,, 程光远, 杨宗桃, 王彤, 刘淑娴, 商贺阳, 赵贺, 徐景升,*福建农林大学国家甘蔗工程技术研究中心 / 农业农村部福建甘蔗生物学与遗传育种重点实验室 / 教育部作物遗传育种与综合利用重点实验室, 福建福州 350002Cloning of sugarcane ScCRT1 gene and its response to SCMV infection
ZHANG Hai,, CHENG Guang-Yuan, YANG Zong-Tao, WANG Tong, LIU Shu-Xian, SHANG He-Yang, ZHAO He, XU Jing-Sheng,*National Engineering Research Center for Sugarcane / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China通讯作者:
收稿日期:2020-07-14接受日期:2020-09-13网络出版日期:2021-01-12
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Received:2020-07-14Accepted:2020-09-13Online:2021-01-12
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张海, 程光远, 杨宗桃, 王彤, 刘淑娴, 商贺阳, 赵贺, 徐景升. 甘蔗ScCRT1基因克隆及其应答SCMV侵染分子机制的研究[J]. 作物学报, 2021, 47(1): 94-103. doi:10.3724/SP.J.1006.2021.04156
ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, WANG Tong, LIU Shu-Xian, SHANG He-Yang, ZHAO He, XU Jing-Sheng.
钙网蛋白(calreticulin, CRT)在真核生物中高度保守并广泛表达[1,2,3,4], 主要定位于内质网(endoplasmic reticulum, ER)和胞间连丝(plasmodesmata, PD), CRT是内质网中主要的Ca2+结合蛋白, 具有分子伴侣功能[5,6,7,8]。Ca2+是细胞内重要的第二信使, 在细胞信号转导中具有重要作用[5,9]。因此, CRT参与调控Ca2+稳态、信号转导、内质网质控(endoplasmic reticulum quality control, ERQC)、生长发育和免疫应答等多种生物学过程[2-4,10-12]。CRT在哺乳动物中的研究较为深入, 该蛋白除了上述基本生物功能外, 还参与了癌症等疾病的发生[13,14]。植物中CRT的研究相对滞后[15], 研究表明CRT广泛应答生物和非生物胁迫, 参与干旱、低温、盐胁迫、真菌、细菌、病毒的侵染等逆境胁迫反应[2,4,8,16-21]。
甘蔗(Saccharum spp. hybrid)是我国和世界上最重要的糖料作物和能源作物[22,23,24]。甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)属于马铃薯Y病毒科(Potyviridae) Y病毒属(Potyvirus), 是甘蔗花叶病的主要病原之一, 严重危害甘蔗生产[25,26,27,28]。SCMV为单链正义RNA病毒, 长度约为10 kb, 编码2个多聚蛋白, 经自身编码的蛋白酶裂解后形成11个成熟的功能蛋白, 从N端至C端分别为P1、HC-Pro、P3、P3N-PIPO、6K1、CI、6K2、VPg、NIa、NIb和CP蛋白[29,30,31,32]。其中, 6K2蛋白参与病毒的复制、胞内与胞间移动, 在马铃薯Y病毒属病毒建立系统性侵染过程中起着至关重要的作用[33,34,35,36,37]。
本课题组在前期研究中, 以SCMV编码的6K2蛋白为诱饵, 从甘蔗品种ROC22叶片cDNA酵母文库中筛选到了具有完整开放读码框(open reading frame, ORF)的CRT基因, 其长度为1281 bp。在本研究中, 我们进一步从Badila叶片中克隆了该基因, 命名为ScCRT1。本研究利用RT-qPCR分析了ScCRT1基因在甘蔗不同组织中的表达特异性以及应答SCMV侵染的表达情况; 利用酵母双杂交(yeast 2 hybrid, Y2H)和双分子互补(bimolecular fluorescence complementation, BiFC)技术验证了ScCRT1蛋白与SCMV-6K2的互作关系; 利用瞬时表达试验, 研究了ScCRT的亚细胞定位。本研究为研究SCMV侵染甘蔗的分子机制提供了实验证据, 并为甘蔗抗花叶病育种提供了基础数据和实验依据。
1 材料与方法
1.1 材料及处理方法
SCMV病毒、甘蔗品种Badila组培苗、本氏烟(Nicotiana benthamiana)苗由福建农林大学农业农村部福建甘蔗生物学与遗传育种重点实验室提供。2019年11月, 待组培苗长至15~25 cm、完全展开叶出现4~5片时, 摩擦接种SCMV, 设置3个重复, 每个重复5株, 对照植株使用磷酸缓冲液(pH 7.0)摩擦接种, 使用SCMV-CP基因特异引物(表1)检测接种是否成功。分别在接种后0 h、4 h、8 h、12 h、18 h、1 d、2 d、3 d、4 d取样, 研究目的基因应答SCMV侵染的表达模式。在隔离网室内随机选取9株处于伸长期且长势一致健康的Badila植株, 分成3组, 每组3株。取其白色嫩根、心叶(未展开的叶)、正一叶(甘蔗最高可见肥厚带的第一叶)、正四叶、第四节间、第八节间, 用于研究目的基因的组织表达特性。样品采集后用液氮速冻, 置于-80℃冰箱保存备用。Table 1
表1
表1本研究使用的引物
Table 1
| 引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 用途 Strategy |
|---|---|---|
| ScCRT1-F | ATGGCGATCCTCGAGAGG | 基因克隆 |
| ScCRT1-R | CTAGAGCTCATCATGTTTA | Gene cloning |
| BD-ScCRT1-F | ATTAACAAGGCCATTACGGCCATGGCGATCCTCGAGAGGTC | 酵母双杂交诱饵载体构建 |
| BD-ScCRT1-R | AACTGATTGGCCGAGGCGGCCCCGAGCTCATCATGTTTAGCATC | Vector generation for Y2H |
| 221-ScCRT1-F | GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGATCCTCGAGAGGTC | 亚细胞定位 |
| 221-ScCRT1-R | GGGGACCACTTTGTACAAGAAAGCTGGGTCGAGCTCATCATGTTTAGCAT | Subcellular localization |
| ScCRT1-qF | CGCCAAGAAGTTAGCAGAGGAGAC | 定量PCR |
| ScCRT1-qR | CCTTGTCATCGTCCGCATCATCC | Real-time-qPCR |
| GAPDH-F | CACGGCCACTGGAAGCA | 内参基因 |
| GAPDH-R | TCCTCAG GGTTCCTGATGCC | Reference gene |
| eEF-1α-F | TTTCACACTTGGAGTGAAGCAGAT | 内参基因 |
