Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.     

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.     

Detection of plum pox virus by isolation of double-stranded ribonucleic acid (dsRNA)

Double-stranded RNA (dsRNA) associated with plum pox virus (PPV) in Nicotiana clevelandii and Prunus domestica has been isolated. While dsRNA was detected in N. clevelandii in considerable amounts by electrophoresis, only small amounts were found in P. domestica. This may be due to viscous substances in the leaves of this woody host. Different PPV strains (NAT - not aphid-transmissible; AT - aphid-transmissible) showed specific patterns in electrophoresis gels. When PPV was assayed in N. clevelandii by dsRNA detection or by standard ELISA or ISEM, all three methods were found to be efficient, with none being superior. ELISA, as a simple and fast routine method, is still the method of choice. DsRNA detection will be suitable for plant disease agents undetectable by ELISA and ISEM.     

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.     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats

ABSTRACT Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.     

Detection of Potyviral Nuclear Inclusion b Proteins by Monoclonal Antibodies Raised to Synthetic Peptides

Potyviral nuclear inclusion b protein (NIb), the RNA-dependent RNA polymerase, contains three highly conserved regions. Peptides corresponding to these regions were synthesised and used for immunisation. A panel of monoclonal antibodies was obtained. Most of the MAbs reacted with the peptides and a recombinant NIb of PVY in PTA-ELISA. Two of them specifically detected native NIb of potato A, potato V, potato Y, plum pox and turnip mosaic potyviruses in extracts of infected plants in Western blots. Time course experiments revealed that NIb protein can be first detected on the fifth day after infection.     

Differences in cultivar response and complete sequence analysis of two isolates of wheat yellow mosaic bymovirus in China

Seeds of selected European and Japanese winter wheat cultivars were grown at two experimental sites in China, namely Yaan, Sichuan province (YA), and Yangzhou, Jiangsu province (YZ), where wheat yellow mosaic bymovirus (WYMV) was severe. There were some differential responses of the cultivars to the virus isolates present at the two sites. The complete nucleotide sequence of both RNAs of both virus isolates was determined. Their genome organization was identical to that reported for a Japanese isolate and the sizes were very similar. Nucleotide comparisons demonstrated that parts of the CI and NIa coding regions on RNA1 and the N-terminal part of the P2 coding region on RNA2 were particularly variable, while substantially conserved regions occurred in the 3' UTR of RNA2, the 7K, one part of the CI and parts of the NIb and coat protein. It seems unlikely that differences in the 7K and NIa-VPg proteins are responsible for virulence differences and the CI and NIb regions were considered the most promising for further study.     

A Natural Population of Recombinant Plum Pox Virus is Viable and Competitive under Field Conditions

The isolate BOR-3, collected in Slovakia in 1996, was recently identified as a natural recombinant between an M and D type of Plum pox virus (PPV). Biological assays demonstrated its capacity to be aphid- and graft-transmitted to various Prunus spp. hosts. A study was carried out to determine the further presence of PPV recombinants in two epidemiologically distinct areas – Slovakia and France. Tools based on PPV-M and D subgroup typing, targeting P3–6K₁, CI and CP regions of the PPV genome were used for recombinant identification. Closely related recombinant variants were detected in different Prunus spp. during a survey conducted in Slovakia in 2001, but not within a set of selected PPV isolates from France collected between 1985 and 2001. Sequence analysis of the (Cter)NIb–(Nter)CP region of 10 recombinant isolates from Slovakia showed their high homology, reaching more than 98%. All the recombinant isolates shared the same recombination breakpoint situated in the C terminus of the NIb gene. Our study demonstrates that the PPV recombinants are viable and competitive with conventional PPV-M and D isolates. The present work indicates that the occurrence of recombinants within PPV isolates might be more common than previously assumed.     

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.     

A potyvirus isolated from Senna occidentalis

A potyvirus causing severe mosaic symptoms was isolated from Senna occidentalis (syn. Cassia occidentalis ) in the Yemen Republic and Ethiopia. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. The flexuous, rod-shaped particles had a mean length of 830 nm, and pinwheels and scrolls were observed by electron microscopy of thin sections of infected Nicotiana clevelandii leaves. Its host range was narrow with only a few legume species, Nicotiana clevelandii and N. benthamiana susceptible to experimental infection. This virus was purified from N. clevelandii and the coat protein had a molecular mass of 34-5 kDa. It reacted positively in ELISA with monoclonal antibody 197 that is specific for potyviruses, but was not decorated by antibodies to any other potyvirus tested when examined by electron microscopy. The virus has been tentatively named cassia severe mosaic potyvirus.     

白背飞虱VAMP7和Vti1a蛋白的原核表达、抗体制备及应用

白背飞虱Sogatella furcifera的囊泡相关膜蛋白7(VAMP7)和囊泡转运蛋白(Vti1a)隶属于SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor)家族,该家族蛋白主要参与生物体中关键的膜转运过程。前期研究发现这两种蛋白分别与南方水稻黑条矮缩病毒Southern rice black-streaked dwarf virus(SRBSDV)的主要外层衣壳蛋白P10存在显著互作,推测可能协助病毒粒体在介体白背飞虱内的转运和扩散。为了进一步利用血清学技术研究VAMP7和Vti1a在传毒过程中的功能,本研究克隆了白背飞虱编码这两种蛋白的基因,成功构建了VAMP7和Vti1a基因的原核表达载体,并将载体分别转入大肠杆菌中进行诱导表达,得到了相应的原核表达蛋白。在蛋白纯化后,将纯化蛋白注射于新西兰大白兔体内进行免疫,分别制备得到VAMP7和Vti1a的抗体。两种抗体经Western blot检测发现,均可分别与白背飞虱体内的VAMP7和Vti1a特异性结合。利用制备的抗体对白背飞虱的肠道进行免疫标记,激光共聚焦显微镜观察发现所制备抗体能够在白背飞虱中肠上皮细胞的胞质中特异性标记到VAMP7和Vti1a,表明制备的抗体能够成功用于这两种蛋白的体内外检测,为阐明这两种蛋白参与传播SRBSDV的机制研究奠定了基础。     

