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Western blot analysis of zucchini squash plants infected with ZYMV infectious clones, using the polyclonal anti- body to ZYMV (Lin et al., 1998). Extracts (5 μl) from equal amounts of leaf tissue (0.05 g /500 μl) collected at 7 days after inoculation (0.5 cm diameter of disk from three different leaves ground in 500 μl extraction buffer) were loaded in each lane. Lane 1, leaf extract from a plant infected with ZYMV TW-TN3; Lanes 2 and 3, leaf extracts from plants infected with in vitro transcript of pT7ZYMV2-5 by mechanical inoculation and infected by the plasmid of p35SZYMV2-26 by particle bombardment, respectively; Lane 4, an uninfected plant as control; M, protein markers.
Source publication
The full-length cDNA of a Taiwan strain of Zucchini yellow mosaic virus (ZYMV TW-TN3) was con- structed from five overlapping cDNA clones downstream from the bacteriophage T7 promoter in plasmid pT7ZYMV2- 5. The plasmid was able to generate an in vitro transcript corresponding to TW-TN3 (9591 nt) with one extra guanosine residue at the 5´ terminus...
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... while the uninfected control or plants inoculated with Papaya ringspot virus (PRSV) showed nega- tive reactions. By immunoblotting analysis, a 33 kDa protein corresponding to the CP of ZYMV was detected in the plants infected by the in vitro transcripts derived from pT7ZYMV2-5 or in vivo transcripts derived from p35ZYMV2-26. The quantity of the CP in plants infected with both of them was similar to that infected with wild- type ZYMV ( Figure 4). To confirm that the development of symptoms in zucchini squash plants was induced by p35SZYMV2-26, the crude sap from leaf tissues of infected squash plants was examined by electron microscopy. Numerous filamentous virus particles of 750 nm in length were observed in the samples from infected plants (Figure 5A). When the crude sap was further analyzed by immunogold labeling, the gold particles were specifically located along the entire length of the virions decorated by ZYMV CP antiserum (Figure 5B). Similarly, filamentous particles decorated by gold particles were also found in the samples infected with capped in vitro transcripts derived from pT7ZYMV2-5. In this investigation, full-length cDNA clones of ZYMV TW-TN3 were constructed downstream from a bacteriophage T7 RNA polymerase promoter or a CaMV 35S promoter. The transcripts produced either by in vitro transcription driven by the T7 promoter or by in vivo transcription derived from the 35S promoter were proven infectious when introduced to the systemic host zucchini squash and the local lesion host C. quinoa. In addition, ELISA, western blotting, and electron microscopy confirmed that the arti- ficial viruses created from both in vitro and in vivo transcripts were similar to the native ZYMV. Typical symptoms of ZYMV infection appeared on plants of zucchini squash and C. quinoa inoculated with infectious in vitro and in vivo transcripts of ZYMV at about the same time as those induced by native RNA or virus particles of ZYMV. These results are similar to the infectivity assay of in vitro and in vivo infectious transcripts of PRSV (Chiang and Yeh, 1997). However, our results are different from those induced by the other six infectious potyviral transcripts, which have a slight lag of 1 to 13 days for symptom development following mechanical inoculation (Domier et al., 1989; Riechmann et al., 1990; Gal-On et al., 1991; Dolja et al., 1992; Jakab et al., 1997; Sanchez et al., 1998; Yang et al., 1998). The infectivity of a number of viral 35S-cDNA clones by manual inoculation onto plants has been reported, including Pea early browning viru s (PEBV, Tobravirus ) (MacFarlane et al., 1992); Tobacco mosaic virus L strain (ToMV-L, Tobamovirus ) (Weber et al., 1992); Plum poxvi- rus (PPV, Potyviridae ) (Maiss et al., 1992); Cowpea mosaic virus (CPMV, Comoviridae ) (Dessens and Lomonossoff, 1993); Alfalfa mosaic virus (AlMV, Bromoviridae ) (Neeleman et al., 1993); Cucumber mosaic virus (CMV, Bromoviridae ) (Ding et al., 1995); ZYMV ( Potyviridae ) (Gal-On et al., 1995); and PRSV ( Potyviridae ) (Chiang and Yeh, 1997). However, low infectivity following the mechanical inoculation of plants using intact plasmid was noticed (Neeleman et al., 1993; Ding et al., ...
