2023, Vol. 39中国科学院微生物研究所、中国微生物学会主办
文章信息
- 周涛, 叶梅燕, 刘天瑶, 兰胡娇, Hashimi Said Masoud, 郭威, 刘建中
- ZHOU Tao, YE Meiyan, LIU Tianyao, LAN Hujiao, HASHIMI Said Masoud, GUO Wei, LIU Jianzhong
- 沉默GmATG10导致大豆免疫反应的激活
- Silencing GmATG10 results in activation of immune responses in soybean
- 生物工程学报, 2023, 39(2): 586-602
- Chinese Journal of Biotechnology, 2023, 39(2): 586-602
- 10.13345/j.cjb.220545
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文章历史
- Received: July 13, 2022
- Accepted: August 31, 2022
- Published: September 5, 2022
引用本文
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2. 浙江师范大学生命科学学院 浙江省特色经济植物生物技术重点实验室, 浙江 金华 321004
2. Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants, College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
自噬是生物体降解和循环细胞质物质如蛋白质、脂类、聚集体乃至整个细胞器的主要途径[1]。自噬的机制依赖于被称为自噬体(autophagosomes)的特化的双层膜囊泡；其专门用于将细胞质中不需要或受到损伤的组分运至溶酶体(动物)或液泡(酵母和植物)中降解，为细胞提供能源和新的合成原料。酸性的液泡中含有大量各种水解酶，可降解几乎所有胞内组分[2]。在营养缺乏及胁迫条件下，通过自噬途径大量降解蛋白、碳水化合物及脂类为机体存活和新的生长提供核苷酸、氨基酸等基本单元[2]。
自噬途径需要40多种自噬相关基因(autophagy-related genes, ATGs)编码的蛋白协同完成[3]。以前人们认为自噬是一个在营养胁迫条件下非专一性的大体量(bulky)分解与循环途径，但最新研究表明自噬也可作为选择性机制对非必要的毒性细胞成分进行降解以达到质量控制及胁迫应答的目的[4]。自噬不但可以专一性地清除泛素化的蛋白聚集体、植物受体蛋白[5]、病原物蛋白[6]、还可以降解受损的细胞器如叶绿体[7-9]、线粒体[10-11]、内质网(endoplasmic reticulum, ER)[12]、过氧化物酶体(peroxisome)[13]、核糖体[14]及蛋白酶体(26S proteasome)[15]。
ATG10是一种E2结合酶(E2-conjugating enzyme)，与E1激活酶(E1-activating enzyme) ATG7依次作用后催化ATG12和ATG5之间的偶联反应；ATG12-ATG5偶联物与ATG16L相互作用，形成ATG12-ATG5-ATG16L多聚复合物[16-18]；该多聚复合物作为ATG8特异性E3连接酶介导磷脂酰乙醇胺(phosphatidylethanoamine, PE)结合于ATG8的C端，形成ATG8-PE；脂化的ATG8-PE可嵌入自噬体膜内、外膜上，从而促进自噬体形成的起始、延伸、封合以及与液泡膜的融合[19-21]。
与其他拟南芥ATG突变体如atg2、atg5及atg7相似，atg10突变体植株对氮或碳饥饿高度敏感并出现衰老和程序性细胞死亡现象[19]。atg10-1和atg5-1植株液泡中检测不到GFP-ATG8a所标记的自噬小体的积累[19]，表明ATG10对于自噬体的形成以及进入液泡是不可或缺的[19]。拟南芥atg5、atg10和atg18a的突变体对死体营养型真菌的抗性降低，但对生体营养型细菌的抗性则增强[22-23]，说明自噬途径负调控依赖于水杨酸的抗性[24]。
尽管在模式植物拟南芥中有大量有关自噬途径研究的报道，但在农作物如大豆中的研究则十分有限。本研究利用大豆豆荚斑驳病毒(bean pod mottle virus, BPMV)介导的基因沉默(virus-induced gene silencing, VIGS)技术成功地同时沉默了大豆中GmATG10的两个同源基因(GmATG10a和GmATG10b)。GmATG10a/10b沉默植株对黑暗处理诱导的碳饥饿高度敏感，同时细胞内积累过量ATG8，说明同时沉默GmATG10a/10b造成大豆自噬途径受损；此外，通过3, 3-二氨基联苯胺四盐酸盐(3, 3-diaminobenzidine, DAB)染色发现GmATG10a/10b沉默植株叶片中过氧化氢(hydrogen peroxide, H2O2)的积累量显著高于空载体对照株；沉默植株中病程相关基因(pathogenesis related gene, PR) GmPR1、GmPR5的表达量也显著高于对照株，说明沉默GmATG10a/10b可激活免疫反应；与此相一致，抗病性分析表明GmATG10a/10b沉默植株对大豆斑疹病菌(Xanthomonas campestris pv. glycine, Xag)以及对大豆花叶病毒(soybean mosaic virus, SMV)的抗性较空载体对照植株显著增强；最后，研究发现GmATG10a/10b沉默植株中flg22 (细菌鞭毛蛋白N端保守的22个氨基酸肽)诱导的丝裂原活化蛋白激酶3/6 (mitogen-activated protein kinase 3/6, MAPK3/6)激活程度显著高于空载体对照植株，暗示GmATG10a/10b沉默植株中免疫反应的激活可能依赖于GmMPK3/6的激活。
