Ustilago maydis Virulence Assays in Maize

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The Plant Cell
Apr 2015



The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection symptoms are seen in the form of prominent tumors on all aerial parts of maize, after the establishment of a biotrophic interaction with the host usually around 5-6 days post infection (dpi). The fungus colonizes the various developmentally distinct aerial organs at different stages of development to form these prominent symptoms. Although being a biotrophic plant pathogen, U. maydis can easily be cultivated under axenic conditions to produce a standardized inoculum. The infections can be carried out under laboratory conditions by syringe inoculation on all the aerial organs of maize. This protocol has been successfully utilized to infect all the aerial organs of maize and formulate the virulence assays in U. maydis making it an excellent model system to study phyto-pathological investigations (Schilling et al., 2014; Redkar et al., 2015).

Keywords: Ustilago (黑粉菌), Maize (玉米), Tumors (肿瘤), Disease (病)

Materials and Reagents

  1. 1 ml syringes (Henke Sass-Wolf, catalog number: 5100.200V0 )
  2. Needles (16 G, 40 mm) (Premier Healthcare & Hygiene, BD microlance, catalog number: 300637 )
  3. 50 ml Falcon tubes (Sigma-Aldrich, catalog number: T2318 )
  4. Glass test tubes (Carl Roth GmbH + Co, catalog number: HA79.1 )
  5. Soil (Frühstorfer Pikiererde T type soil) and Pots
  6. Ustilago maydis strains
    Note: The U. maydis wild-type or any transgenic strains are maintained as glycerol stock in -80 °C for long term storage. Every strain can be retrieved on a PDA plate.
  7. Maize seeds (e.g., variety Early Golden Bantam or Gaspe Flint)
    Note: The maize variety Early Golden Bantam is a standard maize line that requires around 4 weeks for the floral switch and development of the male inflorescence. The maize variety Gaspe Flint is a mutant maize that completes its life cycle in 35-38 days and hence has an early floral switch with the development of male inflorescence around 15 days after sowing. Hence this variety is suitable for floral infections with U. maydis.
  8. Yeast extract (BD, Difco, catalog number: 210934 )
  9. Peptone (BD, Difco, catalog number: 211830 )
  10. Sucrose (Carl Roth GmbH + Co, catalog number: 9097.2 )
  11. Double distilled water
  12. 2.4% Potato Dextrose Broth (BD, Difco, catalog number: 254920 )
  13. 2% agar (BD, Difco, catalog number: 214010 )
  14. YEPSL medium (modified from Tsukada et al., 1988) (see Recipes)
  15. Potato Dextrose Agar (PDA) plates (see Recipes)


  1. Greenhouse
  2. Erlenmeyer flasks (250 ml)
  3. Disposable cuvettes (Carl Roth GmbH + Co, catalog number: Y195.1 )
  4. Incubator [e.g., Certomat BS-1 (B. Braun Biotech International)]
  5. Centrifuge (Thermo Fisher Scientific)
  6. Spectrophotometer [e.g. Ultrospec 3000 proUV/Visible (GE Healthcare)]
  7. Knife


  1. Cultivation of maize plants
    1. Maize varieties (that may have different lengths of developmental switch) (e.g. cv Early Golden Bantam or cv Gaspe Flint, or your maize cultivar of choice) are grown in round pots of 20 cm with 4 seeds in each pot at equal spacing. The pots are kept in a temperature controlled greenhouse with the following conditions (14-h/10-h light/dark cycle, 28/22 °C. Depending on the growth behavior of the plants, it must be ensured that the right stage is used for infection. Seedling infections are regularly done at the three leaf stage. Tassel infections are done when the tassel floral meristem is in the mitotic phase before the pollen formation (tassel size of 1-1.5 cm).
    2. Both varieties can be grown in soil in approximately 15 cm round pots with 4 plants each. The variety Gaspe Flint can mainly be used for tassel (male inflorescence) infections as they have an early floral switch (15 days) as compared to the standard maize (early golden Bantam which is approx. 4 weeks).