| eEF-1α-R | GACTTCCTTCACAATCTCATCATAA | Reference gene |
| SCMV-CP-F | TACAGAGAGACACACAGCTG | SCMV检测 |
| SCMV-CP-R | ACGCTACACCAGAAGACACT | Detection of SCMV |
新窗口打开|下载CSV
1.2 总RNA提取和cDNA合成
使用TRIzol (Invotrigen, USA)试剂, 按照说明书要求提取样品总RNA, 用1.5%琼脂糖凝胶电泳检测RNA质量。使用Nanodrop (Thermo Scientific, USA)测定RNA的浓度, 按照Prime Script RT Reagent Kit使用说明书, 将RNA反转录成cDNA。1.3 ScCRT1基因的克隆及生物信息学分析
以筛选酵母文库获得的CRT基因序列为参考, 通过同源克隆的方法, 在起始密码子和终止密码子附近设计ScCRT1基因的特异扩增引物(表1)。以反转录获得的cDNA为模板, 使用PrimeSTAR GXL DNA polymerase (TaKaRa Bio, Japan)高保真酶, 参照Shinohara等[38]的方法克隆ScCRT1基因, 并送生工生物工程(上海)股份有限公司测序。将测序获得目的基因序列, 利用NCBI中的ORF finder (
1.4 ScCRT1的RT-qPCR表达分析
根据ScCRT1基因的ORF序列, 设计特异性实时荧光定量PCR引物(表1), 以GAPDH基因和eEF-1α 基因(表1)作为内参基因[39]。按照SYBR Green PCR Master Mix (Roche)说明书配制定量反应体系, 在荧光定量PCR仪上完成定量PCR扩增。每个样品设置3次技术重复, 以无菌水作对照, 扩增程序为50℃ 2 min, 95℃ 10 min; 95℃ 15 s, 60℃ 1 min, 40个循环。反应结束后, 采用2-ΔΔCt算法分析获得的数据[40]。1.5 ScCRT1的亚细胞定位
参照Cheng等[29]的方法, 利用Gateway方法将ScCRT1构建到pEarlyGate101载体中, 获得ScCRT1亚细胞定位质粒ScCRT1-YFP并转入农杆菌EHA105中。取健康的本氏烟植株, 参照Cheng等[29]农杆菌侵染方法, 将带有重组质粒的农杆菌注射入健康的本氏烟叶片。48 h后在激光共聚焦显微镜(Leica TCS SP5II)下观察本氏烟叶片表皮细胞中ScCRT1蛋白的定位, YFP的激发光波长为514 nm, 捕获波长为530~590 nm。1.6 Y2H验证ScCRT1与SCMV-6K2的互作
参照Zhang等[34]的方法构建ScCRT1基因的诱饵载体pBT3-SET-ScCRT1, 利用Y2H技术验证其与pPR3-N-SCMV-6K2的互作关系。pTSU2-APP和pNubG-Fe65组合作为阳性对照, pNubG-Fe65和pPR3-N组合作为阴性对照, pBT3-SET-ScCRT1和pPR3-N组合为自激活验证。1.7 BiFC验证ScCRT1与SCMV-6K2的互作
参照Cheng等[29]的方法构建ScCRT1-YC载体, 利用BiFC技术验证其与YN-SCMV-6K2的互作。YFP 的激发光波长为514 nm, 捕获波长为530~590 nm; 叶绿素自荧光的激发光波长为552 nm, 采集波长为650~680 nm。2 结果与分析
2.1 ScCRT1基因的克隆与生物信息学分析
经PCR扩增和测序, 本研究从Badila中克隆了1条ORF长度为1281 bp的CRT基因, 该基因编码426个氨基酸。核苷酸序列比对表明, 该CRT序列与高粱的CRT1 (Sorghum bicolor, XM_021451918.1)同源性高达96.49%, 将该基因命名为ScCRT1, 并提交GenBank, 登录号为MT635395。ProtParam分析表明, ScCRT1的分子量为48.22 kD, 等电点为4.43; 不稳定系数为35.67, 为稳定蛋白; 脂溶指数为63.29, 总平均亲水性是-1.023, 可能是亲水性蛋白。GOR4预测表明, ScCRT1中无规则卷曲占比最高, 达到60.09%; 其次是延伸链和α螺旋, 占比分别为20.19%和19.72%; 无β-折叠结构。跨膜结构域预测结果表明, ScCRT1具有1个跨膜结构域, 其分布在13~35, 膜向性为i→o。SignalP 5.0分析表明,ScCRT1蛋白含有信号肽, 且信号肽的剪切位点位于第31和32号氨基酸之间(图1), 为分泌蛋白。图1
新窗口打开|下载原图ZIP|生成PPT图1甘蔗钙网蛋白(ScCRT1)氨基酸序列比对与结构域
高粱: SbCRT1 (XP_021307591.1); 玉米: ZmCRT1 (XP_008670080.1); 谷子: SiCRT1 (XP_004981159.1); 二穗短柄草: BdCRT1 (XP_003563166.1); 水稻: OsCRT1 (XP_015628738.1); 拟南芥: AtCRT1 (NP_176030.1)。黑色背景中的蛋白残基为相同的部分。箭头表示3个结构域(N, P和C)的起始位置。预测的信号肽、保守的CRT家族基序(CRT motif 1: KLDCGGGYVKLL; CRT motif 2: IMFGPDICG)、3个重复的残基(A: PXXIXDPXX KKPEXWDD; B: GXWXAXXIXNPXYK, X为任意氨基酸)[1]和内质网滞留信号HDEL用黑色下划线标记。假定的核靶向序列(PPKKIKDPE)用红色下画线标记。
Fig. 1Amino acid sequence alignment and domains of Saccharum calreticulin (ScCRT1)
Sorghum bicolor: SbCRT1 (XP_021307591.1); Zea mays: ZmCRT1 (XP_008670080.1); Setaria italic: SiCRT1 (XP_004981159.1); Brachypodium distachyon: BdCRT1 (XP_003563166.1); Oryza sativa: OsCRT1 (XP_015628738.1); Arabidopsis thaliana: AtCRT1 (NP_176030.1). Protein residues on the black background in all of these proteins are identical. The arrows indicate the approximate positions of the three domains (N, P and C). The predicted signal peptide, conserved CRT family motifs (CRT motif 1: KHEQKLDCGGGYVKLL; CRT motif 2: IMFGPDICG), triplicate repeats (A: PXXIXDPXX KKPEXWDD; B: GXWXAXXIXNPXYK, X represents arbitrary residue) [1] and the endoplasmic reticulum retention sequence HDEL was underlined in black. The putative nuclear targeting sequence (PPKKIKDPE) was underlined in red.