1 材料与方法 1.1 材料 1.1.1 植物材料本研究中使用的大豆(Glycine max Wm.a2.v1/4.v1)。大豆植株在温室或生长室中保持在22 ℃条件下，光周期为16 h。
1.1.2 菌种大肠杆菌(Escherichia coli) DH5α/TOP10、Xag、SMV-N-GUS[25]以及农杆菌GV3101。
1.2 方法 1.2.1 BPMV介导的VIGS技术对于BPMV、BPMV-VIGS载体系统以及使用PDS-1000/He系统(Bio-Rad实验室)通过基因枪微粒轰击将基于DNA的BPMV载体接种到大豆幼苗上在此前已有描述[25-26]。通过在Phytozome基因组数据库(https://phytozome-next.jgi.doe.gov/Phytozome)中进行关键词搜索，确定了2个大豆ATG10同源基因Glyma.07G124300 (GmATG10a)和Glyma.03G097000 (GmATG10b)。用GmATG10a的扩增引物(表 1)通过反转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)扩增出300 bp的GmATG10a片段用于构建沉默载体。用BPMV病毒载体上的特异性引物及GmATG10a/10b特异性沉默验证引物(表 1)通过RT-PCR进行沉默验证。以GmELF1b (Glyma.02G276600)作为内参基因。
| Primer name | Primer sequence (5′→3′) | Size (bp) | |
| Primers for amplifying silent fragments | |||
| GmATG10a-F | gggGGATCCCATGTGCCATATCAAATCAAGTG | 32 | |
| GmATG10a-R | cgcGGTACCCATGAGATATAAATGTCCATTTGGAT | 35 | |
| Primers for silencing examination | |||
| GmELF1b-F | ACCGAAGAGGGCATCAAATCCC | 22 | |
| GmELF1b-R | CTCAACTGTCAAGCGTTCCTC | 21 | |
| GmATG10a/10b-F | GGTGCTGTTAAGGACATAATTG | 22 | |
| GmATG10a/10b-R | AACTACAGTATCCAGCATTTCC | 22 | |
| BPMV-F | CAAGAGAAAGATTTGTTGGAGGGA | 24 | |
| BPMV-R | ACAAGGAAATAAGGTACGCTT | 21 | |
| Primers for RT-qPCR | |||
| GmELF1b-F-RT-qPCR | ACCGAAGAGGGCATCAAATCCC | 19 | |
| GmELF1b-R-RT-qPCR | TCTTACCCCTTGAGCGTGG | 19 | |
| GmPR1-F-RT-qPCR | CAACGCTGCAAGATCACAGG | 20 | |
| GmPR1-R-RT-qPCR | GTTTGCAGTCACCTTTGCGT | 19 | |
| GmPR5-F-RT-qPCR | GGAATAGACATTTCGGTGG | 19 | |
| GmPR5-R-RT-qPCR | TTGTGCGACTGGCTTCA | 17 | |
| GmSAG12-F-RT-qPCR | ACAGGCAAGTTGGTTTCA | 18 | |
| GmSAG12-R-RT-qPCR | GTATTCTGCTTCGGTTTG | 17 | |
| The lowercase sequence is the protective base. The bold sequences are BamH Ⅰ and Kpn Ⅰ restriction sites, for cloning PCR fragments into BPMV-VIGS (ia-d35)[26]. Primers for RT-qPCR are used to detect the expression levels of GmPR1 (Glyma.15G062500), GmPR5 (Glyma.07G095800) and GmSAG12 (Glyma.02G140000). GmELF1b (Glyma.02G276600) is used as an internal reference gene. | |||
将离体叶片分别放置在含有2.78 mmol/L的DAB染液中染色5 h。通过在乙醇: 乙酸: 甘油=3:1:1的比例配制脱色液煮沸10 min以清除叶绿素，然后将其置于96%乙醇(体积分数)中。叶片中棕褐色沉淀物即为H2O2的积累处[27]。
1.2.3 蛋白质印迹分析从BPMV-0和BPMV-GmATG10植株的叶片中提取蛋白质，在黑暗中处理不同时间。免疫印迹如前所述进行[28]。抗ATG8 (Agrisera, AS142769)用于蛋白质印迹。
