Transcriptome analysis of the plant pathogen Sclerotinia sclerotiorum interaction with resistant and susceptible canola (Brassica napus) lines.

Sclerotinia stem rot is an economically important disease of canola (Brassica napus) and is caused by the fungal pathogen Sclerotinia sclerotiorum. This study evaluated the differential gene expression patterns of S. sclerotiorum during disease development on two canola lines differing in susceptibility to this pathogen. Sequencing of the mRNA libraries derived from inoculated petioles and mycelium grown on liquid medium generated approximately 164 million Illumina reads, including 95 million 75-bp-single reads, and 69 million 50-bp-paired end reads. Overall, 36% of the quality filter-passed reads were mapped to the S. sclerotiorum reference genome. On the susceptible line, 1301 and 1214 S. sclerotiorum genes were differentially expressed at early (8-16 hours post inoculation (hpi)) and late (24-48 hpi) infection stages, respectively, while on the resistant line, 1311 and 1335 genes were differentially expressed at these stages, respectively. Gene ontology (GO) categories associated with cell wall degradation, detoxification of host metabolites, peroxisome related activities like fatty acid ß-oxidation, glyoxylate cycle, oxidoreductase activity were significantly enriched in the up-regulated gene sets on both susceptible and resistant lines. Quantitative RT-PCR of six selected DEGs further validated the RNA-seq differential gene expression analysis. The regulation of effector genes involved in host defense suppression or evasion during the early infection stage, and the expression of effectors involved in host cell death in the late stage of infection provide supporting evidence for a two-phase infection model involving a brief biotrophic phase during early stages of infection. The findings from this study emphasize the role of peroxisome related pathways along with cell wall degradation and detoxification of host metabolites as the key mechanisms underlying pathogenesis of S. sclerotiorum on B. napus.

Microscopic, Biochemical, and Molecular Comparisons of Moderately Resistant and Susceptible Populus Genotypes Inoculated with Sphaerulina musiva.

Sphaerulina musiva, the causal agent of Septoria leaf spot and stem canker, is responsible for mortality and yield loss in Populus plantations. However, little is known about the mode of infection and the mechanisms of resistance in this pathosystem. To characterize these phenomena, microscopic, biochemical, and transcriptome comparisons were performed between leaves of moderately resistant and susceptible genotypes of Populus inoculated with S. musiva conidia. Using scanning electron, cryofracture, and laser-scanning confocal microscopy, the infection and colonization of Populus leaves by S. musiva were examined across five time points (48 h, 96 h, 1 week, 2 weeks, and 3 weeks). The infection process was similar regardless of the host genotype. Differences in host colonization between susceptible and moderately resistant genotypes were apparent by 1 week postinoculation. However, the germination of conidia was greater on the susceptible than on the moderately resistant genotype (P < 0.008). Diaminobenzidine staining, a measure of hydrogen peroxide accumulation, was different (P < 0.001) between the host genotypes by 2 weeks postinoculation. Transcriptome differences between genotypes indicated that the speed and amplitude of the defense response were faster and more extensive in the moderately resistant genotype. Changes in gene expression support the microscopic and biochemical observations.

Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to a late diagnosis and poor response to available treatments. There is a need to identify complementary treatment strategies that will enhance the efficacy and reduce the toxicity of currently used therapeutic approaches. We investigated the ability of a known ROS inducer, piperlongumine (PL), to complement the modest anti-cancer effects of the approved chemotherapeutic agent gemcitabine (GEM) in PDAC cells in vitro and in vivo. PDAC cells treated with PL + GEM showed reduced cell viability, clonogenic survival, and growth on Matrigel compared to control and individually-treated cells. Nude mice bearing orthotopically implanted MIA PaCa-2 cells treated with both PL (5 mg/kg) and GEM (25 mg/kg) had significantly lower tumor weight and volume compared to control and single agent-treated mice. RNA sequencing (RNA-Seq) revealed that PL + GEM resulted in significant changes in p53-responsive genes that play a role in cell death, cell cycle, oxidative stress, and DNA repair pathways. Cell culture assays confirmed PL + GEM results in elevated ROS levels, arrests the cell cycle in the G0/G1 phase, and induces PDAC cell death. We propose a mechanism for the complementary anti-tumor effects of PL and GEM in PDAC cells through elevation of ROS and transcription of cell cycle arrest and cell death-associated genes. Collectively, our results suggest that PL has potential to be combined with GEM to more effectively treat PDAC.

Flaxseed Consumption Inhibits Chemically Induced Lung Tumorigenesis and Modulates Expression of Phase II Enzymes and Inflammatory Cytokines in A/J Mice.

Flaxseed consumption is associated with reduced oxidative stress and inflammation in lung injury models and has shown anticancer effects for breast and prostate tissues. However, the chemopreventive potential of flaxseed remains unexplored for lung cancer. In this study, we investigated the effect of flaxseed on tobacco smoke carcinogen (NNK)-induced lung tumorigenesis in an A/J mouse model. Mice exposed to NNK were fed a control diet or a 10% flaxseed-supplemented diet for 26 weeks. Flaxseed-fed mice showed reduced lung tumor incidence (78%) and multiplicity, with an average of 2.7 ± 2.3 surface lung tumor nodules and 1.0 ± 0.9 H&E cross-section nodules per lung compared with the control group, which had 100% tumor incidence and an average of 10.2 ± 5.7 surface lung tumor nodules and 3.9 ± 2.6 H&E cross-section nodules per lung. Furthermore, flaxseed-fed mice had a lower incidence of adenocarcinomas compared with control-fed mice. Western blotting performed on normal lung tissues showed flaxseed suppressed phosphorylation (activation) of p-AKT, p-ERK, and p-JNK kinases. RNA-Seq data obtained from normal lung and lung tumors of control and flaxseed-fed mice suggested that flaxseed intake resulted in differential expression of genes involved in inflammation-mediated cytokine signaling (IL1, 6, 8, 9, and 12α), xenobiotic metabolism (several CYPs, GSTs, and UGTs), and signaling pathways (AKT and MAPK) involved in tumor cell proliferation. Together, our results indicate that dietary flaxseed supplementation may be an effective chemoprevention strategy for chemically induced lung carcinogenesis by altering signaling pathways, inflammation, and oxidative stress. Cancer Prev Res; 11(1); 27-37. ©2017 AACR.

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70-90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean.