Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

Degree of RNA silencing and the ability of a viral suppressor vary depending on the cell species in a protoplast system

RNA silencing is a sequence-specific defense mechanism against viruses. As a counterdefense, viruses evolved silencing suppressors to interfere with host silencing. In analyses using protoplasts prepared from cultured cells (BY-2) and mesophyll cells of Nicotiana tabacum and N. benthamiana, viral suppressors differentially functioned in different cell types. This phenomenon has not been discussed in earlier papers on protoplast systems and RNA silencing. In investigations of the cellular activities of viral suppressors and their role in the RNA-silencing pathway, assays with host protoplasts offer many advantages and can complement other in planta assays such as Agrobacterium-mediated transient expression.     

Geminivirus C2 protein represses genes involved in sulphur assimilation and this effect can be counteracted by jasmonate treatment

Geminiviruses are plant viruses that infect a broad range of crops and cause extensive losses worldwide, having an important economic impact. C2, a multifunctional pathogenicity factor encoded by geminiviruses, has been recently shown to suppress the responses to jasmonates in the host plant, which might at least partially explain its well-established role in pathogenicity. Sulphur is one of the essential macro-elements for plant life, and is considered to have a role in plant defence, in a phenomenon named sulphur-induced resistance (SIR) or sulphur-enhanced defence (SED). In this work, we show that geminivirus C2 protein represses the expression of genes involved in the sulphur assimilation pathway in Arabidopsis, but, interestingly, this effect can be neutralized by exogenous jasmonate treatment. These preliminary results may raise the idea that geminiviruses might be affecting sulphur metabolism, and maybe counteracting SIR/SED, through the manipulation of the jasmonate signalling pathway, which would define a novel strategy in plant-virus interactions and may unveil SIR/SED as an important player in the plant defence against viruses.     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.     

Soilborne viruses: advances in virus movement,virus induced gene silencing,and engineered resistance

Until recently soilborne plant viruses were considered important only because they are causative agents for agricultural diseases. In recent years, soilborne plant viruses have played a significant role in advancing research into mechanisms of plasmodesmata transport, gene silencing, and engineered resistance to plant pathogens. Three different mechanisms by which viruses move through plasmodesmata have been identified using dianthoviruses, nepoviruses, and benyviruses. The infectious clone of the tobravirus Tobacco rattle virus (TRV) has become an important tool for studying virus induced gene silencing and may be a tool to silence meristematic gene expression. Investigations of soilborne viruses may enable the development of new strategies for control of soilborne diseases. The potential use of pathogen-derived resistance to control soilborne viruses is currently being explored in several laboratories.     

植物病毒基因沉默抑制因子研究进展

RNAi是近年来发现的一种重要的基因沉默现象,可以介入植物的整体防御体系,在植物细胞中产生一种不确定的流动信号,使远距离组织的特异RNA序列得到降解。为利于病毒的侵染,植物、动物和昆虫的病毒同时也编码一种蛋白来对抗RNAi,这类蛋白可以抑制RNA沉默的各个步骤,称为RNAi抑制因子,本文对几个研究较清楚的植物病毒抑制因子,从其发现到主要特点、作用机制等方面进行了阐述,并且依据其特点及前景进行归类与展望。     

Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3

GLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3’ end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed suppressor activity demonstrated in diverse silencing inducing systems. This suppressor is able to overcome strong silencing inducers and shares several properties with the BYV p21-like family of suppressors of the closteroviruses. This is the first report of an RNA silencing suppressor in the genus Ampelovirus.     

Sequence analysis and classification of apparent recombinant begomoviruses infecting tomato in the nile and mediterranean basins

ABSTRACT Numerous whitefly-transmitted viral diseases of tomato have emerged in countries around the Nile and Mediterranean Basins the last 20 years. These diseases are caused by monopartite geminiviruses (family Gemini viridae) belonging to the genus Begomovirus that probably resulted from numerous recombination events. The molecular biodiversity of these viruses was investigated to better appreciate the role and importance of recombination and to better clarify the phylogenetic relationships and classification of these viruses. The analysis partitioned the tomato-infecting begomoviruses from this region into two major clades, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus. Phylogenetic and pairwise analyses together with an evaluation for gene conversion were performed from which taxonomic classification and virus biodiversity conclusions were drawn. Six recombination hotspots and three homogeneous zones within the genome were identified among the tomatoinfecting isolates and species examined here, suggesting that the recombination events identified were not random occurrences.     

