,1, 刘大群,2Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31
QI Yue1, Lü JunYuan1, ZHANG Yue1, WEI Jie1, ZHANG Na1, YANG WenXiang,1, LIU DaQun,2通讯作者:
责任编辑: 杨鑫浩
收稿日期:2019-12-26网络出版日期:2020-06-16
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Received:2019-12-26Online:2020-06-16
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齐悦,E-mail：908952718@qq.com。
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齐悦, 吕峻元, 张悦, 韦杰, 张娜, 杨文香, 刘大群. 小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应[J]. 中国农业科学, 2020, 53(12): 2371-2384 doi:10.3864/j.issn.0578-1752.2020.12.006
QI Yue, Lü JunYuan, ZHANG Yue, WEI Jie, ZHANG Na, YANG WenXiang, LIU DaQun.
0 引言
【研究意义】由小麦叶锈菌（Puccinia triticina）引起的小麦叶锈病严重影响小麦生产[1],该病害在世界范围分布广泛[2],严重发生时可造成15%—45%甚至更高的经济产量损失[3]。小麦叶锈菌致病性多样化且致病类型频繁发生变异,导致小麦抗叶锈性不断丧失。因此,研究小麦叶锈菌致病的分子机制对控制小麦叶锈病尤为重要。【前人研究进展】植物的免疫机制主要由两层防御体系构成[4]。第一层次防御系统是由植物细胞膜表面的模式识别受体（pattern recognition receptors,PRRs）识别病原微生物保守的模式分子（microbial- or pathogen-associated molecular patterns,MAMPs or PAMPs）引发的抗病反应,称之为PTI（PAMPs-triggered immunity）[5]。第二层的防御系统是由植物细胞内的抗病蛋白（disease resistance protein,R protein）,主要是NB-LRR类型的抗病蛋白直接或间接地识别病原微生物分泌到植物细胞内的效应因子,引发更为强烈的免疫反应,称之为ETI（effector-triggered immunity）[6]。植物的PTI又称为基础抗性,是植物通过细胞膜表面的受体激酶（receptor-like kinases,RLKs）或受体蛋白（receptor- like proteins,RLPs）感受病原微生物的PAMPs从而触发植物细胞的一系列反应[7],比如细胞膜离子通道的激活,钙依赖蛋白激酶（calcium-dependent protein kinases,CPKs）的激活,细胞质内钙离子浓度的变化,胼胝质的积累,MAPK级联反应的激活,病程相关基因PRs的上调表达等。植物的ETI由病原菌分泌到寄主细胞中的一些被称为“效应蛋白”的蛋白触发[8],效应蛋白能够通过多种方式抑制植物免疫反应,包括抑制PTI[9],促使病原成功侵染植物[10,11,12,13]。ETI往往在受侵染的植物细胞部位产生大量的过氧化物（如H2O2）,并且诱导植物的过敏性坏死反应（hypersensitive response,HR）[14]。锈菌是专性寄生真菌,主要通过吸器从寄主细胞内吸收营养,进而有效的在寄主中扩展,产生孢子[15]。病原菌侵染时向寄主细胞分泌效应蛋白[16],通过效应蛋白调控寄主的免疫防御反应,造成植物感病。锈菌效应蛋白在寄主中的作用机理尚不清楚,基因组、转录基因组测序成为探明锈菌效应蛋白功能的重要研究途径。至今已完成多种植物病原锈菌的基因组测序和转录组测序,包括杨树锈菌（Melampsora larici-populina）[17]、亚麻锈菌（Melampsora lini）[18]、小麦秆锈菌（Puccina graminis f. sp. tritici,Pgt）[19]、小麦条锈菌（Puccinia striiformis f. sp. tritici,Pst）[19,20,21]和小麦叶锈菌[22]。通过基因组测序,从杨树锈菌的1 898个分泌蛋白中鉴定出1 184个CSEP（candidate effectors protein）[23];从亚麻锈菌的1 085个分泌蛋白中鉴定出了762个CSEP[24];在小麦秆锈菌的1 934个分泌蛋白中筛选出1 106个CSEP[19];在条锈菌CY-32中筛选出2 029个CSEP[25];在叶锈菌中预测到1 358个效应蛋白[26]。然而,锈菌中已经克隆出的效应蛋白数量非常有限,这些效应蛋白分别来源于亚麻锈菌的AvrM[27]、AvrL567[28]、AvrP123[29]、AvrP4[30]、AvrL2[31]和AvrM14[31];蚕豆单胞锈菌（Uromyces fabae）的RTP1[32],小麦秆锈菌的PGTAUSPE-10-1[33]、AvrSr35[34]、AvrSr50[35]和小麦条锈菌的Pst8853[36]、Pst23616[37]、Pstha5a23[38]、Pst87[39]、Pec6[40]、HASP2[41]、Pst30[42]、Hasp58[43]、PstGSRE1[44]、Pst8713、Pst12806[45]和Pst18363[46]。而对小麦叶锈菌效应蛋白的研究仍处于起始阶段。BRUCE等[22]利用6个小麦叶锈菌菌株与小麦互作中的吸器形成阶段开展转录组分析,获得了222 571个锈菌中的序列,共筛选出532个候选效应蛋白;SEGOVIA等[47]在2016年发现Pt3和Pt27可分别在含有抗性基因Lr9或Lr24,Lr26的近等基因系中抑制GUS的表达;2017年,澳大利亚获得了小麦叶锈菌中与抗病基因Lr20对应的候选效应蛋白,但并未开展相关功能的进一步分析。CUOMO等[26]在2017年对小麦秆锈菌、小麦条锈菌和小麦叶锈菌的基因组进行了测序结果比较,发现小麦叶锈菌基因组在这3个基因组中最大（估计为135 Mb）,且基因组高度杂合,通过全基因组和RNA-seq在小麦叶锈菌的5个发育阶段中总计预测到1 358个效应子,但是尚无相关功能验证的报道。【本研究切入点】中国小麦叶锈菌受种植小麦品种及地理环境的影响,具有与其他国家菌株不同的致病力,自2012年开始河北农业大学小麦叶锈病研究中心开展了小麦叶锈菌的效应蛋白的预测和相关分析,利用小麦叶锈菌与寄主互作的转录组筛选得到635个候选效应蛋白。其中Pt18906在小麦叶锈菌吸器形成阶段显著高表达,关于该基因的特性及其在致病中的作用尚不明确。【拟解决的关键问题】对小麦叶锈菌效应蛋白Pt18906的基本特性进行分析,测试其在小麦抗叶锈近等基因系中对寄主胼胝质和活性氧（reactive oxygen species,ROS）的影响,明确被诱导发生防御反应的近等基因系,揭示Pt18906在小麦近等基因系中对应的基因,探测该效应蛋白与对应抗叶锈基因的关系,为深入揭示Pt18906在致病中的作用及其调控机理打下基础。1 材料与方法