  2. U. maydis culture preparation
    1. U. maydis strains growing on a fresh PDA plate (no older than 1 week) are used for preparation of pre-inoculum. The strains are inoculated in a 3 ml YEPSL medium in a sterile glass test tube and incubated overnight at 28 °C with shaking at 200 rpm.
    2. The pre-inoculum (40 µl) is used for inoculation of the large media (50 ml) in a 250 ml sterile Erlenmeyer flask and then incubated at 28 °C with shaking at 200 rpm for around 10-12 h.
    3. The cultures are then harvested when the optical density (OD600) reaches to 0.6 to 0.8 (cultures should not be below 0.5, nor above 1.0). This range of OD ensures the growth of the U. maydis sporidia in the active dividing phase. The cultures are then centrifuged in 50 ml Falcon tubes at 900 x g for 5 min at room temperature.
    4. The harvested cells are resuspended and washed in sterile water to remove all the media remains. The step B4 of centrifugation is repeated.
    5. The cells are then resuspended by pipetting or vortexing in water to OD600 = 1.0. This inoculum is used to infect the maize plants.
      Note: For infection with wild type U. maydis strains (e.g., FB1 and FB2 which are the two compatible mating types) are to be mixed together in equal volume at OD600 =1.0 for a successful infection. In case of the solopathogenic strain SG200 (Kämper et al., 2006) or in case of any mutant resulting from this background one cultural strain can be used as inoculum.

  3. U. maydis virulence assays
    In seedlings:
    1. Seedling infections are done with the maize plantlets that normally show three juvenile leaf stage. For most of the cultivars of maize, the plants are 7 days old when this developmental stage is reached (Figure 1).

      Figure 1. Maize plants cv. Early Golden Bantam and their inoculation with U. maydis. A. Maize seedling at the three leaf stage is inoculated with U. maydis for seedling leave infection. The syringe points into the center of the leaf whirl. B. A maize plant is inoculated with U. maydis for tassel infection. The syringe hits the tassel inside the stem. Inset: the maize tassel at the stage of fungal infection.

    2. The seeding infections are carried out by syringe with 300-500 μl of the inoculum cell suspension into the interior of the leaf whorl. The injection site is chosen approx. 1 cm above the soil, which is about 2.5 to 3 cm above the basal plant meristem and is the juvenile stem.
    3. The leaf sheaths of the first and second leaf and the leaf blades of the third and fourth leaf which are immersed into the whorl are pierced by the syringe half way onto the center of the stem cylinder, which later show an infection mark after the symptom development.
    4. Once the inoculum is seen on the inner whorl of leaves the seedling is known to be successfully infected.
    5. Plants are kept in controlled growth conditions at 28/22 °C. Successful infection will lead to tumor appearance after 4-6 dpi.
    6.  The tumors formed by the artificial inoculation of seedlings are scored at 12 dpi by a modified standard scheme as described previously (Kämper et al., 2006) which is shown in Table 1 and shown below (Figure 2).

      Figure 2. Morphological symptoms in seedling (left) and tassel tissues (right) of maize after infection by U. maydis. Photographs were taken at 10 dpi.

      Table 1. Classification of symptoms of infected maize seedlings
      Plant symptoms
      No symptom
      The plant shows no signs of infection
      The plant shows chlorotic discolouration of the infected leaves (third leaf and younger)
      Small tumors
      The largest tumors of the plant are <1.5 mm
      Normal tumors
      Tumors of the plant are 2-4 mm in diameter
      Heavy Tumors
      Very strong tumors leading to a stunted growth of stem
      Dead Plant
      The plant is dead and looks necrotic after infection with U. maydis

