Unravelling the Prunus/Plum pox virus interactions

Plum pox virus (PPV) belongs to the genus Potyvirus that contains the largest number of virus species infecting plants. Its virus genome has been extensively characterised and sequenced. However, few data are available on its interactions with woody host plants. We therefore focused, in the past 4 years, both on the cellular and molecular aspects of the compatible and incompatible Prunus /PPV interactions. GFP (Green fluorescent protein)-tagged PPV and in situ hybridisation were used to compare the localization of viral particles in stems and leaves of susceptible and resistant apricot cultivars. In parallel, molecular tools were developed through the cloning and characterization of polymorphic, homologous resistance genes and of candidate genes involved in the expression of Prunus /PPV interactions. Candidate genes are currently used to target genomic regions involved in resistance or susceptibility and to identify molecular markers indispensable for further marker assisted selection for resistance to sharka disease.     

The use of Plum pox virus as a plant expression vector

Among the biotechnological uses of plant viruses, the expression of foreign sequences through virus-based vectors represents a promising research area. The potyvirus Plum pox virus (PPV) has been used to design expression vectors which have allowed successful expression of foreign sequences in plants, either in the form of small peptides fused to the viral coat protein, or as whole independent proteins inserted on different points of the genome. The present review describes the different PPV-based vectors that have been produced, including information regarding relevant aspects of their use, such as the optimal location of peptides or the stability of inserts. Recent developments, like the expression of proteins on stone fruit trees by using PPV-based vectors capable to infect woody plants, are also described.     

Advances and prospects in potato virology with special reference to virus resistance

Knowledge of the nucleotide sequences in the genomic nucleic acid of several potato viruses has enabled the open reading frames to be identified. These open reading frames are expressed by a variety of strategies, to produce proteins with functions in virus nucleic acid replication, virus particle production, cell-to-cell transport of virus and virus transmission by vectors. The activity of such proteins depends on their interactions with other viral or non-viral materials.Several other biological properties of plant viruses can also be related to individual viral gene products. For example, in plants co-infected with a specific pair of unrelated viruses, one virus can benefit from an ability to use the gene product of the second virus in replication, cell-to-cell transport or transmission by vectors. Similarly, different host resistance genes are targeted against viral replicase, movement protein or coat protein. Thus it is becoming possible to relate gene-for-gene (or more accurately, viral gene domain-host gene) interactions to events at the molecular level. Genetically engineered resistance to plant viruses likewise can be targeted against individual viral genes, and probably also against viral regulatory sequences. Such transgenic resistance seems likely to be as durable as conventional host resistance but durability should be improved by producing plants with combinations of resistances of different kinds, either conventional or genetically engineered, or both.     

Post-translational modifications in effectors and plant proteins involved in host–pathogen conflicts

Molecular interplay between two species is largely driven by protein–protein interactions and protein modifications that set the pace of co-evolution in these species. During host–pathogen interactions, proteins involved in virulence and defence impart tempospatial dynamic post-translational modifications (PTMs) to gain advantage for the causative species. Pathogens mainly cause disease in plant hosts by secreting elicitors (peptides and small molecules) or proteins in the inter- and intracellular space of host cells. These pathogen proteins have evolved a wide array of sophisticated mechanisms to manipulate host responses, including resistance. Through a set of diverse events ranging from PTMs to post-translational oligomerization, these proteins are able to enhance virulence and suppress the otherwise elaborate plant immune system. Similarly, PTMs adapted by host proteins often lead to the activation of a robust defence response. Insights into the PTMs of pathogen and host proteins are therefore germane to the understanding of the co-evolutionary arms race. This review summarizes the characterization of PTMs in pathogen effectors and their target host proteins. Based on this, a metaphorical view of host–pathogen conflicts is proposed, where PTMs act as molecular pivots in a 3D combinatorial game model – a novel abstraction of the arms race, where these molecular pivots restore the balance of competition between the two organisms.     

