,西南大学农学与生物科技学院,重庆 400715Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS
YUAN JunHu, DING YiJuan, YANG WenJing, YAN BaoQin, CHAI YaRu, MEI JiaQin, QIAN Wei,College of Agronomy and Biotechnology, Southwest University, Chongqing 400715通讯作者:
责任编辑: 岳梅
收稿日期:2019-05-28接受日期:2019-08-14网络出版日期:2019-12-01
| 基金资助: |
Received:2019-05-28Accepted:2019-08-14Online:2019-12-01
作者简介 About authors
远俊虎,E-mail：jhyuan1998@163.com
丁一娟,E-mail：dding1989@163.com
摘要
关键词：
Abstract
Keywords：
PDF (1652KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
远俊虎, 丁一娟, 杨文静, 闫宝琴, 柴亚茹, 梅家琴, 钱伟. 利用TRV-HIGS技术鉴定核盘菌致病相关的分泌蛋白基因[J]. 中国农业科学, 2019, 52(23): 4274-4284 doi:10.3864/j.issn.0578-1752.2019.23.008
YUAN JunHu, DING YiJuan, YANG WenJing, YAN BaoQin, CHAI YaRu, MEI JiaQin, QIAN Wei.
0 引言
【研究意义】核盘菌（Sclerotinia sclerotiorum）是一种寄主范围十分广泛的病原真菌,可以侵染包括油菜、马铃薯、棉花、番茄、大豆、烟草等多种重要作物在内的400多种植物[1],造成作物产量损失,且防治困难。研究表明,除了草酸等致病因子外[2],分泌蛋白（secretory protein）在核盘菌与植物互作中也起着重要作用[3,4,5,6,7,8,9,10,11],但大多数分泌蛋白在核盘菌致病方面的功能还亟待确定。利用烟草脆裂病毒（Tobacco rattle virus,TRV）介导的寄主诱导基因沉默（host-induced gene silencing,HIGS）技术快速挖掘核盘菌致病相关分泌蛋白基因,对明确核盘菌致病机理,进一步安全防控菌核病具有重要意义。【前人研究进展】目前,针对核盘菌致病性的研究主要集中于草酸等致病因子[2]。分泌蛋白是一类由内质网/高尔基体途径分泌到细胞外,具有信号肽结构,且在生物体发育及防御等生理过程中起着重要作用的蛋白质[12]。近年来研究发现,核盘菌的一些分泌蛋白参与了其与寄主的互作过程,如SSCP1和SsSSVP1可分别与寄主植物中的PR1和QCR8蛋白互作,促进寄主植物细胞的死亡,从而利于核盘菌的侵染[3,4];SSITL蛋白参与了核盘菌抑制寄主JA/ET信号途径介导的局部和系统性抗病反应[5]。另外有研究表明一些分泌蛋白可能参与了核盘菌的生长发育过程,比如SsCVNH在核盘菌菌核发育中发挥重要作用[6],Ss-Rhs1和Ss-Caf1则参与了核盘菌复合侵染垫的形成[7,8]。DERBYSHIRE等分别通过生物信息学的方法预测到核盘菌中存在78个和70个候选分泌蛋白[9,10];DING等[11]利用RNA-seq鉴定到在核盘菌侵染寄主过程中有93个含有信号肽的差异表达基因。这些研究表明核盘菌基因组中存在大量编码分泌性效应蛋白的基因,但是它们是否都参与核盘菌致病性的生物学功能仍不清楚。RNA干扰（RNA interference,RNAi）技术是揭示生物体基因功能的一种反向遗传学研究手段。早期有****分别利用携带特定基因片段的不同病毒载体侵染本氏烟（Nicotiana benthamiana）,导致特定靶基因的沉默[13],从而建立了病毒介导的基因沉默（virus-induced gene silencing,VIGS）技术。由于VIGS技术操作简便,周期性短,能在当代快速获取表型,继而对基因功能进行分析[14],故该技术在植物基因功能研究中展示出广阔的应用前景[15]。目前常用的病毒载体有PVXB[16]、PGMV[17]、TRV[18]及BSMV[19]等。其中TRV载体与其他病毒载体相比具有诱导基因沉默效率高,持久性长,对宿主不会造成明显伤害等优点,因而被广泛应用[20]。HIGS技术是在VIGS技术上发展而来的,它通过将含有病原物目的基因片段的反义发夹结构的载体转染到寄主植物中,产生病原物特定序列的双链RNA（dsRNA）,经过植物体加工后产生siRNA,从而在病原菌侵染过程中沉默病原菌特定基因[21]。近年来,HIGS技术在植物-病原菌互作研究中已被大量应用,并且成功获得了一系列抗性增强的寄主材料[22,23]。由于病毒载体介导的基因瞬时沉默技术周期短,体系成熟,因此多数研究将VIGS与HIGS技术结合起来,利用病毒介导的HIGS技术来快速鉴定病原菌致病基因的功能[24]。QI等[22]首先利用BSMV介导的HIGS技术,在大麦中瞬时表达BSMV-PsCPK1,初步确定了PsCPK1在增强大麦条锈病抗性中的作用,随后利用HIGS技术获得持久稳定抗性的转基因大麦株系;XU等[23]利用TRV介导的HIGS技术在棉花中成功鉴定了黄萎病G蛋白信号基因VdRGS1的功能;ANDRADE等[25]在烟草中表达了核盘菌几丁质合成酶基因的dsRNA,获得了菌核病抗性增强的T1代转基因烟草,证实了HIGS技术在核盘菌基因功能研究中的可行性。因此,利用病毒介导的HIGS技术可以作为鉴定核盘菌致病基因功能的有效手段。【本研究切入点】笔者实验室前期研究中鉴定到核盘菌在侵染过程中有93个具有信号肽结构的差异表达基因,但其中大多数基因的功能还不清楚[11]。因此,本研究采用TRV介导的HIGS技术对其中8个未知功能基因进行功能鉴定。【拟解决的关键问题】利用TRV介导的HIGS技术在本氏烟中瞬时表达靶向8个未知功能的核盘菌基因的siRNA,通过菌核病抗性评价及基因表达量分析,筛选与核盘菌致病性相关的候选基因,探究利用TRV介导的HIGS技术快速鉴定核盘菌致病相关基因的可行性,为进一步解析核盘菌的致病机制打下基础。1 材料与方法
试验于2018年11月至2019年5月在西南大学农学与生物科技学院油菜资源研究所完成。1.1 试验材料
供试本氏烟、核盘菌野生菌株1980、pTRV1及pTRV2等相关载体均由重庆市油菜工程技术研究中心提供。大肠杆菌（Escherichia coli）DH5α、农杆菌（Agrobacterium tumefaciens）GV3101等购自北京全式金生物技术有限公司（TransGen Biotech）。本氏烟培养于实验室光照培养箱中,参数设定为28℃,18 h光照,光照强度8 000 lx;20℃,6 h黑暗。
1.2 候选基因序列分析
前期对核盘菌侵染甘蓝叶片和茎秆过程中的差异基因进行了分析,发现在侵染茎秆过程中有93个具有分泌信号肽结构的核盘菌差异表达基因[11]。选择其中8个在感病甘蓝的叶片及茎秆中表达量都显著高于抗病甘蓝的基因作为候选基因,并在NCBI数据库（1.3 TRV-HIGS载体的构建
从核盘菌基因组数据库（1.4 TRV-HIGS载体侵染烟草
烟草侵染参照LIU等[26]的方法进行。分别活化pTRV1、pTRV2-Gene及pTRV2-GFP,挑取单克隆预培养后,扩大培养至OD=0.8左右。随后5 000 r/min离心15 min,弃上清,收集菌体后用重悬液（10 mmol·L-1 MgCl2,10 mmol·L-1 MES,150 μmol·L-1 AS）重悬至OD=0.8—1.6。等量混合pTRV1及pTRV2-Gene,pTRV1及pTRV2-GFP,室温静置3 h后,用针筒浸润法对5—6周龄的烟草进行侵染。暗培养48 h后,在正常光照条件下培养7 d。试验重复3次,每次重复侵染3株烟草。1.5 菌核病抗性鉴定
在马铃薯培养基（PDA）平板上活化核盘菌野生型菌株1980,用直径6 mm的打孔器取生长2 d的边缘菌丝用于接种。在TRV-HIGS载体侵染烟草9 d后,将直径6 mm的核盘菌PDA菌丝块接种于侵染后的烟草叶片左右叶腹的中央,带菌面紧贴叶片。接种后,将接种植株置于可控温控湿房间内,其中温度设置为22℃,相对湿度保持在85%左右。接种48 h后统计病斑的长径和短径,利用公式S =π×a×b/4计算病斑面积（其中a代表病斑长轴长度,b代表病斑短轴长度）,并利用SAS软件进行统计学分析。1.6 候选基因的实时荧光定量PCR（qRT-PCR）
