Name: Preparation of Fungal Infected Detached Maize Leaf Sheaths for Live-Cell Imaging
Uploaded: 2023-09-15T14:20:00
Description: Watch this Scientific Journal Video about Preparation of Fungal Infected Detached Maize Leaf Sheaths for Live-Cell Imaging at JoVE.com

preparation of fungal infected detached maize leaf sheaths for live-cell imaging

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Spatial and temporal analyses at the cellular level are critical for understanding the physiology and cytology of fungal-plant interactions. Foliar tissues that have been chemically fixed1,2,3or cleared and stained4, as well as artificial membranes5, have been used in the past to investigate the cytology of foliar pathogen development and plant-fungal interactions. However, investigation of infection events in living host tissues in real-time without fixation or clearing is challenging due to technical issues related to the preparation of optically transparent samples for imaging.
A detached leaf sheath inoculation protocol was developed in the late 1940s for bright field microscopic investigation of resistance of living rice epidermal cells to the rice blast fungus Magnaporthe oryza6. More recently, detailed molecular, physiological, and cytological observations of host colonization by Colletotrichum and Magnaporthe species have been greatly facilitated by combining modified versions of this leaf sheath method with fungal transformants expressing fluorescent proteins, and high-performance live-cell imaging protocols, including epifluorescence and confocal microscopy7,8,9,10,11,12,13.
This paper details an optimized inoculation protocol using detached maize leaf sheaths for observation of infection processes by hemibiotrophic and necrotrophic foliar fungal pathogens. We have specifically used it to study Colletotrichum graminicola (C. graminicola), the causal agent of anthracnose leaf blight and stalk rot, and Stenocarpella maydis, which causes Diplodia leaf blight and stalk rot. However, the method should be applicable to other hemibiotrophic and necrotrophic foliar fungal pathogens. Cytological and physiological responses during infection and colonization events in these excised leaf sheaths are similar to those in entire leaf blades12,14,15. Furthermore, hemibiotrophic colonization of sheath epidermal cells by C. graminicola is similar to colonization of stalk pith cells16,17. Detached sheaths show greater synchronicity and experimental reproducibility of fungal penetration and colonization than leaf blades or stalk pith tissues14,16,17,18. Most maize varieties can be used for this protocol. However, inbreds or hybrids with excessive purple pigments in the sheaths are less suitable since the pigments interfere with imaging. Golden Jubilee sweet corn has been particularly useful for our studies because untreated seeds are commercially available, the plants are highly susceptible to many foliar diseases, and they grow well in the greenhouse. The first epidemics of anthracnose stalk rot in the United States resulted in the total loss of sweet corn crops in Indiana in the 1970s19,20. This leaf sheath inoculation method can be applied to directly observe and quantify fungal growth and development in living vs. locally killed plant cells, to demonstrate resistance reactions in compatible/incompatible responses to fungal infection, and to test interactions between fungal strains on the same sheath in real-time.

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

So our team aims to understand fungal pathogenicity mechanisms in plants to improve disease management. We are currently exploring how the maize autochthonous fungus regulates protein secretion beyond its housekeeping function. Our protocol shows high synchronicity across replications and reproducibility of fungal colonization.Also, with our assays, there is no need for clearing or fixating samples before imaging them in real-time. The method offers a variety of applications in plant-pathogen interactions and contributed to detailed studies on the mechanisms of fungal pathogenicity in maize and comparative genomic analysis.

Watch this Scientific Journal Video about Preparation of Fungal Infected Detached Maize Leaf Sheaths for Live-Cell Imaging at JoVE.com

Preparation of Fungal Infected Detached Maize Leaf Sheaths for Live-Cell Imaging  

To begin, gently rinse the maize leaf sheaths inoculated with fungal spores in deionized water. Place the sheath on a clean glass microscope slide with the adaxial surface facing down and the abaxial surface uppermost. Using a single-edge razor blade, trim approximately one centimeter off the sheath ends and remove most lamina tissue on either side of the midrib.Hold the razor blade at a 90-degree angle to shave tissue from the abaxial midrib, exposing the epidermal layer on the adaxial surface above the inoculated spot. Carefully lift the sheath section from one edge and transfer it onto a new slide with the adaxial surface facing the uppermost closest to the objective lens. Apply 60 microliters of deionized water to the section and place a glass cover slip without introducing air bubbles.Stenocarpella maydis rapidly invaded epidermal cells directly via undifferentiated hyphal swellings. While Colletotrichum graminicola produced melanized appressoria. Trypan blue staining revealed the nature and extent of fungal hyphae colonization, with C.graminicola initially invading via thick primary hyphae and later switching to a necrotrophic stage.C.graminicola invades living maize cells using thick primary hyphae separated from the host cytoplasm by a membrane. In contrast, S.maydis produces a phytotoxin that kills the epidermal cells before invading them. In C.graminicola, the production of dark brown precipitate in the maize leaf sheaths indicated an oxidative burst related to a host-resistance response.

Research

JoVE Journal

Biology

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...