Bioassay to Screen Pathogenesis-associated Genes in Alternaria brassicicola

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PLOS Pathogens
Oct 2012



Alternaria brassicicola is a necrotrophic fungus that causes black spot disease of most plants in the Brassicaceae, including cultivated Brassica species and weedy Arabidopsis species. Since the concept of transformation constructs of linear minimal elements was developed (Cho et al., 2006), we have produced over 200 strains of loss-of-function mutants with an aid of selectable marker genes. Pathogenicity assays are a time-consuming step in screening pathogenesis-associated genes among targeted gene mutants. Here we describe a method for pathogenesis assays of A. brassicicola. Using this method, we have discovered pathogenesis-associated genes and were able to further characterize the functions of selected gene (Cho et al., 2013; Cho et al., 2012; Srivastava et al., 2012).

Materials and Reagents

  1. Host plants (Brassica oleraceae and Arabidopsis thaliana)
  2. Fungal conidia (Alternaria Brassicicola)
  3. Potato dextrose agar (PDA)
  4. Miracle-Gro (The Scotts Company LLC, U.S.A.) It is a nitrogen based fertilizer. Any fertilizer would work
  5. PDA with 30 ng/ml Hygromycin B (see Recipes)


  1. Centrifuge (Eppendorf, model: 5180R or equivalent)
  2. Water spray bottle
  3. Hemocytometer
  4. Sterile 50 ml tubes with screw caps
  5. Tube holder(s) for 50 ml-sized tubes
  6. Miracloth (Calbiochem, catalog number: 475855 )
  7. Funnel for filtration of conidia
  8. Glass “hockey stick” for conidial harvest
  9. Petri plates
  10. Petri dish
  11. Fluorescence light, 30 W household lights


  1. Preparation of host plants
    1. Fill a plastic pot (3 inch x 3 inch) with a mix of half local soil and half commercially available potting mix; pack the soil by pushing on the top by hand or using a flat plate.
      Note: Do not autoclave soil mix.
    2. Place 24 pots containing the soil in a plastic tray with small drainage holes in the bottom. Soak the soil with tap water by gently sprinkling it with water. Gentle sprinkling will keep the soil from spilling out of the pot. Let excess water drain from the soil for about 2 h.
    3. Use a stick to make a hole about 1-cm deep in the soil of each pot.
    4. Put one seed of commercially available (e.g., Jonny’s, Winslow, ME) green cabbage (Brassica oleracea) in the hole and cover with soil. For Arabidopsis thaliana, broadcast several seeds on top of the soil and spray water later; gradually remove all but one plant from each pot during growth.
    5. Place the pots at room temperature under fluorescence light with a 14-h light, 10-h dark cycle for the green cabbage, and a 10-h light, 14-h dark cycle for Arabidopsis. The seeds will take about one week to germinate at room temperature. If the tray is covered with semi-transparent plastic cover, they germinate faster.
    6. Grow the plants for 5 to 6 weeks under the same conditions. Keep the soil moist by adding tap water when the soil surface becomes dry.
      Note: Do not overwater or leave the pot sitting in a water-filled tray.
    7. (Optional) If seedlings 4 weeks old or older show symptoms of nitrogen deficiency (yellowing of older leaves), dissolve 1 teaspoon of Miracle-Gro in 1 gallon of water. Pour about 70 ml of the solution or enough amounts to soak the soil in each of 48 pots. Similar nitrogen-based fertilizer would have similar effects.

    A-1 (Option 1) Detached leaf assay. Bioassays are more convenient with detached leaves than whole plants. It is easy to inoculate and measure the size of lesions on detached leaves. In addition, this method requires less space than the other method.
    1. Select 6-week-old cabbage plants of similar heights and with similar-sized leaves for each assay. Harvest healthy leaves from the 4th to 8th position on the stem, counting from the bottom.
    2. Place two disks of paper towel covering the bottom of a Petri dish 150 mm diameter x 15 mm high. Soak the disks with deionized water, creating a mini-moist chamber.
    3. Remove the waxy surface of each leaf by misting with water from a spray bottle. Do not touch the surface of the leaves with bare hands, which results in bigger lesions than usual for the wild type. In addition, nonpathogenic mutants of a gene became pathogenic in our previous work. Place leaves in the mini-moist chambers and randomly arrange them on a laboratory bench for the assay.
    4. After inoculation, cover the Petri dish with a lid to keep the relative humidity close to 100%.
      Note: This assay can be performed with various cabbage species and varieties. Leaves of Arabidopsis thaliana, however, are too thin for this procedure.

