PTEN deficiency induces an extrahepatic cholangitis-cholangiocarcinoma continuum via aurora kinase A in mice.

BACKGROUND & AIMS: The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aimed to evaluate the role of PTEN in the pathogenesis of eCCA and identify novel therapeutic targets for this disease. METHODS: The Pten gene was genetically deleted using the Cre-loxp system in biliary epithelial cells. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry, reverse-transcription PCR, cell culture, and RNA sequencing. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. We also tested the effectiveness of an Aurka inhibitor. RESULTS: We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as 1 month after birth. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated disease progression, potentially by downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expression of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS: Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum that was dependent on Aurka. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS: The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition, cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted disease progression. This model will be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

A small molecule MST1/2 inhibitor accelerates murine liver regeneration with improved survival in models of steatohepatitis.

Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.

Lipoblastoma-Like Tumor and Fibrosarcoma-Like Lipomatous Neoplasm Represent the Same Entity: A Clinicopathologic and Molecular Genetic Study of 23 Cases Occurring in Both Men and Women at Diverse Locations.

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma, and spindle cell lipoma but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of "fibrosarcoma-like lipomatous neoplasm" (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor. We compared the morphologic, immunohistochemical, and genetic features of 23 tumors previously classified as LLT (n = 17) and FLLN (n = 6). The 23 tumors occurred in 13 women and 10 men (mean age, 42 years; range, 17 to 80 years). Eighteen (78%) cases arose in the inguinogenital region, whereas 5 tumors (22%) involved noninguinogenital soft tissue, including the flank (n = 1), shoulder (n = 1), foot (n = 1), forearm (n = 1), and chest wall (n = 1). Microscopically, the tumors were lobulated and septated, with variably collagenized fibromyxoid stroma, prominent thin-walled vessels, scattered univacuolated or bivacuolated lipoblasts, and a minor component of mature adipose tissue. Using immunohistochemistry, 5 tumors (42%) showed complete RB1 loss, with partial loss in 7 cases (58%). RNA sequencing, chromosomal microarray, and DNA next-generation sequencing study results were negative for significant alterations. There were no clinical, morphologic, immunohistochemical, or molecular genetic differences between cases previously classified as LLT or FLLN. Clinical follow-up (11 patients [48%]; range, 2-276 months; mean, 48.2 months) showed all patients were alive without disease, and only one patient had experienced a single local recurrence. We conclude that LLT and FLLN represent the same entity, for which "LLT" seems most appropriate. LLT may occur in either sex and any superficial soft tissue location. Careful morphologic study and appropriate ancillary testing should allow for the distinction of LLT from its potential mimics.

Kidney transcriptome and cystic kidney disease genes in zebrafish.

Introduction: Polycystic kidney disease (PKD) is a condition where fluid filled cysts form on the kidney which leads to overall renal failure. Zebrafish has been recently adapted to study polycystic kidney disease, because of its powerful embryology and genetics. However, there are concerns on the conservation of this lower vertebrate in modeling polycystic kidney disease. Methods: Here, we aim to assess the molecular conservation of zebrafish by searching homologues polycystic kidney disease genes and carrying transcriptome studies in this animal. Results and Discussion: We found that out of 82 human cystic kidney disease genes, 81 have corresponding zebrafish homologs. While 75 of the genes have a single homologue, only 6 of these genes have two homologs. Comparison of the expression level of the transcripts enabled us to identify one homolog over the other homolog with >70% predominance, which would be prioritized for future experimental studies. Prompted by sexual dimorphism in human and rodent kidneys, we studied transcriptome between different sexes and noted significant differences in male vs. female zebrafish, indicating that sex dimorphism also occurs in zebrafish. Comparison between zebrafish and mouse identified 10% shared genes and 38% shared signaling pathways. String analysis revealed a cluster of genes differentially expressed in male vs. female zebrafish kidneys. In summary, this report demonstrated remarkable molecular conservation, supporting zebrafish as a useful animal model for cystic kidney disease.

Differential prognosis of single and multiple TP53 abnormalities in high-count MBL and untreated CLL.

