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Mating and Progeny Isolation in the Corn Smut Fungus Ustilago maydis
玉蜀黎黑粉菌的融合和后代分离   

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Abstract

The corn smut pathogen, Ustilago maydis (U. maydis) (DC.) Corda, is a semi-obligate plant pathogenic fungus in the phylum Basidiomycota (Alexopoulos et al., 1996). The fungus can be easily cultured in its haploid yeast phase on common laboratory media. However, to complete its sexual cycle U. maydis strictly requires its specific plant host, maize (Zea mays). The fungus is an interesting and important model organism for the study of the interactions of fungal biotrophic pathogens with plants. In this protocol we describe the process of plant inoculation, teliospore recovery, germination, progeny isolation and initial mating type analysis. The primary purpose of this protocol is to identify individual progeny strains of U. maydis that can be used for downstream genetic analyses. Generation of targeted mutants to study various processes is a common approach with this and many plant pathogenic fungi. The ability to generate combinations of mutations is facilitated by sexual crossing without the need for additional selectable markers.

Materials and Reagents

  1. Plastic sterile syringes: 3 ml (BD, catalog number: 309657 ) and 10 ml (BD, catalog number: 309604 )
  2. Needles: 22 gauge (BD, EclipseTM, catalog number: 305768 ) and 27 gauge (BD, EclipseTM, catalog number: 305758 )
  3. Conical tubes: Falcon® 50 ml (Corning, catalog number: 352070 )
  4. Test tubes
  5. 1.5 ml Microfuge tubes (Thermo Fisher Scientific, catalog number: 05-408-130 )
  6. Haploid Ustilago maydis tester strains of known mating type
  7. Seeds for maize seedling assay: Variety Golden Bantam (Rich Farm Garden Supply)
  8. Seeds for maize ear inoculation: Variety Tom Thumb (Seed Savers Exchange)
  9. Sterile distilled water (sdH2O)
  10. CuSO4 solution (1%) (Sigma-Aldrich, catalog number: 451657 )
  11. Casamino acids (Becton, Dickinson and Company, catalog number: 223050 )
  12. Ammonium nitrate (Sigma-Aldrich, catalog number: A9642 )
  13. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  14. Activated charcoal (Sigma-Aldrich, catalog number: C9157 )
  15. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  16. Sodium sulfate (Na2SO4) (Sigma-Aldrich, catalog number: 239313 )
  17. Calcium chloride dehydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 223506 )
  18. Boric acid (H3BO3 ) (Sigma-Aldrich, catalog number: B6768 )
  19. Manganese(II) chloride (MnCl2) (Sigma-Aldrich, catalog number: 244589 )
  20. Zinc chloride (ZnCl2 ) (Sigma-Aldrich, catalog number: 746355 )
  21. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: 331058 )
  22. Iron(III) chloride (FeCl3) (Sigma-Aldrich, catalog number: 157740 )
  23. Potato dextrose agar (PDA) 2% agar (Sigma-Aldrich) (see Recipes)
  24. Potato dextrose broth (PDB) (Sigma-Aldrich) (see Recipes)
  25. Charcoal mating plate medium (see Recipes)
  26. U. maydis salt solution (Sigma-Aldrich) (see Recipes)
  27. Trace element solution (Sigma-Aldrich) (see Recipes)

Equipment

  1. Glass stirring rods
  2. Pipettes
  3. Ceramic mortars and pestles
  4. Sterile cheesecloth
  5. Hemocytometer or automated cell counter
  6. Light microscope
  7. Centrifuge holding 50 ml conical tubes (Beckman Coulter, model: J25I ) with a JS7.5 rotor (Beckman Coulter)
  8. Growth chamber (Conviron, model: E15 )
  9. Laminar flow hood
  10. Rotational Incubator (VWR International, New Brunswick model: 12500KC )
  11. Stationary incubator (PrecisionTM, model: 815 )

Procedure

U. maydis teliospores can potentially be produced in any above ground, actively growing tissues. However, for research purposes two plant stages will be described, each with its particular advantages. These commonly used stages are:

  1. Seedlings (7 days old).
    Advantages: Seedlings are convenient for speed and for space conservation.
  2. Maturing ears.
    Advantages: Ears have the advantage of producing copious numbers of teliospores and, although the plants must be older, the time from inoculation to spore production period is similar to that of seedlings.
    Ear inoculation should be performed before pollination occurs; removing tassels may improve gall yield (Snetselaar et al., 2001).