    A-2 (Option 2) Whole plant assay. I did not encounter any mutants that showed significant differences in the results of bioassays between detached leaves and whole plants. I, however, anticipate identifying genes with mutants showing different results. I recommend to perform assays three times on detached leaves and twice on whole plants.
    1. Line a semi-transparent plastic trough (90 cm x 50 cm x 50 cm or similar size) with water-soaked paper towels and mist the inside wall of the trough with water.
    2. Place potted plants in the plastic trough. Any Brassica species or ecotypes of A. thaliana are suitable for whole plant assays.
    3. After inoculation, seal the troughs and plants with plastic wrap to keep the relative humidity close to 100%. Plastic wrap can be purchased at most grocery stores. Keep the trough and plants at room temperature for about a week with periodic observation of lesion development.

  2. Inoculum preparation
    1. Perform two rounds of single-spore isolation for each strain of A. brassicicola. Harvest conidia from the second single-spore isolation in ~3 ml of 20% glycerol. Dispense 50 μl of the glycerol plus conidia (glycerol stock of conidia) into 1.5 ml aliquot tubes. Freeze and maintain the glycerol stock at -80 °C until use. Harvest conidia from PDA plates in a biosafety cabinet to protect researchers and prevent environmental contamination.
    2. Transfer 10 μl of the glycerol stock to a fresh PDA plate with an appropriate selection reagent in a biosafety cabinet.
    3. Incubate plates in the dark for 5 days at 25 °C in a growth chamber. This can be done in a light-tight cardboard box at room temperature.
    4. In a biosafety cabinet, add 5 ml of sterile water to each plate and gently rub the conidial mat with a sterilized glass hockey stick. If the conidia are released gently, aerial hyphae will not contaminate the conidial suspension and you can proceed directly to the next step. If the suspension contains too many hyphal fragments or chains or clumps of conidia, however, it will be necessary to filter the suspension through a layer of Miracloth or cheese cloth until most are removed. A funnel can be used at this step.
    5. Transfer the 3-5 ml of conidia suspended in water to a 50 ml conical tube with a cap. Fill the tube with sterile water.
    6. Centrifuge the tube for 5 min at 3,000 rpm. If a centrifuge is not available, leave the tube on the bench until conidia precipitate. It takes about 15 min.
    7. Replace the water with 50 ml of fresh sterile water and centrifuge for 5 min at 3,000 rpm. Floating water can be removed either by draining or pipetting. The tube can be filled by carefully pouring 50 ml of water.
    8. Count the conidia under a microscope using a hemocytometer. Always agitate the conidial suspension rigorously before loading the hemocytometer.
    9. Repeat step B-6.
    10. Adjust the conidial concentration to 2 x 105 in 1 ml water. Count the number of spores to confirm the concentration. This is a time-consuming process and requires experience. An approximate spore count is usually sufficient for identifying genes that are required for pathogenicity or greatly affect virulence. Accurate quantification of virulence changes requires accurate spore counting. Pathogenicity factor mutants are normally nonpathogenic regardless the numbers of conidia.
    11. Directly inoculate the right side of each leaf with conidial suspension (2,000 conidia in 10 μl of water) of wild-type A. brassicicola and the left side of the same leaf with 2,000 conidia of the experimental mutant strain in 10 μl water. This method aligns the control and experimental specimen in a symmetrical manner on both sides of the central vein. It is important to agitate the inoculum frequently or the internal variation in lesion size will increase. Do not make wound before inoculation. Nine leaves originated in three plants are sufficient to produce statistically robust results. I recommend at least three rounds of bioassay to get reliable and consistent results. Plant conditions and age of conidia are important factors that affect the result of assays.
    12. Spray the inside of the Petri dish lid with water and cover the dish.
    13. Incubate the whole plants in the plastic trough or detached leaves Petri dish inoculated with fungal strains for 5 days at room temperature. Keep the light and dark cycle similar to the one used for plant growth.
    14. Measure lesion diameters at 5 days postinoculation. Calculate the virulence of each mutant relative to the wild type using the formula (∑(Dmi-Dwi)/∑(Dwi)) x 100, where Dwi is the lesion diameter created by the wild type for the ith sample and Dmi is the lesion diameter created by the mutant for the ith sample.
    15. Analyze lesion sizes among the wild type and mutants using various statistics methods, such as the Student t-test, two-way analysis of variance (ANOVA) in Excel or in the Statistical Analysis System (SAS Institute, Cary, NC).