取接种48 h后的病斑及病斑周围组织叶片（距腐烂组织边缘1 cm左右）,采用TRIzol?试剂分离提取总RNA,采用Bio RAD的iScriptTM cDNA Synthesis Kit试剂盒将RNA样品反转录成cDNA。采用Bio-Rad的iTaqTM Universal SYBR? Green Supermix试剂盒,利用CFX96TM real-time PCR仪进行荧光定量扩增,反应程序：95℃预变性30 s,95℃变性5 s,60℃退火20 s,72℃延伸20 s,40个循环。每个样本重复3次,采用2-ΔΔCT法[27]计算目的基因表达量。每个基因的qRT-PCR引物信息见表1,其中内参基因为核盘菌基因Sstub1（SS1G_04652）。Table 1
表1
表1本试验所用引物信息
Table 1
| 基因 Gene | 引物序列 Primer sequence (5′-3′) | 产物大小 Product size (bp) | 用途 Usage |
|---|---|---|---|
| SS1G_00263 | F: CCGGAATTCCGGCAGCGCCTCAAGCTCGACTCAAATC R: CGAGCTCGCTGACGGTAGCGGAACCAACAACGG qF: TCTTTGAGGATGGAACTTGGAC qR: AGCCTGGCAAGCATAATCG | 273 | 基因扩增Gene amplification qRT-PCR |
| SS1G_04945 | F: CCGGAATTCCGGTCTCCTTCCTTCTCGGC R: CGAGCTCGCTCGTAATCGGCACCAT qF: TCTCAACGGTGCTCTTTACTTC qR: TTGCTATCAGGGACCCATCC | 263 | 基因扩增Gene amplification qRT-PCR |
| SS1G_03181 | F: CCGGAATTCCGGTGAGCGATGCTACCAACAGCGCCTAC R: CGAGCTCGCACCAGCACTTTGGAGAGCACCGTAA qF: TGCCGATTACGAGGGAACA qR: TGGAAACATCGACGGTGAAG | 210 | 基因扩增Gene amplification qRT-PCR |
| SS1G_04343 | F: CCGGAATTCCGGTGGTCTTTACGCTGGGTATTTC R: CGAGCTCGCACCGTTGGTTGTGTTTTCATT qF: TGGTCTTTACGCTGGGTATTTC qR: CAGACACTTGCGAATGGAGC | 284 | 基因扩增Gene amplification qRT-PCR |
| SS1G_03146 | F: CCGGAATTCCGGACATTTCCTCTTGAACCATCCCGTA R: CGAGCTCGTTTATGACACCCTTGTTTCCAGCGA qF: GCACATTTCCTCTTGAACCATC qR: GCAGTGTCACTTCCCACCATT | 309 | 基因扩增Gene amplification qRT-PCR |
| SS1G_02250 | F: CCGGAATTCCGGTCACACTTTTGGCATT R: CGAGCTCGATCAGCACGTTTTTCT qF: AAGCCAACACCAACCTCATC qR: CACTGGAGCGTAGTTCTCGTAG | 272 | 基因扩增Gene amplification qRT-PCR |
| SS1G_11912 | F: CCGGAATTCCGGTCAGCAGCTCCACCTCCACCACC R: CGAGCTCGATTCGCCTTTCCAAAATCCGTAT qF: TTCCCGAAACCGTTCCTAGT qR: TCACCATTGCTACTGCCACTT | 257 | 基因扩增Gene amplification qRT-PCR |
| SS1G_07655 Sstub1 （内参基因Actin gene） | F: CCGGAATTCCGGGCTACTGTTCCTTTGGACTACGCT R: CGAGCTCGTTACTGAGGAGTGAGTCGTGTCGG qF: AATATGCCAGAGCCATCACA qR: CAGCGTAGTCCAAAGGAACAG qF: GTGAGGCTGAGGGCTGTGTGA qR: CCTTTGGCGATGGGACG | 322 | 基因扩增Gene amplification qRT-PCR qRT-PCR |
新窗口打开|下载CSV
2 结果
2.1 候选基因表达及蛋白结构域分析
图1为8个候选基因在侵染感病、抗病甘蓝叶片上的表达热图。为了进一步了解这8个候选基因的功能,分析了其蛋白质结构域。SMART分析显示,这8个具有信号肽结构的候选基因具有不同类型的SCOP功能域,其中有4个候选基因（SS1G_04945、SS1G_03181、SS1G_11912和SS1G_07655）具有不同类型的PDB功能域,此外有4个基因具有不同类型Pfam功能域,其中SS1G_04945具有Glyco_hydro_7结构域,SS1G_03181具有ASP结构域,SS1G_11912具有NPP1结构域,SS1G_07655具有Pro-kuma_activ结构域。SS1G_02250则包含重复结构域（表2）。将SMART分析得到的候选基因的结构域分别在Pfam、PDB以及SCOP数据库中进行了功能注释,随后对这8个候选可能参与的功能进行了预测。SS1G_00263可能参与核盘菌对药物耐受性的调节,SS1G_04945可能参与多糖的水解,SS1G_03181可能参与蛋白的水解,SS1G_04343可能参与诱导植物的病原相关分子模式（pathogen-associated molecular pattern,PAMP）引发的免疫反应（PAMP-triggered immunity,PTI）或物质的异构化过程,SS1G_03146可能参与生物素合成,SS1G_02250可能参与核酸分子磷酸基团的水解,SS1G_11912可能参与坏死诱导蛋白或毒素的产生从而诱导植物的效应蛋白所引发的免疫反应（effector-triggered immunity,ETI）,SS1G_07655可能参与肽链或蛋白质的水解过程（表2）。图1
新窗口打开|下载原图ZIP|生成PPT图1候选基因在侵染抗、感病甘蓝过程中的表达热图
核盘菌候选基因在接种抗、感甘蓝茎秆（A）0、12、24 h和叶片（B） 0、6、12 h后的表达量Relative expression levels of candidate genes in the susceptible and resistant B. oleracea after inoculation with S. sclerotiorum in stem (A) post 0, 12, 24 h and leaf (B) post 0, 6, 12 h。Rs：抗病甘蓝材料的茎秆Resistant B. oleracea stem;Ss：感病甘蓝材料的茎秆Susceptible B. oleracea stem;Rl：抗病甘蓝材料的叶片Resistant B. oleracea leaf;Sl：感病甘蓝材料的叶片Susceptible B. oleracea leaf。以lg (FPKM+1)值绘制基因表达量的热图,红色表示高表达基因,蓝色表示低表达基因。颜色从红到蓝,表示lg (FPKM+1)从大到小。基因表达热图构建方法见文献[11] The lg (FPKM+1) value was used for the heatmap. Red indicated high expression genes and blue indicated low expression genes. The colors ranged from red to blue, indicating that lg (FPKM+1) from large to small. The heatmap was conducted according to reference [11]
Fig. 1Heatmap of candidate genes of S. sclerotiorum in the process of infecting the resistant and susceptible B. oleracea
Table 2
表2
表2候选基因的结构域功能分析
Table 2
| 基因 Gene | 结构域名称 Domain name | 结构域位置 Location (aa) | E期望值 E-value | 可能的功能或特性 Putative function or feature |
|---|---|---|---|---|
| SS1G_00263 | SCOP:d1hw1a2 | 14-79 | 0.77 | 调节药物的耐受性 Regulation of drug tolerance |