  1. Plant inoculation for U. maydis teliospore production
    1. Streak U. maydis compatible mating strains onto fresh potato dextrose agar (PDA) plates (from older PDA plates or glycerol stocks) and incubate at 30 °C until individual colonies appear (Figure 1). Pick a single colony of compatible mating strains into separate test tubes containing 5 ml of potato dextrose broth (PDB) and grow overnight in a rotary shaker set at 250 rpm and 30 °C. Carefully label each tube with the corresponding mating type strain growing in each tube. PDA U. maydis plates can be stored sealed with parafilm and upside down at 4 °C for 2-3 weeks. Re-streak onto fresh PDA plates prior to future use.
    2. Make 1/100 dilutions of the overnight cultures and estimate cell concentrations using a hemocytometer or automated cell counter. U. maydis cells are cigar-shaped and measure between 2 and 4 μm in width and 20 and 30 μm in length.
    3. Prepare 50 ml of cell suspension as follows. Dilute and combine each strain to a final concentration of 106 cells/ml using sdH2O. This mixture will be used to inoculate plants and it is important that compatible U. maydis strains are in roughly equal proportions in the cell mixture to achieve optimal teliospore yield. Cell mixtures are stable for inoculations for at least several hours.
    4. Inoculation of maize plants
      1. Maize seedling inoculation (7 days old). One effective injection site per seedling should suffice. However, there is frequently a need to move the needle if injection flow is inhibited. Inoculate each seedling with the cell mixture using a 3 ml disposable plastic sterile syringe fitted with a 27 gauge needle. Insert the needle about half the diameter (ca. 2.5 mm) into the pseudo-stem (the internal cavity formed by growing leaves) about 1 cm above the soil line (Figure 2). Inject enough mixture until the liquid can be observed to rise to the top of the area where leaves begin to expand (between 1-2 ml of cell suspension).
      2. Maize ear inoculation. Grow ‘Tom Thumb’ maize plants in the greenhouse for approximately 40 days or until the ears begin to develop. Before inoculation, cut silks with scissors to top of ear husks and check young ear (by feeling the exterior of the ears to check if grains are present) to ensure that grain filling had not yet taken place. Using a 10 ml hypodermic syringe with a 22 gauge needle inject cell suspension at multiple locations in the ear until liquid is observe to overflow (3-10 ml) from the point where the silks were cut. Injecting liquid into an ear often requires more force than seedlings; move the needle around to find a suitable spot for efficient injections.
    5. For all inoculated tissues, allow galls to develop and teliospores to form. This step will take about 3-4 weeks for either tissue (Figure 3).
    6. Harvest fully developed darkly pigmented galls (coloration is from the teliospores).
    7. Teliosopores can be extracted immediately, or alternatively galls can be let to dry in a paper bag at room temperature for later extractions.

  2. Teliospore isolation
    1. Grind and sterilize gall tissue [4-5 small or 1 large fresh or dried gall(s)] in a mortar containing 30 ml 1% CuSO4. Watch for the release of black teliospores during grinding. Teliospores cell walls are extremely tough and therefore will not be damaged during the grinding process.
    2. Filter the macerated tissue through several layers of sterile cheesecloth into a sterile 50 ml conical tube. Incubate flow-through for 30 min at room temperature to allow antibacterial action of CuSO4.
    3. Pellet teliospores in centrifuge set at 4,000 x g for 5 min.
    4. Resuspend the pellet in 50 ml sdH2O.
    5. Repeat steps B3 through B4 once more.
    6. Resuspend the washed teliospore pellet in 1 ml sdH2O.
    7. Spread roughly 50-150 μl of the spore suspension onto two PDA plates amended with 50 μg/ml each of streptomycin and ampicillin (PDA-Strep-Amp).
    8. Incubate overnight at 30 °C.
    9. When microcolonies are visible (<0.5 mm diameter) under a stereo microscope, add 1 ml of sdH2O to the plate. Slide a sterilized bent glass rod across the plate to suspend microcolony cells. Pipette the sporidial (cell) suspension into a sterile microfuge tube.
    10. Plate dilutions (10-1, 10-2, 10-3 dilutions work well) onto fresh PDA-Strep-Amp plates.
    11. Pick progeny colonies onto fresh plates and analyze for the segregation of the traits of interest.

