植物病毒及其卫星RNA在寄主体内的移动和系统分布

植物病毒与动物病毒有多方面的不同,主要包 括:大多数植物病毒的感染需要微伤口(少数靠内 吞作用;包膜病毒靠融合方式),而动物病毒的感染 则需要受体。在植物病毒进入细胞或从一个细胞扩 散到周围未被感染的细胞时都会遇到一些障碍,如 细胞壁和细胞质膜。到目前为止,尚未在植物细胞 中发现有病毒受体参与侵染的证据。植物病毒需要 藉由运动蛋白(MP)进行胞间移动,动物病毒则 无。植物病毒常包被数个颗粒,动物病毒一般只包 被一个颗粒,而昆虫病毒NPV则有多个包埋型。 植物病毒出现卫星RNA的频率高,动物病毒则频 率低。     

植物病毒的卫星病毒和卫星RNA

植物病毒的卫星病毒和卫星ＲＮＡ陈金标（南京农业大学微生物系２１００９５）病毒是一种无细胞结构的生物体,个体极小,约为２０—４００ｕｒｎ。Ｍａｔｔｈｅｗｓ将它定义为：病毒是丁组或多组核酸分子、它通常祛外完蛋白包裹,且只在适合的寄主细胞里复制。在这种细胞...     

竹花叶病毒卫星RNA及其编码卫星蛋白的结构与功能

竹花叶病毒(Bamboo mosaic virus,BaMV)是目前为止被发现感染竹类惟一的病毒,除了巴西、夏威夷及琉球有过零星报道外,台湾对此病毒有较深入的研究[1～13].BaMV在台湾地区的竹类栽培区普遍发生,可危害4个属、14个种、3个变种和3个栽培种[14,15],其中麻竹(Dendrocalamus latiflorus Munro)和绿竹(Bambusa oldhamii Munro)遭受危害最为严重,主要竹产地染病率可高达90%以上,给台湾的竹产业造成了严重危害[16].     

竹花叶病毒卫星RNA及其编码卫星蛋白的结构与功能

竹花叶病毒(Bamboo mosaic virus,BaMV)是目前为止被发现感染竹类惟一的病毒,除了巴西、夏威夷及琉球有过零星报道外,台湾对此病毒有较深入的研究[1~13]。BaMV在台湾地区的竹类栽培区普遍发生,可危害4个属、14个种、3个变种和3个栽培种[14,15],其中麻竹(Dendrocalamuslatifloru     

病毒在植物体内的运转

病毒能否引致植物发病,取决于病毒侵入植物后能否运转到植物的其它部分.一般认为病毒是通过由生物介体或机械磨擦造成的机械损伤而侵入植物细胞的.从初始侵染的细胞开始,大多数病毒在植物体内有两种运转方式:在薄壁细胞间进行的缓慢的短距离运转;在输导组织间进行的快速的长距离运转.80年代中期认识到病毒的体内运转需要其基因产物(运动蛋白,movement protem,MP)的参与,证实了烟草花叶病毒(TMV)的30kD蛋白即为TMV的MP[1,2].之后有关病毒MP及对病毒如何在植物体内进行运转的研究取得很大的进展.有关这方面的综述文章有Hull R、Atabekov等、Lucas等和Carrington等[3-6]的.本文主要综述近五年来的研究进展,但为了其完整性,也包含了一些上述综述的主要有关内容.     

RNA沉默与植物病毒

植物中RNA沉默（RNAsilencing)亦称为转录后基因沉默（PTGS）或共抑制,是植物抵抗外来核酸（转座子、转基因或病毒）入侵,并保护自身基因组完整性的一种防御机制。RNA沉默是近十年来发现的植物界中普遍存在的现象,已成为植物分子生物学领域的一个新的研究方向。对RNA沉默特点和机制的研究表明,植物病毒与（转基因）植物内发生的RNA沉默有着密切的联系,作者从病毒对RNA沉默的诱导、抑制、防御等方面,简述了RNA沉默与病毒的关系。并对病毒载体所诱导的RNA沉默在植物发育和基因组功能分析等方面的应用价值进行了讨论。     

系统侵染寄主中黄瓜花叶病毒及其卫星RNA的动态变化

以³²P标记的黄瓜花叶病毒（CMV）RNA3 cDNA片段和卫星RNA全长cDNA作为探针,定量测定CMV基因组RNA和卫星RNA的含量变化,结果显示：二者均具有明显的寄主效应和时间效应.在16～20℃条件下,接种不携带卫星RNA的分离物CMV-R3,15天、30天和75天时,CMV基因组RNA负荷量呈显著下降的趋势.在第15天,RNA3的负荷量以烟草>心叶烟>克里夫兰烟>番茄的顺序表现为不同寄主的显著性差异.相同条件下接种携带高拷贝卫星RNA的分离物CMV-RS,在5天和15天之间基因组RNA和卫星RNA负荷量均呈现上升的趋势,同时测得其基因组RNA和卫星的负荷量具有相似的寄主效应和时间效应,但程度不同.第15天时,二者负荷量以烟草＞心叶烟＞番茄的顺序表现寄主效应的显著性差异.在18～21℃条件下,接种携带坏死卫星RNA的CMV强毒株HC4,第5天、第10天和第15天时,基因组RNA和卫星RNA的负荷量均以番茄＞心叶烟＞烟草的顺序表现出显著性差异,并表现出明显的时间效应.不同来源CMV分离物还存在寄主选择性差异.     