Citations
... The presence of potyviruses, including ZYMV, has been associated with the formation of cylindrical inclusions, which serve as diagnostic markers for group-level infections (Lisa et al., 1981, Shukla and Ward 1989, Vega et al., 1995, Abdel-Ghaffar et al., 1998. Additionally, electron microscopy studies have identified filamentous flexuous particles in partially purified ZYMV preparations (Lin et al., 2002, Pospieszny et al., 2003. ...
... The ZYMV-cp gene was amplified by reverse transcripti on -polym erase chain reaction (RT -PCR), sequenced and analyzed compared to those strains or isolates documented in GenBank (Usher et al., 2012, Romay et al., 2014, Khalifa et al., 2015, Spadotti et al., 2015, Nasr-Eldin et al., 2016, Ali et al., 2023 in which identities ranging from 82 to 99% were obtained. The molecular weight of ZYMV-CP among different strains ranged from 34 to 36 kDa (Lisa et al., 1981, Lin et al., 2002. ...
In this study, the focus was on the bioinformatics analysis of the coat protein (cp) gene of a specific Egyptian isolate of Zucchini yellow mosaic virus (ZYMV), which was identified based on both biological and molecular characteristics. The presence of ZYMV was confirmed in 27 squash samples using DAS-ELISA and RT-PCR techniques employing ZYU-F and ZYD1186-R primers that flank the cp gene of ZYMV. Transmission experiments involving mechanical and aphid transmission, specifically through Aphis gossypii, were carried out on various hosts, including Chenopodium amaranticolor, which served as an indicator necrotic local lesion host. Using light and electron microscopies, amorphous and cylindrical inclusions (pinwheels and scrolls) were observed, respectively. A partially purified virus preparation of ZYMV was obtained from infected squash leaves using a protocol involving polyethylene glycol purification and ultracentrifugation, revealing the presence of filamentous viral particles that were negatively stained with 2% uranyl acetate. Subsequently, the ZYMV-cp gene was amplified via RT-PCR for sequencing following the purification of PCR products from the agarose gel. The nucleotide sequences obtained, consisting of 831 base pairs, and encoding a 236 amino acid open reading frame, were deposited under the ID: LC778450.1 as the ZYMV-Shrouk-23 strain in GenBank. Bioinformatics analyses were conducted, comparing the Egyptian isolate to 25 overseas isolates of ZYMV. The resulting phylogenetic analysis dendrogram revealed a distinct clade with identities ranging from 91.94% to 99.16% based on nucleotide sequences and from 96.19% to 100.00% based on deduced amino acid sequences.
... The GFP fluorescent spots of a non-HR infection were generally less intense than those of the WT ZG infection (Figure 1a Table S1) containing 30 bp overlap were used to combine fragments by overlapping extension PCR with primer pair P-ZYMV--139/M-ZYMV-1413 to obtain F1 fragment without GFP gene (Table S1). Other genome fragments of F2, F3, and F4 were amplified using corresponding primers (F2 fragment: P-ZYMV-EGFP/M-ZYMV-5262; F3: P-ZYMV-3531/M-ZYMV-7597; F4: P-ZYMV-7001/18T-NotI, as listed in Table S1) and ligated to the backbone of p35SZYMV2-26 (Lin et al., 2002) with suitable restriction sites as shown in the diagram. (Yeh & Gonsalves, 1984), while seven PVY attenuated mutants were identified after transferring 168 local lesions (Ogawa et al., 2013). ...
... Then the overlapping fragments were amplified by PCR using primers P-ZYMV-139/M-ZYMV-1413, while other parts of the mutant genome were amplified using primers P-ZYMV-EGFP/M-ZYMV-5262, P-ZYMV-3531/M-ZYMV-7597, and P-ZYMV-7001/18 T-NotI (Figure 6a and Table S1). After treating individual fragments with corresponding restriction enzymes NheI-PacI, PacI-AgeI, AgeI-StuI, or StuI-NotI, each was used to replace the corresponding WT sequence of p35SZYMV2-26 (Lin et al., 2002) to generate the infectious clone of pZ4-10. The construct of pZ4-10 was introduced to C. quinoa and squash plants for infectivity assay. ...