TP53 aberrations, including mutations and deletion of 17p13, are important adverse prognostic markers in chronic lymphocytic leukemia (CLL) but are less studied in high count monoclonal B-cell lymphocytosis (HCMBL), an asymptomatic pre-malignant stage of CLL. Here we estimated the prevalence and impact of TP53 aberrations in 1,230 newly diagnosed treatment-naïve individuals (849 CLL, 381 HCMBL). We defined TP53 state as: wild-type (no TP53 mutations and normal 17p), single-hit (del(17p) or one TP53 mutation), or multi-hit (TP53 mutation and del(17p), TP53 mutation and loss of heterozygosity, or multiple TP53 mutations). Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time to first treatment and overall survival by TP53 state. We found 64 (7.5%) CLL patients and 17 (4.5%) HCMBL individuals had TP53 mutations with variant allele fraction >10%. Del(17p) was present in 58 (6.8%) of CLL and 11 (2.9%) of HCMBL cases. Most individuals had wild-type (N=1,128, 91.7%) TP53 state, followed by multi-hit (N=55, 4.5%) and then single-hit (N=47, 3.8%) TP53 state. The risk of shorter time to therapy and death increased with the number of TP53 abnormalities. Compared to wild-type patients, multi-hit patients had 3-fold and single-hit patients had 1.5-fold increased risk of requiring therapy. Multi-hit patients also had 2.9-fold increased risk of death compared to wild-type. These results remained stable after accounting for other known poor prognostic factors. Both TP53 mutations and del(17p) may provide important prognostic information for HCMBL and CLL that would be missed if only one were measured.

Transcriptomic landscapes of effective and failed liver regeneration in humans.

Background & Aims: Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Methods: Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Results: Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Conclusions: Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Impact and implications: Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Tissue Eosinophilia is Superior to an Analysis by Polyp Status for the Chronic Rhinosinusitis Transcriptome: An RNA Study.

OBJECTIVE: RNA sequencing (transcriptomics) is used to study biological pathways. However, the yield of data depends on comparing well-characterized cohorts. We compared tissue eosinophilia versus nasal polyp (NP) status as the metric to characterize transcriptomic mechanisms at play in eosinophilic and non-eosinophilic chronic rhinosinusitis (CRS) versus controls. METHODS: RNA sequencing was conducted on sinonasal tissue samples of CRS and controls. Analyses were conducted based on polyp status [with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP)] as well as tissue eosinophil levels per high power field (eos/hpf)[non-eosinophilic (<10 eos/hpf, neCRS) or eosinophilic (≥10 eos/hpf, eCRS)]. The yield of differentially expressed genes (DEGs) and biological pathways through Ingenuity Pathway Analysis (IPA) were compared. RESULTS: CRS tissue differed from controls by 736 statistically significant DEGs. Both NP status and tissue eosinophilia were effective in differentiating CRS from controls and into two distinct subgroups. Statistically significant DEGs identified when comparing CRS by NP status were 60, whereas 110 DEGs were identified using eosinophil cutoff ≥10 and <10 eos/hpf. Additionally, heatmaps showed greater homogeneity within each CRS subgroup when analyzed by tissue eosinophilia versus NP status. On IPA, the IL-17 signaling pathway was significantly different only by tissue eosinophilia status, not NP status, being higher in CRS <10 eos/hpf. CONCLUSION: Tissue eosinophilia is superior to an analysis by NP status for the study of CRS transcriptome by RNA sequencing in identifying DEGs. Classification of CRS samples by eosinophil counts agnostic of NP status may offer advantageous insights into CRS pathogenetic mechanisms. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2480-2489, 2023.

LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma.

BACKGROUND & AIMS: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity. METHODS: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. RESULTS: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth. CONCLUSIONS: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models. IMPACT AND IMPLICATIONS: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Virus induced gene silencing confirms oligogenic inheritance of brown stem rot resistance in soybean.

Brown Stem Rot (BSR), caused by the soil borne fungal pathogen Phialophora gregata, can reduce soybean yields by as much as 38%. Previous allelism studies identified three Resistant to brown stem Rot genes (Rbs1, Rbs2, and Rbs3), all mapping to large, overlapping regions on soybean chromosome 16. However, recent fine-mapping and genome wide association studies (GWAS) suggest Rbs1, Rbs2, and Rbs3 are alleles of a single Rbs locus. To address this conflict, we characterized the Rbs locus using the Williams82 reference genome (Wm82.a4.v1). We identified 120 Receptor-Like Proteins (RLPs), with hallmarks of disease resistance receptor-like proteins (RLPs), which formed five distinct clusters. We developed virus induced gene silencing (VIGS) constructs to target each of the clusters, hypothesizing that silencing the correct RLP cluster would result in a loss of resistance phenotype. The VIGS constructs were tested against P. gregata resistant genotypes L78-4094 (Rbs1), PI 437833 (Rbs2), or PI 437970 (Rbs3), infected with P. gregata or mock infected. No loss of resistance phenotype was observed. We then developed VIGS constructs targeting two RLP clusters with a single construct. Construct B1a/B2 silenced P. gregata resistance in L78-4094, confirming at least two genes confer Rbs1-mediated resistance to P. gregata. Failure of B1a/B2 to silence resistance in PI 437833 and PI 437970 suggests additional genes confer BSR resistance in these lines. To identify differentially expressed genes (DEGs) responding to silencing, we conducted RNA-seq of leaf, stem and root samples from B1a/B2 and empty vector control plants infected with P. gregata or mock infected. B1a/B2 silencing induced DEGs associated with cell wall biogenesis, lipid oxidation, the unfolded protein response and iron homeostasis and repressed numerous DEGs involved in defense and defense signaling. These findings will improve integration of Rbs resistance into elite germplasm and provide novel insights into fungal disease resistance.