| SS1G_04945 | Pfam:Glyco_hydro_7 | 21-446 | 2.6e-200 | O型糖苷水解酶 O-glycoside hydrolase |
| PDB:3PL3|A | 19-446 | 0 | 纤维二糖水解酶 Cellobiohydrolase | |
| SCOP:d1gpia_ | 19-450 | 0 | 伴刀豆球蛋白A样凝集素/葡聚糖酶 Concanavalin A/Glucanase | |
| SS1G_03181 | Pfam:ASP | 91-399 | 3.2e-69 | 天冬氨酰蛋白酶（酸性蛋白酶）Aspartyl proteases (acid proteases) |
| SCOP:d1bxoa_ | 79-399 | 1e-81 | 酸性蛋白酶 Acid proteases | |
| PDB:3APP|A | 83-398 | 1e-90 | 酸性蛋白酶 Acid proteases | |
| SS1G_04343 | SCOP:d1gh8a_ | 339-377 | 0.39 | 翻译延长因子 Translation elongation factor |
| SCOP:d1fuia2 | 411-457 | 0.68 | 异构酶 Isomerase | |
| SS1G_03146 | SCOP:d1h4vb2 | 18-62 | 1.8 | 生物素合成酶 Biotin synthetase |
| SS1G_02250 | SCOP:d1ed8a_ | 43-90 | 5.7 | 碱性磷酸酶 Alkaline phosphatase |
| SCOP:d1dofa_ | 215-241 | 0.86 | L型天冬氨酸 L-aspartic acid | |
| SS1G_11912 | Pfam:NPP1 | 52-242 | 4.5e-61 | 坏死诱导蛋白 Necrosis inducing protein |
| PDB:3GNZ|P | 32-242 | 4e-65 | 毒素 Toxin | |
| SCOP:d1fuia2 | 188-240 | 0.58 | 异构酶 Isomerase | |
| SS1G_07655 | Pro-kuma_activ | 35-181 | 3.25e-19 | 肽酶 Peptidase |
| PDB:3EDY|A | 35-581 | 2e-67 | 肽酶 Peptidase | |
| SCOP:d1gt91_ | 36-581 | 9e-19 | 枯草杆菌蛋白酶类 Subtilisins |
新窗口打开|下载CSV
2.2 TRV-HIGS载体构建及侵染
扩增8个基因的片段,克隆入载体pTRV2,并利用EcoRI/SacI对pTRV2-Gene进行酶切检测,结果显示在8个pTRV2-Gene质粒的泳道均能检测到目的条带大小的片段（图2）,表明成功构建了8个基因的TRV-HIGS载体。利用相同方法克隆GFP（绿色荧光蛋白）片段,并成功构建了阳性对照载体pTRV2-GFP。图2
新窗口打开|下载原图ZIP|生成PPT图2利用EcoRI/SacI双酶切验证pTRV2-Gene载体
M: Marker; 1: SS1G_00263; 2: SS1G_04945; 3: SS1G_03181; 4: SS1G_04343; 5: SS1G_03146; 6: SS1G_02250; 7: SS1G_11912; 8: SS1G_07655
Fig. 2Verification of pTRV2-Gene vectors by EcoRI/SacI digestion
分别将pTRV1、pTRV2-GFP及pTRV2-Gene的重组质粒转化农杆菌 GV3101后,侵染5—6周龄的烟草。提取侵染9 d后携带TRV-Gene重组质粒的烟草植株叶片的DNA,利用表1中的引物进行目的片段的PCR检测。结果显示,侵染烟草叶片部位均能检测到相应大小的目的基因片段（图3-A）。同时将侵染9 d后的携带TRV-GFP载体的烟草植株叶片进行荧光信号观察,发现叶片组织存在绿色荧光信号（图3-C）,而侵染不含重组质粒的重悬液的烟草没有检测到荧光信号（图3-B）。
图3
新窗口打开|下载原图ZIP|生成PPT图3TRV-HIGS转化烟草植株的鉴定
A：注射含有TRV-Gene重组质粒9 d后的烟草植株的PCR鉴定The identification of N. benthamiana at 9 days post TRV-Gene inoculation. M: Marker; 1: SS1G_00263; 2: SS1G_04945; 3: SS1G_03181; 4: SS1G_04343; 5: SS1G_03146; 6: SS1G_02250; 7: SS1G_11912; 8: SS1G_07655;B：注射不含重组质粒的重悬液9 d后的烟草植株的GFP信号观察GFP signal observation of N. benthamiana at 9 days after suspension without recombinant vectors inoculation;C：注射TRV-GFP载体9 d后的烟草植株的GFP信号观察GFP signal observation of N. benthamiana at 9 days post TRV-GFP inoculation。标尺Bar = 30 μm
Fig. 3Identification of TRV-HIGS N. benthamiana
2.3 目的基因的表达分析
核盘菌接种TRV-HIGS烟草植株48 h后,提取病斑及周围组织RNA,进行目的基因的qRT-PCR分析。结果显示,与对照植株中的目的基因表达量相比,SS1G_07655在TRV-HIGS烟草植株上基因表达水平与对照差异不显著（P>0.05）,其余7个基因在所对应的TRV-HIGS烟草植株上的基因表达水平均显著低于对照（P≤0.05）,基因表达水平较对照相比下降了26.0%—89.3%（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4接种48 h后TRV-HIGS侵染的烟草中核盘菌目的基因相对TRV-GFP转化植株的表达情况
*：差异显著Significant difference (P≤0.05);ns：差异不显著No significant difference (P>0.05)
Fig. 4Relative expression level of the target gene in the N. benthamiana lines carrying TRV-HIGS vector to those carrying TRV-GFP vector at 48 hours post inoculation of S. sclerotiorum
2.4 TRV-HIGS烟草植株的菌核病抗性
为了分析这8个基因对核盘菌致病性的影响,在农杆菌侵染烟草9 d后进行核盘菌活体接种。结果发现在接种48 h后,除SS1G_07655的TRV-HIGS烟草植株的病斑面积与对照差异不显著外（P>0.05）,其余7个基因所对应的TRV-HIGS烟草植株的病斑面积相比对照植株均显著减小（P≤0.05）（图5-A、5-B）。其中对照TRV-GFP植株的平均病斑面积为3.44 cm2,而携带7个有差异基因的烟草植株的病斑面积介于1.63—2.61 cm2,SS1G_03146的TRV-HIGS烟草植株上的平均病斑面积最小（图5-B）。图5
新窗口打开|下载原图ZIP|生成PPT图5TRV-HIGS侵染的烟草的菌核病抗性鉴定
TRV-HIGS转基因烟草活体接种核盘菌48 h后的发病情况（A）及病斑大小统计（B）,柱上不同字母表示差异显著（P≤0.05）
Fig. 5Resistance identification of N. benthamiana plants carrying TRV-HIGS vector to Sclerotinia stem rot
Phenotypes (A) and lesion sizes (B) of TRV-HIGS N. benthamiana at 48 hours post inoculation with S. sclerotiorum in vivo. Different letters on the columns indicate significant difference (P≤0.05)
3 讨论