高通量测序技术在鉴定木本植物双生病毒中的应用

高通量测序技术(next-generation sequencing,NGS)平台提供了一种高效、快速、低成本、深度测序DNA的解决方案,2009年该技术开始被应用于植物病毒学领域,包括新病毒的发现,病害病原的鉴定,病毒基因组多样性及进化的研究,显著加快了植物病毒学的发展进程。迄今,应用高通量测序技术已经成功鉴定了上百种新的植物病毒和类病毒。双生病毒是一类对多种作物造成毁灭性危害的DNA病毒,多发生于草本作物。然而利用高通量测序技术,从柑橘、葡萄、苹果和桑树等多种多年生木本植物中检测到了新的双生病毒,显示出了高通量测序技术所独有的、传统检测技术所不具备的优势。本文围绕高通量测度技术在植物病毒学领域的应用进行概述,重点阐述NGS用于检测木本植物双生病毒的几个实例。     

Diverse conserved domains and a positively selected site in the sugarcane streak mosaic virus P1 protein are essential for RNA silencing suppression and protein stability

RNA silencing is one of the conserved antiviral mechanisms in plants, and viruses encode RNA silencing suppressors (RSS) to overcome host RNA silencing and facilitate virus infection. Sugarcane streak mosaic virus (SCSMV; species Sugarcane streak mosaic virus, genus Poacevirus, family Potyviridae) is a major causal agent of sugarcane mosaic disease in many countries in Asia, including China. In this study, we used Agrobacterium co-infiltration to show that the SCSMV P1 protein, rather than the helper component-proteinase (HC-Pro), functions as a strong RSS to suppress local RNA silencing in Nicotiana benthamiana. Mutational analysis indicated that the 15 amino acids (aa; aa 1–15) of the SCSMV P1 N-terminus were not important for RNA silencing suppression, but rather another 15 aa domain (aa 108–122) containing a conserved motif (LFR/KNKQAYIST) was essential for efficient silencing suppression by P1. In addition to the 15 aa (aa 344–358) domain in the P1 N-terminus, another 15 aa domain (aa 65–79) of P1, containing the LXKA motif and one conserved aa (D78), were associated with P1 protein stability. Furthermore, substituting the histidine (H263) residue in P1 with threonine (H263T) or alanine (H263A) also affected P1 protein stability. Notably, the H263 residue is both a positively selected site and part of the serine protease catalytic triad (HDS). Taken together, our data demonstrate that SCSMV P1, and not HC-Pro, plays a functional role in suppressing RNA silencing, and also show that some conserved motifs and a positivelyselected site in the P1 protein are associated with RSS activity and protein stability.     

RNA silencing against viruses: molecular arms race between Cucumber mosaic virus and its host

Plants have developed RNA silencing as an antiviral defense mechanism. To escape from the plant host’s defenses, viruses have countered their host’s antiviral silencing by producing RNA silencing suppressor proteins (RSSs). Although the mode of action of the majority of viral RSSs has been found to be through double-stranded RNA-binding, viruses have different strategies to counteract the host’s antiviral silencing pathways. The 2b protein of Cucumber mosaic virus, which is one of the most extensively studied viral RSSs, is reviewed here to provide insights on the molecular arms race between viruses and their host plants.     

双生病毒复制增强蛋白C3/AC3的研究进展

双生病毒作为全球范围内引起作物严重病害的单链DNA病毒，编码多种多功能蛋白，其帮助自身完成复制、转录、组装、移动和致病等生命过程，其中双生病毒的复制增强蛋白C3/AC3在病毒侵染初期可促进病毒复制，提高病毒积累水平以帮助其快速建立侵染。为深入解析C3/AC3蛋白的功能和双生病毒致病机制，该文概述双生病毒C3/AC3蛋白的基本生物学特性及其在病毒侵染过程中的功能，重点阐述C3/AC3蛋白与其他蛋白及寄主因子互作以促进病毒复制的分子机制，同时针对双生病毒复制机制和C3/AC3蛋白的功能解析进行展望。     

Comparing p20’s RNA silencing suppressing activity among five phylogenetic groups of <Emphasis Type="Italic">Citrus Tristeza virus</Emphasis>

The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20’s suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don’t know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor’s activity.     