    In tassels (male inflorescence):
    1. Tassel infections are done immediately after floral switch of the plant when the tassel is still in the actively dividing mitotic phase. In cv. Gaspe Flint this is normally after 15 days and after around 4 weeks in cv. Early Golden Bantam after sowing (see Figure 1).
    2. The tassel infections are also performed using a syringe based method. The immature tassels location is identified without cutting the plant by pressing the stem. The tassel meristem embedded in the stem has an air space surrounding it which makes the stem hollower. Above the tassel which terminates the shoot, the leaf sheaths overlap, but there is no solid center and this region contains the developing inflorescence.
    3. Prior to injection, one or two plants are cut open with a sharp knife from the base, to understand the position of the tassel meristem that is embedded in the whorl of leaves.
    4. Based on this height, the tassel location is approximately identified on all plants. The standard injection protocol involves inoculation of 1 ml of culture injected all at once, taking care to penetrate only halfway across the plant diameter. This ensures the inoculum to the entire floral meristem and fills the air space around the tassel.
    5. Plants are kept in a controlled greenhouse at 28 °C which show successful tumor appearance after 8-10 dpi (Figure 2).
    6. Disease symptoms in the tassels are scored 10 days after infection in cv. Gaspe Flint and after 14 days in cv. Early Golden Bantam by the scoring scheme described previously (Schilling et al., 2014, Table 2) and shown below.

      Table 2. Classification of symptoms of infected maize tassels
      Tassel Symptom
      Tumors<50% of tassel, small
      The part of the tassel is converted into small tumors in range of 2-4 mm
      Tumors<50% of tassel, large
      Less than half of the tassel is converted into large tumors >4 mm
      Tumors>50% of tassel, small
      The entire tassel inflorescence is covered with small tumors.
      Tumors>50% os tassel, large
      The complete tassel is converted into heavy tumors by U. maydis
      Tassel growth stunted
      The tassel is arrested at the developmental time point at which it was infected and this 1-3 cm stunted tassel becomes tumorous.


  1. YEPSL medium
    This medium consists of 0.4% (w/v) yeast extract, 0.4% (w/v) peptone, 2% (w/v) sucrose in double distilled water.
  2. PDA plates
    The PDA medium consists of 2.4% Potato Dextrose Broth and 2% agar in double distilled water. After autoclaving approx. 18 ml of media in each petri plate.


Our work was funded by the Max Planck Society, The Deutsche Forschungsgemeinschaft (DFG), The Deutscher Akademischer Austauschdienst (DAAD) and the Cluster of Excellence on Plant Science (CEPLAS). The protocol is adapted from Kämper et al. (2006); Schilling et al. (2014) and Redkar et al. (2015).


  1. Kamper, J., Kahmann, R., Bolker, M., Ma, L. J., Brefort, T., Saville, B. J., Banuett, F., Kronstad, J. W., Gold, S. E., Muller, O., Perlin, M. H., Wosten, H. A., de Vries, R., Ruiz-Herrera, J., Reynaga-Pena, C. G., Snetselaar, K., McCann, M., Perez-Martin, J., Feldbrugge, M., Basse, C. W., Steinberg, G., Ibeas, J. I., Holloman, W., Guzman, P., Farman, M., Stajich, J. E., Sentandreu, R., Gonzalez-Prieto, J. M., Kennell, J. C., Molina, L., Schirawski, J., Mendoza-Mendoza, A., Greilinger, D., Munch, K., Rossel, N., Scherer, M., Vranes, M., Ladendorf, O., Vincon, V., Fuchs, U., Sandrock, B., Meng, S., Ho, E. C., Cahill, M. J., Boyce, K. J., Klose, J., Klosterman, S. J., Deelstra, H. J., Ortiz-Castellanos, L., Li, W., Sanchez-Alonso, P., Schreier, P. H., Hauser-Hahn, I., Vaupel, M., Koopmann, E., Friedrich, G., Voss, H., Schluter, T., Margolis, J., Platt, D., Swimmer, C., Gnirke, A., Chen, F., Vysotskaia, V., Mannhaupt, G., Guldener, U., Munsterkotter, M., Haase, D., Oesterheld, M., Mewes, H. W., Mauceli, E. W., DeCaprio, D., Wade, C. M., Butler, J., Young, S., Jaffe, D. B., Calvo, S., Nusbaum, C., Galagan, J. and Birren, B. W. (2006). Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444(7115): 97-101.
  2. Redkar, A., Hoser, R., Schilling, L., Zechmann, B., Krzymowska, M., Walbot, V. and Doehlemann, G. (2015). A secreted effector protein of Ustilago maydis guides maize leaf cells to form tumors. Plant Cell 27(4): 1332-1351.
  3. Schilling, L., Matei, A., Redkar, A., Walbot, V. and Doehlemann, G. (2014). Virulence of the maize smut Ustilago maydis is shaped by organ-specific effectors. Mol Plant Pathol 15(8): 780-789.
  4. Tsukada, T., Carleton, S., Fotheringham, S. and Holloman, W. K. (1988). Isolation and characterisation of an autonomously replicating sequence from Ustilago maydis. Mol Cell Biol 8: 3703-3709.