目前基于转录后基因沉默（post-transcriptional gene silencing,PTGS）原理研究寄主-病原菌互作主要集中于两个方面,一方面是利用VIGS等技术筛选寄主的抗病基因。VIGS是指利用重组病毒载体沉默植物内源基因的技术[14],目前利用该技术已经成功挖掘出了一系列植物抗病基因[15,28-30]。在植物抗菌核病研究中,也有研究者利用VIGS技术在烟草及番茄中筛选到了抗菌核病基因,如NbCML1[29]、SlCDPK[30]、SlCRK[30]等;另一方面利用HIGS等技术鉴定病原菌致病基因。HIGS是通过寄主植物作为媒介间接沉默病原菌内源基因的技术,主要用于创制抗性材料和病原菌致病基因的功能鉴定两个方面[18,19]。由于病毒载体介导的HIGS技术具有快速、操作简单等优点,因而在鉴定病原菌致病基因的研究中得到越来越多的应用[19,22,24]。其中,BSMV介导的HIGS技术在小麦条锈病、叶锈病等病原菌未知功能基因的鉴定中发挥着巨大作用[19,22]。本研究利用TRV载体,以GFP基因作为对照,采用农杆菌注射的方法在烟草上建立了利用TRV介导的HIGS鉴定核盘菌致病相关基因的技术体系。菌核病抗性鉴定结果显示7个携带候选基因的烟草植株的病斑面积与对照相比显著减小,表现为烟草的抗病性增强。qRT-PCR结果显示在侵染TRV-HIGS烟草植株过程中,核盘菌这7个候选基因的表达被显著抑制。研究结果证实了TRV介导的HIGS技术可以用于快速筛选核盘菌致病相关基因。分泌蛋白是核盘菌致病的关键“武器”之一[31]。目前针对分泌蛋白的研究主要集中在两个方面：一方面分泌蛋白与核盘菌自身菌核、附着胞结构形成相关[6,7,8];另一方面分泌蛋白可参与抑制宿主植物的抗病反应,诱导宿主植物细胞性坏死[5]。因此挖掘核盘菌致病相关的分泌蛋白对于解析核盘菌致病机理十分重要。本研究中,通过蛋白质保守结构域预测到SS1G_03146可能参与生物素合成。生物素为生物体内关键酶发挥作用所必需的辅因子,有研究显示鸭疫里默氏杆菌（Riemerella anatipestifer）生物素突变菌株的致病力及生长速度较野生型菌株显著减弱[32]。本研究结果显示携带SS1G_03146的TRV-HIGS烟草植株病斑面积减少到对照的47.4%。结合这些研究,推测生物素可能是核盘菌致病力的重要因子。此外,本研究预测到SS1G_00263具有GntR家族成员的保守结构域。HAYDON等[33]研究表明,GntR家族成员能够利用其保守结构域通过变构效应与代谢产物结合来调控基因转录;曾洁[34]证明了重组耻垢分枝杆菌（Mycobacterium smegmatis）对抗生素万古霉素非常耐受。本研究中发现SS1G_00263的沉默显著影响了核盘菌的致病性,因而推测SS1G_00263编码的分泌蛋白在侵染过程中可能参与了核盘菌对药物或植物抗菌物质等的调节过程。
植物的免疫系统可分为两道防线[35]：第一道防线是病原相关分子模式引发的免疫反应（PTI）;第二道防线是病原菌产生的效应蛋白所引发的免疫反应（ETI）。有研究证实SsCP1是核盘菌编码的一个可以被植物识别的保守PAMP[4],而病原菌侵染过程需要多种PAMP协同作用。在本研究中,预测到SS1G_04343具有翻译延伸因子的保守结构域,同时验证了其与核盘菌致病力相关。KUNZE等[36]利用细菌的翻译延伸因子EF-Tu 的保守肽段elf18处理植物后能引起植物产生PTI反应。根据这些研究,推测SS1G_04343可能是与核盘菌致病力相关的一种PAMP,是提高宿主菌核病抗性的关键因子。此外,预测SS1G_11912可能编码坏死诱导蛋白和毒素,坏死诱导蛋白能够触发植物体的ETI反应,诱导植物体产生强烈的过敏反应,引起植物细胞死亡[35]。菌核病抗性鉴定结果也证实SS1G_11912参与影响了核盘菌的致病性,故推测SS1G_11912通过影响植物的ETI反应,从而参与核盘菌的致病过程。
除了参与物质合成及药物耐受性调节、植物免疫反应相关外,这7个与核盘菌致病力相关的候选基因中还有部分参与编码水解酶类,主要包括酸性蛋白酶（SS1G_03181、SS1G_02250）和细胞壁降解酶（SS1G_04945）。后续将利用基因敲除、超表达等技术获得核盘菌转化子来深入解析这7个基因的功能,以期拓宽目前对分泌蛋白功能的认知,进一步解析核盘菌的致病机理。同时,这些致病相关的基因将为下一步利用HIGS技术创制持久稳定的高抗菌核病油菜材料提供重要靶标。
4 结论
利用TRV介导的HIGS技术成功筛选到了7个与核盘菌致病性相关的编码分泌蛋白的基因,其中SS1G_03146对核盘菌致病性影响最大。此外,预测SS1G_03146可能参与核盘菌生物素合成,SS1G_04343及SS1G_11912可能参与影响植物的免疫反应。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1094/PDIS-06-19-1147-REURLPMID:31746694 [本文引用: 1]
Armillaria root rot (ARR) is a serious disease of woody plants caused by several species of Armillaria. Armillaria isolates from diagnostic samples received in 2017 were identified by genus- and species-specific PCR and compared with isolates from an earlier survey (2004 to 2007). The results were comparable and, therefore, were combined for further analysis. Three species were identified: Armillaria mellea (83%), A. gallica (15%), and A. ostoyae (2%). Their wide host range makes choice of resistant plants in management of the disease difficult. We used the Royal Horticultural Society diagnostic dataset of ARR records from U.K. gardens to compare the susceptibility of different host genera to the disease. The dataset was compared with an earlier experiment at the University of California. An index-based approach was used to separate genera into three categories: 77 low-index (<0.99), 37 medium-index (0.99 to 1.76), and 56 high-index (>1.76) genera were recorded. All three species were associated with both angiosperms and gymnosperms; moreover, A. ostoyae did not show the host preference for gymnosperms that has been reported elsewhere. A. gallica was particularly common on herbaceous perennials and showed a trend to occur on resistant hosts that may be under other stress, supporting its description as an opportunistic pathogen. Four monocotyledons grown as trees or shrubs in U.K. gardens had a very low ARR index according to indices associated with A. mellea and A. ostoyae. Genera in the order Myrtales were almost always low index, while those in the Saxifragales and Fagales were mostly high index. These results provide confidence in the use of host resistance as part of the integrated management of ARR.
DOI:10.1186/s12864-017-3642-5URLPMID:28356071 [本文引用: 2]