  3. Mating assay
    The plate mating assay is used during isolation of strains from teliospores to establish the mating type of progeny strains (Andrews et al., 2000). U. maydis has a tetrapolar mating system governed by two independently assorting loci: a and b (Bölker et al., 2012). Mating and further filamentous development require strains to differ at both mating type loci. This assay is also used to test the ability of mutant strains to mate.
    1. Pick genetically pure progeny colonies isolated from teliospores (described above) into 5 ml PDB and grow overnight as described above.
    2. Grow tester U. maydis strains of known mating type (e.g. a1b1; a2b1; a1b2; a2b2).
    3. Spotting strains onto charcoal plate.
      1. Tester strains are spotted first. For each tester spot 3-5 μl onto charcoal forming a column (Figure 4a).
      2. Let inoculum droplets dry (in a laminar flow hood).
      3. On top of each tester strain, spot 3-5 μl of each of the progeny strains to be tested (as shown in Figure 4a).
    4. Incubate plates in the dark at room temperature for 1-2 days. Positive mating events are evidenced by white fuzzy growth due to the development of short filaments indicative of the formation of the dikaryon after mating (Figure 4b).
      Occasionally, mutants are isolated which lose their potential to complete part or all of the mating process on charcoal plates. In those cases, confrontation (Snetselaar et al., 1996; Gold et al., 1997) and cytoduction assays (Trueheart and Herskowitz, 1992; Mayorga and Gold, 1999) can be used to test the ability of these strains to undergo the first stages of mating response.

Representative data


Figure 1. U. maydis haploid colonies. Typical cream colored colonies of U. maydis grown on PDA medium. Size bar is 2 mm.


Figure 2. Inoculation of maize seedlings with mixture of compatible U. maydis haploid cells. Insert the syringe’s needle half the diameter into the maize seedling pseudo-stem at about 1 cm above the soil line. Inject enough cell mixture to fill the seedling pseudo-stem cavity; observe for liquid expression from top of leaf whorl.


Figure 3. Gall production and teliospore development in maize seedlings and ears inoculated with compatible U. maydis strains. a. Large galls have developed on the lower portion of the seedling stems (arrowheads), close to the soil. b. Series of small galls formed at the distal portion of seedling stem and leaf ligule (arrowhead). c. Young ear galls cut transversely to show teliospore production; note small black areas corresponding to mature teliospores (arrows). d. Mature ear galls; teliospore production increases at later stages of gall development (arrows) and galls become darker in color due to the abundant melanized teliospores.


Figure 4. Plate mating assay. a). Representation of suggested mating plate organization. Each isolated strain is co-spotted with tester strains of known mating type to determine mating type of progeny. b). Mating plate results with progeny of previously determined mating type. Four autophagy deletion mutant strains (Nadal and Gold, 2010) (MN8.1; MN8.4; MN8.11 and MN8.12) were tested for their ability to mate. White fuzzy growth indicates positive mating events. ɸ indicates mock (water) inoculation for the corresponding row showing the morphology of unmated strains.

Notes

  1. Charcoal mating plates should be poured when relatively cool and well mixed in a single layer in direct contact with bench. This is necessary because with prolonged solidification the charcoal will fall out of suspension and reduce the efficiency of the mating reactions.
  2. For those items without catalog numbers we have used multiple suppliers with equal success.
  3. Do not run inoculation experiments in areas where fungicides are sprayed routinely.

Recipes

Note: Chemical reagents were obtained from Sigma-Aldrich.

  1. Potato dextrose agar (PDA) 2% agar
    39 g potato dextrose agar (PDA) powder
    5 g supplemental agar
    1 L H2O
    Autoclave at 121 °C, 15 psi for 15 min
  2. Potato dextrose broth (PDB)
    24 g potato dextrose broth powder
    1 L H2O
    Autoclaved at 121 °C, 15 psi for 15 min
  3. Charcoal mating plate medium
    10 g casamino acids
    3 g ammonium nitrate
    20 g yeast extract (5% or 5 g yeast extract works even better)
    125 ml Ustilago maydis salt solution (see below)
    H2O to 1 L (pH 7.0)
    15 g agar
    5 g activated charcoal and stir
    Autoclave at 121 °C, 15 psi for 15 min
    After autoclaving add:
    10 ml filter sterilized 50% glucose
  4. Ustilago maydis salt solution
    16 g KH2PO4
    4 g Na2SO4
    8 g KCl
    2 g MgSO4.7H2O
    1 g CaCl2.2H2O
    8 ml trace element solution
    H2O to 1 L
    Storage at 4 °C
  5. Trace element solution
    30 mg H3BO3
    70 mg MnCl2
    200 mg ZnCl2
    20 mg Na2MO4.2H2O
    50 mg FeCl3
    200 mg CuSO4
    H2O to 500 ml
    Storage at 4 °C