... PCR product of 1137 bp represents protective attenuated ZYMV mutants, while the product of 423 bp represents challenged virus TW-TN3 without GFP gene. TW-TN3(Lin et al., 2002) without GFP and ZG(Hsu et al., 2004) with GFP were used as controls. (c) Bioassay of protection effectiveness on Chenopodium quinoa leaves. ...
Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%-100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.
... However, the construction of infectious clones for Potyviruses is particularly challenging due to the presence of special viral sequences that have a toxic effect on Escherichia coli (Chikh Ali et al., 2011;Johansen and Lund, 2008); consequently, only a few reports have described the construction of such clones. The successful generation of infectious clones for Potyviruses has now been described for tobacco vein mottling virus (TVMV) (Domier et al., 1989;Nicolas et al., 1997), zucchini yellow mosaic virus (ZYMV) (Gal-On et al., 1991;Lin et al., 2002), tobacco etch virus (TEV) (Bedoya and Daròs, 2010;Dolja et al., 1992), peanut stripe virus (PStV) (Flasinski et al., 1996), potato virus A (PVA) (Puurand et al., 1996;Rajamäki and Valkonen, 2009), papaya ringspot virus (PRSV) (Chiang and Yeh, 1997), potato virus Y (PVY) (Chikh Ali et al., 2011;Jakab et al., 1997), turnip mosaic virus (TuMV) (Sánchez et al., 1998), johnsongrass mosaic virus (JGMV) (Kim et al., 2003), PMV (Lee et al., 2011), bean common mosaic virus (BCMV) (Naderpour and Johansen, 2011), and maize dwarf mosaic virus (MDMV) (Stewart et al., 2012). Although an infectious clone was successfully constructed for SMV in 1996 (Seo et al., 2009), the construction method was time-consuming and laborious. ...
Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the crossfamily infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.
... Comparisons of the original clones of BR1, BR2, and BR3 showed that only two nt in the 3′ UTR differentiated the three isolates (G9395A in BR2, and T9537C in BR3 compared to the consensus); it is unlikely that these changes explain the differences in infectivity, whereas the importance of a longer poly(A) tract has been established for several viral systems. Substitution of a 66 nt poly(A) for the initial 48 nt was reported for the first infectious ZYMV clone, but both were infectious by mechanical inoculation of capped RNA transcripts (Gal-On et al. 1991); a Taiwanese infectious ZYMV clone had a 95 residue poly(A) tail (Lin et al. 2002). In comparison, a tobacco vein mottling virus clone with only 12 A residues was not infectious, but infectivity was observed with 37 nt, and much higher infectivity with a 96 nt poly(A) tract (Domier et al. 1989); the first infectious clone of plum pox virus had approximately 100 A residues (Riechmann et al. 1990). ...
The incidence of plant virus diseases infecting important cucurbit vegetables in Korea has increased as new isolates have been introduced, associated with warming temperatures and vector movement caused by climate change. Transcript infectivity of full-length infectious clones of three new ZYMV isolates was dependent upon the length of the poly(A) tract; transcripts with 55 A residues were inefficiently infectious, whereas 60 A residues resulted in highly efficient infection and significantly reduced time to production of systemic symptoms. Sequences of isolates BR1 (MH042024), BR2 (MH042025), and BR3 (MH042026) showed 99% pair-wise identity and differed at only five amino acid positions in: HC-Pro (D134N in BR2), CI (F31 L in BR1), and 6 K2 (A24V in BR3), and two positions in NIb (T300S in BR2, H429Q in BR3). Cucurbita pepo plants inoculated with transcripts of clones with these amino acid differences showed symptoms that ranged from mild to severe. Phylogenetic analysis of these new ZYMV isolates with previously characterized isolates indicated that the new isolates had 87.8–97.5% identity to other ZYMV isolates and were most closely related to recent ZYMV isolates from Australia and Spain.
... In contrast, the CaMV 35S promoter is usually used with the in vivo infectious clone and the promoter is recognized by host RNA polymerase II (pol II) in the plant nucleus to transcribe viral RNA in vivo. (Angenent et al., 1989;Heaton et al., 1989;Janda et al., 1987;Lin et al., 2002;Melton et al., 1984). Once the viral RNA is transcribed from DNA, the virus life cycle is initiated, and consequent infectivity is established. ...