Sclerotinia sclerotiorum causes stem rot in Brassica napus, which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection.
DOI:10.1111/nph.14842URLPMID:29076546 [本文引用: 2]
Cerato-platanin proteins (CPs), which are secreted by filamentous fungi, are phytotoxic to host plants, but their functions have not been well defined to date. Here we characterized a CP (SsCP1) from the necrotrophic phytopathogen Sclerotinia sclerotiorum. Sscp1 transcripts accumulated during plant infection, and deletion of Sscp1 significantly reduced virulence. SsCP1 could induce significant cell death when expressed in Nicotiana benthamiana. Using yeast two-hybrid, GST pull-down, co-immunoprecipitation and bimolecular florescence complementation, we found that SsCP1 interacts with PR1 in the apoplast to facilitate infection by S.?sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S.?sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several plant pathogens (namely Botrytis cinerea, Alternaria brassicicola and Golovinomyces orontii). Our results suggest that SsCP1 is important for virulence of S.?sclerotiorum and that it can be recognized by plants to trigger plant defense responses. Our results also suggest that the SA signaling pathway is involved in CP-mediated plant defense .
DOI:10.1371/journal.ppat.1005435URLPMID:26828434 [本文引用: 3]
Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1?SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1?SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1?SP had significant effects on its homo-dimer formation, SsSSVP1?SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1?SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1?SP might be caused by the SsSSVP1?SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction.
DOI:10.1371/journal.pone.0053901URLPMID:23342034 [本文引用: 3]
SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection.
DOI:10.1038/srep15565URLPMID:26531059 [本文引用: 3]
Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.
DOI:10.1111/mpp.12459URLPMID:27392818 [本文引用: 3]
Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S.?sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.
DOI:10.1094/MPMI-05-13-0145-RURLPMID:24299212 [本文引用: 3]
To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca(2+)-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.
DOI:10.1093/gbe/evx030URLPMID:28204478 [本文引用: 2]
DOI:10.1186/1471-2164-15-336URLPMID:24886033 [本文引用: 2]
The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.
DOI:10.1094/PHYTO-06-18-0204-RURLPMID:30265202 [本文引用: 6]
White mold disease caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops. Here, we simultaneously assessed the transcriptome changes from lesions produced by S. sclerotiorum on disease-resistant (R) and -susceptible (S) B. oleracea pools bulked from a resistance-segregating F2 population. Virulence genes of S. sclerotiorum, including polygalacturonans, chitin synthase, secretory proteins, and oxalic acid biosynthesis, were significantly repressed in lesions of R B. oleracea at 12 h postinoculation (hpi) but exhibited similar expression patterns in R and S B. oleracea at 24 hpi. Resistant B. oleracea induced expression of receptors potentially to perceive Sclerotinia signals during 0 to 12 hpi and deployed complex strategies to suppress the pathogen establishment, including the quick accumulation of reactive oxygen species via activating Ca2+ signaling and suppressing pathogen oxalic acid generation in S. sclerotiorum. In addition, cell wall degradation was inhibited in the resistant B. oleracea potentially to prevent the expansion of Sclerotinia hyphae. The transcriptome changes in S. sclerotiorum and host revealed that resistant B. oleracea produces strong responses against S. sclerotiorum during early infection.