卫星病毒和卫星RNA的分子结构及遗传工程

自60年代发现卫星病毒、70年代发现卫星RNA以来,已发现10个病毒组中的31种植物病毒含有卫星病毒或卫星RNA(统称卫星)。植物病毒卫星是指依赖于植物病毒进行复制的核酸分子,如核酸分子含有编码外壳蛋白的遗传信息,并能包裹成形态学和血清学与辅助病毒不同的颗粒,称卫星病毒;如本身没有编码外壳蛋白的遗传信息,而是装配于辅助病毒的外壳蛋白中,则称卫星RNA。     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Global Analyses of Small Interfering RNAs Derived from Bamboo mosaic virus and Its Associated Satellite RNAs in Different Plants

Background

Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.

Methodology/Principal Findings

Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.

Conclusions/Significance

The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.     

Appearance of a Host Protein in Cucumber Plants Infected with Viruses, Bacteria and Fungi

Electrophoretic analyses of extracts of cucumber leaves infectedwith Colleiotrichum lagenarium, Fusarium oxysporum f. sp. cucumerinum,Pseudomonas lachrymans, Erwinia tracheiphila, tobacco necrosisvirus or cucumber mosaic virus revealed the presence of a proteinband with an R_F value of 0.55–0.60 (based on mobilityof bromophenol blue) on 10% polyacrylamide gel. This band wasnot evident in extracts of healthy or mechanically wounded leaves.The protein was not detected in uninfected leaves of infectedplants, but it was detected in similar amounts in infected leavesand in secondarily challenged leaves of infected plants eventhough symptoms were not apparent on the latter. The proteinhad a molecular weight of approximately 16 000 d, was adsorbedon DEAE-cellulose, did not react with Schiff's reagent, anddid not have ribonuclease activity. When injected into cucumberleaves, it did not inhibit germination of conidia of C. lagenariumor induce resistance against disease caused by the fungus.     

Plant Host Finding by Parasitic Plants: A New Perspective on Plant to Plant Communication