2.2 ScCRT1的氨基酸序列同源性分析和系统进化树分析
典型的CRT蛋白含有3个不同的结构和功能域: 高度保守的球状N-结构域, 其末端含有一个信号肽序列; P-结构域, 具有高亲和力和低Ca2+结合能力; C-结构域表现出低亲和力和高的Ca2+结合能力, 其末端包含有(K/H)DEL内质网滞留信号[1,2]。在ScCRT1氨基酸中发现具有上述典型的CRT的3个结构域组成, 残基1~31的疏水信号肽序列, 残基32~216的球形N-结构域, 残基217~313的富含脯氨酸P-结构域, 残基314~426的多酸性氨基酸C-结构域并含有内质网滞留信号HDEL基序(图1)。通过NCBI网站的 Blastp 和 Phytozome 网站的 Proteome Blastp 搜索ScCRT1同源序列, 结果显示, ScCRT1蛋白与高粱(XP_021307591.1)、玉米(Zea mays, XP_008670080.1)、二穗短柄草(Brachypodium distachyon, XP_003563166.1)、谷子(Setaria italic, XP_004981159.1)、水稻(Oryza sativa, XP_01562 8738.1)和拟南芥(Arabidopsis thaliana, NP_176030.1)的CRT蛋白相似度分别为96%、90%、92%、86%、78%、72%。对甘蔗及其他物种CRT蛋白的氨基酸序列同源性分析表明, 在其N-结构域和P-结构域氨基酸序列高度保守, 信号肽序列和C-结构域氨基酸序列因物种的不同表现出明显的差异(图1)。使用ClustalX和MEGA 7.0的ML (maximum likelihood, LG+G)法构建系统进化树, 分析ScCRT1蛋白与其他物种CRT蛋白的进化关系。结果表明, 植物CRTs蛋白主要分为2种亚型: CRT1/CRT2亚型和CRT3亚型。在拟南芥中CRT含有AtCRT1 (AT1G56340.2)、AtCRT2 (AT1G09210.1)和AtCRT3 (AT1G08450.1) 3种蛋白, 包括2个蛋白亚型; 其中AtCRT1与AtCRT2相似性很高, 为CRT1/CRT2亚型; AtCRT3为CRT3亚型, 该亚型与CRT1/CRT2亚型存在着较大差异[41,42]。基于进化树分析, 我们克隆的ScCRT1蛋白属于CRT1/CRT2亚型(图2)。
图2
新窗口打开|下载原图ZIP|生成PPT图2ScCRT1与其他物种CRT 蛋白的系统进化树分析
Fig. 2Phylogenetic tree analysis of ScCRT1 protein and CRT proteins from other plant species
单子叶植物甘蔗、高粱、柳枝稷(Panicum virgatum)、水稻、谷子、大麦(Hordeum vulgare)、二穗短柄草形成群I; 双子叶植物烟草(Nicotiana tabacum)、葡萄(Vitis vinifera)、三叶杨(Populus trichocarpa)、大豆(Glycine max)、苜蓿(Medicago sativa)、拟南芥形成群II (图2)。在遗传进化上, CRT蛋白在单子叶植物和双子叶植物之间存在明显的分化。
2.3 ScCRT1的亚细胞定位
在激光共聚焦显微镜下观察烟草瞬时表达ScCRT1-YFP融合蛋白的黄色荧光在细胞内的分布(图3); ScCRT1-YFP融合蛋白的黄色荧光信号为典型的内质网定位信号, 表明其主要定位于内质网。图3
新窗口打开|下载原图ZIP|生成PPT图3ScCRT1-YFP在本氏烟表皮细胞中的定位
A: ScCRT1-YFP荧光信号; B: 明场; C: 合并。标尺为 25 μm。
Fig. 3Subcellular localization of ScCRT1 fused with YFP in the epidermal cells of N. benthamiana
A: fluorescent signal of ScCRT1-YFP; B: bright field; C: merged pictures. Bar = 25 μm.
2.4 ScCRT1基因的组织特异性表达及应答SCMV侵染的表达模式
基于RT-qPCR技术, 以根中ScCRT1基因的表达量为参照基准, 采用2-ΔΔCt法对甘蔗各组织ScCRT1基因表达量的分析结果(图4)表明, ScCRT1基因的表达具有明显的组织特异性, 在不同组织中的表达量差异较显著, 其在心叶中的相对表达量最高, 根和正一叶次之, 第八节间中表达量最少。使用SCMV-CP基因特异引物(表1)检测SCMV接种的甘蔗, 选出扩增出目的片段的样品, 进行ScCRT1基因应答SCMV侵染的RT-qPCR试验。结果显示SCMV 的侵染对ScCRT1基因表达的影响显著, 与对照相比, ScCRT1基因在侵染早期显著上调, 在12 h时表达量达到最大值, 然后下调表达, 但仍显著高于对照(图5)。图4
新窗口打开|下载原图ZIP|生成PPT图4ScCRT1基因在甘蔗不同组织中的表达模式
误差线为每组处理的标准误差(n = 3)。Root: 根; Leaf roll: 心叶; 1st leaf: 正一叶; 4th leaf: 正四叶; 4th internode: 第四节间; 8th internode: 第八节间。柱上不同的小写字母表示在P < 0.05时显著性的差异。
Fig. 4Expression profile of ScCRT1 in different sugarcane tissues
The error bars represent the standard error of each treatment group (n = 3). Bars superscripted by different lowercase letters are significantly different at P < 0.05.
图5
新窗口打开|下载原图ZIP|生成PPT图5 ScCRT1基因应答SCMV侵染的表达模式
误差线为每组处理的标准误差(n = 3)。柱上不同的小写字母表示在P < 0.05时显著性的差异。
Fig. 5Expression profile of ScCRT1 under the infection of SCMV
The error bars represent the standard error of each treatment group (n = 3). Bars superscripted by different lowercase letters are significantly different at P < 0.05.