Microsatellites reveal widespread predominance of an invasive over an indigenous <Emphasis Type="Italic">Bemisia tabaci</Emphasis> in Venezuela

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an important pest due to its capacity for producing strong infestations and transmitting plant viruses. The New World putative species of B. tabaci complex (NW) is the indigenous whitefly of the Americas, but only the invasive Middle East-Asia Minor 1 putative species of B. tabaci complex (MEAM1), commonly referred to as the “B biotype” was identified in a limited scope sampling in Venezuela. Similarly to MEAM1 invasions elsewhere, in this South American country there has been an increase in the geographic range and abundance of B. tabaci, and in the number of viruses that it transmits since the late 1980s. We estimated the diversity of B. tabaci to elucidate their role in the epidemiology of geminiviruses in Venezuela. Thirteen microsatellite loci were screened in samples collected from 19 localities in ten major agricultural states. A Bayesian clustering method (Structure) grouped the samples into two genetic groups. Control samples from whiteflies NW and MEAM1 and partial sequencing of the mitochondrial cytochrome oxidase I gene showed that our samples of B. tabaci populations from Venezuela fall within NW and MEAM1 groups. In this survey, MEAM1 was predominant over NW whitefly in a proportion of 35:1. No evidence was observed for gene flow between indigenous and invader whitefly. Altogether, our results stress the urgency for controlling the proliferation of the invasive whitefly.     

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

Attenuated viruses have been isolated and studied not only as a practical means of controlling virus diseases but also to gain a molecular understanding of viral virulence and cross protection. They have been isolated from crop fields and generated through high/low temperature treatment or by mutagens such as nitrous acid and ultraviolet irradiation. Some viruses have been beneficially used in fields and evaluated for one or more decades. Molecular genetic studies on attenuated viruses have revealed that amino acid substitutions are located in replicase and the movement protein in tobamovirus, protein 2b for cucumovirus, and P1 and HC-Pro for potyvirus. In most cases, with a few exceptions, symptom attenuation is positively correlated with a reduced level of RNA silencing suppression. Molecular mechanisms underlying virus attenuation and cross protection and the rationale for practical use of attenuated viruses for effective virus disease control are discussed.     

中国番木瓜曲叶病毒南宁分离物的基因组结构特征

 从广西南宁田间表现曲叶症状的番木瓜植株上分离到病毒分离物G4,经三抗体夹心ELISA (TAS-ELISA)检测,G4与粉虱传双生病毒的抗体呈阳性反应。对G4 DNA-A全序列测定和分析表明,G4 DNA-A全长2 748个核苷酸,共编码6个ORFs。同源性比较及系统进化关系分析表明,G4 DNA-A与在亚洲发现的粉虱传双生病毒关系较近,其中与我国报道的中国番木瓜曲叶病毒(PaLCuCNV)同源性最高,达到98.0%。进一步比较发现,G4 DNA-A编码的AV1、AV2、AC1、AC2、AC3和AC4与PaLCuCNV相应ORFs的氨基酸同源性分别为98.4%、95.7%、97.5%、97.8%、94.1%和94.6%,表明G4应属于PaLCuCNV的一个分离物。G4编码的ORFs与中国胜红蓟黄脉病毒(AYVCNV)、辣椒曲叶病毒(PepLCV)及烟草曲茎病毒(TbCSV)有较高的氨基酸同源性,可能起源于共同的祖先。利用DNA-B及卫星DNAβ的保守引物均未能从G4分离物中扩增出相应的组分。     

Serological relationships of geminivirus isolates from Gramineae in Australia

Three distinct viruses, named chloris striate mosaic virus (CSMV), paspalum striate mosaic virus (PaSMV) and digitaria didactyla striate mosaic virus (DDSMV), have been identified among the five Gramineae-infecting geminiviruses from Australia using polyclonal antisera. An isolate from Microlaena was confirmed as a strain of CSMV, and an isolate from Bromus catharticus was identified as PaSMV-BC. Enzyme-linked immunosorbent assay (ELISA) detected relationships between all but one of the viruses tested, the exception being miscanthus streak virus (MiSV) from Japan. The Australian viruses proved to be distantly related to similar viruses from Africa, digitaria streak virus (DSV) from Vanuatu, and wheat dwarf virus (WDV) from Europe. Three distinct groups of viruses from Africa, Australia and Europe were distinguished by phylogenetic analysis.     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.     

A geminivirus causing vein yellowing of Ageratum conyzoides in Singapore

A vein-yellowing disease of Ageratum conyzoides in Singapore was shown to be caused by a geminivirus, here named ageratum yellow vein virus (AYVV), which was transmitted by the whitefly Bemisia tabaci but not by inoculation with sap or through seed. AYVV particles (30 × 20 nm) are serologically related to those of other whitefly-transmitted geminiviruses, and reacted with some monoclonal antibodies elicited by particles of African cassava mosaic or Indian cassava mosaic geminiviruses. However, the epitope profile of AYVV differed from the profiles of these viruses, and from those of geminiviruses from vein yellowing-affected A. conyzoides from India and from yellow leaf curl-affected tomato from either Singapore or India. The results provide further evidence of antigenic differences among geminiviruses that cause similar diseases in the same plant species in different geographical regions.