Acknowledgments

We are grateful to our funding sources including the US Departiment of Agriculture and National Science Foundation competitive grant programs. The methods described here for teliospore harvesting and germination and progeny isolation were adapted from standard protocols (Holliday, 1974).

References

  1. Alexopoulos, C. J. and Mims, C. W. et al. (1996). Chapter 21. Phyllum Basidiomycota. Order Ustilaginales. The Smut Fungi. In: Alexopoulos, C. J., Mims, C. W. and Blackwell, M. (eds). Introductory Mycology. Wiley, 639-657.
  2. Andrews, D. L., Egan, J. D., Mayorga, M. E. and Gold, S. E. (2000). The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth. Mol Plant Microbe Interact 13(7): 781-786.
  3. Bölker, M., Genin, S., Lehmler, C. and Kahmann, R. (1995). Genetic regulation of mating and dimorphism in Ustilago maydis. Can J Bot 73(S1), 320-325.
  4. Holliday, R. (1974). Ustilago maydis. Chapter: Bacteria, Bacteriophages, and Fungi, In: King, R. C. (ed.) Handbook of genetics. Springer, 575-595.
  5. Nadal, M. and Gold, S. E. (2010). The autophagy genes ATG8 and ATG1 affect morphogenesis and pathogenicity in Ustilago maydis. Mol Plant Pathol 11(4): 463-478.
  6. Snetselaar, K. M., Carfioli, M. A. and Cordisco, K. M. (2001). Pollination can protect maize ovaries from infection by Ustilago maydis, the corn smut fungus. Can J Bot 79(12): 1390-1399.

简介

科斯达(Corda)是在担子菌门(Basidiomycota)中的半必需的植物致病真菌(Alexopoulos等人)中的玉米黑斑病病原体( al。,1996)。真菌可以容易地在其单倍体酵母相中在普通实验室培养基上培养。然而,为了完成其性周期,U. maydis严格要求其特定的植物宿主,玉米( Zea mays )。真菌是研究真菌生物营养性病原体与植物相互作用的有趣和重要的模式生物体。在这个协议中,我们描述植物接种,冬孢子恢复,萌发,后代分离和初始交配型分析的过程。本协议的主要目的是鉴定个体的子代菌株。 maydis ,可用于下游遗传分析。生成目标突变体研究各种过程是这种和许多植物致病真菌的常见方法。通过有性杂交促进产生突变组合的能力,而不需要额外的选择性标记。

材料和试剂

  1. 塑料灭菌注射器:3ml(BD,目录号:309657)和10ml(BD,目录号:309604)
  2. 针:22号(BD,Eclipse TM ,目录号:305768)和27号(BD,Eclipse TM ,目录号:305758)
  3. 锥形管:Falcon 50ml(Corning,目录号:352070)
  4. 试管
  5. 1.5ml Microfuge管(Thermo Fisher Scientific,目录号:05-408-130)
  6. 单倍体 Ustilago maydis 已知交配型的试验菌株
  7. 玉米幼苗测定种子:品种金色矮脚鸡(Rich Farm Garden Supply)
  8. 玉米穗接种种子:品种汤姆拇指(种子交换)
  9. 无菌蒸馏水(sdH 2 O)
  10. CuSO 4溶液(1%)(Sigma-Aldrich,目录号:451657)
  11. 酪氨酸(Becton,Dickinson and Company,目录号:223050)
  12. 硝酸铵(Sigma-Aldrich,目录号:A9642)
  13. 酵母提取物(Sigma-Aldrich,目录号:Y1625)
  14. 活性炭(Sigma-Aldrich,目录号:C9157)
  15. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  16. 硫酸钠(Na 2 SO 4)(Sigma-Aldrich,目录号:239313)
  17. 氯化钙脱水物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:223506)
  18. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  19. 氯化锰(II)(MnCl 2)(Sigma-Aldrich,目录号:244589)
  20. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:746355)
  21. 无水钼酸钠(Na 2 MoO 4·2H 2 O)(Sigma-Aldrich,目录号:331058)
  22. 氯化铁(III)(FeCl 3)(Sigma-Aldrich,目录号:157740)
  23. 马铃薯葡萄糖琼脂(PDA)2%琼脂(Sigma-Aldrich)(参见Recipes)
  24. 马铃薯葡萄糖肉汤(PDB)(Sigma-Aldrich)(参见Recipes)
  25. 木炭配对板培养基(见配方)
  26. U。 maydis盐溶液(Sigma-Aldrich)(参见Recipes)
  27. 微量元素溶液(Sigma-Aldrich)(参见配方)