2.5 ScCRT1与SCMV-6K2的互作验证
Y2H试验结果显示, pBT3-SET-ScCRT1和pPR3-N共转酵母细胞NMY51和阴性对照在添加了5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-Gal)的SD/-Leu/- Trp (DDO)培养基上生长, 但在添加了X-Gal的SD/- Leu/-Trp/-His/-Ade (QDO)培养基上不生长; pBT3- SET-ScCRT1和pPR3-N-SCMV-6K2共转的酵母菌株NMY51和阳性对照在添加了X-Gal的DDO和QDO培养基上可以生长并呈现蓝色(图6)。表明pBT3- SET-ScCRT1不具有自激活性; ScCRT1与SCMV- 6K2蛋白在体外互作。图6
新窗口打开|下载原图ZIP|生成PPT图6Y2H检测ScCRT1与SCMV-6K2的互作
pNubG-Fe65和pTSU2-APP组合作为阳性对照, pNubG-Fe65和pPR3-N组合作为阴性对照。DDO+X-Gal: 添加了5-溴-4-氯-3-吲哚-β-D-半乳糖苷的缺少亮氨酸(Leu)和色氨酸(Trp)的酵母合成限定基本培养基; QDO+X-Gal: 添加了X-Gal的缺少亮氨酸(Leu)、色氨酸(Trp)、组氨酸(His)和腺嘌呤(Ade)的酵母合成限定基本培养基。
Fig. 6Protein-protein interactions between ScCRT1 and SCMV-6K2 by Y2H assay
The positive and negative controls are yeast cotransformants with plus pNubG-Fe65 pTSU2-APP and pNubG-Fe65 plus pPR3-N, respectively. DDO+X-Gal: synthetic defined yeast minimal medium lacking Leu and Trp but plus the 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside; QDO+X-Gal: synthetic defined yeast minimal medium lacking Leu, Trp, His, and Ade but plus the X-Gal.
BiFC试验结果表明, 共注射的YN-6K2和ScCRT1-YC在本氏烟叶片表皮细胞中产生黄色荧光信号(图7), 说明ScCRT1与SCMV-6K2互作, 这与Y2H试验结果一致, 进一步表明ScCRT1与SCMV-6K2互作。
图7
新窗口打开|下载原图ZIP|生成PPT图7BiFC检测ScCRT1与SCMV-6K2的互作
ScCRT1融合于YFP的C末端, SCMV-6K2融合于YFP的N末端, 在本氏烟叶片中瞬时表达, 48 h后激光共聚焦观察(标尺为25 μm)。
Fig. 7Interactions between ScCRT1 and SCMV-6K2 by BiFC assay
ScCRT1 was fused to the C-terminal half of YFP, while SCMV-6K2 was fused to the N-terminal half of YFP. ScCRT1-YC and YN- SCMV-6K2 were transiently co-expressed in the epidermal cells of N. benthamiana. The fluorescent signal was monitored by confocal microscopy at 48 hours post inoculation (Bar = 25 μm).
3 讨论
尽管病毒与寄主相融, 但是病毒侵染仍然会对寄主造成胁迫, 导致寄主在转录组水平对基因表达进行重新编程, 产生大量差异表达基因[43,44,45,46]。本课题组前期研究表明, 甘蔗花叶病另外1个主要病原甘蔗条纹花叶病毒(Sugarcane streak mosaic virus, SCSMV, Poacevirus)侵染甘蔗品种FN40导致CRT基因表达上调[45]。本研究中, ScCRT1基因在SCMV侵染早期表达上调, 随着时间加长逐渐下降, 但仍显著高于对照。该结果与番木瓜PaCRT基因应答番木瓜环斑花叶病毒(Papaya ringspot virus, PRSV, Potyvirus)侵染的表达模式相似[21]。这些研究结果表明, CRT应答病毒侵染的分子机制可能相同, 这也与CRT蛋白结构保守的特性相符。ScCRT1主要定位在内质网中(图3)。定位于内质网的CRT参与了Ca2+稳态调控和内质网质控, 对于Ca2+依赖的信号传导和新合成蛋白的正确折叠具有重要作用[9,10]。病毒侵染会造成内质网胁迫, 影响蛋白的正确折叠[45,47]。因此我们推测SCMV侵染甘蔗也会造成内质网胁迫, 干扰ScCRT1作为分子伴侣协助蛋白折叠的功能, 为此寄主上调ScCRT1表达以应答胁迫。植物病毒与寄主长期协同进化[48]且结构简单, 必须与寄主因子互作才能建立系统性侵染[49]。本研究利用Y2H和BiFC试验证明了SCMV-6K2与ScCRT的互作(图6和图7), 互作位于内质网和细胞膜, 并且在细胞膜上出现点状结构。我们推测细胞膜上的互作位点有可能位于胞间连丝, 这种互作可能在侵染早期阻碍SCMV胞间移动。马铃薯Y病毒的胞间移动是以6K2诱导内质网形成的囊泡的形式进行的[36,37], SCMV-6K2与ScCRT1互作于胞间连丝, 可能由于CRT的Ca2+调节功能, 导致胞间连丝位置Ca2+浓度升高, 激活胼胝质合成酶Cals [50], 促进胼胝质合成积累, 进而减小胞间连丝的通透性。在烟草应答烟草花叶病毒(Tobacco mosaic virus, TMV, Tobamovirus)侵染的研究中, 也有类似研究报道。烟草的NtCRT与TMV的运动蛋白互作, 过表达NtCRT严重影响TMV的胞间移动[8]。在非生物逆境铝离子胁迫下, 小麦、烟草和苜蓿的CRT上调表达, 与胼胝质共定位沉积于胞间连丝, 阻碍共质体运输[51,52]。这些研究结果表明, CRT参与调节胞间连丝的通透性。Badila极易感染SCMV, 在侵染后期, ScCRT1的表达量下调, 推测胞间连丝胼胝质积累下降, 通透性增加, 便于SCMV胞间移动, 最终建立系统性侵染。
病毒在寄主体内建立系统性侵染是一个动态的生物学过程, 6K2不但参与病毒的复制和胞间移动, 还参与了自噬调节[53], 6K2除了与病毒自身编码蛋白CI、P3互作[36,54-55]互作外, 还与很多寄主编码蛋白互作[34]。而CRT可能还与病毒编码的其他蛋白互作, 比如番木瓜的PaCRT与番木瓜环斑病毒(Papaya ring spot virus, PRSV, Potyvirus)的HC-Pro互作[21]。因此, 阐明ScCRT1在SCMV侵染中的作用还需要进一步深入研究, 如Ca2+浓度的变化, 过表达或抑制ScCRT1的表达对SCMV的复制和胞间移动的影响等。
4 结论
从甘蔗叶片中克隆到钙网蛋白基因ScCRT1, GenBank登录号为MT635395。ScCRT1的ORF长度为1281 bp, 编码426 aa。亚细胞定位试验表明, ScCRT1蛋白定位于内质网; RT-qPCR分析显示, 该基因在心叶等幼嫩组织中表达量较高; 在SCMV侵染条件下, ScCRT1基因的表达呈先上调后下调的表达模式; Y2H与BiFC试验表明, ScCRT1与SCMV-6K2蛋白互作。参考文献 原文顺序
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URLPMID:10567207 [本文引用: 4]
The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology.