落叶青霉菌(Basidiomycetous smut fungus)Ustilago maydis( U。maydis )感染其宿主植物玉米的所有地上部分( Zea mays 感染症状在与宿主生物营养相互作用通常在感染后约5-6天(dpi)建立后以玉米的所有地上部分的突出肿瘤的形式见到。真菌在不同发育阶段定居各种发育不同的气生器官以形成这些突出的症状。虽然是一种生物营养性植物病原体, maydis 可以容易地在无菌条件下培养以产生标准化接种物。感染可以在实验室条件下通过注射器接种在玉米的所有气生器官上进行。该方案已经成功地用于感染玉米的所有气生器官并且在U中制定毒力测定。 maydis 使其成为研究植物病理学研究的优良模型系统(Schilling等人,2014; Redkar等人,2015)。

关键字:黑粉菌, 玉米, 肿瘤, 病


  1. 1ml注射器(Henke Sass-Wolf,目录号:5100.200V0)
  2. 针(16G,40mm)(Premier Healthcare& Hygiene,BD microlance,目录号:300637)
  3. 50ml Falcon管(Sigma-Aldrich,目录号:T2318)
  4. 玻璃试管(Carl Roth GmbH + Co,目录号:HA79.1)
  5. 土壤(FrühstorferPikiererde T型土)和罐
  6. Ustilago maydis 菌株
  7. 玉米种子(例如品种Early Golden Bantam或Gaspe Flint)
    注意:玉米品种Early Golden Bantam是标准的玉米品系,需要大约4周的花卉开关和雄性花序的发育。玉米品种Gaspe Flint是一种突变体玉米,其在35-38天内完成其生命周期,因此在播种后约15天具有早期花卉开关,雄性花序发育。因此,这种品种适用于U.canis的花卉感染。
  8. 酵母提取物(BD,Difco,目录号:210934)
  9. 蛋白胨(BD,Difco,目录号:211830)
  10. 蔗糖(Carl Roth GmbH + Co,目录号:9097.2)
  11. 双蒸水
  12. 2.4%土豆葡萄糖肉汤(BD,Difco,目录号:254920)
  13. 2%琼脂(BD,Difco,目录号:214010)
  14. YEPS L medium(从Tsukada 修改,1988)(参见配方)
  15. 马铃薯葡萄糖琼脂(PDA)平板(见配方)


  1. 温室
  2. 锥形烧瓶(250ml)
  3. 一次性比色皿(Carl Roth GmbH + Co,目录号:Y195.1)
  4. 孵化器[例如 Certomat BS-1(B. Braun Biotech International)]
  5. 离心机(Thermo Fisher Scientific)
  6. 分光光度计[例如 Ultrospec 3000 proUV/Visible(GE Healthcare)]
  7. 刀具


  1. 玉米植物的栽培
    1. 玉米品种(可能有不同长度的发育 开关)( cv Early Golden Bantam或cv Gaspe Flint(或您的玉米 选择的品种)在20cm的圆形罐中生长,4粒种子 每个罐以相等的间距。将罐保持在温度下 控制温室,具有以下条件(14小时/10小时 亮/暗循环,28/22℃。取决于生长行为 植物,必须确保正确的阶段用于感染。 幼苗感染在三叶期定期进行。流苏 当流苏花分生组织在有丝分裂中时进行感染 在花粉形成之前的阶段(流苏大小为1-1.5cm)
    2. 都 ?品种可以在土壤中在具有4的约15cm圆形花盆中生长 ?植物。品种Gaspe Flint主要用于流苏 (男性花序)感染,因为他们有早期花开关(15 天)与标准玉米(早期金黄矮脚鸡)相比 约。 4周)。