设备

  1. 玻璃搅拌棒
  2. 移液器
  3. 陶瓷砂浆和杵
  4. 无菌奶酪布
  5. 血细胞计数器或自动细胞计数器
  6. 光学显微镜
  7. 用JS7.5转子(Beckman Coulter)装有50ml锥形管(Beckman Coulter,型号:J25I)的离心机
  8. 生长室(Conviron,型号:E15)
  9. 层流罩
  10. 旋转培养箱(VWR International,New Brunswick,型号:12500KC)
  11. 固定培养箱(Precision TM ,型号:815)

程序

U。可能在任何地上,活跃生长的组织中潜在地产生冬孢子。然而,为了研究目的,将描述两个工厂阶段,每个具有其特别的优点。这些常用的阶段是:

  1. 幼苗(7日龄)。
    优点:幼苗方便快速和节省空间。
  2. 成熟的耳朵。
    优点:穗具有产生大量冬孢子的优点,尽管植物必须更大,从接种到孢子生产期的时间与幼苗的时间相似。
    在授粉前应进行耳朵接种;去除流苏可以提高胆汁产量(Snetselaar等人,2001)。

  1. 植物接种U。 maydis 冬孢子生产
    1. Streak U。 maydis 相容的交配菌株接种到新鲜马铃薯葡萄糖上 琼脂(PDA)平板(来自较老的PDA平板或甘油储液)和 在30℃孵育直至单个菌落出现(图1)。选择一个 单菌落的兼容交配菌株分成单独的试管 含有5ml马铃薯葡萄糖肉汤(PDB),并在37℃下生长过夜 旋转振荡器设定在250rpm和30℃。仔细标记每个管 相应的交配型菌株在每个管中生长。 PDA maydis 板可以用石蜡膜密封并在4℃下颠倒储存 2-3周。在将来使用前将其重新划线到新鲜的PDA平板上。
    2. 使1/100稀释的过夜培养物和估计细胞 浓度使用血细胞计数器或自动细胞计数器。 U。 maydis 细胞是雪茄形的,并且测量宽度在2和4μm之间 和20和30μm的长度。
    3. 如下制备50ml的细胞悬浮液。稀释和组合 菌株,使用sdH 2 O,终浓度为10 6个细胞/ml。这个 混合物将用于接种植物,重要的是 兼容 maydis 菌株在大致相同的比例 细胞混合物以达到最佳冬孢子产量。细胞混合物 对于接种至少几个小时是稳定的。
    4. 玉米植株的接种
      1. 玉米幼苗接种(7日龄)。一个有效的注射部位 每幼苗应该足够了。然而,经常需要移动 ?如果注射流被抑制则针。接种每个幼苗 ?使用3ml一次性塑料无菌注射器装配细胞混合物 ?用27号针。将针头插入直径一半左右。 2.5 mm)插入假茎(通过生长形成的内腔) 叶)在土壤线上约1厘米(图2)。注入足够的混合物 ?直到液体可以观察到上升到区域的顶部 叶开始扩增(1-2ml细胞悬浮液之间)。
      2. 玉米穗接种。成长'汤姆拇指'玉米植物在温室 约40天或直到耳朵开始发育。之前 接种,用剪刀剪丝到耳壳顶部并检查年轻 ?耳朵(通过感觉耳朵的外部检查谷物是否 存在),以确保还没有发生谷物灌装。使用a 10ml皮下注射器用22号针注射细胞悬浮液 在耳朵的多个位置,直到液体观察到溢出 (3-10ml)从丝切割的点。注入液体 ?耳朵通常比幼苗需要更多的力;移动针 ?找到合适的点有效注射。
    5. 对于所有接种的组织,允许疱发展和冬孢子 形成。这个步骤将需要大约3-4周的任一组织(图3)。
    6. 收获完全发育的黑色色素痣(着色是从冬孢子)。
    7. 可以立即提取立毛孔,或者可以是疱疹 ?在室温下在纸袋中干燥以备后续提取