URLPMID:18349049 [本文引用: 4]
DOI:10.4161/psb.20721URLPMID:22827946 [本文引用: 1]
Since its initial discovery as a high affinity Ca ( 2+) -binding protein in the sarcoplasmic reticulum and endoplasmic reticulum (ER), calreticulin (CRT) has been documented to be a multifunctional protein in both animal and plant cells. This protein is well recognized as a Ca ( 2+) -binding molecular chaperone that facilitates the folding of newly synthesized glycoproteins and regulates the Ca ( 2+) homeostasis in the ER lumen. However, functional relevance associated with its localization in other cellular compartments has also been reported. Recent studies suggest that both isoforms of plant CRTs (AtCRT1/2 and AtCRT3) are involved in regulating plant defense against biotrophic pathogens. Here we discuss the cellular functions of CRT and its connection to the emerging functions of AtCRTs in plant immunity.
URLPMID:31351124 [本文引用: 3]
DOI:10.1016/s0143416002001884URLPMID:12543089 [本文引用: 2]
The endoplasmic reticulum is a centrally located organelle which affects virtually every cellular function. Its unique luminal environment consists of Ca(2+) binding chaperones, which are involved in protein folding, post-translational modification, Ca(2+) storage and release, and lipid synthesis and metabolism. The environment within the lumen of the endoplasmic reticulum has profound effects on endoplasmic reticulum function and signaling, including apoptosis, stress responses, organogenesis, and transcriptional activity. Calreticulin, a major Ca(2+) binding (storage) chaperone in the endoplasmic reticulum, is a key component of the calreticulin/calnexin cycle which is responsible for the folding of newly synthesized proteins and glycoproteins and for quality control pathways in the endoplasmic reticulum. The function of calreticulin, calnexin and other endoplasmic reticulum proteins is affected by continuous fluctuations in the concentration of Ca(2+) in the endoplasmic reticulum. Thus, changes in Ca(2+) concentration may play a signaling role in the lumen of the endoplasmic reticulum as well as in the cytosol. Recent studies on calreticulin-deficient and transgenic mice have revealed that calreticulin and the endoplasmic reticulum may be upstream regulators in the Ca(2+)-dependent pathways that control cellular differentiation and/or organ development.
DOI:10.1016/j.biocel.2004.02.030URLPMID:15474971 [本文引用: 1]
Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in
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DOI:10.1042/BJ20081847URLPMID:19133842 [本文引用: 2]
Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by calreticulin might be the key to explain its multi-process property.
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DOI:10.2174/1389203718666170111123253URLPMID:28079009 [本文引用: 1]
Calreticulin (CRT), initially identified as a ubiquitous calcium-binding protein in the endoplasmic reticulum, has emerged as a multifunctional protein with roles in calcium homeostasis, molecular chaperoning and cell adhesion. Emerging evidence suggests its involvement in tumorigenesis facilitating proliferation, migration, and adhesion. CRT translocated to the cell surface (ecto-CRT) serves as a phagocytic signal for immunogenic cell death (ICD) mediated through dendritic cells (DCs) and cytotoxic T-cell activation thereby making tumors susceptible to immunotherapy-based anti-cancer strategies. CRT is now regarded as one of the most potent danger-associated molecular patterns (DAMPs) with the ecto-CRT triggering restoration of homeostasis by immune stimulation. A recently identified novel transacetylase activity of CRT adds a new dimension to its multi-faceted involvement in cancer by virtue of polyphenolic acetates (PA): CRT transacetylase (CRTase) system which results in hyperacetylation of target proteins, thereby mimicking the effects of Histone deacetylase inhibitors (HDACi). Since protein acetylation is one of the crucial post-translational modifications (PTMs) influencing the epigenetic regulation and signal transduction, CRT can be a potential target for developing anticancer therapeutics and preventive strategies by employing pharmacologically compatible semi-synthetic acetyl donors like polyphenolic acetates and other agents.
DOI:10.1006/bbrc.1993.1167URLPMID:8439313 [本文引用: 1]
In a search for the plant equivalent of calsequestrin or calreticulin, the high capacity, low affinity Ca2+ binding proteins of muscle and non-muscle cells thought to play important roles in Ca2+ storage, we purified two Ca(2+)-binding proteins from spinach leaves. The proteins had apparent molecular weights of 55 and 53 kDa. On Western blot, they did not react either with anti-rabbit skeletal muscle, anti-dog cardiac muscle calsequestrin or anti-rabbit or anti-rat liver calreticulin antibodies, indicating that they were antigenically distinct. Periodic acid Schiff staining (PAS) revealed that the larger protein was glycosylated while the 53 kDa one was PAS-negative. When the proteins were subjected to NH2-terminus amino acid sequencing, the 55 and 53 kDa proteins turned out to be identical, thus probably representing different isoforms of the same protein. Comparison with published amino acid sequences of calreticulin reveals regions of similarity indicating that the plant Ca(2+)-binding proteins probably belong to the calreticulin family.
DOI:10.1248/bpb.26.256URLPMID:12576690 [本文引用: 1]
Calreticulin is an abundant endo/sarcoplasmic reticulum Ca2+-binding protein. To investigate whether calreticulin (CRO1) is involved in the cold-stress response in rice, a transgenic plant was constructed. The transcriptional level was decreased within 30 min and recovered within 2 h of a cold treatment. The calreticulin protein was shifted from a soluble fraction to an insoluble fraction by cold stress. Endogenous abscisic acid (ABA) is an important factor in cold response, and the synthesis of ABA was strongly induced in CRO1-sense transgenic rice, the same as in cold-sensitive rice. The phosphorylation of calreticulin increased after cold treatment. Over-expression of calreticulin enhanced the activities of 47 kDa Ca2+-dependent protein kinase (CDPK) that had been induced by cold treatment. The 47-kDa CDPK activity increases more in the cold sensitive variety IR36 and the sense transgenic rice than it does in other varieties. The synthesis of ABA, phosphorylation of calreticulin and 47-kDa CDPK activity induced in sense transgenic rice were the same as in cold-sensitive rice and the phosphorylation of antisense transgenic rice was similar to that of cold-tolerant rice. These results suggest that the calreticulin is involved in the signaling pathway leading to response to cold stress.