DOI:10.1016/j.bbapap.2013.03.024URLPMID:23557863 [本文引用: 1]
Recent studies suggest that plants secrete a large number of proteins and peptides into the extracellular space. Secreted proteins play a crucial role in stress response, communication and development of organisms. Here we review the current knowledge of the secretome of more than ten plant species, studied in natural conditions or during (a)biotic stress. This review not only deals with the classical secretory route via endoplasmic reticulum and Golgi followed by proteins containing a known N-terminal signal peptide, but also covers new findings about unconventional secretion of leaderless proteins. We describe alternative secretion pathways and the involved compartments like the recently discovered EXPO. The well characterized secreted peptides that function as ligands of receptor proteins exemplify the biological significance and activity of the secretome. This article is part of a Special Issue entitled: An Updated Secretome.
DOI:10.1073/pnas.92.5.1679URLPMID:7878039 [本文引用: 1]
The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.
DOI:10.1111/j.1365-313X.2004.02158.xURLPMID:15315635 [本文引用: 2]
Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.
DOI:10.1146/annurev.arplant.55.031903.141803URLPMID:15377229 [本文引用: 2]
The discovery that plants recognize and degrade invading viral RNA caused a paradigm shift in our understanding of viral/host interactions. Combined with the discovery that plants cosuppress their own genes if they are transformed with homologous transgenes, new models for both plant intercellular communication and viral defense have emerged. Plant biologists adapted homology-based defense mechanisms triggered by incoming viruses to target individual genes for silencing in a process called virus-induced gene silencing (VIGS). Both VIGS- and dsRNA-containing transformation cassettes are increasingly being used for reverse genetics as part of an integrated approach to determining gene function. Virus-derived vectors silence gene expression without transformation and selection. However, because viruses also alter gene expression in their host, the process of VIGS must be understood. This review examines how DNA and RNA viruses have been modified to silence plant gene expression. I discuss advantages and disadvantages of VIGS in determining gene function and guidelines for the safe use of viral vectors.
DOI:10.1105/tpc.10.6.937URLPMID:9634582 [本文引用: 1]
The phytoene desaturase (PDS) gene of Nicotiana benthamiana was silenced in plants infected with potato virus X (PVX) vectors carrying PDS inserts, and a green fluorescent protein (GFP) transgene was silenced in plants infected with PVX-GFP. This virus-induced gene silencing (VIGS) is post-transcriptional and cytoplasmic because it is targeted against exons rather than introns of PDS RNA and against viral RNAs. Although PDS and GFP RNAs are most likely targeted through the same mechanism, the VIGS phenotypes differed in two respects. PDS mRNA was targeted by VIGS in all green tissue of the PVX-PDS-infected plant, whereas PVX-PDS was not affected. In contrast, VIGS of the GFP was targeted against PVX-GFP. Initially, VIGS of the GFP was initiated in all green tissues, as occurred with PDS VIGS. However, after 30 days of infection, the GFP VIGS was no longer initiated in newly emerging leaves, although it was maintained in tissue in which it had already been initiated. Based on these analyses, we propose a model for VIGS in which the initiation of VIGS is dependent on the virus and maintenance of it is virus independent.
DOI:10.1046/j.1365-313X.1998.00101.xURLPMID:15494056 [本文引用: 1]
The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.
DOI:10.1111/mpp.12500URLPMID:27749994 [本文引用: 2]
Verticillium wilt, caused by soil-borne fungi of the genus Verticillium, is an economically important disease that affects a wide range of host plants. Unfortunately, host resistance against Verticillium wilts is not available for many plant species, and the disease is notoriously difficult to combat. Host-induced gene silencing (HIGS) is an RNA interference (RNAi)-based process in which small RNAs are produced by the host plant to target parasite transcripts. HIGS has emerged as a promising strategy for the improvement of plant resistance against pathogens by silencing genes that are essential for these pathogens. Here, we assessed whether HIGS can be utilized to suppress Verticillium wilt disease by silencing three previously identified virulence genes of V. dahliae (encoding Ave1, Sge1 and NLP1) through the host plants tomato and Arabidopsis. In transient assays, tomato plants were agroinfiltrated with Tobacco rattle virus (TRV) constructs to target V. dahliae transcripts. Subsequent V. dahliae inoculation revealed the suppression of Verticillium wilt disease on treatment with only one of the three TRV constructs. Next, expression of RNAi constructs targeting transcripts of the same three V. dahliae virulence genes was pursued in stable transgenic Arabidopsis thaliana plants. In this host, V. dahliae inoculation revealed reduced Verticillium wilt disease in two of the three targets. Thus, our study suggests that, depending on the target gene chosen, HIGS against V. dahliae is operational in tomato and A. thaliana plants and may be exploited to engineer resistance in Verticillium wilt-susceptible crops.
DOI:10.1007/s11103-013-0022-7URL [本文引用: 4]
Rust fungi are devastating plant pathogens and several Puccinia species have a large economic impact on wheat production worldwide. Disease protection, mostly offered by introgressed host-resistance genes, is often race-specific and rapidly overcome by newly-emerging virulent strains. Extensive new genomic resources have identified vital pathogenicity genes but their study is hampered because of the biotrophic life styles of rust fungi. In cereals, Barley stripe mosaic virus (BSMV)-induced RNAi has emerged as a useful tool to study loss-of-function phenotypes of candidate genes. Expression of pathogen-derived gene fragments in this system can be used to obtain in planta-generated silencing of corresponding genes inside biotrophic pathogens, a technique termed host-induced gene silencing (HIGS). Here we test the effectiveness of BSMV-mediated HIGS in the wheat leaf rust fungus Puccinia triticina (Pt) by targeting three predicted pathogenicity genes, a MAPK, a cyclophilin, and a calcineurin regulatory subunit. Inoculation of BSMV RNAi constructs generated fungal gene-specific siRNA molecules in systemic leaves of wheat plant. Subsequent Pt inoculation resulted in a suppressed disease phenotype and a reduction in endogenous transcript levels of the targeted fungal genes indicating translocation of siRNA molecules from host to fungal cells. Efficiency of this host-generated trans-specific RNAi was enhanced by using BSMV silencing vectors defective in coat protein coupled with introducing fungal gene sequences simultaneously in sense and antisense orientation. The disease suppression indicated the likely involvement of these fungal genes in pathogenicity. This study demonstrates that BSMV-mediated in planta-generated RNAi is an effective strategy for functional genomics in rust fungi.