  2. Teliospore隔离
    1. 研磨和消毒胆组织[4-5小或1大新鲜或干燥 胆汁]在含有30ml 1%CuSO 4的研钵中。注意发布的 ?黑色冬孢子。电穿孔细胞壁极其 ?坚韧,因此在研磨过程中不会被损坏。
    2. 通过几层无菌过滤浸软组织 干酪包布放入无菌的50ml锥形管中。孵育流过 ?30分钟,以允许CuSO 4的抗菌作用。
    3. 将离心机中的颗粒冬孢子放置在4,000×g下5分钟。
    4. 将沉淀重悬在50ml sdH 2 O中。
    5. 再次重复步骤B3至B4。
    6. 在1ml sdH 2 O中重悬悬浮的冬孢子丸。
    7. 将大约50-150μl的孢子悬浮液扩散到两个PDA平板上 用50μg/ml链霉素和氨苄青霉素修饰 (PDA-Strep-Amp)。
    8. 在30℃下孵育过夜。
    9. 当在立体声下可见小菌落时(<0.5mm直径) 显微镜,向板中加入1ml sdH 2 O 2。滑动灭菌弯头 玻璃棒穿过板悬浮微菌落细胞。吸移 孢子囊(细胞)悬浮液加入无菌微量离心管中。
    10. 板稀释(10-1,10-2,10-3稀释效果良好)到新鲜的PDA-Strep-Amp平板上。
    11. 挑选后代菌落到新鲜的平板上,并分析感兴趣的性状的分离

  3. 配对测试
    在从冬孢子分离菌株期间使用板交配测定以建立子代菌株的交配型(Andrews等人,2000)。 U。 maydis 具有由两个独立分类位点控制的四极配合系统:a和b(B?lker等人,2012)。交配和进一步的丝状发育需要菌株在两种交配型基因座上不同。该测定也用于测试突变株交配的能力。
    1. 挑选从冬孢子分离的遗传纯的子代殖民地 (如上所述)到5ml PDB中并如上所述生长过夜。
    2. 成长测试员maydis 菌株(例如 a1b1; a2b1; a1b2; a2b2)。
    3. 点样菌株在炭板上。
      1. 首先检测测试仪菌株。对于每个测试点3-5μl到炭上形成柱(图4a)。
      2. 让接种液滴干燥(在层流罩中)。
      3. 在每个测试菌株的顶部,点样3-5μl待测试的每个后代菌株(如图4a所示)。
    4. 在黑暗中室温孵育平板1-2天。正面的 交配事件由于发展的白色模糊增长证明 ?的短细丝指示后的二核的形成 (图4b)。
      偶尔,突变体被隔离失去 他们完成部分或全部配对过程的潜力 木炭板。在这些情况下,对抗(Snetselaar等人,1996; ?Gold等人,1997)和细胞凋亡测定(Trueheart和Herskowitz, 1992; Mayorga和Gold,1999)可以用于测试这些的能力 菌株经历交配反应的第一阶段

代表数据


图1. maydis 单倍体菌落。 典型的乳白色菌落。 maydis 生长在PDA培养基上。尺寸栏为2 mm。


图2.用相容的U的混合物接种玉米幼苗。 单倍体细胞。将直径一半的注射器针头插入玉米幼苗假茎中距离土壤线约1厘米处。注入足够的细胞混合物填充幼苗假茎腔;观察从叶轮顶部的液体表达。


图3.在用相容的U接种的玉米幼苗和穗中的gall生产和冬孢子发育。 maydis 菌株。 a。在幼苗茎部(箭头)的下部,靠近土壤形成大的疱。 b。在幼茎和叶片的末梢部分形成一系列小疱(箭头)。 C。幼穗横切,显示冬孢子的生产;注意对应于成熟冬孢子(箭头)的小黑色区域。 d。成熟耳s由于丰富的黑化冬孢子,蒴果发育的后期阶段的冬孢子产量增加(箭头)和gall s变得更暗。