DOI:10.1371/journal.pone.0140591URLPMID:26469859
Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.
URLPMID:33131209
To better understand the mechanisms regulating plant carotenoid metabolism in staple crop, we report the map-based cloning and functional characterization of the Glycine max carotenoid cleavage dioxygenase 4 (GmCCD4) gene, which encodes a carotenoid cleavage dioxygenase enzyme involved in metabolizing carotenoids into volatile beta-ionone. Loss of GmCCD4 protein function in four Glycine max increased carotenoid content (gmicc) mutants resulted in yellow flowers due to excessive accumulation of carotenoids in flower petals. The carotenoid contents also increase three times in gmicc1 seeds. A genome-wide association study indicated that the GmCCD4 locus was one major locus associated with carotenoid content in natural population. Further analysis indicated that the haplotype-1 of GmCCD4 gene was positively associated with higher carotenoid levels in soybean cultivars and accumulated more beta-carotene in engineered E. coli with ectopic expression of different GmCCD4 haplotypes. These observations uncovered that GmCCD4 was a negative regulator of carotenoid content in soybean, and its various haplotypes provide useful resources for future soybean breeding practice.
DOI:10.1038/emboj.2009.263URLPMID:19763087
Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe-associated molecular patterns shared by many microbe species. The Leu-rich repeat receptor-like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor-Tu, respectively. We describe Arabidopsis 'priority in sweet life' (psl) mutants that show de-repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP-glucose:glycoprotein glycosyltransferase that act in concert with STT3A-containing oligosaccharyltransferase complex in an N-glycosylation pathway in the endoplasmic reticulum. However, EFR-signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid-induced, but EFR-independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N-glycosylation for different layers of plant immunity, likely through quality control of membrane-localized regulators.
DOI:10.1016/j.chom.2009.10.005URL
SummaryMounting an effective innate immune response against pathogens requires the rapid and global reprogramming of host cellular processes. Here we employed complementary proteomic methods to identify differentially regulated proteins early during a plant's defense response. Besides defense-related proteins, constituents of the largest category of upregulated proteins were cytoplasmic- and ER-residing molecular chaperones. Investigating the significance of upregulated ER chaperones, we find that silencing of ER-resident protein disulfide isomerases NbERp57 and NbP5 and the calreticulins NbCRT2 and NbCRT3 led to partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 were required for the expression of a previously uncharacterized induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for N-mediated hypersensitive response, programmed cell death, and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane-bound or secreted proteins during plant innate immunity.]]>
DOI:10.1111/j.1364-3703.2009.00606.xURLPMID:20447282 [本文引用: 3]
Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in plant-virus interactions. In this study, we constructed a Carica papaya L. plant cDNA library to investigate the host factors interacting with Papaya ringspot virus (PRSV) HC-Pro using a Sos recruitment two-hybrid system (SRS). We confirmed that the full-length papaya calreticulin, designated PaCRT (GenBank accession no. FJ913889), interacts specifically with PRSV HC-Pro in yeast, in vitro and in plant cells using SRS, in vitro protein-binding assay and bimolecular fluorescent complementation assay, respectively. SRS analysis of the interaction between three PaCRT deletion mutants and PRSV HC-Pro demonstrated that the C-domain (residues 307-422), with a high Ca(2+)-binding capacity, was responsible for binding to PRSV HC-Pro. In addition, quantitative real-time reverse transcriptase-polymerase chain reaction assay showed that the expression of PaCRT mRNA was significantly upregulated in the primary stage of PRSV infection, and decreased to near-basal expression levels in noninoculated (healthy) papaya plants with virus accumulation inside host cells. PaCRT is a new calcium-binding protein that interacts with potyviral HC-Pro. It is proposed that the upregulated expression of PaCRT mRNA may be an early defence-related response to PRSV infection in the host plant, and that interaction between PRSV HC-Pro and PaCRT may be involved in plant calcium signalling pathways which could interfere with virus infection or host defence.
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DOI:10.1016/j.cropro.2012.07.005URL [本文引用: 1]
Sugarcane mosaic virus (SCMV) infects maize, sorghum, sugarcane and other poaceous species throughout the world. SCMV is an important virus pathogen, especially in European and Chinese maize production, causing serious losses in grain and forage yields in susceptible cultivars. Like other potyviruses, SCMV is a positive-sense single-stranded RNA virus with a genome size of approximately 10 kb in length. SCMV is naturally transmitted by aphids in a non-persistent manner. Control of the aphid vectors is not effective because of the non-persistent mode of virus transmission. Therefore, cultivation of resistant maize varieties is the preferred way to control SCMV infections. The high incidence of co-infection and the occurrence of new strains or genome variations indicate that SCMV will continue to be a threat to industry. Aspects concerning virus structure and genome organization, geographic distribution, diagnosis and strain characterization, and genetic variation are reviewed. Special emphasis is placed on the control of SCMV disease. (C) 2012 Elsevier Ltd.
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DOI:10.3389/fmicb.2013.00351URLPMID:24409170 [本文引用: 1]
To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs). However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV) induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked toward the plasma membrane and were associated with plasmodesmata (PD). We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP) to visualize how 6K2 moved intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.
DOI:10.1104/pp.17.01484URLPMID:29089395 [本文引用: 3]
Plant viruses move from the initially infected cell to adjacent cells through plasmodesmata (PDs). To do so, viruses encode dedicated protein(s) that facilitate this process. How viral proteins act together to support the intercellular movement of viruses is poorly defined. Here, by using an infection-free intercellular vesicle movement assay, we investigate the action of CI (cylindrical inclusion) and P3N-PIPO (amino-terminal half of P3 fused to Pretty Interesting Potyviridae open reading frame), the two PD-localized potyviral proteins encoded by Turnip mosaic virus (TuMV), in the intercellular movement of the viral replication vesicles. We provide evidence that CI and P3N-PIPO are sufficient to support the PD targeting and intercellular movement of TuMV replication vesicles induced by 6K2, a viral protein responsible for the generation of replication vesicles. 6K2 interacts with CI but not P3N-PIPO. When this interaction is impaired, the intercellular movement of TuMV replication vesicles is inhibited. Furthermore, in transmission electron microscopy, vesicular structures are observed in connection with the cylindrical inclusion bodies at structurally modified PDs in cells coexpressing 6K2, CI, and P3N-PIPO. CI is directed to PDs through its interaction with P3N-PIPO. We hypothesize that CI serves as a docking point for PD targeting and the intercellular movement of TuMV replication vesicles. This work contributes to a better understanding of the roles of different viral proteins in coordinating the intercellular movement of viral replication vesicles.