TuMV management for brassica crops through host resistance: retrospect and prospects

Turnip mosaic virus (TuMV), the only potyvirus known to infect brassicas, is a devastating virus threatening many economically important brassica crops, including cabbage, Chinese cabbage, oilseed rape and mustard. TuMV disease, which was first discovered in the United States, is now found worldwide, especially in Europe, Asia and North America. TuMV results in a yield loss of up to 70% and has a wide host range, infecting most cruciferous plants, as well as many non-cruciferous species. This virus is also characterized by high pathotype diversity because of its highly variable genome structure and has been divided into 12 pathotypes. These characteristics, as well as its nonpersistent transmission mode by as many as 89 aphid species, mean the disease is difficult to prevent through traditional methods such as the application of chemicals, prompting researchers to seek host resistance for effective control. During the last decade, extensive studies have been conducted to investigate inheritance, mapping and cloning of the TuMV resistance genes, and several NB-LRR- or eIF-encoding loci with divergent molecular mechanisms have been uncovered. These studies have greatly facilitated resistance breeding for brassica crops and have advanced our understanding of virus−host interactions.     

An infectious cDNA clone of a radish-infecting <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> strain

Turnip Mosaic Virus (TuMV) is an economically important potyvirus for which hundreds of hosts have been reported, thus making it a rather exceptional case in the genus. Several viral infectious clones have been generated over the years, which have been useful in deciphering the viral elements involved in the interactions of this virus with the host plant, such as different forms of resistance, gene silencing suppression, host range or host developmental alterations. However, all infectious clones obtained so far correspond to viral isolates within the same phylogenetic cluster, a circumstance biasing our understanding of the peculiarities of this potyvirus. In particular, members of one viral cluster of radish-infecting isolates have been especially reluctant to be copied into infectious clones. This paper reports the construction of an infectious clone of the TuMV isolate JPN 1, belonging to this cluster. The infectious clone maintains all the distinctive biological properties previously described for this viral isolate. The availability of this infectious clone opens the door to many additional studies on the virus, which should allow a deeper understanding of the differential responses to different strains of TuMV in several different hosts.     

Hosts and symptoms of Plum pox virus: Herbaceous hosts

Plum pox virus (PPV) is polyphagous and epidemic. Apart from cultivated and wild Prunus species, a large number of herbaceous plants can be hosts of the virus. New herbaceous host species are continuously being reported following artificial inoculation studies. Some of these herbaceous hosts, Chenopodium foetidum , Nicotiana clevelandii , N. benthamiana and Pisum sativum are very useful for concentrating and purifying the virus. The list of plants that have been found to be infected with PPV in their natural environment is shorter than the list of plants which can be experimentally infected. The role of weed species in PPV survival and spread in orchards is poorly understood. It is widely accepted that annual plants or weeds are not important in the epidemiology of PPV.     

Hosts and symptoms of Plum pox virus: woody species other than fruit and ornamental species of Prunus

The presence of Plum pox virus (PPV) in woody hosts other than in fruit and ornamental Prunus species was evaluated. PPV symptoms, and their variability and intensity, are described in leaves and fruits of Prunus cerasifera , Prunus spinosa , Ligustrum vulgare and Euonymus europaea . Juglans regia is not confirmed as a new host of PPV.     

葡萄浆果内坏死病毒侵染的‘贝达'葡萄cDNA文库构建及其利用

 葡萄浆果内坏死病毒(grapevine berry inner necrosis virus, GINV)侵染葡萄可引起葡萄叶片表现明显的褪绿斑驳症状,其致病的分子机制,特别是病毒与寄主互作蛋白的研究尚未见报道。本研究构建了GINV侵染的‘贝达'葡萄叶片的cDNA文库,并以GINV 外壳蛋白(coat protein,CP)为诱饵筛选文库中与其互作的寄主因子。本研究构建的cDNA文库库容量达1.6×10⁷,插入片段平均大小在1.0 kb以上,质量符合cDNA文库筛库要求。筛选到了55个候选寄主蛋白与GINV CP在酵母中互作。经测序分析及回转验证,结果确定了17个寄主互作蛋白,其涉及叶绿体相关类蛋白、泛素化相关蛋白、防御相关蛋白等。本研究可为深入研究GINV致病性及其与寄主互作机制提供依据。     

荧光蛋白mNeonGreen、mKOk作为FRET传能对在研究植物蛋白质互作中的应用分析

蛋白质相互作用是蛋白质组学研究的重要方向之一,对解析植物病原微生物与寄主植物之间的博弈具有十分重要的意义。荧光共振能量转移(fluorescence resonance energy transfer,FRET)作为一种新技术,能够在活细胞的正常生理条件下进行检测,并具有灵敏度高,假阳性较低,实验周期短的优势,在研究蛋白质相互作用过程中得到了广泛的应用。本研究选取了2种新型的荧光蛋白mNeonGreen/mKOk作为FRET传能对,并与传统的传能对CFP/YFP相比较,证实mNeonGreen/mKOk背景干扰较低。通过构建并表达荧光蛋白与目标蛋白的融合蛋白,利用FRET技术成功地在植物细胞中验证了蛋白质间的相互作用。本研究为FRET技术在植物细胞中的应用提供了更多的技术支持和借鉴。     

Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees

ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.     