1.2.4 RNA分离将大豆叶片剪下放入盛有液氮研钵中充分研磨，研磨后的粉末装入1.5 mL离心管中。加入1 mL的TRIpure，在涡旋混合器中与粉末充分混匀，4 ℃、12 000 r/min离心10 min。吸取上清于新的离心管中，室温放置5 min，加入200 μL氯仿，涡旋振荡15 s，室温放置10 min后，4 ℃、12 000 r/min离心15 min。吸取最上层透明上清于新的离心管中，加入500 μL异丙醇，上下翻转混匀，室温放置10 min，4 ℃、12 000 r/min离心10 min。弃上清，加入75%的酒精，轻弹离心管使沉淀悬浮，4 ℃、7 500 r/min离心5 min。弃上清，在超净台吹干沉淀，加入20 μL用焦碳酸二乙酯(diethyl pyrocarbonate, DEPC)处理并经过高温高压灭菌的水溶解沉淀。检测RNA浓度后保存在–80 ℃超低温冰箱中备用。
1.2.5 cDNA合成及实时荧光定量聚合酶链式反应(real time-quantitative PCR, RT-qPCR)(1) cDNA的合成
根据(ReverTra Ace qPCR RT, TOYOBO)试剂盒提供的步骤，将RNA进行逆转录反应：
先将RNA在65 ℃水浴中热变性5 min，后放置冰上。参照说明书中的反应体系：在离心管中加入2.0 μL的5×RT缓冲液，0.5 μL的Primer Mix，0.5 μL的RT enzyme Mix，1 μg的RNA，最后加Nuclease-free water定容至10 μL。逆转录的PCR程序设置为37 ℃，15 min；98 ℃，5 min；PCR结束后，将所得的cDNA存于4 ℃或者–20 ℃冰箱中保存。
(2) RT-qPCR
将cDNA稀释一倍，根据qPCR扩增酶2×SYBR Green qPCR Mix (High ROX)说明书中的反应体系：在离心管中加入5.0 μL的2×SYBR Green qPCR Mix (High ROX)，0.8 μL的稀释一倍后的cDNA，10 μmol/L的正反向引物(表 1)各0.4 μL，3.4 μL的去离子水，配制反应溶液。并根据说明书中的三步法程序进行PCR扩增：95 ℃ 2 min；95 ℃ 15 s；56 ℃ 30 s；72 ℃ 20 s，40个循环。
1.2.6 接种大豆斑疹病病菌Xag培养按先前文献中所述方法进行[29]。先将Xag在28 ℃培养约36 h至OD600为1.2。将细菌培养物于5 500 r/min离心10 min，并将菌种重悬至OD600为1。在空载体对照植株和GmATG10a/1b沉默植物叶片上下两面均匀喷洒重悬的菌液，然后用塑料袋保湿。在侵染后不同时间段进行打孔取样，然后按先前描述方法进行菌落计数分析[29]。
1.2.7 SMV-N-GUS接种、GUS染色和GUS病灶测量之前对SMV-N-GUS侵染、GUS (β-葡萄糖苷酸酶)染色和GUS病灶测量已有描述[30]。通过基因枪法将与GUS报告基因融合的SMV-N菌株(SMV-N-GUS)分别接种到空载对照株(BPMV-0)与BPMV-GmATG10a/10b沉默株离体叶片中[25-26]；接着将被侵染的离体叶片置于装有湿润滤纸的培养皿中培养3 d后进行GUS染色[31]。使用立体显微镜(Olympus SZX16)对GUS染色情况进行观察并拍照；对GUS病灶进行计数，并使用Image J软件测量GUS病灶直径。
1.2.8 免疫印迹法分析检测磷酸化GmMPK3/6大豆叶片组织中蛋白质提取按所述方法进行[32]。对所提取的蛋白通过聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)进行分离，并通过半干电转移装置(Bio-Rad)将胶中的蛋白转移至PVDF膜上(Millipore)；先在1×三羟甲基氨基甲烷吐温缓冲液(tris buffered saline with tween, TBST)中封闭，然后用1:3 000稀释的检测磷酸化MAPK的p44/p42抗体(cell signaling technology)进行孵育；用1×TBST洗3次后，用1:7 500稀释的二级抗体孵育。最后，用化学发光辣根过氧化物酶(horseradish peroxidase, HRP)底物(Millopore)通过曝光检测条带。
1.2.9 GmATG10a的亚细胞定位首先将GmATG10a的全长cDNA克隆至带有黄色荧光标签的双元载体(pGDY载体)中[33]构建成能共表达黄色荧光蛋白(yellow fluorescent protein, YFP)的YFP-GmATG10a融合载体；然后通过农杆菌注射法在本生烟叶片上瞬时表达；最终通过激光共聚焦显微镜(Leica TCS SP5 AOBS)观察并对YFP-GmATG10a在表皮细胞中的表达情况进行拍照。
2 结果与分析 2.1 GmATG10序列分析以及沉默植株表型ATG10在自噬体的形成过程中扮演着至关重要的角色[34]。为了研究ATG10在大豆免疫防御反应中的作用，通过在Phytozome植物基因组数据库(https://phytozome-next.jgi.doe.gov/Phytozome)中进行关键词ATG10搜索，找到了大豆(Glycine max Wm82.a2/4.v1)基因组中2个ATG10的同源基因：Glyma.07G124300和Glyma.03G097000；两者核苷酸序列的同源性高达93.3%，因此将其分别命名为GmATG10a和GmATG10b。通过序列比对发现，GmATG10a、GmATG10b与拟南芥ATG10 (At3G07525)的同源性分别为68.8%和69.4%。利用病毒诱导的基因沉默技术可同时沉默同源性高于85%的两个或多个基因[35]。因此我们选取了与GmATG10b同源性高达93%的300 bp GmATG10a片段克隆到BPMV-RNA2载体上，然后通过基因枪轰击法侵染大豆幼苗。侵染14 d后，GmATG10a/10b沉默植株虽然出现肉眼可见的病毒症状，但在表型上与对照植株并无显著差异(图 1A)，表明沉默GmATG10a/ 10b在正常条件下对大豆的生长发育没有显著影响。通过用GmATG10a/10b特异性沉默验证引物(表 1)进行RT-PCR分析，发现沉默株中GmATG10a/10b的转录水平较空载体对照株显著降低(图 1B)，证明GmATG10a/10b成功被沉默。同时利用BPMV病毒载体上设计的特异性引物(表 1)进行RT-PCR分析发现，从沉默株中扩增出的片段长度大于从空载体对照株中扩增出的片段，且增加的片段长度与插入靶基因片段的大小相符(图 1C)。以上结果表明GmATG10a/ 10b已被成功沉默。