试验于2019年在河北农业大学植物保护学院完成。1.1 供试植物
供试烟草为本氏烟（Nicotiana benthamiana）。39个以Thatcher为遗传背景的小麦抗叶锈病近等基因系包括TcLr1、TcLr2a、TcLr2c、TcLr3、TcLr3Ka、TcLr9、TcLr10、TcLr11、TcLr14a、TcLr16、TcLr17、TcLr18、TcLrB、TcLr24、TcLr26、TcLr30、TcLr2b、TcLr14b、TcLr15、TcLr19、TcLr20、TcLr21、TcLr23、TcLr25、TcLr27+31、TcLr28、TcLr29、TcLr32、TcLr33、TcLr33+ 34、TcLr36、TcLr38、TcLr41、TcLr42、TcLr44、TcLr45、TcLr50、TcLr51和TcLr53,由CIMMTY惠赠,笔者实验室保存。1.2 供试菌株及培养条件
大肠杆菌（Escherichia coli）菌株DH5α购自天根（TIANGEN）公司,使用LB培养基在37℃条件下培养;农杆菌（Agrobacterium tumefaciens）菌株GV3101由中国农业大学孙文献教授惠赠,在28℃条件下培养;酵母突变株为YTK12;荧光假单胞菌（Pseudomonas fluorescens）菌株EtHAn 由西北农林科技大学康振生院士惠赠。1.3 供试载体
烟草瞬时过表达载体PVX病毒载体pGR107由中国农业大学孙文献教授惠赠,信号肽分泌功能验证载体pSUC2和细菌三型分泌系统载体pEDV6由西北农林科技大学康振生院士惠赠。1.4 Pt18906的克隆和载体构建
以小麦叶锈菌菌株13-5-72（THSN）侵染小麦叶片的RNA反转录产物为模板,克隆效应蛋白基因,分别利用引物（表1）构建到pGR107、pSUC2和pEDV6载体上,构建完成的载体用于烟草瞬时表达、信号肽分泌功能验证、亚细胞定位和利用细菌三型系统开展的免疫反应测定。Table 1
表1
表1本试验所用引物
Table 1
| 引物类型 Primer type | 引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 序列长度 Sequence length (bp) |
|---|---|---|---|
| Pt18906的ORF引物 Primer for Pt18906 ORF 信号肽分泌功能验证 Function verification of signal peptide secretion 异源瞬时表达 Heterogeneous transient expression 亚细胞定位 Subcellular localization 毒性结构域验证 Verification of virulent domain 细菌三型分泌系统 Bacterial type Ⅲ secretion system | ORFPt18906-F ORFPt18906-R SPPt18906-F SPPt18906-R Pt18906-F ΔSPPt18906-F Pt18906-R LBA LBB DPt18906-F DPt18906-R qPt18906-F1 qPt18906-R1 qPt18906-R2 qPt18906-R3 qPt18906-R4 PPt18906-F PPt18906-R | ATGTTTTCAGCAAGTTCAAT CTACTTACCCTTCTCCTTAG CCGGAATTCATGTTTTCAGCAAGTTCAAT CCGCTCGAGAAGCTCAACGGGTGGTAAGC TCCCCCGGGATGTTTTCAGCAAGTTCAAT TCCCCCGGGGCCGAAGTCCAACGACACGC GCGTCGACCTACTTACCCTTCTCCTTAG CAATCACAGTGTTGGCTTGC GACCCTATGGGCTGTGTTG CCCATCGATGCCGAAGTCCAACGACACGC TCCCCCGGGCTTACCCTTCTCCTTAGGAT CCCATCGATGCTCCACTCAAAAACGGTGA TCCCCCGGGTTGGTTGTTAACGTCCTCAA TCCCCCGGGGACCGCCATGTATCCGGCTA TCCCCCGGGGGGGTCTATCTCTGTAGGAT TCCCCCGGGTATGCTCCCGTATTTGCATG CACCATGTTTTCAGCAAGTTCAAT CTACTTACCCTTCTCCTTAG | 20 20 29 29 29 29 28 21 20 29 29 29 29 29 29 29 24 20 |
新窗口打开|下载CSV
1.5 农杆菌介导的烟草瞬时转化
将构建的ΔSPPT18906-pGR107转入农杆菌感受态细胞GV3101中,倒置培养2 d后挑取阳性克隆接种到含有100 μg·mL-1 Kan和100 μg·mL-1 Rif的5 mL LB培养基中。28℃,200 r/min 摇床中振荡培养2—3 d至菌液浑浊后,用5 mL农杆菌缓冲液（100 mL灭菌水+1 mL MgCl2（10 mmol·L-1）+1 mL 2-（N-吗啉）乙磺酸（10 mmol·L-1））洗涤3次,稀释至OD600=0.3,Bax和INF1稀释至OD600=0.3避光静置2 h;选择6—8周龄本氏烟草植物叶的第2—4叶接种,并使用1 mL的无针注射器渗透入叶中。在注射之前将效应蛋白基因构建体和Bax或INF1构建体以1﹕1比例混合,同时注射,设定为0 h抑制试验;将含有效应蛋白的农杆菌菌株在造成细胞死亡的Bax或INF1构建体前24 h注射烟草,定为24 h抑制试验。在烟草叶上测试效应蛋白,并将该试验重复3次,在5—7 d内观察细胞死亡情况,接种后7 d对叶片进行拍照。1.6 Pt18906信号肽分泌功能的验证
采用酵母信号肽筛选（yeast signal peptide screen,YSST）验证效应蛋白信号肽的分泌功能。试验中选用的酵母菌株为缺失蔗糖转化酶基因、在以蔗糖为唯一碳源的培养基上不能生长的YTK12。选用的载体质粒为含有色氨酸合成基因和一个缺失信号肽与起始密码子ATG的蔗糖转化酶基因（SUC2）的pSUC2。1.6.1 酵母感受态的制备与转化 在YPDA固体培养基上28℃活化酵母菌株YTK12;2—3 d后刮取适量的酵母菌,配置反应体系（10XLiAc 36 μL,50% PEG3500 240 μL,鲑鱼精ssDNA（2 ng·mL-1）50 μL,酵母感受态细胞50 μL,pSUC2-效应基因的重组载体5 μL,ddH2O 80 μL）,混匀,放置42℃ 50 min;9 000 r/min离心1 min,弃上清;加入150 μL水,吹吸混匀,涂布于SD-Trp的培养基平板上,28℃培养2—3 d。
