Borowicz, P. | Search bioRxiv

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

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↗