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| 图 1 BPMV-VIGS介导的GmATG10a/10b沉默 Fig. 1 Silencing of GmATG10a/10b mediated by BPMV-VIGS. A: Phenotype comparison between vector control plants and the GmATG10a/10b-silenced plants two weeks post inoculation. B: Examination of silencing effects by RT-PCR using primers that amplify the full length cDNA of GmATG10a/10b. GmELF1b (Glyma.02G276600) is used as an internal reference gene. C: The RT-PCR fragment amplified from the GmATG10a/10b-silenced plants is larger in size than that of amplified from the vector control plants using primers flanking the insertion site of the BPMV-RNA2 vector. |
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拟南芥中ATG相关突变体的显著特征之一是对营养饥饿的敏感性显著提高[2, 12, 36]。在连续黑暗条件下生长的植物由于缺乏光合作用而导致碳水化合物合成的减少[19, 37]。自噬途径的受损则导致植物叶片黄化及加速衰老。拟南芥ATG相关突变体在自然生长条件下表现出衰老加速的黄化表型[19]。在正常生长条件下，BPMV-0对照株和GmATG10a/10b沉默株在表型上没有显著差异(图 2A)。猜测这可能与GmATG10a/10b未完全沉默有关[38]，未被沉默的GmATG10a/10b足以维持在正常生长条件下的需要。因而对沉默株及对照株进行黑暗处理。将BPMV-0对照株和GmATG10a/10b沉默株从上至下的第二层叶片剪下，放在铺有湿润滤纸的培养皿中，然后置于黑暗培养室中培养。经过9 d的黑暗培养后，发现BPMV-0对照株的叶片仍然健康鲜绿，而GmATG10a/10b沉默叶片出现明显的衰老加速的黄化表型(图 2B)，说明GmATG10a/10b沉默植株中自噬途径可能受损。SAG12是一个典型的与衰老相关的基因，编码一种半胱氨酸蛋白酶，参与拟南芥叶片叶绿体蛋白质的降解。SAG12在衰老过程中被诱导表达，是衰老的标记基因[39-40]。因而对GmATG10a/10b沉默株与对照株中GmSAG12的表达情况进行了分析。如图 2C所示，相对于BPMV-0对照株，GmATG10a/10b沉默株中GmSAG12的表达量显著升高。综上所述，GmATG10a/10b沉默株中自噬途径受损，导致对营养缺陷敏感及加速衰老。
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| 图 2 黑暗处理条件下GmATG10a/10b沉默株叶片出现加速衰老表型 Fig. 2 An accelerated senescence phenotype was observed for the dark-treated GmATG10a/10b-silenced leaves. A: Comparison of leaf phenotypes between control plants and the GmATG10a/10b-silenced plants before dark treatment. B: Comparison of leaf phenotypes between control plants and the GmATG10a/10b-silenced plants after 9 d of dark treatment. C: The expression of GmSAG12 was significantly induced in the leaves of GmATG10a/10b-silenced plants relative to vector control plants. ***: P < 0.001, unpaired t-test; GmELF1b (Glyma.02G276600) is used as an internal reference gene. The experiment was done in triplicates. At least 3 plants were used in each repeat. |