1.6.2 信号肽分泌功能的检测 将SD-Trp培养基上生长的阳性克隆转接到YPRAA培养基上,28℃倒置培养,如果能在YPRAA培养基上继续生长,则证明候选信号肽序列具有分泌功能;若不能生长则证明候选信号肽序列无分泌功能。
1.7 Pt18906的实时荧光定量分析
将小麦叶锈菌菌株13-5-72（THSN）接种在小麦Thatcher完全展开的第一叶上,黑暗条件下保湿（保湿温度16℃,相对湿度100%）,接种后分别采取0、6、12、18、24、36、48、72、96 h和6、9、12 d的样品,将上述12个时间点的样品按照TaKaRa MiniBEST Plant RNA Extraction Kit试剂盒提供的方法提取总RNA,反转录成cDNA。使用荧光定量PCR仪（罗氏LightCycler480）,以各取样点侵染的叶片cDNA为模板,以叶锈菌夏孢子的肌动蛋白β-Actin为内参基因,进行PCR扩增引物为RTPt18906-F：ATACGATGGTCTGGGTT;RTPt18906-R：TTGTTTT GTCTGGTGGA,每个反应生物学重复3次。PCR反应结果按照2-ΔΔCT法计算目的基因在不同时间点的相对表达量。1.8 Pt18906抑制Bax诱导的细胞程序性死亡（programmed cell death,PCD）功能motif分析
为进一步确定在候选效应基因中负责抑制PCD过程的功能结构域,采用氨基酸逐步缺失的方法,将缺失部分氨基酸的候选效应基因的序列构建到PVX载体上,利用农杆菌侵染烟草细胞试验来验证突变的候选效应基因对Bax诱导的PCD功能,从而鉴定其功能结构域。1.9 Pt18906亚细胞定位
将ΔSP Pt18906-pGR107重组载体电转入农杆菌感受态细胞GV3101中,倒置培养2 d后挑取阳性克隆接种到含有相应抗性的5 mL LB培养基中。28℃,200 r/min摇床中振荡培养至菌液浑浊后,用5 mL农杆菌缓冲液洗涤3次;稀释至OD600=0.2,避光静置2 h;选择8叶左右的本氏烟草植物叶的第2—4叶接种,并使用无针注射器渗透入叶中,以GFP为对照。农杆菌注射后2 d,剪取注射的烟草叶片,利用荧光显微镜观察效应蛋白的表达量以及定位;之后撕取烟草叶片下表皮在0.8 mol·L-1的山梨醇中浸泡15 min,荧光显微镜下观察。1.10 Pt18906的无毒性功能分析
将重组质粒ORFPt18906-pGR107利用农杆菌转化法转到农杆菌GV3101中。Pt18906构建体稀释至OD600=1.0,静置于28℃ 3 h,后用于注射全套的近等基因系。观察效应蛋白在不含抗病基因和含有不同抗病基因的小麦中过表达能否激发过敏性细胞坏死反应（HR）。选取感病材料Thatcher作为对照,在39个小麦抗叶锈病近等基因系中瞬时表达效应蛋白Pt18906,注射5 d后观察表型,7 d拍照,并取样利用DAB进行染色,观察过氧化氢（H2O2）积累情况。1.11 细菌三型分泌系统介导的小麦瞬时转化
将质粒转化进入荧光假单胞感受态EtHAn中。挑取菌落接种到含50 μg·mL-1庆大霉素和30 μg·mL-1氯霉素抗性的培养基中,菌液浑浊后,用10 mmol·L -1 MgCl2溶液洗涤3次,将处理好的菌液稀释至OD600=1.0,28℃避光放置2 h,用于注射小麦叶片。Pt18906诱导的胼胝质的观察：采集含有Pt18906载体荧光假单胞菌株EtHAn注射后12、24、36、48和72 h的小麦叶片,将叶片浸泡在脱色液（乙醇和冰醋酸（1﹕1）混合）中,完全脱色后转入水合氯醛中处理过夜;加入0.05%苯胺蓝染液染色,在荧光显微镜下观察胼胝质的积累情况并随机选取50个视野,统计胼胝质的数目。
Pt18906诱导的活性氧的观察：采集EtHAn注射后0、5、10、15、20和30 min的小麦叶片,用1%硝基蓝四氮唑染液染色超氧阴离子,强光下光照4—8 h;使用无水乙醇染色完毕后脱色,在水合氯醛溶液中过夜处理。利用ASSESS软件统计每个叶片的染色面积占总注射叶片面积的百分比,每个样品生物学重复3次。
2 结果
2.1 Pt18906的序列及结构分析
通过克隆获得一个开放阅读框长为672 bp的Pt18906,编码223个氨基酸,具有8个半胱氨酸残基,与小麦叶锈菌BBBD的PTTG_11719同源性达到99%。利用SignalP 4.1在线软件预测,发现该效应蛋白的1—27 aa 为信号肽区域（图1-A）。使用Target P预测编码的蛋白,发现该蛋白不含有线粒体定位信号序列,利用TMHMM 2.0在线软件预测无跨膜结构;利用Pfam在线软件分析该效应蛋白无保守基序;利用MEME在线软件分析目的基因编码的蛋白无保守结构域;Pt18906编码的蛋白等电点（pI）为7.535,证明此效应蛋白的工作环境偏向于碱性;利用在线软件Swiss-Model预测得出效应蛋白的三级结构（图1-B）,Pt18906的二级结构含有12.56%的α-螺旋,并且存在13%的延伸链、4.48%的转角和69.96%的无规则卷曲。图1
新窗口打开|下载原图ZIP|生成PPT图1Pt18906的序列分析
A：Pt18906信号肽预测Pt18906 signal peptide prediction;B：Pt18906蛋白的三级结构Tertiary structure of Pt18906 protein
Fig. 1Sequence analysis of Pt18906
2.2 效应蛋白Pt18906抑制Bax和INF1诱导的细胞程序性死亡
为了测试效应蛋白是否能抑制PCD,Bax和INF1在候选效应基因注射后24 h注射到4—6周龄的本氏烟草植物中,注射排布如图2-A。从表型可以看到单独注射载体GFP的位点没有出现PCD现象,证明载体对PCD的产生没有任何影响。注射GFP和Bax/INF1的位点出现坏死,单独注射Bax/INF1的位点在5 d左右出现了坏死,而单独注射Pt18906的位点没有出现坏死,但在注射Pt18906后24 h注射Bax/INF1的位点没有出现坏死,说明该基因抑制了Bax/INF1引起的PCD（图2 B-1/B-3）。为了排除先注射基因可能会占据Bax/INF1诱导PCD的作用位点,将Bax、INF1和效应蛋白按照1﹕1（V/V）混合后注射到烟草叶片中,从表型结果,看到Pt18906依然能够有效抑制住Bax/INF1激发的PCD（图2B-2/B-4）。图2
新窗口打开|下载原图ZIP|生成PPT图2Pt18906抑制Bax和INF1诱导的细胞程序性死亡
A：验证Pt18906抑制Bax和INF1注射模式图Verify that Pt18906 inhibits Bax and INF1 (injection pattern figure);B：验证Pt18906抑制Bax和INF1诱导细胞程序性死亡的结果（1：注射后24 h有效抑制Bax造成的坏死;2：注射后0 h有效抑制Bax造成的坏死;3：注射后24 h有效抑制INF1造成的坏死;4：注射后0 h有效抑制INF1造成的坏死）
Fig. 2Pt18906 inhibits PCD induced by Bax and INF1
