Protocols to enable fluorescence microscopy of microbial interactions on living maize silks (style tissue) that balance the requirements for containment, biological relevance, viability, and experimental accessibility

ABSTRACT There is interest in studying microbes that colonize maize silks (style tissue, critical for reproduction) including the fungal pathogen Fusarium graminearum ( Fg ) and its interactions with the microbiome and biocontrol agents. In planta imaging of these interactions on living silks using confocal fluorescence microscopy would provide key insights. However, newly discovered microbes have unknown effects of on human health, and there are regulatory requirements to prevent the release of fluorescently-tagged microbes into the environment. Therefore, the microbe infection, colonization, and interaction stages on silks prior to microscopy must be contained. At the same time, silk viability must be maintained and experiments conducted that are biologically relevant (e.g. silks should remain attached to the cob), yet the silk tissue must be accessible to the researcher (i.e. not within husk leaves) and allow for multiple replicates. Here we present methods that meet these five contrasting criteria. We tested these methods using Fg and four silk-derived bacterial endophytes. The endophytes were previously known to have anti- Fg activity in vitro , but in planta observations were lacking. In Method 1, a portion of the tip of a cob was dissected, and silks remained attached to the cob in a Petri dish. The cob was placed on a water agar disc to maintain hydration. DsRed-tagged bacteria and GFP-tagged Fg were inoculated onto the silks and incubated, allowing the two microbes to grow towards one another before staining with propidium iodide for confocal microscopy. A variation of the protocol was presented in Method 2, where detached silk segments were placed directly on water agar where they were inoculated with bacteria and Fg to promote dense colonization, and to allow for many replicates and interventions such as silk wounding. The bacterial endophytes were successfully observed colonizing Fg hyphae, silk trichomes, and entering silks via cut ends and wounds. These protocols can be used to study other silk-associated microbes including several globally important fungal pathogens that enter maize grain through silks.

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