DOI:10.12677/BR.2014.33014URL [本文引用: 1]
DOI:10.12677/BR.2014.33014URL [本文引用: 1]
DOI:10.1105/tpc.110.077040URLPMID:20884801 [本文引用: 1]
Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.
DOI:10.1111/pbi.12829URLPMID:28881438 [本文引用: 4]
Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)-mediated host-induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T3 to T4 generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down-regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal-derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.
DOI:10.1111/pbi.12900URLPMID:29431919 [本文引用: 2]
Verticillium wilt (VW), caused by soil-borne fungi of the genus Verticillium, is a serious disease affecting a wide range of plants and leading to a constant and major challenge to agriculture worldwide. Cotton (Gossypium hirsutum) is the world's most important natural textile fibre and oil crop. VW of cotton is a highly devastating vascular disease; however, few resistant germplasms have been reported in cotton. An increasing number of studies have shown that RNA interference (RNAi)-based host-induced gene silencing (HIGS) is an effective strategy for improving plant resistance to pathogens by silencing genes essential for the pathogenicity of these pathogens. Here, we have identified and characterized multifunctional regulators of G protein signalling (RGS) in the Verticillium dahliae virulence strain, Vd8. Of eight VdRGS genes, VdRGS1 showed the most significant increase in expression in V.?dahliae after treating with the roots of cotton seedlings. Based on the phenotype detection of VdRGS1 deletion and complementation mutants, we found that VdRGS1 played crucial roles in spore production, hyphal development, microsclerotia formation and pathogenicity. Tobacco rattle virus-mediated HIGS in cotton plants silenced VdRGS1 transcripts in invaded V.?dahliae strains and enhanced broad-spectrum resistance to cotton VW. Our data demonstrate that VdRGS1 is a conserved and essential gene for V.?dahliae virulence. HIGS of VdRGS1 provides effective control against V.?dahliae infection and could obtain the durable disease resistance in cotton and in other VW-susceptible host crops by developing the stable transformants.
DOI:10.3864/j.issn.0578-1752.2015.07.07URL [本文引用: 2]
【Objective】 The objective of this study is to screen glycometabolism related genes from Verticillium dahliae, confirm the relationship between those genes and the growth and pathogenicity of V. dahliae, and to provide a theoretical basis for the control of plant Verticillium wilt. 【Method】 The glycometabolism related genes of V. dahliae were screened using host-induced gene silencing (HIGS) method. The Tobacco rattle virus vectors containing target genes were injected into tobacco for transient expression of those genes, then V.d991 was inoculatedto those transgenic tobacco using the root-dip approach, PDS would be whitened 7-10 days later, and their disease indexes were counted after 14 days. The fluorescence quantitative PCR was used to detect the amount of V.d991 biomass and the expression of VDPDF-1, VDEG-1, VDMAN-1and VDPD-1. 【Result】 Four glycometabolism related genes from V. dahliae (VDPDF-1, VDEG-1, VDMAN-1 and VDPD-1) were selected because of the great change of their disease indexes. Transgenic tobacco disease indexes of these four genes were reduced by 35%, 30%, 20%, and 10%, respectively, compared with the negative control. After injected with the mixture of VDEG-1/VDPDF-1, VDPD-1/VDPDF-1/ VDMAN-1, VDPD-1/VDMAN-1, VDPD-1/VDPDF-1,and VDEG-1/VDPD-1, transgenic tobacco disease indexes were reduced by 45%, 45%, 40%, 40%, and 35%. In addition to VDPD-1 transgenic tobacco, the transgenic tobacco disease indexes had significant differences compared with the control. After inoculation withV.d991, the V.d991 biomass and expression of target gene were decreased, mixed gene injection had better interference effect than the single gene. The most obvious biomass reduction wereEG/PDF (VDEG-1/VDPDF-1)and EG/PD (VDEG-1/VDPD-1), reached to 0.94, and the most significant interference effect was VDPDF-1, from PD/PDF (VDPD-1/VDPDF-1),reduced by 0.91. 【Conclusion】 Four genes were selected from V. dahliae by HIGS method. When these genes were interfered, those plants had high resistance to disease, mixed injection of multiple gene vectors were more significant than the single gene. These genes were confirmed with the glycometabolism in V.d991, probably related with V. dahliae pathogenicity.
DOI:10.3864/j.issn.0578-1752.2015.07.07URL [本文引用: 2]
【Objective】 The objective of this study is to screen glycometabolism related genes from Verticillium dahliae, confirm the relationship between those genes and the growth and pathogenicity of V. dahliae, and to provide a theoretical basis for the control of plant Verticillium wilt. 【Method】 The glycometabolism related genes of V. dahliae were screened using host-induced gene silencing (HIGS) method. The Tobacco rattle virus vectors containing target genes were injected into tobacco for transient expression of those genes, then V.d991 was inoculatedto those transgenic tobacco using the root-dip approach, PDS would be whitened 7-10 days later, and their disease indexes were counted after 14 days. The fluorescence quantitative PCR was used to detect the amount of V.d991 biomass and the expression of VDPDF-1, VDEG-1, VDMAN-1and VDPD-1. 【Result】 Four glycometabolism related genes from V. dahliae (VDPDF-1, VDEG-1, VDMAN-1 and VDPD-1) were selected because of the great change of their disease indexes. Transgenic tobacco disease indexes of these four genes were reduced by 35%, 30%, 20%, and 10%, respectively, compared with the negative control. After injected with the mixture of VDEG-1/VDPDF-1, VDPD-1/VDPDF-1/ VDMAN-1, VDPD-1/VDMAN-1, VDPD-1/VDPDF-1,and VDEG-1/VDPD-1, transgenic tobacco disease indexes were reduced by 45%, 45%, 40%, 40%, and 35%. In addition to VDPD-1 transgenic tobacco, the transgenic tobacco disease indexes had significant differences compared with the control. After inoculation withV.d991, the V.d991 biomass and expression of target gene were decreased, mixed gene injection had better interference effect than the single gene. The most obvious biomass reduction wereEG/PDF (VDEG-1/VDPDF-1)and EG/PD (VDEG-1/VDPD-1), reached to 0.94, and the most significant interference effect was VDPDF-1, from PD/PDF (VDPD-1/VDPDF-1),reduced by 0.91. 【Conclusion】 Four genes were selected from V. dahliae by HIGS method. When these genes were interfered, those plants had high resistance to disease, mixed injection of multiple gene vectors were more significant than the single gene. These genes were confirmed with the glycometabolism in V.d991, probably related with V. dahliae pathogenicity.