Hosts and symptoms of Plum pox virus: ornamental and wild Prunus species

Several ornamental and wild Prunus species have been identified as natural and/or experimental hosts for Plum pox virus (PPV). The significance of natural vs. experimental hosts, graft or bud-transmitted infections vs. aphid-transmitted infections in ornamental or wild Prunus hosts, and their relevance in the field situation, are not clearly understood. However, since PPV is aphid-transmitted, any host in the field or nursery serves as a potential reservoir and source of inoculum and must be monitored and controlled in any PPV eradication or management programme.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Control and monitoring: quarantine situation of Plum pox virus in New Zealand

Biosecurity New Zealand recognizes that Plum pox virus (PPV) is one of the most destructive diseases of stone fruit and aims to keep New Zealand free from it. PPV has never been detected in New Zealand orchards during national surveys. but it has been detected in post-entry quarantine. Biosecurity New Zealand has analysed the potential pathways for PPV to enter the country, and developed phytosanitary regulations and prevention control measures to reduce the risks. A contingency plan, a response programme, and diagnostic protocols have been developed and simulation exercises based on a hypothetical PPV incursion have been carried out.     

昆虫与弹状病毒互作的研究进展

近年来宏基因组学研究的普及大大丰富了人们对RNA病毒多样性的认识,但对这些新发现病毒的生物学特性却所知甚少。本文围绕RNA病毒中一类重要的负单链RNA病毒--弹状病毒与其昆虫寄主互作的研究进行综述,总结已发现的弹状病毒及其昆虫寄主类型,共有20个属144种弹状病毒可以感染14个属的昆虫;根据已有的系统进化研究对弹状病毒的寄主起源进行推测;并以感染黑腹果蝇Drosophila melanogaster的sigma病毒(Drosophila melanogaster sigma virus,DMelSV)为主要对象,就弹状病毒引起的CO2麻痹致死症状以及昆虫寄主对其的免疫反应研究进行总结,而在对黑腹果蝇的研究中发现很多非经典免疫通路中的新抗病毒基因,暗示存在新的抗病毒免疫通路;通过飞虱、叶蝉与其传播的植物弹状病毒以及长须罗蛉Lutzomyia longipalpis与其传播的脊椎动物病毒的互作研究,发现Toll、IMD信号通路、细胞自噬及小RNA干扰(small interfering RNA,siRNA)通路等可能与昆虫对弹状病毒的免疫反应相关。昆虫是弹状病毒主要的寄主和媒介,也是病毒遗传多样性的储主,因此更好地研究和了解昆虫寄主与弹状病毒的相互关系,有助于病毒致病和传播机制以及昆虫抗病毒免疫机理的深入研究。     

Detection and characterization of Plum pox virus: serological methods

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.     

The coat proteins and putative movement proteins of isolates of Prunus necrotic ringspot virus from different host species and geographic origins are extensively conserved

A complete sequence for the RNA 3 of Prunus necrotic ringspot virus (PNRSV) is described (Genbank Accession U57046). Primers from this sequence were used to amplify both the movement protein and coat protein genes of 3 other isolates of PNRSV originating from different host species and geographic locations. Comparisons of these sequences with those of other published sequences for PNRSV and the closely related apple mosaic virus (ApMV) showed that both the movement proteins and coat proteins of isolates of PNRSV are extensively conserved irrespective of either the original host or the geographic origin. The movement protein and coat protein of ApMV and PNRSV are sufficiently conserved to suggest that these two viruses may have evolved from a common ancestor. The amino acid sequence of the two coat proteins shows areas of similarity and difference that would explain the serological continuum reported to occur among isolates of these two viruses. Nevertheless, the movement protein and coat protein of the two viruses are sufficiently different so that ApMV and PNRSV should be considered to be distinct viruses.