Molecular,biological and serological variability of Zucchini yellow mosaic virus in Tunisia

A study was conducted to better understand the population structure of Zucchini yellow mosaic virus (ZYMV), a severe virus affecting cucurbit crops worldwide, in Tunisia and to estimate whether the use of resistant cultivars may provide durable control. Analysis of the polymerase and coat protein (NIb‐CP) partial sequences of 83 isolates collected in the three main cucurbit‐growing areas in Tunisia showed that ZYMV grouped into two distinct clusters within ZYMV molecular group A. An important variability was observed in the MREK motif of the P3 protein, a motif associated with tolerance breaking in ZYMV‐tolerant zucchini squash cultivars. Interestingly, significant differences were found in the distribution of the MREK variants in the two clusters defined by the partial NIb‐CP sequences, MREK and MKEK sequences being more common in cluster 1 and cluster 2, respectively. When combining NIb‐CP and P3 sequence information, ZYMV molecular variability was shown to be significantly higher in the Cap Bon region than in the Bizerte area. An important biological variability was observed in a subset of 23 isolates regarding symptomatology in susceptible or resistant cucurbits. Some isolates overcame ZYMV tolerance or resistance in zucchini squash and melon, but not in cucumber. Three serotypes were differentiated using a set of 13 monoclonal antibodies (MAbs). Seven parameters characterizing the 23 isolates, including molecular, serological and biological properties, were used for a multiple component analysis (MCA). This analysis revealed that symptom intensity of a given isolate was similar in different susceptible cucurbit hosts, suggesting similar degrees of aggressiveness in different hosts.     

The ultrastructure of germinating conidia of Penicillium digitatum inhibited by sec-butylamine

Electron micrographs of germinating conidia of P. digitatum showed osmiophilic inclusions scattered in the ground plasm or located within membrane-bound vacuoles where they were associated with various membranes and “myelin-like” structures. Cytochemical studies suggested that the osmiophilic inclusions within the vacuoles contained phospholipids and proteins. These phospholipoprotein inclusions may provide precursors needed for the assembly of membrane-bound organelles formed during germination. In conidia germinated in the presence of fungistatic concentrations of sec-butylamine (SBA) the vacuoles enlarged and the phospholipoprotein inclusions decreased in size and appeared to disintegrate and give rise to membrane-bound vesicles. SBA may deprive the conidium of materials normally utilized for membrane formation by the developing organelles.     

水稻条纹叶枯病细胞病理变化的观察

 利用透射电子显微镜和免疫胶体金标记技术,观察水稻条纹叶枯病病叶细胞病理变化,病毒粒体、病毒外壳蛋白和病害特异蛋白在寄主细胞中的分布。结果表明,病叶叶肉细胞中线粒体增多,细胞核变大,几乎每个细胞中都有蛋白体,叶绿体基粒片层变稀疏和变粗,或被挤压和变细,或叶绿体解体,基粒片层残留于细胞质中。在叶肉细胞和维管束筛管细胞中发现一些内含体和电子密度较高的物体,在高放大倍数下,可以清楚的看出为点状和丝状结构,其直径约为8nm。免疫胶体金标记显示,病毒外壳蛋白和病害特异蛋白存在于叶绿体、细胞质和细胞核以及细胞壁上,但线粒体中没有发现。     

Expression of the mature form of plum pox potyvirus helper component1

Polyclonal antibodies raised to the plum pox potyvirus (PPV) helper component (HC) have been produced for the specific detection of HC protein in PPV-infected plants. These experiments suggested that PPV HC is a single soluble protein of 48 kDa. The independent expression of the first two and half cistrons of the PPV genome in transgenic plants suggests that the P1 and P2 (HC-Pro) proteases are involved in the processing of the mature form of HC.     

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic -(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.     

Light and electron microscopy of red clover vein mosaic virus in pea (Pisum sativum)

Epidermal strips of pea infected with the RK31 isolate of the red clover vein mosaic virus were found to contain many diagnostic crystalline inclusions, recognizable two, but more clearly three weeks after inoculation. They could be easily detected by light microscopy, even at low magnification, when stained with phloxine and methylene blue, but more rapidly with a phase-contrast microscope in unstained preparations in water. They were always found after the onset of external symptoms. With the pea streak strain (P42) they were usually absent. In ultrathin sections made 22 days after inoculation or later, virus distribution and accumulation were comparable to those of other members of the carlavirus group. Sometimes extensive irregular bundles were observed. These were distinct from the crystals seen by light microscopy. Sections of the three-dimensional crystals studied in the electron microscope showed a very regular internal striation with a periodicity of about 11 nm, or were sometimes composed of spherical particles in looser array. When crystals were isolated intact and stained negatively, the majority of the material appeared to consist of spherical or polyhedral particles 11–12 nm in diameter.