... The RNase contamination specifically indicated that the operation with the in vitro transcript that was exposed to an RNase-rich environment might damage the viral RNA during the inoculation. In a previous study, plants of Chenopodium quinoa were inoculated with a DNA form of an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) by mechanical inoculation for local lesion formation, and the generated virus was then transferred from a C. quinoa plant to a zucchini squash plant (Lin et al., 2002). It has been reported that the host characteristics of squash cannot cause initial infectivity by direct mechanical inoculation with the in vivo infectious clone of ZYMV (Lin et al., 2002). ...
... In a previous study, plants of Chenopodium quinoa were inoculated with a DNA form of an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) by mechanical inoculation for local lesion formation, and the generated virus was then transferred from a C. quinoa plant to a zucchini squash plant (Lin et al., 2002). It has been reported that the host characteristics of squash cannot cause initial infectivity by direct mechanical inoculation with the in vivo infectious clone of ZYMV (Lin et al., 2002). The C. quinoa plants can be directly inoculated with the DNA of an in vivo infectious clone, with high efficiency of the infectivity and without the risk of RNase contamination. ...
The in vivo infectious clone of Turnip mosaic virus (TuMV), p35S-TuMV, was used on plant pathology research for many years. To activate p35S-TuMV, the plasmid was mechanically introduced to the local lesion host Chenopodium quinoa. However, low infectivity occurred when the TuMV from C. quinoa was transferred to the systemic host Nicotiana benthamiana.
To increase the efficiency of initial infectivity on N. benthamiana, the expression of the TuMV infectious clone by a binary vector that directly activates viral RNA through agro-infiltration is considered to be a good alternative. The size of the binary vector by agro-infiltration is usually large and its backbone has numerous restriction sites that increase difficulty for construction. In this study, we attempted to construct a mini binary vector (pBD003) with less restriction sites. The full-length cDNA of TuMV genome, with or without green fluorescence protein, was inserted in pBD003 to generate pBD-TuMV constructs, which were then individually introduced to N. benthamiana plants by agro-infiltration. Symptom development and ELISA positivity with TuMV antiserum indicated that the pBD-TuMV constructs are infectious. Moreover, the initial infectivity of a mild strain TuMV-GK, which contains an R182K mutation on HC-Pro, constructed in the pBD003 vector was significantly increased by agro-infiltration.
Thus, we concluded that the newly constructed mini binary vector provides a more feasible tool for TuMV researches in areas, such as creating a mild strain for cross-protection, or a viral vector for foreign gene expression. In addition, the multiple cloning sites will be further cloned in pBD003 for convenience in constructing other viral infectious clones.
... For all constructs using the ZYMV viral vector, p35SZYMVNIbMCS was used as the template (Lin et al., 2002). For N-or C-terminal c-Myc-tagged proteins, the ZYMV vector was re-engineered using the primers listed in Table S1 as described in Methods S1. ...
Cucurbita moschata FLOWERING LOCUS T-LIKE 2 (hereafter FTL2) and Arabidopsis thaliana (Arabidopsis) FT, components of the plant florigenic signaling system, move long-distance through the phloem from source leaves to the vegetative apex where they mediate floral induction. The mechanisms involved in long-distance trafficking of FT/FTL2 remains to be elucidated. In this study, we found the critical motifs on both FT and FTL2 required for cell-to-cell trafficking through mutant analyses using a Zucchini yellow mosaic virus (ZYMV) expression vector. Western blot analysis, carried out on phloem sap collected from just beneath the vegetative apex of C. moschata plants, established that all mutants tested had retained the capacity to enter the phloem translocation stream. In contrast, immunolocalization studies revealed that a number of these FTL2/FT mutants were defective in the post-phloem zone, suggesting that regulation mechanism for FT trafficking exists in post-phloem unloading step. The selective movements of FT/FTL2 were further observed by microinjection and trichome rescue studies, which revealed that FT/FTL2 has the capacity to dilate plasmodesmata (PD) microchannels during the process of its cell-to-cell trafficking, and various mutants were compromised in their capacity to traffic through PD. Based on these finding, a model was presented to account for the mechanism by which FT/FTL2 enters the phloem translocation stream and subsequently exits the phloem to traffic into the apical tissue, where it initiates the vegetative to floral transition. This article is protected by copyright. All rights reserved.
