Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.
- MeSH
- biotechnologie metody MeSH
- genetické inženýrství metody MeSH
- odolnost vůči nemocem * MeSH
- slivoň genetika virologie MeSH
- transgeny MeSH
- umlčování genů * MeSH
- virové plášťové proteiny genetika metabolismus MeSH
- virus šarky švestky genetika patogenita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.
- MeSH
- genom virový * genetika MeSH
- Hemiptera virologie MeSH
- jedlá semena virologie MeSH
- nemoci rostlin virologie MeSH
- rostlinné viry * genetika MeSH
- viry mozaiky * genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko MeSH
- Švédsko MeSH
The plant genome can produce long non-coding RNAs (lncRNAs), some of which have been identified as important regulators of gene expression. To better understand the response mechanism of rice plants to Rice black-streaked dwarf virus (RBSDV) infection, we performed a comparative transcriptome analysis between the RBSDV-infected and non-infected rice plants. A total of 1342 mRNAs and 22 lncRNAs were identified to be differentially expressed after RBSDV infection. Most differentially expressed transcripts involved in the plant-pathogen interaction pathway were upregulated after RBSDV infection, indicating the activation of rice defense response by RBSDV. A network of differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) was then constructed. In this network, there are 56 plant-pathogen interaction-related DEmRNAs co-expressing with 20 DElncRNAs, suggesting these DElncRNAs and DEmRNAs may play essential roles in rice innate immunity against RBSDV. Moreover, some of the lncRNA-mRNA regulatory relationships were experimentally verified in rice calli by a quick and effective method established in this study. Three DElncRNAs were selected to be tested, and the results indicated that five mRNAs were found to be regulated by them. Together, we give a whole landscape of rice mRNAs and lncRNAs in response to RBSDV infection, and a feasible method to rapidly verify the lncRNA-mRNA regulatory relationship in rice.
- MeSH
- messenger RNA genetika metabolismus MeSH
- nemoci rostlin genetika virologie MeSH
- regulace genové exprese u rostlin * MeSH
- RNA dlouhá nekódující genetika metabolismus MeSH
- RNA rostlin genetika metabolismus MeSH
- rostlinné viry genetika fyziologie MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The bird cherry-oat aphid (Rhopalosiphum padi (L.)) is one of the most detrimental pests of cereals, causing harm mainly by transmitting Barley yellow dwarf virus (BYDV). R. padi migration has been monitored since 1992 using suction traps at five sites in the Czech Republic. The count data were subjected to the following different analyses: (i) the minimum temperature thresholds for the aphids to take off were determined; (ii) a partial redundancy analysis using the minimum, average, and maximum temperatures, as well as the wind speed, the precipitation total, and past aphid migration descriptors was performed to explain the relationship between aphid occurrences and weather patterns; and (iii) three types of models from the field of machine learning were used to predict aphid occurrences. According to our findings, (i) in Central Europe, 8°C is the temperature threshold for R. padi migration unless insufficient daylight delays the take-off; (ii) weather conditions occurring roughly 9 months before R. padi migration influence the migration size; (iii) the duration of the summer migration influences the autumn migration size; and (iv) the daytime and nighttime temperatures in the autumn determine the summer peak, whereas winter frosts and precipitation influence the autumn peak.
- MeSH
- ječmen (rod) * virologie MeSH
- Luteovirus * fyziologie MeSH
- migrace zvířat * MeSH
- mšice * fyziologie virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Many plant viruses are vectored by insects in a persistent circulative manner. The insect gut and salivary gland are important barriers limiting virus spread, but the mechanisms by which viruses are able to cross the gut escape barriers of the insect remain largely unknown. Wheat dwarf virus (WDV), transmitted by Psammotettix alienus in a persistent, circulative, and nonpropagative manner, causes the most economically important virus disease in wheat. In this study, ADP ribosylation factor 1 (ARF1) was found to interact with the coat protein of WDV in a yeast two-hybrid, pull-down assay and to colocalise with virions in the gut and salivary glands of P. alienus. When transcription of ARF1 was suppressed by RNA interference, the WDV titre decreased in the haemolymph and salivary glands, and transmission efficiency decreased, but titre in the gut did not differ from that of the control. These data suggest that ARF1 of P. alienus binds to the WDV virion and helps virus spread from gut to haemolymph. Our study provides direct experimental evidence that WDV can use the existing membrane trafficking mechanism to aid its spread within the insect vector. This first analysis of the molecular interaction between WDV and its vector P. alienus contributes to understanding the mechanisms involved in circulative transmission of the virus by the leafhopper vector.
