Biology subjects

Solanki, S.

Publications and source records attributed to Solanki, S..

23 records · Page 2Linked to original sources

bioRxiv2020

Localization of hyphal growth associated with mycotoxin production during the malting of Fusarium head blight infected grains

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Objectives of the current study were to localize the growth of Fusarium within FHB infected kernels and to associate it with the production of DON that occurred during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in Fusarium Tri5 DNA and trichothecene mycotoxins following malting, were screened for hyphal localization. The growth of hyphae, both on the surface of kernels and within tissues of grain and malt was, imagined by scanning electron microscopy and confocal laser scanning microscopy assisted with WGA-Alexa Fluor 488 pre-staining, respectively. In barley, hyphae were primarily present on or within husk, vascular bundle, and pericarp cavities. Following malting, large amounts of hyphal growth were observed in not only these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. Interestingly, these grains already had an extensive internal presence of hyphae in unmalted grain, occurring in the pericarp, testa, vascular bundle, nucellar projection, aleurone layer, endosperm, pericarp and endosperm cavities, and embryo. Shotgun sequencing followed by metagenomics analysis verified that Fusarium spp. accounted for above 90% of the fungal hyphae growing in the interior of grains during malting, which coincided with the significant production of mycotoxins.

plant biology↗

bioRxiv2020

rcs5-mediated spot blotch resistance in barley is conferred by wall-associated kinases that resist pathogen manipulation

Plant biotrophic pathogen disease resistances rely on immunity receptor-mediated programmed cell death (PCD) responses, but specialized necrotrophic/hemi-biotrophic pathogens hijack these mechanisms to colonize the resulting dead tissue in their necrotrophic phase. Thus, immunity receptors can become necrotrophic pathogen dominant susceptibility targets but resistance mechanisms that resist necrotroph manipulation are recessive resistance genes. The barley rcs5 QTL imparts recessive resistance against the disease spot blotch caused by the hemi-biotrophic fungal pathogen Bipolaris sorokiniana. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four wall-associated kinase (WAK) genes, designated HvWak2, Sbs1, Sbs2 (susceptibility to Bipolaris sorokiniana 1&2), and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in susceptible barley cultivars resulted in resistance showing dominant susceptibility function. Allele analysis of Sbs1&2 from resistant and susceptible barley cultivars identified sequence polymorphisms associated with phenotypes in their primary coding sequence and promoter regions, suggesting differential transcriptional regulation may contribute to susceptibility. Transcript analysis of Sbs1&2 showed nearly undetectable expression in resistant and susceptible cultivars prior to pathogen challenge; however, upregulation of both genes occurred specifically in susceptible cultivars post-inoculation with a virulent isolate. Apoplastic wash fluids collected from barley infected with a virulent isolate induced Sbs1, suggesting regulation by an apoplastic-secreted effector. Thus, Sbs1&2 function as B. sorokiniana susceptibility targets and non-functional alleles or alleles that resist induction by the pathogen mediate rcs5-recessive resistance. The sbs1&2 alleles underlying the rcs5 QTL that the pathogen is unable to manipulate are the first resistance genes identified against spot blotch. SIGNIFICANCE STATEMENTThe rcs5 locus in barley confers a high level of seedling resistance and a moderate level of adult plant resistance to spot blotch. It is part of a complex that has provided durable spot blotch resistance in many North American barley cultivars (cv) for more than 50 years. Genetic characterization and positional cloning of rcs5 identified the dominant susceptibility genes, Sbs1 and Sbs2 (susceptibility to Bipolaris sorokiniana 1 and 2) as wall-associated kinases. These genes are hijacked by the hemibiotrophic pathogen in its necrotrophic phase to induce programmed cell death, facilitating disease development. We report the first spot blotch resistance/susceptibility genes cloned that function via alleles that cannot be specifically induced and hijacked by virulent isolates of the pathogen.

plant biology↗

bioRxiv2020

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAcope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Thus, we optimized the RNAscope detection method in plants to visualize the spatial expression and semi-quantification of target RNAs which can be employed in other plants such as the widely utilized model dicot plant Arabidopsis.

plant biology↗

bioRxiv2019

Long-term metabolic consequences of acute dioxin exposure differ between male and female mice

Exposure to environmental pollutants is consistently associated with increased diabetes risk in humans. In male mice, acute dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) exposure supresses insulin secretion. This study investigated the long-term effects of a single TCDD injection (20 {micro}g/kg) on glucose metabolism and beta cell function in male and female mice. TCDD-exposed males displayed modest fasting hypoglycemia for [~]4 weeks post-injection, reduced fasting insulin levels for up to 6 weeks, increased insulin sensitivity, and decreased beta cell area. TCDD-exposed females also had long-term suppressed basal plasma insulin levels, and abnormal insulin secretion for up to 6 weeks. Unlike males, TCDD did not impact insulin sensitivity or beta cell area in females, but did cause transient glucose intolerance 4 weeks post-exposure. Our results show that a single exposure to dioxin can supress basal insulin levels long-term in both sexes, but effects on metabolism are sex-dependent.

pharmacology and toxicology↗

bioRxiv2017

Cadmium exposure inhibits branching morphogenesis and causes alterations consistent with HIF-1α inhibition in human primary breast organoids

BackgroundDevelopmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cell development have not been measured.\n\nObjectiveThe objective of this study was to quantify the effects of cadmium exposure on normal breast stem cell proliferation and differentiation.\n\nMethodsWe tested the effects of two physiologically relevant doses of cadmium: 250M and 2.5M on reduction mammoplasty patient-derived breast cells using the mammosphere assay, organoid formation in 3D hydrogels, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a HIF-1 transcription factor activity reporter line and pharmaceutical inhibition of HIF-1 in mammosphere and organoid formation assays.\n\nResults2.5M cadmium reduced primary and secondary mammosphere formation and branching structure organoid formation rates by 33%, 40%, and 83%, respectively. Despite no changes in mammosphere formation, 0.25M cadmium treatment inhibited branching organoid formation in hydrogels by 68%. RNA-seq revealed that cadmium treatment downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium treatment also downregulated HIF-1 target genes including LOXL2, ZEB1, and VIM. Cadmium treatment significantly inhibited HIF-1 activity in a luciferase assay, and the HIF-1 inhibitor acriflavine ablated mammosphere and organoid formation.\n\nDiscussionThese findings show that cadmium, at doses relevant to human exposure, inhibited human mammary gland development, potentially through disruption of HIF-1 activity. These findings do not support cadmium being a breast cancer initiator via induction of stem cell proliferation, but instead implicate cadmium as an inhibitor of mammary gland morphogenesis.

pharmacology and toxicology↗