  2. U。 maydis 文化准备
    1. U。 maydis 菌株在新鲜PDA平板上生长(不超过1周) 用于制备预接种物。将菌株接种在a ?3ml YEPSL培养基中并在无菌玻璃试管中孵育过夜 在28℃下以200rpm振荡。
    2. 预接种物(40μl) 用于在250ml无菌培养基中接种大培养基(50ml) 锥形瓶中,然后在28℃下以200rpm振荡温育 ?约10-12小时。
    3. 然后当光学时收获培养物 ?密度(OD 600)达到0.6至0.8(培养物不应低于) 0.5,也不高于1.0)。这个范围的OD确保了em的生长。 可能在活跃分裂阶段的孢子。然后培养 在50ml Falcon管中在900×g离心5分钟 温度
    4. 将收获的细胞重悬浮并洗涤 无菌水除去所有的培养基。步骤B4 重复离心。
    5. 然后将细胞重悬浮 移液或在水中涡旋至OD 600 = 1.0。该接种物用于 ?感染玉米植物 注意:对于野生型U maydis菌株(例如FB1和FB2是两种相容的配对 类型)在OD 600 = 1.0时以等体积混合在一起 成功感染。对于solopathogenic菌株SG200(K?mper ?et al。,2006)或在由此背景产生的任何突变体的情况下 一个文化菌株可以用作接种物。

  3. U。 maydis 毒力测定
    1. 幼苗感染用通常显示的玉米植株进行 ?三个幼叶阶段。对于大多数玉米品种 当达到这个发育阶段时植物是7天大 1)。

      图1.玉米植物cv。早期黄金矮脚鸡和他们 接种U。 maydis 。 A.玉米幼苗在三叶期是 ?接种U。 maydis 用于幼苗感染。注射器 指向叶片旋转的中心。 B.接种玉米植物 ?与。 maydis 为流感感染。注射器撞在里面的流苏 ?茎。插图:玉米穗在真菌感染阶段。

    2. 接种感染通过注射器用300-500μl的进行 ?接种细胞悬浮液进入叶轮内部。的 注射部位选择约。在土壤上方1厘米,约为2.5厘米 ?至基部植物分生组织3cm以上并且是幼年茎。
    3. 第一叶片和第二叶片的叶鞘和叶片 浸入螺旋中的第三和第四叶片被刺穿 通过注射器一半到杆柱的中心,这 后来在症状发展后显示感染标记
    4. 一旦在叶子的内部叶子上看到接种物,就知道幼苗被成功感染
    5. 将植物保持在28/22℃的受控生长条件下。 成功的感染会导致4-6 dpi后出现肿瘤。
    6.  对通过人工接种幼苗形成的肿瘤进行评分 ?(K?mper等人,2006)的改进的标准方案在12dpi下进行,如表1所示和下面显示的(图2)。

      图2.幼苗(左)和雄穗组织中的形态症状 (右)玉米感染后的U。 maydis 。拍摄了照片 ?10 dpi。

      植物是死的,感染后感染后看起来坏死。 maydis

    1. 流感感染在植物花开关后立即进行 当雄穗仍在有效分裂有丝分裂期。在cv。 Gaspe Flint这通常是在15天后和大约4周后 简历。早播金万丹播种后(见图1)。
    2. 流苏 感染也使用基于注射器的方法进行。不成熟 ?流苏位置被识别,而不通过按压切割植物 茎。嵌入茎中的雄穗分生组织具有空气空间 包围它使茎干燥。上面的流苏哪 终止苗,叶鞘重叠,但没有固体 中心,该区域包含发育中的花序。
    3. 在注射之前,用锋利的刀切开一个或两个植物 从基地,了解缨子分生组织的位置 ?嵌入叶子的轮生。
    4. 基于这个高度, 流苏位置在所有植物上大致确定。标准 注射方案包括接种1ml全部注射的培养物 同时,注意只穿透植物的一半 直径。这确保接种到整个花分生组织和 填充流苏周围的空气空间。
    5. 将植物保持在28℃的受控温室中,其在8-10dpi后显示成功的肿瘤外观(图2)
    6. 流感病毒症状在感染后10天评分 在cv。 Gaspe Flint和14天后在cv。早期黄金矮脚鸡由 前述的评分方案(Schilling等人,2014,表2) 如下所示。