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ATG8泛素样蛋白在自噬途径中发挥多种作用，它们存在于自噬体的膜上并最终在液泡中降解[12, 36]。如果自噬途径受损，ATG8蛋白的积累水平就会升高。为了进一步检测GmATG10a10/b沉默株中的自噬途径是否受损，用拟南芥ATG8的专一性抗体(AS14 2769, Agrisera)对经黑暗处理的对照植株以及GmATG10a/10b沉默株离体叶片中提取的蛋白质样品进行Western blotting印迹分析。Western blotting分析结果表明，GmATG8蛋白在GmATG10a/10b沉默株叶片中的积累水平显著高于BPMV-0对照株叶片中的积累水平(图 3A、3B)，进一步说明GmATG10a/10b的沉默导致自噬降解途径缺陷，同时说明GmATG10a/10b在大豆自噬途径中起着至关重要的作用。
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| 图 3 沉默GmATG10a/10b导致GmATG8在叶片中的积累水平升高 Fig. 3 Silencing of GmATG10a/10b resulted in increased accumulation of GmATG8. A: Comparison of GmATG8 protein accumulation level between BPMV-0 control plants and the BPMV-GmATG10a/10b silenced plants treated under dark for different time. B: Comparison of the ratios of the band intensities of the GmATG10a/10b-silenced plants over vector control plants quantified using Image J at different time points of dark treatment. **: P < 0.01; ***: P < 0.001; unpaired t-test. The experiment was done in triplicates. At least 3 plants were used in each repeat. |
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活性氧(reactive oxygen species, ROS)是胁迫响应中最重要的信号分子之一，在免疫防御中发挥着重要的作用[41]。在外界病原物侵染下，植株体内会积累ROS来抵御病原物侵染。为了探究沉默GmATG10a/10b对细胞中ROS积累的效应，通过DAB染色法对空载体对照株及GmATG10a/10b沉默株叶片中H2O2的积累水平进行了染色比较(图 4A)。结果表明对照株叶片上H2O2的积累水平(图 4B)远低于GmATG10a/10b沉默株(图 4C)，说明GmATG10a/10b沉默株叶片上出现的加速衰老的黄化表型可能与H2O2的过量积累相关。
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| 图 4 DAB染色检测GmATG10a/10b沉默株中的H2O2的积累水平 Fig. 4 Detection of H2O2 accumulation in GmATG10a/10b-silenced plants by DAB staining. A: DAB staining was performed to detect the accumulation of H2O2 on the leaves of both vector control plants (BPMV-0) and the GmATG10a/10b silenced plants. B: Enlarged portion of the vector control leaf under stereomicroscope. C: Enlarged portion of the GmATG10a/10b silenced leaf under stereomicroscope. The experiment was done in triplicate. Bars=4 mm. |
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PR基因的诱导表达是植物防御反应激活的重要标志之一[42-43]，其编码的PR蛋白在植物体外表现出潜在的抗菌活性，在植物体内的积累与植物的抗性反应密切相关[42]。为了检测GmATG10a/10b沉默株中GmPR1与GmPR5的表达是否上调，分别从空载对照株和GmATG10a/10b沉默株叶片中提取RNA，然后通过RT-qPCR对GmPR1 (Glyma.15G062500)与GmPR5 (Glyma.07G095800)的表达进行了分析。相对于对照株，GmATG10a/10b沉默株叶片中GmPR1与GmPR5基因的表达量均显著升高(图 5A、5B)，表明沉默GmATG10a/10b可诱导PR基因的表达及激活免疫反应。