Verify that Pt18906 inhibits Bax and INF1-induced PCD (1: The inhibition of Bax-induced PCD 24 h after Pt18906 injection; 2: The inhibition of Bax-induced PCD at 0 h after Pt18906 injection; 3: The inhibition of PCD by INF1 24 h after Pt18906 injection; 4: The effective inhibition of PCD by INF1 0 h after Pt18906 injection)
2.3 Pt18906信号肽分泌功能验证
为检测重组质粒的分泌功能,挑取在SD-Trp培养基上生长的阳性克隆,转接到YPRAA培养基上,检测转化酵母细胞是否能够在YPRAA培养基上能生长。选用大豆疫霉（Phytophthora sojae）中的分泌型效应蛋白Avr1b的信号肽为阳性对照,选用稻瘟病菌（Magnaporthe oryzae）的非分泌性蛋白Mg87为阴性对照。结果发现,含有Avr1b与缺失SUC2的转化子在筛选培养基中能够正常生长,含有Pt18906效应蛋白信号肽及缺失SUC2重组载体的酵母菌株在SD-Trp培养基中能正常生长,分泌型效应蛋白Avr1b能在YPRAA培养基中正常生长,但Pt18906效应蛋白信号肽不能在YPRAA培养基中正常生长,说明效应蛋白的信号肽没有分泌功能,从而确认Pt18906效应蛋白为非分泌型蛋白（图3）。图3
新窗口打开|下载原图ZIP|生成PPT图3Pt18906信号肽分泌功能验证
Fig. 3Verification of signal peptide secretory function of Pt18906
2.4 Pt18906表达量分析
通过对Pt18906在小麦叶锈菌不同生长发育阶段以及小麦叶片侵染过程的表达趋势进行分析,结果表明候选效应蛋白在0—24 h阶段逐渐上调表达,在24 h达到表达高峰,之后表达量呈现出逐渐下调的趋势（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4Pt18906在小麦叶锈菌13-5-72和Thatcher互作中不同时间点的表达量
Fig. 4 Pt18906 expression at different time points in P. triticina 13-5-72 and Thatcher interaction
2.5 效应蛋白Pt18906毒性功能
为探知Pt18906决定相关功能的结构域,构建了6个缺失突变体,包括N-末端缺失20个氨基酸的突变体和C-末端缺失突变体,分别缺失30个（Δ194—224）、60个（Δ164—224）、90个（Δ134—224）和120个（Δ104—224）氨基酸。缺失突变体农杆菌注射烟草的结果发现,从C-末端缺失30、60、90和120个氨基酸的突变体均能抑制细胞坏死,而N-末端缺失20个氨基酸的突变体丧失了抑制细胞坏死的能力（图5）。表明Pt18906效应蛋白28—47位的氨基酸序列对其毒性功能具有重要作用。图5
新窗口打开|下载原图ZIP|生成PPT图5Pt18906毒性功能结构域分析
Fig. 5Pt18906 virulence functional domain analysis
2.6 效应蛋白Pt18906的作用位点
利用GFP对Pt18906进行亚细胞定位分析,质壁分离后发现pGR107-GFP载体（对照）在细胞核及细胞质中均有表达,绿色荧光分布在整个细胞内,Pt18906与GFP融合蛋白表达激发的绿色荧光同样充斥整个细胞内（图6）,证明效应蛋白Pt18906在细胞内起作用。图6
新窗口打开|下载原图ZIP|生成PPT图6Pt18906亚细胞定位
Ⅰ：荧光场Epi-fluorescence;Ⅱ：明场Bright-field;Ⅲ：融合场Merged images;GFP发绿色荧光;标尺为100 μm Green means the fluorescence of the GFP. Scale bars are 100 μm
Fig. 6Subcellular localization of Pt18906 in N. benthamiana
2.7 Pt18906的无毒效应
利用农杆菌侵染的方法检测Pt18906在39个小麦抗叶锈病近等基因系中过表达效应蛋白,注射5 d后观察过敏性坏死反应发生的情况。结果发现携带有空载体pGR107的农杆菌株注射没有引起坏死,Pt18906在近等基因系TcLr27+31和TcLr42上出现了坏死反应,用DAB对小麦叶片进行染色,发现出现坏死反应的部位被DAB染成棕褐色（图7）,在显微镜下观察到棕褐色,说明有H2O2的存在,表明效应蛋白触发了寄主的防御反应,小麦TcLr27+31和TcLr42中存在与效应蛋白互作的分子靶标。图7
新窗口打开|下载原图ZIP|生成PPT图7在小麦中过表达Pt18906激发坏死反应
A：在TcLr27+31过表达Pt18906激发坏死Overexpression of Pt18906 in TcLr27+31 stimulates HR;B：在TcLr42过表达Pt18906激发坏死Overexpression of Pt18906 in TcLr42 stimulates HR
1：注射的表型结果Phenotypic results of injection;2：4倍荧光显微镜下的H2O2 The H2O2 under fluorescence microscope (4×);3：20倍荧光显微镜下的H2O2 The H2O2 under fluorescence microscope (20×)
Fig. 7Overexpression of Pt18906 in wheat stimulates HR
2.8 Pt18906在寄主上诱导的胼胝质分析
采用细菌的三型分泌系统将效应蛋白递送到小麦中,以MgCl2、EtHAn和转化有pEDV6的荧光假单胞菌作为对照。注射后的0、12、24、36、48、72 h采集组织学样品,与对照相比,Pt18906在小麦中激发了胼胝质的产生,从12 h及可以观察到胼胝质的产生,而且随着时间的延长,胼胝质的积累逐渐增强（图8）,表明效应蛋白Pt18906能够促进TcLr27+31胼胝质的积累,触发了寄主的防御反应。图8
新窗口打开|下载原图ZIP|生成PPT图8在TcLr27+31中瞬时表达Pt18906引起胼胝质的积累
A：注射Pt18906后不同时间点胼胝质累积Callose accumulation at different time points after injection of Pt18906;B：胼胝质积累量与注射时间关系Relationship between callose accumulation and injection time
Fig. 8Expression of Pt18906 in TcLr27+31 causes accumulation of callose
2.9 Pt18906在寄主上诱导的活性氧分析