SHP2 inhibition enhances Yes-associated protein-mediated liver regeneration in murine partial hepatectomy models.

Disrupted liver regeneration following hepatectomy represents an "undruggable" clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.

Morphological heterogeneity in beta-catenin-mutated hepatocellular carcinomas: implications for tumor molecular classification.

Beta-catenin (CTNNB1) is commonly mutated in hepatocellular carcinoma (HCC). CTNNB1-mutated HCC has important clinical correlates, such as being immune cold and less likely to respond to immune checkpoint inhibitor therapies. It remains unclear, however, if they are a morphologically homogenous group of tumors. To better understand the association between the morphology, CTNNB1 mutations, and other molecular features, a detailed study of 338 The Cancer Genome Atlas cases was performed. A characteristic histological morphology was strongly associated with CTNNB1 mutations but was present in only 58% of CTNNB1-mutated HCCs. Tumors with APC mutations tended to have the classic morphology; those with AXIN mutations did not. Pseudoglands are a key feature of the classic morphology, and they were associated with CTNNB1 mutations, male gender, specific CTNNB1 mutation site, and lack of TP53 mutations. Differential gene expression analysis stratified by the presence/absence of pseudoglands identified 60 differentially expressed genes (FDR <5%); clustering according to these differentially expressed genes revealed three groups of tumors, one with pseudoglands and a strong association with genes regulated by Wnt signaling; within this group, TP53 mutations were associated with a loss of the typical morphology of CTNNB1-mutated HCCs. When stratified by gender, further differential gene expression showed Wnt-regulated genes were associated with pseudoglands in men but not women. These findings indicate HCC with CTNNB1 mutations are morphologically heterogeneous, with gene penetrance for morphology dependent in part on gender, specific CTNNB1 mutations, and co-occurring TP53 mutations. This heterogeneity has important implications for the classification of HCC.

Interaction between Rag genes results in a unique synergistic transcriptional response that enhances soybean resistance to soybean aphids.

BACKGROUND: Pyramiding different resistance genes into one plant genotype confers enhanced resistance at the phenotypic level, but the molecular mechanisms underlying this effect are not well-understood. In soybean, aphid resistance is conferred by Rag genes. We compared the transcriptional response of four soybean genotypes to aphid feeding to assess how the combination of Rag genes enhanced the soybean resistance to aphid infestation. RESULTS: A strong synergistic interaction between Rag1 and Rag2, defined as genes differentially expressed only in the pyramid genotype, was identified. This synergistic effect in the Rag1/2 phenotype was very evident early (6 h after infestation) and involved unique biological processes. However, the response of susceptible and resistant genotypes had a large overlap 12 h after aphid infestation. Transcription factor (TF) analyses identified a network of interacting TF that potentially integrates signaling from Rag1 and Rag2 to produce the unique Rag1/2 response. Pyramiding resulted in rapid induction of phytochemicals production and deposition of lignin to strengthen the secondary cell wall, while repressing photosynthesis. We also identified Glyma.07G063700 as a novel, strong candidate for the Rag1 gene. CONCLUSIONS: The synergistic interaction between Rag1 and Rag2 in the Rag1/2 genotype can explain its enhanced resistance phenotype. Understanding molecular mechanisms that support enhanced resistance in pyramid genotypes could facilitate more directed approaches for crop improvement.

Comparing Early Transcriptomic Responses of 18 Soybean (Glycine max) Genotypes to Iron Stress.

Iron deficiency chlorosis (IDC) is an abiotic stress that negatively affects soybean (Glycine max [L.] Merr.) production. Much of our knowledge of IDC stress responses is derived from model plant species. Gene expression, quantitative trait loci (QTL) mapping, and genome-wide association studies (GWAS) performed in soybean suggest that stress response differences exist between model and crop species. Our current understanding of the molecular response to IDC in soybeans is largely derived from gene expression studies using near-isogenic lines differing in iron efficiency. To improve iron efficiency in soybeans and other crops, we need to expand gene expression studies to include the diversity present in germplasm collections. Therefore, we collected 216 purified RNA samples (18 genotypes, two tissue types [leaves and roots], two iron treatments [sufficient and deficient], three replicates) and used RNA sequencing to examine the expression differences of 18 diverse soybean genotypes in response to iron deficiency. We found a rapid response to iron deficiency across genotypes, most responding within 60 min of stress. There was little evidence of an overlap of specific differentially expressed genes, and comparisons of gene ontology terms and transcription factor families suggest the utilization of different pathways in the stress response. These initial findings suggest an untapped genetic potential within the soybean germplasm collection that could be used for the continued improvement of iron efficiency in soybean.