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| 图 5 GmATG10a/10b沉默株中GmPR1与GmPR5的表达量上调 Fig. 5 Up-regulation of PR1 and PR5 genes in GmATG10a/10b silenced plants. A: The expression of GmPR1 was increased in GmATG10a/10b-silenced plants relative to control plants. ***: P < 0.001, unpaired t-test. B: The expression of GmPR5 was increased in GmATG10a/10b-silenced plants relative to control plants. ***: P < 0.001, unpaired t-test. GmELF1b (Glyma.02G276600) was used as an internal reference gene. The experiment was done in triplicates. |
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先前的研究表明，MAPK信号途径不受拟南芥中ATG基因缺失的影响[22]。为了研究沉默GmATG10a/10b对大豆中MAPK激活的效应，用10 μmol/L flg22处理BPMV-0和BPMV-GmATG10a/10b沉默株叶片0–6 h，然后用可专一性检测磷酸化的MPK3/6激酶的人源抗体p44/42 MAP Erk1/2进行蛋白质印迹分析。如图 6所示，相对于BPMV-0，flg22诱导的GmMPK3/6激活程度在GmATG10a/10b沉默株中显著增强，表明GmMPK3/6参与了GmATG10a/ 10b沉默植株中的防御激活，GmATG10a/10b负调控大豆GmMPK3/6的激活。
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| 图 6 沉默GmATG10a/10b导致flg22诱导的GmMPK3/6的激活程度增强 Fig. 6 Silencing GmATG10a/10b results in enhanced activation of GmMPK3/6 in response to flg22. Leaf discs collected from BPMV-0 and BPMV-GmATG10a/10b plants respectively, were incubated on wet filter paper for 24 h for recovery from wounding effects followed by treatment with 10 μmol/L flg22 for the specified time. Kinase activity was detected by Western blotting with p44/42 MAP Erk1/2 antibody specifically recognizes the phosphorylated MPK3/4/6 across kingdoms. The Arabidopsis sample treated with wounding was used as a positive control. Coomassie blue staining (CBS) was used for loading control. The experiment was done in triplicates. |
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大豆花叶病毒(SMV)是一种马铃薯Y病毒属，主要侵染豆科植物，由种子传播和蚜虫传播。SMV导致大豆产量严重下降，并影响种子质量[44]。本研究通过基因枪法将与GUS报告基因融合的SMV-N菌株(SMV-N-GUS)[25]分别接种到对照株和GmATG10a/10b沉默株的3片离体叶片上。然后将被侵染的离体叶片置于铺有湿润滤纸的培养皿中于培养箱中培养；在此期间实时观察并补充水分，以防离体叶片因缺水而干枯；侵染3 d后进行GUS染色。GUS编码β-葡萄糖苷酸酶，可以催化底物X-Gluc产生深蓝色化合物。将侵染叶片置于X-Gluc液中孵育16 h，然后进行脱色后发现在侵染叶片上有许多的蓝色斑点，其染色深浅与直径大小可以反映SMV-N-GUS复制与运动能力。因此，可通过测量斑点的直径大小直接反映出植株对SMV抗性的强弱。GUS染色结果如图 7A和7B所示，通过测量蓝色斑点直径表明BPMV-0空载植株叶片上蓝色斑点直径显著大于GmATG10a/10b沉默株叶片上的蓝色斑点直径(图 7C)，说明沉默GmATG10a/10b导致大豆植株对SMV的抗性增强。
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| 图 7 GmATG1010a/10b沉默植株对大豆花叶病毒(SMV)的抗性增强[L4] Fig. 7 Silencing GmATG10a/10b results in increased resistance to SMV-N-GUS infection. A: Visualization of SMV-N-GUS infection on the leaves of empty vector control plants and the GmATG10a/10b-silenced plants under stereomicroscope. B: Comparison of the diameters of the GUS lesion on the leaves of empty vector control plants and the GmATG10a/10b-silenced plants. Bars=4 mm; ***: P < 0.001, unpaired t-test. |