利用植物组织O2-能被捕获剂NBT进行原位捕获和显色,蓝色的位置代表存在O2-。统计结果显示,与对照MgCl2、EtHan和pEDV6相比,Pt18906激发的活性氧面积更大（图9）,并且在效应蛋白注射后活性氧先升高,在注射10 min达到最高,之后降低,表明效应蛋白Pt18906能够引起TcLr27+31活性氧的迸发,触发了寄主的防御反应。图9
新窗口打开|下载原图ZIP|生成PPT图9在TcLr27+31中瞬时表达Pt18906引起活性氧的迸发
A：注射Pt18906 后不同时间点的活性氧情况ROS at different time points after injection of Pt18906;B：量化注射Pt18906后不同时间点的活性氧情况The ROS at different time points after injection of Pt18906
Fig. 9Expression of Pt18906 in TcLr27+31 causes ROS bursts
3 讨论
锈菌作为活体营养寄生真菌可通过形成吸器侵入寄主细胞,参与寄主与病原物之间营养物质和信息的交换与传递。国内外****已在不同锈菌中鉴定出了多种效应蛋白,这些已鉴定的效应蛋白均为编码未知功能的分泌蛋白,多无明显功能结构域及明确的生化功能。本研究对小麦叶锈菌Pt18906进行序列和结构分析,初步确定其为候选效应蛋白,其可抑制Bax和INF1诱导的细胞坏死。目前对于病原菌效应蛋白序列特征的定义都是基于已经发现的效应蛋白的一些特征来总结的,但是由于病原菌效应蛋白本身的多样性与不保守性,如果根据这些特征来进行效应蛋白的筛选会存在一定的局限性。大麦白粉病菌（Blumeria graminis f. sp. hordei,Bgh）中的无毒基因Avra10和Avrk1[48]编码的均为非分泌型蛋白,如果根据大部分效应蛋白为分泌蛋白的特征来定义效应蛋白,会丢失一些非分泌型的效应蛋白。本研究中效应蛋白Pt18906信号肽无分泌功能,为少数的非分泌型效应蛋白,在胞内起作用。对于非分泌型效应蛋白是如何运输到胞内起作用的,有待于进一步研究。另外该效应蛋白的三级结构含有较高的无规则卷曲（69.96%）,推测其可能为具有识别功能的、对其他蛋白质特异的功能性蛋白。Pt18906在24 h表达量最高,此时间点为叶锈菌形成吸器母细胞的关键点,也是寄主有效抵抗病原物的一个关键点,因此推测Pt18906可能在叶锈菌与寄主植物侵入过程中被寄主识别。关于效应蛋白发挥功能的结构域,不同的效应蛋白存在差异,但多在N端起作用。在对小麦条锈菌的研究中发现,通过氨基酸缺失的方法检测效应蛋白的毒性区域,其中小麦条锈菌Pst82、Pst148、Pst227、Pst8853和Pst9302成熟蛋白N末端10—20位、23位、20—40位、20—30位和20—40位的氨基酸序列分别对其毒性功能具有重要作用[36]。本研究中,同样通过氨基酸逐步确实法对效应蛋白Pt18906进行功能区域验证,发现在N末端缺失28—47位的20个氨基酸的突变体无抑制坏死功能,表明28—47位的AEVQRHAFSRRGKENDKPPP氨基酸序列对Pt18906发挥功能具有重要作用。
植物的免疫机制主要由PTI和ETI两层防御体系构成。季森等[41]利用细菌三型分泌系统将HASP2在小麦中过表达,发现其可以抑制寄主PTI相关胼胝质的积累,同时对HASP2过表达的小麦接种无毒性菌系CYR23后,发现HASP2可以抑制寄主ETI相关活性氧积累和减少细胞坏死面积,推测小麦条锈菌效应蛋白HASP2抑制寄主的免疫反应;王力坤等[42]研究表明,小麦条锈菌效应子Pst30抑制植物的胼胝质和活性氧积累,推测Pst30可抑制寄主植物的PTI和ETI,从而促进自身的侵染;陈增菊等[43]研究发现,小麦条锈菌效应蛋白Hasp58抑制荧光假单胞菌引起的胼胝质积累和活性氧的迸发,推测Hasp58同样可以抑制寄主植物的PTI和ETI,并促进自身的侵染。本试验通过细菌三型分泌系统在TcLr27+31上表达效应蛋白Pt18906,发现Pt18906激发了TcLr27+31胼胝质的积累,且该效应蛋白同样可造成活性氧的积累,进而造成O2-的迸发,说明Pt18906触发了寄主的防御反应PTI和ETI,表明近等基因系TcLr27+31在抵抗小麦叶锈菌的侵染过程中行使多种防御系统。另外,胼胝质积累随效应因子激发时间的延长而增加,由图8可见注射效应因子72 h的积累量显著高于12 h的积累量。而活性氧迸发较胼胝质积累更早发生,是因为在防御反应PTI的过程中也会有活性氧的产生,并且活性氧是体内一类氧的单电子还原产物,是电子在未能传递到末端氧化酶之前漏出呼吸链并消耗大约2%的氧生成的,包括氧的电子还原产物超氧阴离子（O2-）、H2O2、羟基自由基（·OH）以及NO等。文中使用的NBT是用来检测O2-的,O2-极其不稳定,会与H2O结合形成H2O2,在小麦上表达效应蛋白5 d后观察到H2O2（图7）。
在小麦条锈菌效应蛋白的研究中发现PstGSRE1定位于细胞质和细胞核,在胞内起作用,能够抑制寄主活性氧积累,小麦锌指结构转录因子TaLOL2是其靶标蛋白,TaLOL2能够促进小麦活性氧积累,是小麦抗条锈病的正向调控因子,而PstGSRE1通过干扰并阻止TaLOL2进核,抑制寄主免疫反应从而利于病菌的侵染[44]。本研究通过亚细胞定位分析发现Pt18906在细胞内起作用,但其作用靶标及作用方式还有待于进一步研究。
SEGOVIA等[47]发现Pt3和Pt27可分别在含有抗性基因Lr9或Lr24,Lr26的近等基因系表现出对GUS的抑制作用,推测具有对Lr9或Lr24,Lr26的无毒性。本研究发现,效应蛋白Pt18906可促进寄主细胞中胼胝质的积累,在近等基因系TcLr27+31上引起活性氧的迸发,并激发坏死反应,表明Pt18906在TcLr27+31上具有无毒作用,并能够触发寄主TcLr27+31的防御反应PTI和ETI。本研究利用全套的抗叶锈病近等基因系进行效应蛋白相应功能的分析,发现该效应蛋白能够在TcLr27+31和TcLr42上诱导产生坏死反应,在其他测试的进等基因系上无诱导坏死反应,而且缺少近等基因系TcLr27和TcLr31,因此,说明该效应蛋白与Lr27或Lr31,Lr42上发生了直接或间接的作用,但不能确定该基因对应的抗病基因是Lr27或Lr31。本研究只涉及了Pt18906在TcLr27+31上引起的胼胝质以及活性氧情况,而在TcLr42上未进行相关试验,有待于后续完成。
4 结论
效应蛋白Pt18906是一个具有223个氨基酸的小分子蛋白,作用于细胞内,信号肽不具有分泌活性,在与小麦互作的初期发生显著的上调反应。能够有效抑制Bax和INF1激发的细胞程序性死亡,其28—47位氨基酸对其毒性功能具有重要作用。能够激发小麦TcLr27+31胼胝质的积累和活性氧的迸发,TcLr27+31对Pt18906表现免疫反应。参考文献 原文顺序
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被引期刊影响因子
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DOI:10.1046/j.1365-313x.2003.01937.xURLPMID:14675431 [本文引用: 1]