DOI:10.3390/ijms19041138URLPMID:29642627 [本文引用: 1]
Zymoseptoria tritici is a hemibiotrophic pathogen which causes Septoria leaf blotch in wheat. The pathogenesis of the disease consists of a biotrophic phase and a necrotrophic phase. The pathogen infects the host plant by suppressing its immune response in the first stage of infection. Hemibiotrophic pathogens of the genus Fusarium cause Fusarium head blight, and the necrotrophic Parastagonosporanodorum is responsible for Septoria nodorum blotch in wheat. Cell wall-degrading enzymes in plants promote infections by necrotrophic and hemibiotrophic pathogens, and trichothecenes, secondary fungal metabolites, facilitate infections caused by fungi of the genus Fusarium. There are no sources of complete resistance to the above pathogens in wheat. Defense mechanisms in wheat are controlled by many genes encoding resistance traits. In the wheat genome, the characteristic features of loci responsible for resistance to pathogenic infections indicate that at least several dozen genes encode resistance to pathogens. The molecular interactions between wheat and Z. tritici, P. nodorum and Fusarium spp. pathogens have been insufficiently investigated. Most studies focus on the mechanisms by which the hemibiotrophic Z. tritici suppresses immune responses in plants and the role of mycotoxins and effector proteins in infections caused by P. nodorum and Fusarium spp. fungi. Trichothecene glycosylation and effector proteins, which are involved in defense responses in wheat, have been described at the molecular level. Recent advances in molecular biology have produced interesting findings which should be further elucidated in studies of molecular interactions between wheat and fungal pathogens. The Clustered Regularly-Interspaced Short Palindromic Repeats/ CRISPR associated (CRISPR/Cas) system can be used to introduce targeted mutations into the wheat genome and confer resistance to selected fungal diseases. Host-induced gene silencing and spray-induced gene silencing are also useful tools for analyzing wheat-pathogens interactions which can be used to develop new strategies for controlling fungal diseases.
DOI:10.1186/1746-4811-4-5URLPMID:18211705 [本文引用: 1]
Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV).
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
DOI:10.1371/journal.pone.0070723URLPMID:23923019 [本文引用: 1]
Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.
DOI:10.3785/j.issn.1008-9209.2014.03.131URL [本文引用: 1]
组合采用药理学、分子生物学和反向遗传学等技术,分析叶位对本氏烟抗核盘菌（Sclerotinia sclerotiorum）的影响及其机制.结果表明,叶位显著影响本氏烟对核盘菌的抗性,随着叶位自上而下,该抗性逐渐增强.药理学分析结果显示,Ca2+通道抑制剂LaCl3和NaVO3处理消除了本氏烟叶片对核盘菌抗性的叶位间差异.定量反转录聚合酶链反应检测结果显示,3个Ca2+信号通路基因NbCNGC20、NbCAMTA3和NbCML1在本氏烟不同叶位叶片中的表达存在显著差异,随着叶位自上而下逐渐增加.病毒诱导的基因沉默（virus-induced gene silencing,VIGS）分析结果表明,钙调素类似蛋白基因NbCML1的沉默导致叶位介导的本氏烟对核盘菌抗病性的丧失.这些结果说明叶位对本氏烟抗核盘菌具有显著影响,揭示了包括NbCML1基因在内的Ca2+信号通路对叶位介导的核盘菌抗性的重要调控作用.
DOI:10.3785/j.issn.1008-9209.2014.03.131URL [本文引用: 1]
组合采用药理学、分子生物学和反向遗传学等技术,分析叶位对本氏烟抗核盘菌（Sclerotinia sclerotiorum）的影响及其机制.结果表明,叶位显著影响本氏烟对核盘菌的抗性,随着叶位自上而下,该抗性逐渐增强.药理学分析结果显示,Ca2+通道抑制剂LaCl3和NaVO3处理消除了本氏烟叶片对核盘菌抗性的叶位间差异.定量反转录聚合酶链反应检测结果显示,3个Ca2+信号通路基因NbCNGC20、NbCAMTA3和NbCML1在本氏烟不同叶位叶片中的表达存在显著差异,随着叶位自上而下逐渐增加.病毒诱导的基因沉默（virus-induced gene silencing,VIGS）分析结果表明,钙调素类似蛋白基因NbCML1的沉默导致叶位介导的本氏烟对核盘菌抗病性的丧失.这些结果说明叶位对本氏烟抗核盘菌具有显著影响,揭示了包括NbCML1基因在内的Ca2+信号通路对叶位介导的核盘菌抗性的重要调控作用.
[D].
[本文引用: 3]
[D].
[本文引用: 3]
[本文引用: 1]
[本文引用: 1]
[D].
[本文引用: 1]
[D].
[本文引用: 1]
DOI:10.1016/j.biochi.2019.11.012URLPMID:31765672 [本文引用: 1]
Molecular chaperones are a conserved family of proteins that are over-expressed in response to heat and other stresses. The regulation of expression of chaperone proteins plays a vital role in pathogenesis of various bacterial pathogens. In M.?tuberculosis, HrcA and HspR negatively regulate heat shock protein operons by binding to their cognate DNA elements, CIRCE and HAIR respectively. In this study, we show that M.?tuberculosis HrcA is able to bind to its cognate CIRCE DNA element present in the upstream regions of groES and groEL2 operons only with the help of other protein(s). It is also demonstrated that M.?tuberculosis HrcA binds to a CIRCE like DNA element present in the upstream region of hrcA gene suggesting its auto-regulatory nature. In addition, we report the presence of a putative HAIR element in the upstream region of groES operon and demonstrate the binding of HspR to it. In?vitro, HrcA inhibited the DNA binding activity of HspR in a dose-dependent manner. The current study demonstrates that M.?tuberculosis HrcA requires other protein(s) to function, and the heat shock protein expression in M.?tuberculosis is negatively regulated jointly by HrcA and HspR.
[D].
[本文引用: 1]
[D].
[本文引用: 1]
DOI:10.1016/j.cell.2006.02.008URLPMID:16497589 [本文引用: 2]
The evolution of the plant immune response has culminated in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as PAMP-triggered immunity (PTI) and has evolved to recognize common features of microbial pathogens. In the coevolution of host-microbe interactions, pathogens acquired the ability to deliver effector proteins to the plant cell to suppress PTI, allowing pathogen growth and disease. In response to the delivery of pathogen effector proteins, plants acquired surveillance proteins (R proteins) to either directly or indirectly monitor the presence of the pathogen effector proteins. In this review, taking an evolutionary perspective, we highlight important discoveries over the last decade about the plant immune response.
DOI:10.1105/tpc.104.026765URLPMID:15548740 [本文引用: 1]
Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.