图4.平板交配测定。 a)。建议的配对板组织的表示。每个分离的菌株与已知交配型的测试菌株共点,以确定子代的交配型。 b)。交配板产生了之前确定的交配类型的后代。测试四个自噬缺失突变株(Nadal和Gold,2010)(MN8.1; MN8.4; MN8.11和MN8.12)的交配能力。白色模糊生长指示阳性交配事件。 ?表示对应行的模拟(水)接种,显示未配对菌株的形态。

笔记

  1. 木炭配对板应在相对冷却时浇注,并在与工作台直接接触的单层中良好混合。这是必要的,因为在长时间凝固时,木炭将从悬浮液中脱落并降低配合反应的效率
  2. 对于没有目录编号的项目,我们使用多个供应商并取得同样的成功
  3. 不要在常规喷洒杀真菌剂的地方进行接种实验。

食谱

注意:化学试剂获自Sigma-Aldrich。

  1. 马铃薯葡萄糖琼脂(PDA)2%琼脂
    39g马铃薯葡萄糖琼脂(PDA)粉末
    5克补充琼脂
    1 L H sub 2 O
    在121℃,15psi下高压灭菌15分钟
  2. 马铃薯葡萄糖液体培养基(PDB)
    24g马铃薯葡萄糖肉汤粉末
    1 L H sub 2 O
    在121℃,15psi下高压灭菌15分钟
  3. 木炭对板培养基
    10克酪蛋白氨基酸
    3克硝酸铵
    20克酵母提取物(5%或5克酵母提取物工作更好)
    125毫升Ustilago maydis 盐溶液(见下文) H 2 O至1L(pH 7.0) 15克琼脂
    5克活性炭,搅拌
    在121℃,15psi下高压灭菌15分钟
    高压灭菌后,加入:
    10ml过滤灭菌的50%葡萄糖
  4. 黑粉菌盐溶液
    16g KH sub 2 PO 4 sub
    4g Na 2 SO 4 SO 4/
    8克KCl
    2g MgSO 4。7H 2 O 2 1克CaCl 2 2H <2> O
    8ml微量元素溶液
    H sub 2 O到1L
    在4°C下存储
  5. 微量元素溶液
    30mg H sub 3 BO Sub 3
    70mg MnCl 2
    200mg ZnCl 2/
    20mg Na 2 MO 4 sub 。 2H O
    50mg FeCl 3
    200mg CuSO 4
    H sub 2 O至500ml
    储存于4°C

致谢

我们感谢我们的资金来源,包括美国农业和国家科学基金会竞争性资助计划。这里描述的用于冬孢子收获和发芽和子代分离的方法改变自标准方案(Holliday,1974)。

参考文献

  1. Alexopoulos,C.J。和Mims,C.W.E。等人(1996)。第21章担子菌门。订单Ustilaginales。 Smut真菌。 In:Alexopoulos,C.J.,Mims,C.W.and Blackwell,M。(eds)。 介绍性真菌学。 Wiley,639-657
  2. Andrews,D.L.,Egan,J.D.,Mayorga,M.E.and Gold,S.E。(2000)。 Ustilago maydis ubc4和ubc5基因编码MAP激酶级联的成员需要用于丝状生长。 Mol Plant Microbe Interact 13(7):781-786。
  3. B?lker,M.,Genin,S.,Lehmler,C。和Kahmann,R。(1995)。 Ustilago maydis的交配和二态性的遗传调控 Can J Bot 73(S1),320-325。
  4. Holliday,R。(1974)。 Ustilago maydis。章节:细菌,噬菌体和真菌,载于:King,R.C。(ed。) Springer,575-595。
  5. Nadal,M.和Gold,S.E。(2010)。 自噬基因ATG8和ATG1会影响黑粉菌的形态发生和致病性。 Mol Plant Pathol 11(4):463-478。
  6. Snetselaar,K.M.,Carfioli,M.A。和Cordisco,K.M。(2001)。 授粉可以保护玉米卵巢免受 Ustilago maydis ,玉米黑麦真菌。 Can J Bot 79(12):1390-1399。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nadal, M., Takach, J. E., Andrews, D. L. and Gold, S. E. (2016). Mating and Progeny Isolation in the Corn Smut Fungus Ustilago maydis. Bio-protocol 6(8): e1793. DOI: 10.21769/BioProtoc.1793.
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