The Lycopersicon esculentum Bs4 resistance (R) gene specifies recognition of Xanthomonas campestris pv. vesicatoria (Xcv) strains that express the cognate AvrBs4 avirulence protein. Bs4 was isolated by positional cloning and is predicted to encode a nucleotide-binding leucine-rich repeat (NB-LRR) protein that is homologous to tobacco N and potato Y-1 resistance proteins. Xcv infection tests demonstrate that Bs4 confers perception of AvrBs4 but not the 97% identical AvrBs3 protein. However, when delivered via Agrobacterium T-DNA transfer, both, avrBs4 and avrBs3 trigger a Bs4-dependent hypersensitive response, indicating that naturally occurring AvrBs3-homologues provide a unique experimental platform for molecular dissection of recognition specificity. Transcript studies revealed intron retention in Bs4 transcripts. Yet, an intron-deprived Bs4 derivative still mediates AvrBs4 detection, suggesting that the identified splice variants are not crucial to resistance. The L. pennellii bs4 allele, which is >98% identical to L. esculentum Bs4, has a Bs4-like exon-intron structure with exception of a splice polymorphism in intron 2 that causes truncation of the predicted bs4 protein. To test if the receptor-ligand model is a valid molecular description of Bs4-mediated AvrBs4 perception, we conducted yeast two-hybrid studies. However, a direct interaction was not observed. Defense signaling of the Bs4-governed reaction was studied in Nicotiana benthamiana by virus-induced gene silencing and showed that Bs4-mediated resistance is EDS1- and SGT1-dependent.
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DOI:10.1111/j.1365-2958.2006.05162.xURLPMID:16677306 [本文引用: 1]
The Pseudomonas syringae pv. glycinea effector protein AvrB induces resistance responses in soybean varieties that contain the resistance gene Rpg1-b and Arabidopsis varieties that carry RPM1. In addition to this avirulence activity, AvrB also enhances bacterial virulence on soybean plants that lack Rpg1-b and induces a chlorotic phenotype on Arabidopsis plants that lack RPM1. We screened a library of avrB mutants for loss of avirulence on soybean and Arabidopsis, and assayed selected avirulence mutants for loss of virulence on both plants. All mutants screened were recognized similarly on both plant species. Nine single-site avrB mutations that affected avirulence localized to a solvent-accessible pocket in the protein structure. Seven of these mutated residues are absolutely conserved between AvrB and its nine homologues. Avirulence mutants generally lost virulence enhancement on susceptible soybean varieties and lost the ability to induce a chlorotic response on the rpm1 null Arabidopsis variety Mt-0. Three of four avirulence mutants tested failed to interact with RIN4, an Arabidopsis protein previously shown to be required for RPM1 function. Our results suggest that soybean and Arabidopsis recognize AvrB in the same manner, and that AvrB enzymatic activity is required for its function as an avirulence and virulence effector on two different plant species.