New tools for characterizing early brown stem rot disease resistance signaling in soybean.

Brown stem rot (BSR) reduces soybean [Glycine max (L.) Merr.] yield by up to 38%. The BSR causal agent is Phialophora gregata f. sp. sojae, a slow-growing, necrotrophic fungus whose life cycle includes latent and pathogenic phases, each lasting several weeks. Brown stem rot foliar symptoms are often misdiagnosed as other soybean diseases or nutrient stress, making BSR resistance especially difficult to phenotype. To shed light on the genes and networks contributing to P. gregata resistance, we conducted RNA sequencing (RNA-seq) of a resistant genotype (PI 437970, Rbs3). Leaf, stem, and root tissues were collected 12, 24, and 36 h after stab inoculation with P. gregata, or mock infection, in the plant stem. By using multiple tissues and time points, we could see that leaves, stems, and roots use the same defense pathways. Our analyses suggest that P. gregata induces a biphasic defense response, with pathogen-associated molecular pattern (PAMP) triggered immunity observed in leaves at 12 and 24 h after infection (HAI) and effector triggered immunity detected at 36 h after infection in the stems. Gene networks associated with defense, photosynthesis, nutrient homeostasis, DNA replication, and growth are the hallmarks of resistance to P. gregata. While P. gregata is a slow-growing pathogen, our results demonstrate that pathogen recognition occurs hours after infection. By exploiting the genes and networks described here, we will be able to develop novel diagnostic tools to facilitate breeding and screening for BSR resistance.

Dynamic gene expression changes in response to micronutrient, macronutrient, and multiple stress exposures in soybean.

Preserving crop yield is critical for US soybean production and the global economy. Crop species have been selected for increased yield for thousands of years with individual lines selected for improved performance in unique environments, constraints not experienced by model species such as Arabidopsis. This selection likely resulted in novel stress adaptations, unique to crop species. Given that iron deficiency is a perennial problem in the soybean growing regions of the USA and phosphate deficiency looms as a limitation to global agricultural production, nutrient stress studies in crop species are critically important. In this study, we directly compared whole-genome expression responses of leaves and roots to iron (Fe) and phosphate (Pi) deficiency, representing a micronutrient and macronutrient, respectively. Conducting experiments side by side, we observed soybean responds to both nutrient deficiencies within 24 h. While soybean responds largely to -Fe deficiency, it responds strongly to Pi resupply. Though the timing of the responses was different, both nutrient stress signals used the same molecular pathways. Our study is the first to demonstrate the speed and diversity of the soybean stress response to multiple nutrient deficiencies. We also designed the study to examine gene expression changes in response to multiple stress events. We identified 865 and 3375 genes that either altered their direction of expression after a second stress exposure or were only differentially expressed after a second stress event. Understanding the molecular underpinnings of these responses in crop species could have major implications for improving stress tolerance and preserving yield.

Leveraging RNA-Seq to Characterize Resistance to Brown Stem Rot and the Rbs3 Locus in Soybean.

Brown stem rot, caused by the fungus Phialophora gregata, reduces soybean yield by up to 38%. Although three dominant resistance loci have been identified (Rbs1 to Rbs3), the gene networks responsible for pathogen recognition and defense remain unknown. Further, identification and characterization of resistant and susceptible germplasm remains difficult. We conducted RNA-Seq of infected and mock-infected leaf, stem, and root tissues of a resistant (PI 437970, Rbs3) and susceptible (Corsoy 79) genotype. Combining historical mapping data with genotype expression differences allowed us to identify a cluster of receptor-like proteins that are candidates for the Rbs3 resistance gene. Reads mapping to the Rbs3 locus were used to identify potential novel single-nucleotide polymorphisms within candidate genes that could improve phenotyping and breeding efficiency. Comparing responses to infection revealed little overlap in differential gene expression between genotypes or tissues. Gene networks associated with defense, DNA replication, and iron homeostasis are hallmarks of resistance to P. gregata. This novel research demonstrates the utility of combining contrasting genotypes, gene expression, and classical genetic studies to characterize complex disease resistance loci.