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轴突黄单胞菌(Xag)是一种革兰氏阴性植物病原菌，可引起细菌性脓疱病，是大豆中最具破坏性的病害之一[45]。为了明确沉默GmATG10a/10b对Xag的抗性效应，通过喷施侵染法将Xag均匀喷洒到BPMV-0对照株与GmATG10a/10b沉默株的叶片上。接种4 d后，发现BPMV-0对照株叶片上的侵染症状较GmATG10a/10b沉默株叶片上严重(图 8A)；对照株叶片上的菌落数显著高于GmATG10a/10b沉默株叶片上的菌落数(图 8B)，这与叶片上病症相一致，说明沉默GmATG10a/10b导致大豆对Xag的抗性增强，进一步说明GmATG10a/10b在大豆免疫反应中起着负调控作用。
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| 图 8 GmATG10a/10b沉默株对大豆斑疹病菌(Xag)的抗性增强 Fig. 8 Silencing GmATG10a/10b results in increased resistance to Xag. A: Comparison of disease symptoms on the leaves of the vector control plants and GmATG10a/10b-silenced plants at 4 d of Xag infection. B: Comparison of colony forming units on the leaves of the vector control plants and the GmATG10a/10b-silenced plants. ***: P < 0.001, unpaired t-test. The experiment was done in triplicates. |
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为了明确GmATG10a在细胞中的定位情况，构建了YFP-GmATG10a融合蛋白的双元载体，并将其转入到农杆菌GV3101菌株中。通过农杆菌注射法将带有双元载体的农杆菌菌株注射到本生烟叶片中瞬时表达；在注射2 d后，将注射区域叶片切下，在激光共聚焦显微镜下进行观察并拍照(图 9)。结果表明GmATG10a不但定位于细胞膜和细胞质，而且定位于细胞核中，说明其在细胞内不同位置发挥作用。
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| 图 9 YFP-GmATG10a的亚细胞定位 Fig. 9 Subcellular localization of YFP-GmATG10a. A: Subcellular localization of free GFP. B: GmATG10a was not only presented in the plasma membrane/cytoplasm, but also in the nucleus. Bar=40 μm. |
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自噬是真核生物中进化上保守的物质降解过程，在植物的生长发育以及免疫防御反应中起着至关重要的作用[46-47]。ATG10是ATG12专一性的E2结合(conjugation)酶，其通过与ATG7的顺次作用催化ATG12蛋白C端的甘氨酸与ATG5蛋白中保守的赖氨酸之间形成异肽键(isopeptide bond)，从而形成ATG12-ATG5的复合体；接着ATG12-ATG5复合体与脚手架蛋白ATG16同源二聚体形成六聚体，并由该六聚体指导与ATG3结合的ATG8的磷脂酰乙醇胺酯化(ATG8-PE)；脂化后的ATG8-PE便可插入自噬体膜[2]。拟南芥ATG10功能丧失突变体中ATG12-ATG5复合体与ATG8的脂化受阻、自噬体的形成受到破坏[19, 48]。ATG10突变体对缺氮和缺碳超敏感，甚至在正常生长条件下出现加速衰老与程序性细胞死亡(programmed cell death, PCD)的表型[19, 49]。ATG10突变体中表现出的这些缺陷与其他自噬关键基因功能丧失突变体如ATG2、ATG5、ATG7以及ATG18的表型非常相似[50-51]，说明ATG10在自噬途径中起着不可或缺的作用。
大豆是古四倍体，Schmutz等[52]研究表明，大豆基因组中75%的基因存在两个或多个拷贝。本研究通过在Phytozome植物基因组数据库中进行搜索，发现在大豆(Glycine max Wm82.a2/a4.v1)基因组中有2个ATG10的同源基因：Glyma.07G124300和Glyma.03G097000，分别命名为GmATG10a和GmATG10b。GmATG10a和GmATG10b在核苷酸水平的同源性高达93.3%。大量研究表明，利用VIGS技术可以同时沉默同源性大于85%的两个或多个基因[29, 32, 53-55]。通过BPMV介导的VIGS技术，同时沉默GmATG10a和GmATG10b。GmATG10a/10b沉默植株在自然生长条件下并没有与拟南芥ATG基因功能丧失突变体类似的表型(图 1A)。仅在经过黑暗处理的沉默植株上观察到加速衰老的黄化表型(图 2A、2B)。这可能与BPMV介导的基因沉默只能导致转录水平的降低(80%−90%)，而不是完全沉默有关[29, 55]。残存的10%−20%的GmATG10a/10b可以维持在正常生长条件下的需要；然而，黑暗条件下光合作用受阻，长时间暗处理会导致碳缺乏，从而诱导大量自噬的发生[53]，在黑暗条件下通过自噬途径分解细胞内储藏的碳水化合物、核酸、蛋白质、脂类物质以及受损的细胞器，在细胞碳源重复利用方面起着关键作用[19]。在这种情况下，GmATG10a/10b沉默植株中残存的GmATG10a/10b则不能组装足够多的自噬体以应对C饥饿，因而表现自噬途径受损的典型表型[53](图 2)。