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DOI:10.1073/pnas.0909705107URLPMID:20018686 [本文引用: 1]
Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucine-rich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr(237) of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling.
DOI:10.1016/j.chom.2014.10.007URLPMID:25525792 [本文引用: 1]
Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.
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DOI:10.1111/nph.13794URLPMID:26680733 [本文引用: 1]
Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.
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DOI:10.1111/j.1558-5646.1991.tb02667.xURLPMID:28564135 [本文引用: 1]
Spatial variation in the resistance structure of Linum marginale (wild flax) populations to the rust fungus Melampsora lini, and in the racial structure of this pathogen, was investigated by sampling 10 populations distributed throughout the Kosciusko National Park, New South Wales, Australia. Considerable differences were found among populations in the structure of both host and pathogen. Host populations were divided into three broad categories: (1) populations susceptible to all testing races; (2) populations containing a strictly limited number of resistant phenotypes; and (3) populations with a considerable diversity of resistant phenotypes. The pathogen populations also showed a range of diversity. The major differences between these populations were determined by the occurrence and frequency of four common races of pathogen (races A, E, K, and N). These differences were apparent both at a regional spatial scale (over the 100 km separation of the most distant populations) and at a local scale where major differences were detected between two populations only 300 m apart. The distribution of the four common races of M. lini was consistent with the hypothesis that a fitness cost was associated with unnecessary virulence. In general, however, differences in the structure of pathogen populations from genetically very similar host populations implied that, in addition to host resistance genes, other evolutionary forces are also important in determining the genetic structure of individual pathogen populations.
DOI:10.1073/pnas.1019315108URLPMID:21536894 [本文引用: 3]
Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.
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DOI:10.1126/science.1236381URLPMID:23765277 [本文引用: 1]
Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important alpha-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.
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DOI:10.1534/g3.116.032797URLPMID:27913634 [本文引用: 2]
Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.
DOI:10.1094/MPMI-23-1-0049URLPMID:19958138 [本文引用: 1]
In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M-AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes.
DOI:10.1105/tpc.020040URLPMID:14973158 [本文引用: 1]
The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus. At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M. lini map to eight independent loci, some of which are complex and encode multiple specificities. We identified an M. lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes. Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C. Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L. usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene. An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L. usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7. The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins. Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane. Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.
DOI:10.1038/nrmicro3119URL [本文引用: 1]
Live-cell imaging assisted by fluorescent markers has been fundamental to understanding the focused secretory 'warfare' that occurs between plants and biotrophic pathogens that feed on living plant cells. Pathogens succeed through the spatiotemporal deployment of a remarkably diverse range of effector proteins to control plant defences and cellular processes. Some effectors can be secreted by appressoria even before host penetration, many enter living plant cells where they target diverse subcellular compartments and others move into neighbouring cells to prepare them before invasion. This Review summarizes the latest advances in our understanding of the cell biology of biotrophic interactions between plants and their eukaryotic filamentous pathogens based on in planta analyses of effectors.
DOI:10.1098/rspb.2009.0328URLPMID:19457888 [本文引用: 1]
Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.
DOI:10.1186/s12864-016-3011-9URL [本文引用: 2]
DOI:10.1080/12265071.2002.9647663URL [本文引用: 1]
DOI:10.3389/fpls.2014.00416URLPMID:25191335 [本文引用: 1]
Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.
DOI:10.1126/science.aao7294URLPMID:29269474 [本文引用: 1]
Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating fungal disease. The Sr35 resistance gene confers immunity against this pathogen's most virulent races, including Ug99. We used comparative whole-genome sequencing of chemically mutagenized and natural Pgt isolates to identify a fungal gene named AvrSr35 that is required for Sr35 avirulence. The AvrSr35 gene encodes a secreted protein capable of interacting with Sr35 and triggering the immune response. We show that the origin of Pgt isolates virulent on Sr35 is associated with the nonfunctionalization of the AvrSr35 gene by the insertion of a mobile element. The discovery of AvrSr35 provides a new tool for Pgt surveillance, identification of host susceptibility targets, and characterization of the molecular determinants of immunity in wheat.
DOI:10.1126/science.aao4810URLPMID:29269475 [本文引用: 1]
Race-specific resistance genes protect the global wheat crop from stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in Pgt, we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of Pgt virulent to Sr50 contained a 2.5 mega-base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers Sr50-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt.
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DOI:10.1111/1462-2920.13610URLPMID:27871149 [本文引用: 1]
During the infection of host plants, pathogens can deliver virulence-associated 'effector' proteins to promote plant susceptibility. However, little is known about effector function in the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici (Pst) that is an important fungal pathogen in wheat production worldwide. Here, they report their findings on an in planta highly induced candidate effector from Pst, PSTha5a23. The PSTha5a23 gene is unique to Pst and shows a low level of intra-species polymorphism. It has a functional N-terminal signal peptide and is translocated to the host cytoplasm after infection. Overexpression of PSTha5a23 in Nicotiana benthamiana was found to suppress the programmed cell death triggered by BAX, PAMP-INF1 and two resistance-related mitogen-activated protein kinases (MKK1 and NPK1). Overexpression of PSTha5a23 in wheat also suppressed pattern-triggered immunity (PTI)-associated callose deposition. In addition, silencing of PSTha5a23 did not change Pst virulence phenotypes; however, overexpression of PSTha5a23 significantly enhanced Pst virulence in wheat. These results indicate that the Pst candidate effector PSTha5a23 plays an important role in plant defense suppression and rust pathogenicity, and also highlight the utility of gene overexpression in plants as a tool for studying effectors from obligate biotrophic pathogens.