... Die entstandenen Konstrukte der Ligationsstrategie oder der fertige Klon können eine toxische Wirkung auf die Bakterien, in denen sie vermehrt werden, haben (Yang et al. 1998;López-Moya and Garcia 2000). Außerdem kann es in Bakterien ein Problem mit der Stabilität der Volllängen cDNA Klone geben (Yang et al. 1998;Ülper et al. 2008 (Maiss et al. 1992;Terauchi et al. 1997;Lin et al. 2002;Hasiów-Jaroszewska et al. 2009 Pflanzenzellschichten ohne größere Schäden zu erreichen (Lamprecht and Jelkmann 1997;Satoh et al. 1999;Lapidot et al. 2007). Somit lassen sich auch mit Volllängen ...
... Ähnliches wurde bei einem Carlavirus, dem 8,7 kb großem Poplar mosaic virus berichtet, dieses ließ sich ebenfalls nicht nach Volllängen PCR in den Vektor ligieren (Naylor et al. 2005). In anderen Strategien wurden in die Primer für die Volllängen PCR Restriktionsschnittstellen eingebaut, die eine Ligation des Amplifikats in den Vektor ermöglichen (Yoon et al. 2002;Rasmussen et al. 2009 (Scholthof 1999;Lin et al. 2002;Huang et al. 2005 (Spetz et al. 2008;Hasiów-Jaroszewska et al. 2009). Durch Volllängen PCR konnte kein cDNA Klon generiert werden, da sich diese großen cDNA Produkte nicht in den Vektor ligieren ließen, was bereits bei einem anderen Carlavirus beobachtet wurde (Naylor et al. 2005). ...
Im Rahmen der vorliegenden Arbeit sollte ein infektiöser Volllängen cDNA Klon des Apple stem pitting virus (ASPV) hergestellt werden, mit dem die Ätiologie der
hervorgerufenen Krankheitsbilder untersucht werden kann. Dazu sollte zum einen die klassische Methode der Ligationsstrategie genutzt werden. Sie basiert auf
spezifischen Restriktionsschnittstellen mit deren Hilfe das Genom aus mehreren
Fragmenten im Zielvektor zusammengesetzt wird (Prüfer et al. 1995). Für diese
Methode ist es unerlässlich die komplette Sequenz des Virus zu kennen, um die
Restriktionsschnittstellen auswählen zu können. Zum anderen sollte eine Methode
entwickelt werden, die weniger zeit- und kostenintensiv ist und die Probleme, die in
der Ligationsstrategie auftreten können, umgeht. Dazu gehört die hohe
Sequenzvariabilität, die bei den latenten Apfelviren auftritt, welche dazu führt, dass
das verwendete Isolat vorher sequenziert werden muss (Yoshikawa 2009). Des
Weiteren können die während der Ligationsstrategie entstandenen Konstrukte in den
Bakterien instabil oder toxisch sein (Ülper et al. 2008). Die entwickelte Methode sollte
auf das Apple stem grooving virus (ASGV) angewendet werden.
Für die Herstellung eines Volllängen cDNA Klons des ASPV wurde das Isolat PB66
verwendet. Die Ligationsstrategie wurde mit Hilfe der Sequenz des Isolates PA66
entwickelt. Bereits während der Amplifikation mittels Polymerase Kettenreaktion
(PCR) traten Probleme auf, die dazu führten, dass die komplette Sequenz des
Isolates PB66 bestimmt wurde. Dabei stellte sich heraus, dass die beiden Isolate
eine Sequenzidentität von 80 % haben. Anhand der neuen Sequenz wurde eine
Ligationsstrategie entwickelt, mit der erfolgreich ein Volllängen cDNA Klon des ASPV
unter der Kontrolle des 35S Promotors hergestellt werden konnte. Das Konstrukt
wurde in einem weiteren Schritt in einen pBin Vektor umligiert. In den
anschließenden Infektionsversuchen mittels mechanischer Inokulation und
Agroinokulation war dieser nicht infektiös. Die Sequenzierung des entstandenen
Volllängen cDNA Klons zeigte ein verändertes 5'- Ende und 19 weitere
Basenaustausche innerhalb der Sequenz.