ATG8在自噬途径中起着核心作用，其不但是自噬体形成所必需的，而且通过与不同自噬受体蛋白相互作用，选择性招募参与不同生物学过程的特定货物蛋白进行自噬降解[24, 56]；且其自身也通过自噬途径最终在液泡中降解[2, 24]；在自噬诱导条件下，自噬相关基因突变体中由于自噬降解受损而积累大量ATG8[19]。与拟南芥ATG基因功能丧失突变体相似，GmATG8的积累水平在暗处理的GmATG10a/10b沉默植株中显著增加(图 3)；说明沉默GmATG10a/10b导致大豆自噬途径受损。水稻T-DNA插入突变体OsATG10b对盐胁迫与可诱导ROS的甲基紫精(methyl viologen, MV)高度敏感。突变体中自噬体的数目显著减少；MV-诱导的氧化蛋白的积累量在突变体中显著降低[48]。以上结果说明ATG10蛋白的功能在不同植物中是保守的。
活性氧迸发(oxidative burst)可诱导过敏性细胞死亡并激活植物的防御反应[57-58]。自噬的主要作用之一是通过降解细胞内的氧化受损的细胞器来清除活性氧以维持细胞内活性氧的稳态平衡[59]。自然条件下，在拟南芥自噬突变体中观察到加速衰老和自体免疫激活的表型，其中包括H2O2和水杨酸(salicylic acid, SA)的积累水平增强、PR基因的诱导表达以及对生体营养型病原菌的抗性增强[60-61]。与拟南芥ATG功能丧失突变体的表型相似，我们发现GmATG10a/10b沉默植株叶片中H2O2的积累水平增加(图 4)、GmPR1/GmPR5以及GmSAG12基因诱导表达水平显著升高(图 2B、2C和图 5A)、对生体营养型大豆斑疹病菌Xag以及SMV-N-GUS的抗性显著增强。PR1蛋白代表了第一个发现的PR家族，早期研究表明，组成型表达烟草PR1基因的转基因植物对卵菌病原体的抗性显著增强[62-63]，此外，PR1基因的表达被广泛认为是激活超敏反应(hypersensitivity, HR)介导的防御途径或建立SA介导的多种植物抗病性的可靠标志[64]。这些结果说明GmATG10a/10b通过参与自噬途径而负调控大豆的免疫反应。我们猜测GmATG10a/10b沉默植株中免疫激活很可能是由于自噬清除氧化受损细胞器功能减弱导致细胞内活性氧的稳态平衡遭受破坏所致。拟南芥自噬相关突变体中的叶绿体自噬(chlorophagy)[24]、过氧化物酶体自噬(pexophagy)[65-68]、线粒体自噬(mitophagy)以及26S蛋白酶体自噬(proteaphagy)[15]均遭到破坏；ATG10功能缺失导致受损叶绿体自噬清除缺陷[69]及受损蛋白酶体自噬清除缺陷[15]。而过氧化物酶体、叶绿体及线粒体是细胞内ROS的主要来源[21]，叶绿体同时还是SA的主要合成场所[70]。自噬途径的受损造成遭受氧化破坏的细胞器无法降解而在细胞内积累，从而导致细胞内H2O2及SA的过量积累。这可能是自噬途径缺陷诱导细胞死亡与激活免疫反应的分子机理。
与其他自噬相关基因突变体相似，ATG10突变体对生体营养型病原菌的抗性增强，但对死体营养型病菌的抗性降低[22, 60]。轴突黄单胞菌(Xag)是一种革兰氏阴性植物病原菌，可引起细菌性脓疱病，是大豆中最具破坏性的病害之一[45]。Xag通过气孔或伤口进入大豆叶片，并在海绵状叶肉的细胞间隙内繁殖，导致被黄色光环包围的浓密脓疱[71]。最初症状是在叶片感染部位可观察到微小绿色斑点，这些斑点随后扩散到叶片表面，呈棕色或黄色，最后在叶面上形成凹凸不平、有褪绿光环的脓疱。该病害严重影响种子的大小和数量[72-73]。GmATG10a/ 10b沉默植株对生体营养型大豆斑疹病菌Xag以及SMV-N-GUS的抗性均显著增强(图 7和图 8)，我们猜测GmATG10a/10b沉默植株对死体营养型的病原菌的抗性会降低，将通过接种大豆死体营养型的病原菌来验证。
定位于质膜上的类激酶受体在识别PAMPs后可激活PTI，其中包括激活MAPK级联反应[74]。flg22是一种保守肽，源自丁香假单胞菌鞭毛蛋白的N端22个氨基酸，是典型的病原物相关的分子模式(pathogen-associated molecular pattern, PAMP)[75]，可被植物受体类激酶FLS2识别并激活防御反应。丝裂原活化蛋白激酶(MAPK)级联途径在受体类激酶(如FLS2)识别PAMP的下游发挥作用，将细胞外信号转化为细胞内适应性反应[76]。与拟南芥中的情况相似，沉默GmFLS2后降低了flg22诱导的GmMPK6的激活[29]。拟南芥ATG突变体植株中flg22诱导的MPK3/4/6的激活与Col-0植株的中并无区别[22]。大豆GmATG2a/2b、GmATG5a/5b及GmATG7a/7b沉默株中flg22诱导的GmMPK3/6的激活均较对照株显著降低[53]。而本研究发现GmATG10a/10b沉默植株中flg22诱导的GmMPK3/6激活程度却显著增强(图 6)，说明沉默不同GmATG基因对GmMPK3/6的激活具截然相反的效应，其原因尚有待进一步研究。而GmATG10a/10b沉默植株中免疫反应的激活及抗病性增强(图 6)可能部分依赖于MAPK途径。
致谢: 感谢美国艾奥瓦州立大学(Iowa State University)的Steve A. Whitham和John Hill教授惠赠的BPMV-VIGS系统以及SMV-N-GUS质粒。
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