  1. PDA with 30 ng/ml Hygromycin B
    Add 19.5 g of potato dextrose agar in 500 ml water
    Shake and mix the powder and autoclave it for 20 min
    Cool down the PDA broth to 50 °C
    Add 300 μl of 50 mg/ml hygromycin B and mix well
    Pour in 90 mm x 15 mm Petri dish


This protocol is adapted from previously published papers (Cho et al., 2013; Cho et al., 2012; Srivastava et al., 2012).


  1. Cho, Y., Ohm, R. A., Grigoriev, I. V. and Srivastava, A. (2013). Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola. Plant J 75(3): 498-514.
  2. Cho, Y., Davis, J. W., Kim, K. H., Wang, J., Sun, Q. H., Cramer, R. A., Jr. and Lawrence, C. B. (2006). A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs. Mol Plant Microbe Interact 19(1): 7-15.
  3. Cho, Y., Srivastava, A., Ohm, R. A., Lawrence, C. B., Wang, K. H., Grigoriev, I. V. and Marahatta, S. P. (2012). Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola. PLoS Pathog 8(10): e1002974.
  4. Srivastava, A., Ohm, R. A., Oxiles, L., Brooks, F., Lawrence, C. B., Grigoriev, I. V. and Cho, Y. (2012). A zinc-finger-family transcription factor, AbVf19, is required for the induction of a gene subset important for virulence in Alternaria brassicicola. Mol Plant Microbe Interact 25(4): 443-452.


是一种坏死性真菌,其引起芸苔科中大多数植物的黑斑病,包括栽培的芸苔属物种和杂草/em>物种。 由于开发了线性最小元件的转化构建体的概念(Cho等人,2006),我们借助选择性标记基因产生了超过200个功能缺失突变体菌株。 病原性测定是筛选目标基因突变体中发病相关基因的耗时步骤。 在这里我们描述的发病机制测定的方法。 使用这种方法,我们已经发现了致病相关基因,并能够进一步表征选择的基因的功能(Cho等人,2013; Cho等人, ,2012; Srivastava 等人,2012)。


  1. 宿主植物(
    Brassica oleraceae 和拟南芥)
  2. 真菌分生孢子( Alternaria Brassicicola )
  3. 马铃薯葡萄糖琼脂(PDA)
  4. Miracle-Gro(The Scotts Company LLC,U.S.A。)它是一种氮基肥料。 任何肥料都会工作
  5. 具有30ng/ml潮霉素B的PDA(参见配方)


  1. 离心机(Eppendorf,型号:5180R或等同物)
  2. 喷水瓶
  3. 血细胞计数器
  4. 无菌50ml带螺帽的管子
  5. 用于50毫升管的管架
  6. Miracloth(Calbiochem,目录号:475855)
  7. 用于过滤分生孢子的漏斗
  8. 用于分生孢子的玻璃"曲棍球棒"
  9. 培养皿
  10. 培养皿
  11. 荧光灯,30W家用灯