Als weitere Methode sollte die Volllängen PCR genutzt werden, dazu wurden Primer
am 5'- und 3'- Ende des ASPV Isolates PB66 abgeleitet. Beim Vergleich von 5'- und
3'- Enden von verschiedenen ASPV Isolaten konnte eine erhöhte Konserviertheit
festgestellt werden, welche es ermöglicht, die erstellten Primer zur Herstellung von
15
Volllängen PCR Produkten von verschiedenen ASPV Isolaten zu nutzen. In der PCR
konnte das komplette Genom amplifiziert werden. Die Ligation des 9 kb großen PCR
Fragmentes gelang nicht. Als Abänderung in der Volllängen PCR wurde dem
'forward' Primer die Sequenz des T7 Promotors angefügt. Dadurch konnten in der T7
PCR infektiöse RNA Transkripte generiert werden. Die Volllängen PCR wurde auf
ASGV angewendet. Es konnte das komplette 6,5 kb große Genom amplifiziert
werden. Die Ligation des PCR Produktes war auch hier nicht erfolgreich. Durch
Hinzufügen des T7 Promotors an den 'forward' Primer konnten mit den Volllängen
PCR Produkten in der T7 PCR infektiöse Transkripte hergestellt werden. Durch
mechanische Inokulation mit den T7 PCR Ansätzen konnten in beiden Fällen in dem
experimentellen Wirt Nicotiana occidentalis 37B Symptome ausgelöst werden. Die
Infektion mit ASPV und ASGV wurde durch PCR Nachweise und elektronenmikroskopische
Untersuchungen belegt.
Als weitere Methode wurde das Circular Polymerase Extension Cloning (CPEC)
verwendet (Quan and Tian 2009). Hierbei wurde an die Primer für die Volllängen
PCR ein Stück Vektorsequenz angehängt. Diese überlappenden Bereiche dienen in
einer Fusions- PCR als Ausgangspunkt für die Polymerase. Mit Hilfe dieser Methode
konnten infektiöse Volllängen cDNA Klone des ASPV und ASGV in einem pBin
Vektor hergestellt werden. Nach Agroinokulation zeigten sich auf den
Versuchspflanzen Symptome. Die Infektion wurde durch PCR und das Elektronenmikroskop
belegt. Die Infektionsrate lag bei dem ASPV Klon bei 3 % und bei dem
ASGV Klon bei 22 %.
In dieser Arbeit wurden infektiöse Volllängen cDNA Klone der latenten Apfelviren
ASPV und ASGV mit der CPEC Methode hergestellt. Beide Klone könnten in
weiteren Versuchen für die Erforschung der Ätiologie der durch die Viren
hervorgerufenen Krankheiten genutzt werden. Des Weiteren wurde ein Versuch zur
Nachweisbarkeit von ASPV über den gesamten Jahresverlauf in verschiedenen
Apfelbaumgeweben durchgeführt, in dem sich Phloem als das zuverlässigste
Gewebe zur Detektion von ASPV gezeigt hat. Die Nachweisbarkeit von ASPV, Apple
chlorotic leaf spot virus (ACLSV) und ASGV in Mischinfektionen wurde ebenfalls über
einen längeren Zeitraum überprüft.
... Identifying SuCMoV domains involved in the interaction with the Rcmo-1 gene and those related to symptom induction and other biological properties requires the availability of infectious clones. Biologically active in vitro transcript RNAs from fulllength cDNA clones with bacterial phage promoters [6,14,15,17,[22][23][24] and in vivo infectious transcripts driven by a cauliflower mosaic virus (CaMV) 35S promoter [6,13,17,19,20,24,25,30] have been reported for several potyviruses. Here, we report the construction of a full-length, stable, cDNA clone of the SuCMoV genome from which Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1497-2) contains supplementary material, which is available to authorized users. ...
... Identifying SuCMoV domains involved in the interaction with the Rcmo-1 gene and those related to symptom induction and other biological properties requires the availability of infectious clones. Biologically active in vitro transcript RNAs from fulllength cDNA clones with bacterial phage promoters [6,14,15,17,[22][23][24] and in vivo infectious transcripts driven by a cauliflower mosaic virus (CaMV) 35S promoter [6,13,17,19,20,24,25,30] have been reported for several potyviruses. Here, we report the construction of a full-length, stable, cDNA clone of the SuCMoV genome from which Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1497-2) contains supplementary material, which is available to authorized users. ...
... Since development of the first infectious clone from a RNA plant virus [2], many other clones have been produced that function after transcription in vitro or in vivo [14,15,17,19,25]. The development of infectious clones is the first step in the design of virus-based expression vectors, which are useful in biotechnological applications [3]. ...
