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Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

ABSTRACT: Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off-target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12-15?°C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect-mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process.

SUBMITTER: Kis A

PROVIDER: S-EPMC6638354 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

Kis András A Tholt Gergely G Ivanics Milán M Várallyay Éva É Jenes Barnabás B Havelda Zoltán Z

Molecular plant pathology 20150730 3

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature ...[more]

PMID: 26136043

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Project description:The deciphering of the epidemiology of a plant virus has long been focused on the study of interactions between partners of one pathosystem. However, plants are exposed to numerous viruses which lead to frequent co-infection scenarios. This can change characteristics of virus-vector-host interactions and could impact the epidemiology of viral diseases. Barley yellow dwarf virus-PAV (BYDV-PAV; species: Luteovirus pavhordei; genus Luteovirus), wheat dwarf virus (WDV; genus Mastrevirus) and their respective vectors (BYDV-PAV: e.g. Rhopalosiphum padi and WDV: Psammotettix alienus) are commonly found in cereal fields. Wheat plants co-infected with BYDV-PAV and WDV have been reported from field surveys, although epidemiological outcomes of BYDV-PAV - WDV interactions in planta have not yet been studied. Experiments were carried out to evaluate and compare, through different competition scenarios (i.e. single- and co- (simultaneous and sequential) inoculations), the efficiency of BYDV-PAV and WDV to infect, to accumulate in and to be spread between wheat plants. Moreover, the impact of competition scenarios on the biological parameters of these two viruses was evaluated at different stages of the infection and with plants at different ages at inoculation. Results showed i) that these viruses achieve their infection cycle and their plant-to-plant transmission with different efficiencies and ii) BYDV-PAV - WDV interactions lead to different phenotypes ranging from antagonism to synergism. Finally, when these two viruses share a host, the nature and strength of virus-virus interactions varied depending on the order of virus arrival, stages of the infection cycle and plant age at inoculation. Precisely, the introduction (i.e. co- and sequential inoculation) and infection process (i.e. virus accumulation) of BYDV-PAV in a wheat benefit from the presence of WDV. For the latter, the sympatry with BYDV-PAV exerts opposite pressure on parameters involved in virus introduction (i.e. benefit during sequential inoculation) and spread (i.e. lower transmission efficiency and virus accumulation in co-infected plants). In the context of increased potential exposure of crops to insect vectors, this study participates in a better understanding of the impact of BYDV-PAV and WDV co-infections on biological and ecological parameters of the diseases induced by these viruses.

Project description:Barley yellow dwarf viruses (BYDVs) are one of the most widespread and economically important plant viruses affecting many cereal crops. Growing resistant varieties remains the most promising approach to reduce the impact of BYDVs. A Recent RNA sequencing analysis has revealed potential genes that respond to BYDV infection in resistant barley genotypes. Together with a comprehensive review of the current knowledge on disease resistance in plants, we selected nine putative barley and wheat genes to investigate their involvement in resistance to BYDV-PAV infection. The target classes of genes were (i) nucleotide binding site (NBS) leucine-rich repeat (LRR), (ii) coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR), (iii) LRR receptor-like kinase (RLK), (iv) casein kinase, (v) protein kinase, (vi) protein phosphatase subunits and the transcription factors (TF) (vii) MYB TF, (viii) GRAS (gibberellic acid-insensitive (GAI), repressor of GAI (RGA) and scarecrow (SCR)), and (ix) the MADS-box TF family. Expression of genes was analysed for six genotypes with different levels of resistance. As in previous reports, the highest BYDV-PAV titre was found in the susceptible genotypes Graciosa in barley and Semper and SGS 27-02 in wheat, which contrast with the resistant genotypes PRS-3628 and Wysor of wheat and barley, respectively. Statistically significant changes in wheat show up-regulation of NBS-LRR, CC-NBS-LRR and RLK in the susceptible genotypes and down-regulation in the resistant genotypes in response to BYDV-PAV. Similar up-regulation of NBS-LRR, CC-NBS-LRR, RLK and MYB TF in response to BYDV-PAV was also observed in the susceptible barley genotypes. However, no significant changes in the expression of these genes were generally observed in the resistant barley genotypes, except for the down-regulation of RLK. Casein kinase and Protein phosphatase were up-regulated early, 10 days after inoculation (dai) in the susceptible wheat genotypes, while the latter was down-regulated at 30 dai in resistant genotypes. Protein kinase was down-regulated both earlier (10 dai) and later (30 dai) in the susceptible wheat genotypes, but only in the later dai in the resistant genotypes. In contrast, GRAS TF and MYB TF were up-regulated in the susceptible wheat genotypes while no significant differences in MADS TF expression was observed. Protein kinase, Casein kinase (30 dai), MYB TF and GRAS TF (10 dai) were all up-regulated in the susceptible barley genotypes. However, no significant differences were found between the resistant and susceptible barley genotypes for the Protein phosphatase and MADS FT genes. Overall, our results showed a clear differentiation of gene expression patterns in both resistant and susceptible genotypes of wheat and barley. Therefore, further research on RLK, NBS-LRR, CC-NBS-LRR, GRAS TF and MYB TF can lead to BYDV-PAV resistance in cereals.

Project description:Wheat (Tritium aestivum L.) production is essential for global food security. Infection of barley yellow dwarf virus-GAV (BYDV-GAV) results in wheat showing leaf yellowing and plant dwarfism symptom. To explore the molecular and ultrastructural mechanisms underlying yellow dwarf symptom formation in BYDV-GAV-infected wheat, we investigated the chloroplast ultrastructure via transmission electron microscopy (TEM), examined the contents of the virus, H?O?, and chlorophyll in Zhong8601, and studied the comparative transcriptome through microarray analyses in the susceptible wheat line Zhong8601 after virus infection. TEM images indicated that chloroplasts in BYDV-GAV-infected Zhong8601 leaf cells were fragmentized. Where thylakoids were not well developed, starch granules and plastoglobules were rare. Compared with mock-inoculated Zhong8601, chlorophyll content was markedly reduced, but the virus and H?O? contents were significantly higher in BYDV-GAV-infected Zhong8601. The transcriptomic analyses revealed that chlorophyll biosynthesis and chloroplast related transcripts, encoding chlorophyll a/b binding protein, glucose-6-phosphate/phosphate translocator 2, and glutamyl-tRNA reductase 1, were down-regulated in BYDV-GAV-infected Zhong8601. Some phytohormone signaling-related transcripts, including abscisic acid (ABA) signaling factors (phospholipase D alpha 1 and calcineurin B-like protein 9) and nine ethylene response factors, were up-regulated. Additionally, reactive oxygen species (ROS)-related genes were transcriptionally regulated in BYDV-GAV infected Zhong8601, including three up-regulated transcripts encoding germin-like proteins (promoting ROS accumulation) and four down-regulated transcripts encoding peroxides (scavenging ROS). These results clearly suggest that the yellow dwarf symptom formation is mainly attributed to reduced chlorophyll content and fragmentized chloroplasts caused by down-regulation of the chlorophyll and chloroplast biosynthesis related genes, ROS excessive accumulation, and precisely transcriptional regulation of the above-mentioned ABA and ethylene signaling- and ROS-related genes in susceptible wheat infected by BYDV-GAV.

Dataset Information

Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

Publications

Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

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