  1. 宿主植物的制备
    1. 填充塑料罐(3英寸x 3英寸)混合半局部土壤和半市售盆栽混合物; 用手推或用平板推上顶部包装土壤。
    2. 将含有土壤的24盆放在塑料托盘中,底部有小排水孔。 用自来水轻轻地用水浸泡土壤。 温和的喷洒将防止土壤溅出锅。 让多余的水从土壤排出约2小时。
    3. 使用棍子在每个盆的土壤中做一个约1厘米深的洞。
    4. 将一种可商购的(例如,Jonny's,Winslow,ME)绿白菜( Brassica oleracea )放入孔中并用土壤覆盖。对于拟南芥(Arabidopsis thaliana),在土壤上撒播几粒种子,然后喷洒水;在生长期间逐渐从每个花盆中除去一株植物
    5. 将盆在室温下在荧光灯下,14小时光照,10小时黑暗周期为绿白菜,和10小时光,14小时黑暗周期为拟南芥。种子将需要大约一个星期在室温下发芽。如果托盘覆盖有半透明塑料盖,它们会更快地发芽。
    6. 在相同条件下生长植物5至6周。当土壤表面变干时,通过加入自来水保持土壤潮湿。
    7. (可选)如果4周龄以上的幼苗出现缺氮症状(老叶发黄),将1茶匙的Miracle-Gro溶解于1加仑水中。倒入约70ml的溶液或足够的量以浸泡土壤在48个盆中的每个盆中。类似的氮基肥料也会产生类似的效果
    A-1(选项1 )分离叶测定。生物测定比完整植物更容易分离叶。容易接种和测量分离的叶上的病变的尺寸。此外,该方法需要较少的空间 比其他方法。
    1. 选择类似高度的6周龄白菜植物,并且对于每个测定具有相似大小的叶。从茎的第4个至第8个位置收获健康叶,从底部开始计数。
    2. 放置两块纸巾覆盖在直径150mm,高15mm的培养皿的底部。用去离子水浸泡圆盘,形成微湿室
    3. 通过喷雾瓶中的水喷雾除去每片叶子的蜡质表面。不要用裸手触摸叶子的表面,这会导致比野生型更大的病变。此外,基因的非致病突变体在我们以前的工作中变得致病。将叶子放在微湿室中并随机排列在实验台上用于测定。
    4. 接种后,用盖子盖住培养皿以保持相对湿度接近100%。