A full-length cDNA clone (p35SuCMoV) of the sunflower chlorotic mottle virus common strain (SuCMoV-C) genomic RNA was constructed. Three cDNA fragments covering the whole genome of SuCMoV-C were cloned between a cauliflower mosaic virus 35S promoter and a nopaline synthase terminator. Mechanical inoculation of sunflower and Nicotiana occidentalis seedlings with p35SuCMoV DNA led to systemic infection. Symptoms induced by p35SuCMoV were similar to those caused by the wild-type SuCMoV-C but appeared four days later. Infection was confirmed by a western blot test, electron microscopy, RT-PCR and inoculation of progeny virions to sunflower seedlings. This is the first report about the construction of a biologically active, full-length cDNA copy of the SuCMoV-C RNA genome.
... For pull-down assays, GFP, Nt-NCAPP1DN 1-22 , PDGLP1, and PDGLP1DSP were subcloned into a Zucchini yellow mosaic virus (ZYMV)based viral vector as described previously (Ma et al., 2010;Li et al., 2011). GST and Cm-PP16-GST were amplified from pGWB24/CmPP16 (Li et al., 2011) and inserted into the modified ZYMV viral vector without a c-MycX4-His 6 tag (Lin et al., 2002). ...
In plants, a population of non-cell-autonomous proteins (NCAPs), including numerous transcription factors, move cell to cell through plasmodesmata (PD). In many cases, the intercellular trafficking of these NCAPs is regulated by their interaction with specific PD components. To gain further insight into the functions of this NCAP pathway, coimmunoprecipitation experiments were performed on a tobacco (Nicotiana tabacum) plasmodesmal-enriched cell wall protein preparation using as bait the NCAP, pumpkin (Cucurbita maxima) PHLOEM PROTEIN16 (Cm-PP16). A Cm-PP16 interaction partner, Nt-PLASMODESMAL GERMIN-LIKE PROTEIN1 (Nt-PDGLP1) was identified and shown to be a PD-located component. Arabidopsis thaliana putative orthologs, PDGLP1 and PDGLP2, were identified; expression studies indicated that, postgermination, these proteins were preferentially expressed in the root system. The PDGLP1 signal peptide was shown to function in localization to the PD by a novel mechanism involving the endoplasmic reticulum-Golgi secretory pathway. Overexpression of various tagged versions altered root meristem function, leading to reduced primary root but enhanced lateral root growth. This effect on root growth was corrected with an inability of these chimeric proteins to form stable PD-localized complexes. PDGLP1 and PDGLP2 appear to be involved in regulating primary root growth by controlling phloem-mediated allocation of resources between the primary and lateral root meristems.
... The host range of the infectious clones appeared similar to that of the natural isolates (data not shown), with FMF00-LL1 being systemic on Ranunculus sardous but not on Chenopodium quinoa, and FMF00-LL2 being systemic on C. quinoa but not on R. sardous (Lecoq et al., 2011). For ZYMV and PRSV, since several infectious intronless clones have already been obtained and are satisfactorily stable (Gal-On et al., 1991;Lin et al., 2002;Chiang and Yeh, 1997;Chen et al., 2008), it could be expected that clones of these viruses would not require introns. Consequently, a simplified cloning strategy was used. ...
The availability of an infectious cDNA clone is a prerequisite for genetic studies on RNA viruses. However, despite important improvement in molecular biology techniques during the last decades, obtaining such clones often remains tedious, time-consuming and rather unpredictable. In the case of potyviruses, cDNA clones are frequently unstable due to the toxicity of some viral proteins for bacteria. The problem can be overcome by inserting introns into the viral sequence but this requires additional steps in the cloning process and depends on the availability of suitable restriction sites in the viral sequence or adjunction of such sites by mutagenesis. Homologous recombination in yeast rather than in vitro restriction and ligation can be used to build infectious clones or other viral constructs. This paper describes how, by using recombination in yeast and fusion PCR, infectious intron-containing clones were obtained within a few weeks for two strains of watermelon mosaic virus (WMV, Potyvirus), whereas previous attempts using "classical" cloning techniques had failed repeatedly. Using the same approach, intronless infectious clones of two other potyviruses, zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), were obtained in less than two weeks.
