of fungal accessory hemicellulases in <i>Arabidopsis thaliana</i> triggers transcriptional patterns related to biotic stress and defense response - Fig 1

<p><b>Venn diagrams for genes with (a) increased and (b) decreased transcript abundance in transgenic plants relative to non-transformed wild-type plants</b>. The blue, red, and green circles represent the <i>PcGCE-7</i>, <i>PcGCE-13</i>, and <i>AnAF54</i> transgenic lines, respectively; while the open and shaded circles represent top and mid stems, respectively. A black circle represents the intersect of all three circles in each Venn diagram; while the overlap between the open and shaded black circles represent the core set genes.</p

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response.

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to the hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses

Relative fold change of genes involved in call wall biosynthesis (GO: 0070592) in top stem samples of arabidopsis transgenic lines.

<p>Relative fold change of genes involved in call wall biosynthesis (GO: 0070592) in top stem samples of arabidopsis transgenic lines.</p

Core set of genes with differential expression in stem tissue of transgenic arabidopsis lines overexpressing fungal carbohydrate-active enzymes.

<p>The heat map represents relative transcript abundance of genes that differ significantly in each of the three transgenic lines (<i>AnAF54</i>, <i>PcGCE-7</i>, and <i>PcGCE-13</i>) relative to the wild-type in both, top stem and mid stem tissue (n = 655, FDR < 0.05). Genes with significantly lower transcript abundance in transgenic lines when compared with non-transformed wild-type are marked with “reduced” (n = 188) and gene with significantly higher transcript abundance are marked with “increased” (n = 467). Data are row normalized.</p

Relative fold change of genes involved in salicylic acid signaling.

<p>Relative fold change of genes involved in salicylic acid signaling.</p

Photosynthesis—antenna protein pathway (KEGG ath00196).

<p>The pathway schematic was modified from the KEGG database with permission [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref064" target="_blank">64</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref065" target="_blank">65</a>]. The heat maps illustrate differential transcript accumulation in transgenic lines relative to wild-type for both top and mid stem samples. The color corresponds to log₂ fold change in transcript accumulation. Rows in each heat map represent genes and the order of genes corresponds to the order given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.s009" target="_blank">S4B Table</a>.</p

Plant-pathogen interaction pathway (KEGG ath04626).

<p>The pathway schematic was adapted from the KEGG database with permission [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref064" target="_blank">64</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref065" target="_blank">65</a>]. The heat maps illustrate differential transcript accumulation in transgenic lines relative to wild-type for both top and mid stem samples. The color corresponds to log₂ fold change in transcript accumulation. Rows in each heat map represent genes and the order of genes corresponds to the order given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.s009" target="_blank">S4A Table</a>.</p