    A-2(选项2 )全植物测定。我没有遇到任何突变体显示在分离的叶和整株植物之间的生物测定的结果的显着差异。然而,我预计识别具有显示不同结果的突变体的基因。我建议对分离的叶子进行三次测定,对整株植物进行两次。
    1. 将半透明塑料槽(90厘米×50厘米×50厘米或类似尺寸)与水浸泡的纸巾在一起,并用水将槽的内壁喷雾。
    2. 将盆栽植物放在塑料槽中。任何 Brassica 物种或生态型。 thaliana 适用于整株植物检测
    3. 接种后,用塑料包封槽和植物以保持相对湿度接近100%。塑料包装可以在大多数杂货店购买。保持槽和植物在室温下大约一个星期,定期观察病变发展
  2. 接种准备
    1. 对每一个菌株进行两轮单孢子分离。 brassicicola 。收获分生孢子从第二次单孢子分离在〜3毫升20%甘油。分配50μl的甘油加分生孢子(分生孢子的甘油原液)到1.5ml等分试管中。冷冻并保持甘油原液在-80℃直到使用。从生物安全柜中的PDA平板收获分生孢子,以保护研究人员和防止环境污染。
    2. 在生物安全柜中,用适当的选择试剂将10μl甘油原液转移到新鲜的PDA板上
    3. 在黑暗中在25℃在生长室中孵育平板5天。这可以在室温下在不透光的纸板盒中进行。
    4. 在生物安全柜中,向每个板中加入5ml无菌水,并用灭菌的玻璃曲棍球棒轻轻地擦拭分生孢子垫。如果分生孢子轻轻释放,气生菌丝不会污染分生孢子悬浮液,您可以直接进行下一步。然而,如果悬浮液含有太多菌丝碎片或分生孢子的链或簇,则需要通过一层Miracloth或奶酪布过滤悬浮液,直到大部分被除去。此步骤可以使用漏斗。
    5. 将3-5毫升悬浮在水中的分生孢子转移到带有盖子的50ml锥形管中。用无菌水填充管。
    6. 以3,000 rpm离心管子5分钟。如果离心机不可用,离开管在台上,直到分生孢子沉淀。大约需要15分钟。
    7. 用50ml新鲜无菌水替换水,并在3,000 rpm离心5分钟。浮动水可以通过排水或移液除去。管可以通过小心地倾倒50ml水来填充。
    8. 使用血细胞计数器在显微镜下计数分生孢子。在装载血细胞计数器之前,必须严格搅拌分生孢子悬浮液。
    9. 重复步骤B-6。
    10. 在1ml水中将分生孢子浓度调节至2×10 5。计数孢子数以确认浓度。这是一个耗时的过程,需要经验。大致的孢子计数通常足以鉴定致病性所需的基因或极大地影响毒力。毒力变化的准确量化需要精确的孢子计数。致病性因子突变体通常是非致病性的,而不管分生孢子的数目
    11. 用野生型A的分生孢子悬浮液(10μl水中的2,000个分生孢子)直接接种每片叶子的右侧。 brassicicola和相同叶的左侧,在10μl水中具有2,000个分生孢子的实验突变株。该方法以对称方式在中心静脉的两侧对准对照和实验样本。重要的是经常搅拌接种物或者病变大小的内部变化将增加。不要在接种前做伤口。源于三株植物的九株叶足以产生统计学上稳定的结果。我建议至少三轮生物测定,以获得可靠和一致的结果。植物条件和分生孢子的年龄是影响测定结果的重要因素
    12. 用水喷洒培养皿盖的内部并盖住培养皿
    13. 孵育整个植物在塑料槽或分离的叶子培养皿中接种真菌菌株,在室温下5天。保持光照和黑暗循环类似于用于植物生长的循环。
    14. 在接种后5天测量病变直径。使用公式计算每个突变体相对于野生型的毒力(Σ(Dm i)-Dw i)/Σ(Dw i,i) )×100,其中Dw em i是由第i个样本的野生型产生的病变直径,D m,i是所创建的病变直径对于 i 样品的突变体
    15. 使用各种统计学方法,例如在Excel中或在统计分析系统(SAS Institute,Inc。)中的Student方差(ANOVA)的Student方差分析(ANOVA),在野生型和突变体之间分析损伤大小, Cary,NC)。


  1. PDA与30ng/ml潮霉素B
    倒入90 mm x 15 mm培养皿


该协议改编自以前发表的论文(Cho等人,2013; Cho等人,2012; Srivastava等人,2012年) )。


  1. Cho,Y.,Ohm,R.A.,Grigoriev,I.V.and Srivastava,A。(2013)。 真菌特异性转录因子AbPf2在链格孢菌中激活致病性。 Plant J 75(3):498-514。
  2. Cho,Y.,Davis,JW,Kim,KH,Wang,J.,Sun,QH,Cramer,RA,Jr.and Lawrence,CB(2006)。使用线性最小元件(LME)构建体的链格孢菌(Alternaria brassicicola)功能性基因组学的高通量靶向基因破坏方法。 Mol Plant Microbe Interact 19(1):7-15。
  3. Cho,Y.,Srivastava,A.,Ohm,R.A.,Lawrence,C.B.,Wang,K.H.,Grigoriev,I.V.and Marahatta,S.P.(2012)。 转录因子Amr1在链格孢菌中诱导黑色素生物合成并抑制毒力。 PLoS Pathog 8(10):e1002974。
  4. Srivastava,A.,Ohm,R.A.,Oxiles,L.,Brooks,F.,Lawrence,C.B.,Grigoriev,I.V.and Cho,Y.(2012)。 锌指家族转录因子AbVf19是诱导重要基因子集所必需的 在链格孢菌中。 Mol Plant Microbe Interact 25(4):443-452。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cho, Y. (2013). Bioassay to Screen Pathogenesis-associated Genes in Alternaria brassicicola. Bio-protocol 3(16): e873. DOI: 10.21769/BioProtoc.873.
  2. Cho, Y., Srivastava, A., Ohm, R. A., Lawrence, C. B., Wang, K. H., Grigoriev, I. V. and Marahatta, S. P. (2012). Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola. PLoS Pathog 8(10): e1002974.