DOI:10.1186/1471-2164-14-270URLPMID:23607900 [本文引用: 1]
BACKGROUND: Wheat yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (PST) is one of the most devastating diseases of wheat worldwide. To design effective breeding strategies that maximize the potential for durable disease resistance it is important to understand the molecular basis of PST pathogenicity. In particular, the characterisation of the structure, function and evolutionary dynamics of secreted effector proteins that are detected by host immune receptors can help guide and prioritize breeding efforts. However, to date, our knowledge of the effector repertoire of cereal rust pathogens is limited. RESULTS: We re-sequenced genomes of four PST isolates from the US and UK to identify effector candidates and relate them to their distinct virulence profiles. First, we assessed SNP frequencies between all isolates, with heterokaryotic SNPs being over tenfold more frequent (5.29 +/- 2.23 SNPs/kb) than homokaryotic SNPs (0.41 +/- 0.28 SNPs/kb). Next, we implemented a bioinformatics pipeline to integrate genomics, transcriptomics, and effector-focused annotations to identify and classify effector candidates in PST. RNAseq analysis highlighted transcripts encoding secreted proteins that were significantly enriched in haustoria compared to infected tissue. The expression of 22 candidate effector genes was characterised using qRT-PCR, revealing distinct temporal expression patterns during infection in wheat. Lastly, we identified proteins that displayed non-synonymous substitutions specifically between the two UK isolates PST-87/7 and PST-08/21, which differ in virulence to two wheat varieties. By focusing on polymorphic variants enriched in haustoria, we identified five polymorphic effector candidates between PST-87/7 and PST-08/21 among 2,999 secreted proteins. These allelic variants are now a priority for functional validation as virulence/avirulence effectors in the corresponding wheat varieties. CONCLUSIONS: Integration of genomics, transcriptomics, and effector-directed annotation of PST isolates has enabled us to move beyond the single isolate-directed catalogues of effector proteins and develop a framework for mining effector proteins in closely related isolates and relate these back to their defined virulence profiles. This should ultimately lead to more comprehensive understanding of the PST pathogenesis system, an important first step towards developing more effective surveillance and management strategies for one of the most devastating pathogens of wheat.
DOI:10.1111/nph.16741URLPMID:32542680 [本文引用: 1]
The plant leaf surface is coated with a waterproof cuticle layer. Cuticle facing the stomatal pore surface needs to be sculpted to form outer cuticular ledge (OCL) after stomatal maturation for efficient gas exchange. Here, we characterized the roles of Arabidopsis GDSL lipase, Occlusion of Stomatal Pore 1 (OSP1), in wax biosynthesis and stomatal OCL formation. OSP1 mutation results in significant reduction in leaf wax synthesis and occlusion of stomata, leading to increased epidermal permeability, decreased transpiration rate, and enhanced drought tolerance. We demonstrated that OSP1 activity is critical for its role in wax biosynthesis and stomatal function. In vitro enzymatic assays demonstrated that OSP1 possesses thioesterase activity, particularly on C22:0 and C26:0 acyl-CoAs. Genetic interaction analyses with CER1 (ECERIFERUM 1), CER3 (ECERIFERUM 3) and MAH1 (Mid-chain Alkane Hydroxylase 1) in wax biosynthesis and stomatal OCL formation showed that OSP1 may act upstream of CER3 in wax biosynthesis, and implicate that wax composition percentage changes and keeping ketones in a lower level play roles, at least partially, in forming stomatal ledges. Our findings provided insights into the molecular mechanism mediating wax biosynthesis and highlighted the link between wax biosynthesis and the process of stomatal OCL formation.
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DOI:10.1016/j.molp.2019.09.010URLPMID:31606466 [本文引用: 2]
Puccinia striiformis f. sp. tritici (Pst), a biotrophic plant pathogen, secretes numerous effectors to modulate host defense systems. Understanding the molecular mechanisms by which Pst effectors regulate wheat immunity is of great importance for the development of novel strategies for durable control of stripe rust. In this study, we identified a glycine-serine-rich effector gene, PstGSRE1, which is highly induced during early infection. Transgenic expression of PstGSRE1 RNAi constructs in wheat significantly reduced virulence of Pst and increased H2O2 accumulation in wheat. PstGSRE1 was shown to target the reactive oxygen species (ROS)-associated transcription factor TaLOL2, a positive regulator of wheat immunity. PstGSRE1 disrupted nuclear localization of TaLOL2 and suppressed ROS-mediated cell death induced by TaLOL2, thus compromising host immunity. This work reveals a previously unrecognized strategy whereby rust fungi exploit the PstGSRE1 effector to defeat ROS-associated plant defense by modulating the subcellular compartment of a host immune regulator and facilitate pathogen infection.
DOI:10.1038/s41467-019-13398-6URLPMID:31822676 [本文引用: 1]
Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.
DOI:10.1111/nph.16199URLPMID:31529497 [本文引用: 1]
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a tremendous threat to the production of wheat worldwide. The molecular mechanisms of Pst effectors that regulate wheat immunity are poorly understood. In this study, we identified an effector Pst18363 from Pst that suppresses plant cell death in Nicotiana benthamiana and in wheat. Knocking down Pst18363 expression by virus-mediated host-induced gene silencing significantly decreased the number of rust pustules, indicating that Pst18363 functions as an important pathogenicity factor in Pst. Pst18363 was proven to interact with wheat Nudix hydrolase 23 TaNUDX23. In wheat, silencing of TaNUDX23 by virus-induced gene silencing increased reactive oxygen species (ROS) accumulation induced by the avirulent Pst race CYR23, whereas overexpression of TaNUDX23 suppressed ROS accumulation induced by flg22 in Arabidopsis. In addition, TaNUDX23 suppressed Pst candidate effector Pst322-trigged cell death by decreasing ROS accumulation in N. benthamiana. Knocking down of TaNUDX23 expression attenuated Pst infection, indicating that TaNUDX23 is a negative regulator of defence. In N. benthamiana, Pst18363 stabilises TaNUDX23. Overall, our data suggest that Pst18363 stabilises TaNUDX23, which suppresses ROS accumulation to facilitate Pst infection.
DOI:10.1080/07060661.2016.1150884URL [本文引用: 2]
DOI:10.1105/tpc.106.043307URLPMID:16905653 [本文引用: 1]
Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.
