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

Mısır kılıfları ve mantarlar arasındaki hücresel etkileşimlerin görselleştirilmesi

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.

Yazar Gündemi: Mısırda Mantar Patojenite Mekanizmalarının Araştırılması 

Mantar yaprakları mısır patojenlerinin neden olduğu enfeksiyonun canlı hücre görüntülemesi için müstakil mısır kılıfları

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  

Canlı Hücre Görüntüleme için Mantar Enfekte Müstakil Mısır Yaprağı Kılıflarının Hazırlanması

Başlamak için, mantar sporları ile aşılanmış mısır yaprağı kılıflarını deiyonize suda nazikçe durulayın. Kılıfı, adaksiyal yüzey aşağı bakacak ve abaksiyal yüzey en üstte olacak şekilde temiz bir cam mikroskop lamına yerleştirin. Tek kenarlı bir tıraş bıçağı kullanarak, kılıf uçlarından yaklaşık bir santimetre kesin ve orta damarın her iki tarafındaki lamina dokusunun çoğunu çıkarın.Abaksiyal orta damardan dokuyu tıraş etmek için tıraş bıçağını 90 derecelik bir açıyla tutun ve aşılanan noktanın üzerindeki adaksiyal yüzeydeki epidermal tabakayı açığa çıkarın. Kılıf bölümünü bir kenardan dikkatlice kaldırın ve adaksiyal yüzey objektif merceğe en yakın olacak şekilde yeni bir kızağa aktarın. Bölüme 60 mikrolitre deiyonize su uygulayın ve hava kabarcığı oluşturmadan bir cam kapak astarı yerleştirin.Stenocarpella maydis, epidermal hücreleri doğrudan farklılaşmamış hif şişlikleri yoluyla hızla istila etti. Colletotrichum graminicola melanize appressoria üretti. Tripan mavisi boyama, C.graminicola başlangıçta kalın primer hifler yoluyla istila etti ve daha sonra nekrotrofik bir aşamaya geçti.C.graminicola konakçı sitoplazmadan bir zarla ayrılmış kalın primer hif kullanarak canlı mısır hücrelerini istila eder. Buna karşılık, S.maydis, epidermal hücreleri istila etmeden önce öldüren bir fitotoksin üretir. C. graminicola'da, mısır yaprağı kılıflarında koyu kahverengi çökelti üretimi, bir konakçı direnci tepkisi ile ilgili oksidatif bir patlamaya işaret etti.

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JoVE Journal

Biology

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

130 Görüntüleme.  University of Kentucky. Başlamak için, mantar sporları ile aşılanmış mısır yaprağı kılıflarını deiyonize suda nazikçe durulayın. Kılıfı, adaksiyal yüzey aşağı bakacak ve abaksiyal yüzey en üstte olacak şekilde temiz bir cam mikroskop lamına yerleştirin. Tek kenarlı bir tıraş bıçağı kullanarak, kılıf uçlarından yaklaşık bir santimetre kesin ve orta damarın her iki tarafındaki lamina dokusunun çoğunu çıkarın.Abaksiyal orta damardan dokuyu tıraş etmek için tır...

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

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

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

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

Biyoloji

Inoculation of Maize Leaf Sheaths with Pathogenic Fungi for Assessing Fungal Pathogenicity

To begin, use heavy duty scissors or shears to cut off the cap end 0.5 millimeters from the conical bottom of a 1.5-milliliter micro-centrifuge tube. Place a piece of glass wool in the cut tube to cover the center hole. Place one assembled glass wool filter unit into a sterile 1.5-milliliter micro-centrifuge tube and label the tube.Next, cut a non-skirted 96-well PCR plate into six two-column support racks. Flip the two-column racks so the conical bottoms of the wells face upwards. Place the rack in a glass Petri dish containing moist filter paper and cover it with the lid to create a humidity chamber for the leaf sheaths.To harvest conidia from the sporulating Colletotrichum graminicola, add three to four milliliters of sterile deionized water into the plate. Using a sterile conical tip pestle, scrape evenly across the entire plate to loosen the spores from the agar. Aseptically add one milliliter of spore suspension onto a glass wool filter unit and allow the spores to flow into the collection tube by gravity.Centrifuge the filtered spore suspension at 3, 500 G for five minutes and pour off the liquid into an autoclavable container. Add one milliliter of sterile deionized water into the collection tube and gently agitate to resuspend the pelleted spores. Again, centrifuge the suspension and resuspend the spores for quantification in 300 to 500 microliters of deionized water.Use a hemocytometer under a compound microscope at 100 times magnification to determine the spore concentration. Then prepare 500, 000 spores per milliliter suspension with deionized water. To remove the sheath from the first true leaf of week two stage seedlings.Run a thumbnail along the overlapping margin of the sheath and gently loosen it from the chute. Cut the recovered leaf sheath into three to five-centimeter segments. Carefully unroll each segment to expose the inner epidermal layer.Apply 20 microliters of fungal spore suspension on the inner surface at the center of the sheath above the mid-rib. Place the inoculated leaf sheaths horizontally, with a mid-rib positioned at the bottom, in a glass Petri plate containing moistened filter paper. Next place the small humidity chambers into a clear storage box lined with moistened germination paper, cover the storage box with a lid, and incubate at 23 degrees Celsius under continuous illumination for the intended time course.Regularly check the boxes for signs of disease on the sheaths.

Mantar Patojenitesinin Değerlendirilmesi için Mısır Yaprağı Kılıflarının Patojenik Mantarlarla Aşılanması

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.