      肿瘤> 50%的流苏,小
      肿瘤> 50%os穗,大
      完整的雄穗通过U转变成重的肿瘤。 maydis
      流苏在发育时间被捕 它被感染的点和这1-3厘米阻碍流苏变成 肿瘤。


  1. YEPSL介质
  2. PDA平板


我们的工作由马克斯普朗克学会,德意志研究所(DFG),德国学术会议(DAAD)和植物科学卓越丛集(CEPLAS)资助。该协议改编自K?mper等人(2006); Schilling (2014年)和Redkar 等人(2015年)。


  1. Kamper,J.,Kahmann,R.,Bolker,M.,Ma,LJ,Brefort,T.,Saville,BJ,Banuett,F.,Kronstad,JW,Gold,SE,Muller, Wosten,HA,de Vries,R.,Ruiz-Herrera,J.,Reynaga-Pena,CG,Snetselaar,K.,McCann,M.,Perez-Martin,J.,Feldbrugge,M.,Basse,CW,Steinberg G.,Ibeas,JI,Holloman,W.,Guzman,P.,Farman,M.,Stajich,JE,Sentandreu,R.,Gonzalez-Prieto,JM,Kennell,JC,Molina,L.,Schirawski,J Mendoza,A.,Greilinger,D.,Munch,K.,Rossel,N.,Scherer,M.,Vranes,M.,Ladendorf,O.,Vincon,V.,Fuchs,U.,Sandrock B.,Meng,S.,Ho,EC,Cahill,MJ,Boyce,KJ,Klose,J.,Klosterman,SJ,Deelstra,HJ,Ortiz-Castellanos,L.,Li,W.,Sanchez-Alonso, P.,Schreier,PH,Hauser-Hahn,I.,Vaupel,M.,Koopmann,E.,Friedrich,G.,Voss,H.,Schluter,T.,Margolis,J.,Platt,D.,Swimmer C.,Gnirke,A.,Chen,F.,Vysotskaia,V.,Mannhaupt,G.,Guldener,U.,Munsterkotter,M.,Haase,D.,Oesterheld,M.,Mewes,HW,Mauceli, EW,DeCaprio,D.,Wade,CM,Butler,J。,Young,S.,Jaffe,DB,Calvo,S.,Nusbaum,C.,Galagan,J.and Birren,BW(2006)。 从生物营养性真菌植物病原体的黑猩猩的基因组的观点 Ustilago maydis 。 自然 444(7115):97-101。
  2. Redkar,A.,Hoser,R.,Schilling,L.,Zechmann,B.,Krzymowska,M.,Walbot,V。和Doehlemann,G。 Ustilago maydis的分泌效应蛋白指导玉米叶细胞形成肿瘤。植物细胞 27(4):1332-1351。
  3. Schilling,L.,Matei,A.,Redkar,A.,Walbot,V.和Doehlemann,G。(2014)。 玉米黑穗病毒的毒力 Ustilago maydis 是由器官特异性效应物。 Mol Plant Pathol 15(8):780-789。
  4. Tsukada,T.,Carleton,S.,Fotheringham,S。和Holloman,W.K。(1988)。 孤立和表征来自Ustilago的自主复制序列。 Mol Cell Biol 8:3703-3709
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引用:Redkar, A. and Doehlemann, G. (2016). Ustilago maydis Virulence Assays in Maize. Bio-protocol 6(6): e1760. DOI: 10.21769/BioProtoc.1760.