DOI:10.1104/pp.18.01342URLPMID:30538165 [本文引用: 2]
Like other positive-strand RNA viruses, the Turnip mosaic virus (TuMV) infection leads to the formation of viral vesicles at the endoplasmic reticulum (ER). Once released from the ER, the viral vesicles mature intracellularly and then move intercellularly. While it is known that the membrane-associated viral protein 6K2 plays a role in the process, the contribution of host proteins has been poorly defined. In this article, we show that 6K2 interacts with RHD3, an ER fusogen required for efficient ER fusion. When RHD3 is mutated, a delay in the development of TuMV infection is observed. We found that the replication of TuMV and the cell-to-cell movement of its replication vesicles are impaired in rhd3 This defect can be tracked to a delayed maturation of the viral vesicles from the replication incompetent to the competent state. Furthermore, 6K2 can relocate RHD3 from the ER to viral vesicles. However, a Golgi-localized mutated 6K2(GV) is unable to interact and relocate RHD3 to viral vesicles. We conclude that the maturation of TuMV replication vesicles requires RHD3 for efficient viral replication and movement.
DOI:10.1111/1574-6968.12295URLPMID:24164561 [本文引用: 1]
The application of entomopathogenic fungi such as Isaria fumosorosea to combat insect pests on plants is complicated by their sensitivity to commonly used fungicides. In this study, I. fumosorosea mutants with enhanced resistance to the fungicide benomyl were induced by irradiation using either ion beams or gamma rays, or a combination of the two. When grown on agar containing benomyl, mycelial growth was observed for five of the six mutant isolates at benomyl concentrations that were more than 2000-fold those observed for the wild-type isolate (EC50 : > 5000 mg L(-1) c.f. EC50 : 2.5 mg L(-1) for the wild-type isolate). The mutant isolates evaluated also showed enhanced resistance to other fungicides at recommended field application rates. No differences were observed at the beta-tubulin locus between the wild-type and the mutant isolates, suggesting that the enhanced benomyl resistance was not attributable to mutations in that gene. Ion beams and gamma rays are thus potentially useful tools for inducing beneficial fungal mutations and thereby improving the potential for application of entomopathogenic fungi as microbial control agents.
DOI:10.1038/srep07042URLPMID:25391499 [本文引用: 1]
Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses.
DOI:10.1007/BF02772676URL [本文引用: 1]
URLPMID:9159940 [本文引用: 1]
DOI:10.1111/j.1399-3054.2009.1223.xURLPMID:19453510 [本文引用: 1]
Calreticulin (CRT) is a key Ca2+-binding protein mainly resident in the endoplasmic reticulum (ER), which is highly conserved and extensively expressed in all eukaryotic organisms investigated. The protein plays important roles in a variety of cellular processes including Ca2+ signaling and protein folding. Although calreticulin has been well characterized in mammalian systems, increased investigations have demonstrated that plant CRTs have a number of specific properties different from their animal counterparts. Recent developments on plant CRTs have highlighted the significance of CRTs in plants growth and development as well as biotic and abiotic stress responses. There are at least two distinct groups of calreticulin isoforms in higher plants. Glycosylation of CRT was uniquely observed in plants. In this article, we will describe our current understanding of plant calreticulin gene family, protein structure, cellular localization, and diverse functions in plants. We also discuss the prospects of using this information for genetic improvements of crop plants.
URLPMID:17073303 [本文引用: 1]
DOI:10.1007/s11103-015-0362-6URLPMID:26358043 [本文引用: 1]
Plant recovery from viral infection is characterized by initial severe systemic symptoms which progressively decrease, leading to reduced symptoms or symptomless leaves at the apices. A key feature to plant recovery from invading nucleic acids such as viruses is the degree of the host's initial basal immunity response. We review current links between RNA silencing, recovery and tolerance, and present a model in which, in addition to regulation of resistance (R) and other defence-related genes by RNA silencing, viral infections incite perturbations of the host physiological state that trigger reprogramming of host responses to by-pass severe symptom development, leading to partial or complete recovery. Recovery, in particular in perennial hosts, may trigger tolerance or virus accommodation. We discuss evidence suggesting that plant viruses can avoid total clearance but persistently replicate at low levels, thereby modulating the host transcriptome response which minimizes fitness cost and triggers recovery from viral-symptoms. In some cases a susceptible host may fail to recover from initial viral systemic symptoms, yet, accommodates the persistent virus throughout the life span, a phenomenon herein referred to as non-recovery accommodation, which differs from tolerance in that there is no distinct recovery phase, and differs from susceptibility in that the host is not killed. Recent advances in plant recovery from virus-induced symptoms involving host transcriptome reprogramming are discussed.
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URLPMID:32979144 [本文引用: 1]
The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the cyanobacterium Synechocystis PCC 6803. Light-induced loss of PSII activity was compared in the presence of chloramphenicol and another protein synthesis inhibitor, lincomycin, by measuring the rate of oxygen evolution in Synechocystis 6803 cells. Our data show that the rate of PSII photodamage was significantly enhanced by chloramphenicol, at the usually applied 200 mug mL(-1) concentration, relative to that obtained in the presence of lincomycin. Chloramphenicol-induced enhancement of photodamage has been observed earlier in isolated PSII membrane particles, and has been assigned to the damaging effect of chloramphenicol-mediated superoxide production (Rehman et al. 2016, Front Plant Sci 7:479). This effect points to the involvement of superoxide as damaging agent in the presence of chloramphenicol also in Synechocystis cells. The chloramphenicol-induced enhancement of photodamage was observed not only in wild-type Synechocystis 6803, which contains both Photosystem I (PSI) and PSII, but also in a PSI-less mutant which contains only PSII. Importantly, the rate of PSII photodamage was also enhanced by the absence of PSI when compared to that in the wild-type strain under all conditions studied here, i.e., without addition and in the presence of protein synthesis inhibitors. We conclude that chloramphenicol enhances photodamage mostly by its interaction with PSII, leading probably to superoxide production. The presence of PSI is also an important regulatory factor of PSII photodamage most likely via decreasing excitation pressure on PSII.
