Name: Inoculation of Maize Leaf Sheaths with Pathogenic Fungi for Assessing Fungal Pathogenicity
Uploaded: 2023-09-15T14:20:00
Description: Watch this Scientific Journal Video about Inoculation of Maize Leaf Sheaths with Pathogenic Fungi for Assessing Fungal Pathogenicity at JoVE.com

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

トウモロコシ鞘と菌類の細胞間相互作用の可視化

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.

著者スポットライト:トウモロコシにおける真菌の病原性メカニズムの調査 

真菌葉トウモロコシ病原体による感染の生細胞イメージングのための分離トウモロコシ鞘

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.

Watch this Scientific Journal Video about Inoculation of Maize Leaf Sheaths with Pathogenic Fungi for Assessing Fungal Pathogenicity at JoVE.com

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

真菌病原性評価のためのトウモロコシ葉鞘の病原性真菌の接種

まず、頑丈なはさみまたははさみを使用して、1.5ミリリットルのマイクロ遠心チューブの円錐形の底から0.5ミリメートルのキャップの端を切り取ります。カットしたチューブにグラスウールを入れて、中央の穴を覆います。組み立てたグラスウールフィルターユニット1つを滅菌済みの1.5ミリリットルのマイクロ遠心チューブに入れ、チューブにラベルを付けます。次に、スカートなしの96ウェルPCRプレートを6つの2列サポートラックに切断します。ウェルの円錐形の底が上を向くように、2列のラックを裏返します。湿らせた濾紙を入れたガラスのシャーレにラックを置き、蓋をして葉鞘用の湿度チャンバーを作ります。胞子を採取するColletotrichum graminicolaから分生子を採取するには、3〜4ミリリットルの滅菌脱イオン水をプレートに加えます。滅菌した円錐形の先端の乳棒を使用して、プレート全体を均等にこすり、寒天から胞子をほぐします。1ミリリットルの胞子懸濁液をグラスウールフィルターユニットに無菌的に添加し、胞子が重力によって収集チューブに流れ込むようにします。ろ過した胞子懸濁液を3, 500Gで5分間遠心分離し、液体をオートクレーブ可能な容器に注ぎます。1ミリリットルの滅菌脱イオン水を収集チューブに加え、静かに攪拌してペレット化した胞子を再懸濁します。ここでも、懸濁液を遠心分離し、胞子を再懸濁して300〜500マイクロリットルの脱イオン水に定量します。100倍の倍率の複合顕微鏡下で血球計算盤を使用して、胞子濃度を決定します。次に、脱イオン水でミリリットル懸濁液あたり500、000個の胞子を準備します。第2段階の苗の最初の本葉から鞘を取り除きます。シースの重なり合う縁に沿ってサムネイルを走らせ、シュートからそっと緩めます。回収した葉鞘を3〜5センチメートルのセグメントにカットします。各セグメントを慎重に広げて、内側の表皮層を露出させます。20マイクロリットルの真菌胞子懸濁液を、中央肋骨の上のシースの中央にある内面に塗布します。接種した葉鞘を水平に置き、中央の肋骨を底に置き、湿らせた濾紙を入れたガラスのペトリプレートに入れます。次に、湿らせた発芽紙で裏打ちされた透明な収納ボックスに小さな湿度チャンバーを入れ、収納ボックスを蓋で覆い、摂氏23度で連続照明の下で目的の時間経過でインキュベートします。鞘に病気の兆候がないか定期的にチェックしてください。

Research

JoVE Journal

Biology

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 ...