- MeSH
- ADP-ribosylační faktor 1 genetika metabolismus MeSH
- buněčné linie MeSH
- Geminiviridae patogenita MeSH
- Hemiptera genetika metabolismus virologie MeSH
- hmyz - vektory genetika MeSH
- nemoci rostlin virologie MeSH
- RNA interference MeSH
- slinné žlázy metabolismus virologie MeSH
- střeva virologie MeSH
- techniky dvojhybridového systému MeSH
- virion metabolismus MeSH
- virové plášťové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Although many genetic manipulations of crops providing biofortified or safer food have been done, the acceptance of biotechnology crops still remains limited. We report on a transgenic barley expressing the multi-functional protein osmotin that improves plant defense under stress conditions. METHODS: An Agrobacterium-mediated technique was used to transform immature embryos of the spring barley cultivar Golden Promise. Transgenic barley plants of the T0 and T1 generation were evaluated by molecular methods. Transgenic barley tolerance to stress was determined by chlorophyll, total protein, malondialdehyde and ascorbate peroxidase content. Methanol extracts of i) Fusarium oxysporum infected or ii) salt-stressed plants, were characterized by their acute toxicity effect on human dermal fibroblasts (HDF), genotoxicity and affection of biodiversity interactions, which was tested through monitoring barley natural virus pathogen-host interactions-the BYDV and WDV viruses transmitted to the plants by aphids and leafhoppers. RESULTS: Transgenic plants maintained the same level of chlorophyll and protein, which significantly declined in wild-type barley under the same stressful conditions. Salt stress evoked higher ascorbate peroxidase level and correspondingly less malondialdehyde. Osmotin expressing barley extracts exhibited a lower cytotoxicity effect of statistical significance than that of wild-type plants under both types of stress tested on human dermal fibroblasts. Extract of Fusarium oxysporum infected transgenic barley was not able to damage DNA in the Comet assay, which is in opposite to control plants. Moreover, this particular barley did not affect the local biodiversity. CONCLUSION: Our findings provide a new perspective that could help to evaluate the safety of products from genetically modified crops.
- MeSH
- bezpečnost potravin * MeSH
- biologická adaptace MeSH
- ektopická exprese * MeSH
- fyziologický stres genetika MeSH
- geneticky modifikované rostliny MeSH
- interakce hostitele a patogenu genetika MeSH
- ječmen (rod) genetika MeSH
- lidé MeSH
- rekombinantní proteiny * MeSH
- rostlinné proteiny genetika MeSH
- tabák genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Wheat streak mosaic virus (WSMV) causes wheat streak mosaic, a disease of cereals and grasses that threatens wheat production worldwide. It is a monopartite, positive-sense, single-stranded RNA virus and the type member of the genus Tritimovirus in the family Potyviridae. The only known vector is the wheat curl mite (WCM, Aceria tosichella), recently identified as a species complex of biotypes differing in virus transmission. Low rates of seed transmission have been reported. Infected plants are stunted and have a yellow mosaic of parallel discontinuous streaks on the leaves. In the autumn, WCMs move from WSMV-infected volunteer wheat and other grass hosts to newly emerged wheat and transmit the virus which survives the winter within the plant, and the mites survive as eggs, larvae, nymphs or adults in the crown and leaf sheaths. In the spring/summer, the mites move from the maturing wheat crop to volunteer wheat and other grass hosts and transmit WSMV, and onto newly emerged wheat in the fall to which they transmit the virus, completing the disease cycle. WSMV detection is by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) or quantitative RT-PCR (RT-qPCR). Three types of WSMV are recognized: A (Mexico), B (Europe, Russia, Asia) and D (USA, Argentina, Brazil, Australia, Turkey, Canada). Resistance genes Wsm1, Wsm2 and Wsm3 have been identified. The most effective, Wsm2, has been introduced into several wheat cultivars. Mitigation of losses caused by WSMV will require enhanced knowledge of the biology of WCM biotypes and WSMV, new or improved virus detection techniques, the development of resistance through traditional and molecular breeding, and the adaptation of cultural management tactics to account for climate change.
- MeSH
- ELISA MeSH
- nemoci rostlin virologie MeSH
- Potyviridae patogenita MeSH
- pšenice virologie MeSH
- roztoči fyziologie virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
A rapid method for detection, discrimination and quantification of wheat and barley strains of wheat dwarf virus (WDV) was successfully developed. The sensitivity of quantification of the wheat and barley strains of WDV ranged from an average of 1.2 × 10(7)-1.2 × 10(2) and from an average of 1.4 × 10(7)-1.4 × 10(4) copies of viral genome, respectively. These standard serial dilutions were applied to plant and vector tissues for virus titer calculations. Both strains of WDV were clearly discriminated by specific probes and melting curve analysis. Both TaqMan(®) and SYBR(®) Green technologies provided accurate and reliable methods for monitoring, detection, discrimination, and quantification of WDV.
- MeSH
- Geminiviridae klasifikace genetika izolace a purifikace MeSH
- ječmen (rod) virologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nemoci rostlin virologie MeSH
- pšenice virologie MeSH
- senzitivita a specificita MeSH
- virová nálož metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A SYBR Green(®)-based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and β-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.
- Publikační typ
- časopisecké články MeSH
A quantitative PCR, real-time RT-PCR, and one-step real-time RT-PCR using SYBR Green-based tools for reliable detection and relative quantitation of Prune dwarf virus (PDV) in stone fruits are described. The assay reliability was tested on 55 samples from different hosts and regions. The sensitivity of the assay was also compared with other assays with different primers. Two plant-expressed genes, actin and 18S rRNA, were used as housekeeping genes for accurate quantitation of PDV in stone fruit trees. The expression of the gene for actin and the 18S ribosomal RNA gene corresponded with each other accurately, with standard deviation values of 1.905 cycles on average, 1.36 for Prunus persica, and 2.45 for other Prunus species tested. The results of this study support the need to use more than one housekeeping gene as an internal control to avoid possible errors caused by unstable internal control gene mRNA expression when quantifying the extent of PDV infection.
- MeSH
- aktiny genetika MeSH
- barvení a značení metody MeSH
- fluorescenční barviva MeSH
- Ilarvirus genetika izolace a purifikace MeSH
- organické látky MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody normy MeSH
- referenční standardy MeSH
- RNA ribozomální 18S genetika MeSH
- RNA rostlin genetika MeSH
- RNA virová genetika MeSH
- rostlinné proteiny genetika MeSH
- slivoň virologie MeSH
- virová nálož MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