DOI:10.1016/j.coviro.2016.01.018URLPMID:26871502 [本文引用: 1]
The endoplasmic reticulum and Golgi network (ERGN) is vital to most cellular biosynthetic processes. Many positive strand RNA viruses depend upon the ERGN for replication, maturation, and egress. Viruses induce changes in ER architecture and stimulate fatty acid synthesis to create environments that can scaffold replication complexes, plant virus movement complexes, or virion maturation. Potato virus X (PVX) and Turnip mosaic virus (TuMV) each encode small membrane binding proteins that embed in the ERGN and activate the unfolded protein response (UPR). The UPR ensures ERGN homeostasis in the face of environmental assaults that could negatively impact the biosynthetic functions of the ERGN. This article explores the relationship between ER stress, the UPR, and membrane synthesis occurring during virus infection.
DOI:10.1016/S0065-3527(10)76001-2URLPMID:20965070 [本文引用: 1]
Virus infection may damage the plant, and plant defenses are effective against viruses; thus, it is currently assumed that plants and viruses coevolve. However, and despite huge advances in understanding the mechanisms of pathogenicity and virulence in viruses and the mechanisms of virus resistance in plants, evidence in support of this hypothesis is surprisingly scant, and refers almost only to the virus partner. Most evidence for coevolution derives from the study of highly virulent viruses in agricultural systems, in which humans manipulate host genetic structure, what determines genetic changes in the virus population. Studies have focused on virus responses to qualitative resistance, either dominant or recessive but, even within this restricted scenario, population genetic analyses of pathogenicity and resistance factors are still scarce. Analyses of quantitative resistance or tolerance, which could be relevant for plant-virus coevolution, lag far behind. A major limitation is the lack of information on systems in which the host might evolve in response to virus infection, that is, wild hosts in natural ecosystems. It is presently unknown if, or under which circumstances, viruses do exert a selection pressure on wild plants, if qualitative resistance is a major defense strategy to viruses in nature, or even if characterized genes determining qualitative resistance to viruses did indeed evolve in response to virus infection. Here, we review evidence supporting plant-virus coevolution and point to areas in need of attention to understand the role of viruses in plant ecosystem dynamics, and the factors that determine virus emergence in crops.
DOI:10.1146/annurev-phyto-080614-120001URLPMID:25938276 [本文引用: 1]
A successful infection by a plant virus results from the complex molecular interplay between the host plant and the invading virus. Thus, dissecting the molecular network of virus-host interactions advances the understanding of the viral infection process and may assist in the development of novel antiviral strategies. In the past decade, molecular identification and functional characterization of host factors in the virus life cycle, particularly single-stranded, positive-sense RNA viruses, have been a research focus in plant virology. As a result, a number of host factors have been identified. These host factors are implicated in all the major steps of the infection process. Some host factors are diverted for the viral genome translation, some are recruited to improvise the viral replicase complexes for genome multiplication, and others are components of transport complexes for cell-to-cell spread via plasmodesmata and systemic movement through the phloem. This review summarizes current knowledge about host factors and discusses future research directions.
URLPMID:19284621 [本文引用: 1]
DOI:10.1104/pp.124.3.991URLPMID:11080277 [本文引用: 1]
Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.
DOI:10.1016/j.jplph.2018.05.014URLPMID:30025219 [本文引用: 1]
Aluminum (Al) toxicity limits growth and symbiotic interactions of plants. Calcium plays essential roles in abiotic stresses and legume-Rhizobium symbiosis, but the sites and mechanism of Ca(2+) mobilization during mycorrhizae have not been analyzed. In this study, the changes of cytoplasmic Ca(2+) and calreticulin (CRT) in Medicago truncatula mycorrhizal (MR) and non-mycorrizal (NM) roots under short Al stress [50 muM AlCl3 pH 4.3 for 3h] were analyzed. Free Ca(2+) ions were detected cytochemically by their reaction with potassium pyroantimonate and anti-CRT antibody was used to locate this protein in Medicago roots by immunocytochemical methods. In MR and NM roots, Al induced accumulation of CRT and free Ca(2+). Similar calcium and CRT distribution in the MR were found at the surface of fungal structures (arbuscules and intercellular hyphae), cell wall and in plasmodesmata, and in plant and fungal intracellular compartments. Additionally, degenerated arbuscules were associated with intense Ca(2+) and CRT accumulation. In NM roots, Ca(2+) and CRT epitopes were observed in the stele, near wall of cortex and endodermis. The present study provides new insight into Ca(2+) storage and mobilization in mycorrhizae symbiosis. The colocalization of CRT and Ca(2+) suggests that CRT is essential for calcium mobilization for normal mycorrhiza development and response to Al stress.
URLPMID:33259640 [本文引用: 1]
* In their natural environment along coast lines, date palms are exposed to seawater inundation and, hence, combined stress by salinity and flooding. * To elucidate the consequences of this combined stress on foliar gas exchange and metabolite abundances in leaves and roots, date palm seedlings were exposed to flooding with seawater and its major constituents under controlled conditions. * Seawater flooding significantly reduced CO2 assimilation, transpiration and stomatal conductance, but did not affect isoprene emission. A similar effect was observed upon NaCl exposure. In contrast, flooding with distilled water or MgSO4 did not affect CO2 /H2 O gas exchange or stomatal conductance significantly, indicating that neither flooding itself, nor seawater sulfate, contributed greatly to stomatal closure. Seawater exposure increased Na and Cl contents in leaves and roots, but did not affect sulfate contents significantly. Metabolite analyses revealed reduced abundances of foliar compatible solutes, such as sugars and sugar alcohols, whereas nitrogen compounds accumulated in roots. * Reduced transpiration upon seawater exposure may contribute to controlling the movement of toxic ions to leaves and, therefore, can be seen as a mechanism to cope with salinity. The present results indicate date palm seedlings are tolerant towards seawater exposure to some extent, and highly tolerant to flooding.
DOI:10.1371/journal.ppat.1000962URLPMID:20585568 [本文引用: 1]
Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.
URLPMID:31969439 [本文引用: 1]