Plants release airborne chemicals that can convey ecologically relevant information to other organisms. These plant volatiles are known to mediate a large array of, often complex, interactions between plants and insects. It has been suggested that plant volatiles may have similar importance in mediating interactions among plant species, but there are few well-documented examples of plant-to-plant communication via volatiles, and the ecological significance of such interactions has been much debated. To date, nearly all studies of volatile-mediated interactions among plant species have focused on the reception of herbivore-induced volatiles by neighboring plants. We recently documented volatile effects in another system, demonstrating that the parasitic plant Cuscuta pentagona uses volatile cues to locate its hosts. This finding may broaden the discussion regarding plant-to-plant communication, and suggests that new classes of volatile-meditated interactions among plant species await discovery.Key Words: chemical communication, Cuscuta pentagona, host fiding, host selection, plant-plant communication, plant volatiles, parasitic plantsFor nearly 25 years, the ecological importance of plant-to-plant communication through volatiles has remained an open and much debated question. Plants exchange gases with the atmosphere and, in so doing, release plumes of volatile chemicals that can convey ecologically important information to other organisms. The potential ecological significance of these volatile cues is demonstrated by the large and growing array of interactions between plants and arthropods known to be mediated by plant volatiles. Volatiles serve as foraging cues both for insects that are beneficial to plants, such as pollinators,¹ and those that are harmful such as herbivores.^2,3 Because the volatile blends released by plants exhibit variation in response to environmental stimuli, volatiles can convey detailed information about the status of the emitting plant. Predatory and parasitic insects that feed on herbivorous insects respond preferentially to plant volatiles that are induced by insect feeding,⁴ while female herbivores use such cues to avoid laying their eggs on already-infested plants.^3,5 Moreover, the volatile blends released in response to herbivory can differ between individual herbivore species, providing highly specific cues to specialist parasitoids.⁶ Thus, plant volatiles are known to mediate complex interactions among plants and insects across multiple trophic levels.It has long been speculated that plant volatiles might have similar significance for interactions among plant species, yet there are few well-documented examples of communication between plants by way of volatile signals. Essentially all previous work on plant-to-plant communication has focused on the reception of herbivore-induced volatile signals by neighboring plants, which may use them as early warning signals to initiate their own direct and indirect defense responses. The first studies claiming to document such effects were published almost 25 years ago.^7,8 But issues with the experimental design of these early experiments and the availability of alternative explanations for their results led many ecologists to disregard the phenomenon.^9–11 Later, a number of studies demonstrated that direct and indirect plant defenses could be elicited by exposure to certain induced plant volatiles.^12–15 But many of these effects were demonstrated in airtight chambers with volatile concentrations far higher than those likely experienced in natural settings, again raising doubts about the ecological significance of plant-plant communication.^16–18 Still more recently, some researchers have provided evidence that more realistic volatile concentrations likely induce priming of the defenses of receiving plants, rather than the initiation of full scale responses,¹⁵ while others have documented volatile effects under natural conditions.^19–21 Thus, despite continuing caution about the interpretation of experiments in this area,^17,18 there is mounting evidence that plant herbivore-induced volatiles can serve as early warning signals to neighboring plants.We recently documented an entirely new class of volatile mediated interactions among plants: the role of plant volatiles in host location by parasitic plants that attach to above ground shoots of other plants. Plant parasites are important components of natural and agricultural ecosystems and play important roles in determining community structure and dynamics.^22,23 We are exploring the mechanisms of host-location and other interactions between parasitic plants in the genus Cuscuta (dodder) and their host plants. Dodder vines germinate from seeds containing limited energy reserves and, as the parasites have no roots and little photosynthetic ability, must quickly locate and attach to suitable hosts in order to survive (Fig. 1). Thus, there is presumably significant selection pressure for dodder vines to employ efficient strategies for host location, and host plant volatiles may be expected to provide relevant directional cues. Dodder seedlings exhibit a rotational growth habit (circumnutation) following germination and previous researchers have suggested that host-finding might involve random growth²⁴ or the exploitation of light cues.²⁵Open in a separate windowFigure 1Seedling of Cuscuta pentagona (A) foraging toward a 20-day-old tomato plant, (B) attaching to and beginning to grow from stems of tomato seedlings and (C) close up of C. pentagona attachment.Using a very simple experimental design, we explored the possibility that host-plant volatiles might mediate host-location by seedlings of C. pentagona. We placed a germinated seedling in a vial of water located at the center of a dry filter paper disk. A host plant (a 20-day old tomato seedling) was placed near the edge of the disk and the dodder seedling was allowed to forage for four days. By the end of the experiment the seedling would lay horizontally on the disk and we traced its position on the filter paper in order to assess the directionality of growth relative to the host plant. This experiment was replicated 30 times and our results clearly indicated directional growth toward the tomato plant (80% of the tested seedlings grew into the disk half nearest the host) demonstrating that C. pentagona seedlings were perceiving some host-derived cue.We did not observe directed growth when we tested dodder seedling response to alternative targets including pots of moist soil, artificial plants, and vials of colored water intended to mimic possible light cues. In order to confirm a role for plant volatiles in host location by C. pentagona, we tested seedling response to host plant volatiles extracted from filtered air in a volatile collection system and then released from rubber septa in the absence of any other host-derived cues. Here we observed a directed growth response similar to that exhibited toward an intact tomato seedling, confirming that host plant volatiles do provide a cue used for host location by C. pentagona. In subsequent experiments we found directed growth toward impatiens and alfalfa plants, which are attractive hosts for C. pentagona and also toward wheat plants which are poor hosts, suggesting that the host-location mechanisms operate over a wide range of host species.Since discriminating between more and less desirable host species is likely to be important in natural settings, we next explored whether dodder seedlings could distinguish volatile signals from host and nonhost plants. Cuscuta pentagona seedlings exhibited directional growth toward tomato plants in preference to wheat plants and also to extracted volatiles from tomato in preference to those from wheat, demonstrating an ability to distinguish and choose among volatiles from more and less preferred hosts.When we tested seedling responses to individual compounds from the wheat and tomato blends, we found that three compounds from tomato, α-pinene, β-myrcene, and β-phellandrene elicited directed growth. β-myrcene was also present in the wheat blend. Unexpectedly, we also found that one compound present in the wheat blend, (Z)-3-hexenyl acetate, was repellent, providing a plausible explanation for the lower attractiveness of the wheat blend. It is interesting to note that (Z)-3-hexenyl acetate is also released by tomato in response to feeding by herbivores, and we have some data suggesting that C. pentagona seedlings may find tomato seedlings infested by Heliothis virescens caterpillars less attractive than un-attacked plants (unpublished data).The discovery that some parasitic plants exploit host plant volatiles for host location provides a new perspective on volatile mediated interactions among plant species, demonstrating that plant volatiles play a role in mediating ecologically significant interactions in at least one system other than the transfer of herbivore-induced warning signals. We think it is quite likely that plant volatiles will be found to play a role in host location by other parasitic plants and perhaps even by vining plants generally. Moreover, we think it is more likely than not that more classes of volatile mediated interactions among plants remain to be discovered given the potential availability of volatile cues and the fitness benefits to be derived by plants using such cues to gather information about the identity and condition of their neighbors.     

Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

Background

Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.

Principal Findings

Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.

Significance

Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.     

Movement of Viruses between Biomes

Viruses are abundant in all known ecosystems. In the present study, we tested the possibility that viruses from one biome can successfully propagate in another. Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil. The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-μm-pore-size filter). Virus-like particle abundances were then monitored using direct counting. Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate. These results imply that viruses can laterally transfer DNA between microbes in different biomes.