inoculation of maize leaf sheaths with pathogenic fungi for assessing fungal pathogenicity

玉米鞘和真菌之间细胞相互作用的可视化

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.

作者聚焦：研究玉米中的真菌致病机制 

用于真菌叶面玉米病原体感染的活细胞成像的分离玉米鞘

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.

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

用致病真菌接种玉米叶鞘以评估真菌致病性

首先，使用重型剪刀或剪刀从 1.5 毫升微量离心管的锥形底部切下 0.5 毫米的盖端。将一块玻璃棉放入切割管中以覆盖中心孔。将一个组装好的玻璃棉过滤装置放入无菌的 1.5 毫升微量离心管中并标记管。接下来，将无裙边的 96 孔 PCR 板切成六个两柱支撑架。翻转两柱架，使孔的锥形底部朝上。将架子放入装有湿滤纸的玻璃培养皿中，并用盖子盖住它，为叶鞘创建一个湿度室。要从孢子形成的 Colletotrichum graminicola 中收获分生孢子，请在平板中加入三到四毫升无菌去离子水。使用无菌锥形尖杵，均匀地刮过整个平板，以松开琼脂中的孢子。无菌地将一毫升孢子悬浮液添加到玻璃棉过滤装置上，并让孢子通过重力流入收集管。将过滤后的孢子悬浮液以3， 500G离心五分钟，然后将液体倒入可高压灭菌的容器中。向收集管中加入一毫升无菌去离子水，轻轻搅拌以重悬沉淀的孢子。再次，离心悬浮液并将孢子重悬于300至500微升去离子水中进行定量。在复合显微镜下以100倍放大倍率使用血细胞计数器来测定孢子浓度。然后用去离子水制备每毫升悬浮液500， 000个孢子。从第二周幼苗的第一片真叶上取下鞘。沿着护套的重叠边缘运行缩略图，然后轻轻地将其从滑槽上松开。将回收的叶鞘切成三到五厘米的段。小心地展开每个部分以露出内表皮层。在中肋上方鞘中心的内表面上涂抹 20 微升真菌孢子悬浮液。将接种的叶鞘水平放置，中间肋位于底部，在装有湿润滤纸的玻璃培养皿中。接下来，将小型湿度室放入衬有湿发芽纸的透明储物盒中，盖上盖子，并在 23 摄氏度的连续照明下在预定的时间范围内孵育。定期检查护套上的方框是否有疾病迹象。

inoculation-maize-leaf-sheaths-with-pathogenic-fungi-for-assessing

Research

JoVE Journal

Biology

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

144 次观看.  University of Kentucky. 首先，使用重型剪刀或剪刀从 1.5 毫升微量离心管的锥形底部切下 0.5 毫米的盖端。将一块玻璃棉放入切割管中以覆盖中心孔。将一个组装好的玻璃棉过滤装置放入无菌的 1.5 毫升微量离心管中并标记管。接下来，将无裙边的 96 孔 PCR 板切成六个两柱支撑架。翻转两柱架，使孔的锥形底部朝上。将架子放入装有湿滤纸的玻璃培养皿中，并用盖子盖住它，为叶鞘创建一个湿?...

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

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 ...

科研

生物学

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.

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.