Barley Stripe Mosaic Virus Research Articles

Expression of the p24 silencing suppressor of Grapevine leafroll-associated virus 2 from Potato virus X or Barley stripe mosaic virus vector elicits hypersensitive responses in Nicotiana benthamiana

The 24-kDa protein (p24) encoded by Grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor (RSS), but its effect on active viral infection is unclear. Using a Potato virus X (PVX)-based expression system, we demonstrated that p24 elicits lethal systemic necrosis in Nicotiana benthamiana, sharing typical characteristics of the hypersensitive response (HR), and that NbRAR1 (a cytoplasmic Zn2+-binding protein) is involved in the PVX–p24-mediated systemic necrosis. Moreover, expression of p24 from Barley stripe mosaic virus (BSMV) vector triggered local necrosis in infiltrated patches of N. benthamiana, likely inhibiting viral systemic spread. By deletion analysis, we demonstrated that amino acids (aa) 1 to 180, which are located in the region (aa 1–188) previously shown to be necessary for p24's RSS activity, is sufficient for p24 to elicit systemic necrosis in the context of PVX infection. Using substitution mutants, we revealed that silencing-suppression-defective mutants R2A and W54A induce only a mild necrotic response; two mutants without self-interaction ability previously shown to lose or retain weak suppression function also displayed decreased pathogenicity: W149A without RSS activity elicited a mild necrotic response, whereas V162H/L169H/L170H which retains weak RSS activity was able to induce systemic necrosis, but with a 1- to 2-day delay. Taken together, p24 plays an important role in GLRaV-2 pathogenesis, triggering HR-like necrosis in N. benthamiana plants when expressed from PVX or BSMV vector; both the silencing suppression and self-interaction are crucial for p24's pathogenicity activity, and the region of p24 for determining systemic necrosis is mapped to aa 1–180.

TaAMT2;3a, a wheat AMT2-type ammonium transporter, facilitates the infection of stripe rust fungus on wheat

BackgroundAmmonium transporters (AMTs), a family of proteins transporting ammonium salt and its analogues, have been studied in many aspects. Although numerous studies have found that ammonium affects the interaction between plants and pathogens, the role of AMTs remains largely unknown, especially that of the AMT2-type AMTs.ResultsIn the present study, we found that the concentration of ammonium in wheat leaves decreased after infection with Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust. Then, an AMT2-type ammonium transporter gene induced by Pst was identified and designated as TaAMT2;3a. Transient expression assays indicated that TaAMT2;3a was located to the cell and nuclear membranes. TaAMT2;3a successfully complemented the function of a yeast mutant defective in NH4+ transport, indicating its ammonium transport capacity. Function of TaAMT2;3a in wheat-Pst interaction was further analyzed by barley stripe mosaic virus (BSMV)-induced gene silencing. Pst growth was significantly retarded in TaAMT2;3a-knockdown plants, in which ammonium in leaves were shown to be induced at the early stage of infection. Histological observation showed that the hyphal length, the number of hyphal branches and haustorial mother cells decreased in the TaAMT2;3a knockdown plants, leading to the impeded growth of rust pathogens.ConclusionsThe results clearly indicate that the induction of AMT2-type ammonium transporter gene TaAMT2;3a may facilitates the nitrogen uptake from wheat leaves by Pst, thereby contribute to the infection of rust fungi.

Non-structural Functions of Hordeivirus Capsid Protein Identified in Plants Infected by a Chimeric Tobamovirus.

Capsid proteins (CPs) of (+)RNA-containing plant viruses are multifunctional proteins involved in many stages of viral infection cycle, in addition to their main function of virus capsid formation. For example, the tobamoviral CP ensures virus systemic transport in plants and defines the virus-host interactions, thereby influencing the virus host range, virus infectivity, pathogenicity, and manifestation of infection symptoms. Hordeiviruses and tobamoviruses belong to the Virgaviridae family and have rod-shaped virions with a helical symmetry; their CPs are similar in structure. However, no non-structural functions of hordeiviral CPs have been described so far. In this study, we assayed possible non-structural functions of CP from the barley stripe mosaic virus (BSMV) (hordeivirus). To do this, the genome of turnip vein clearing virus (TVCV) (tobamovirus) was modified by substituting the TVCV CP gene with the BSMV CP gene or its mutants. We found that BSMV CP efficiently replaced TVCV CP at all stages of viral infection. In particular, BSMV CP performed the role of tobamoviral CP in the long-distance transport of the chimeric virus, acted as a hypersensitive response elicitor, and served as a pathogenicity determinant that influenced the symptoms of the viral infection. The chimeric tobamovirus coding for the C-terminally truncated BSMV CP displayed an increased infectivity and was transported in plants in a form of atypical virions (ribonucleoprotein complexes).

Virus-Induced Gene Silencing (VIGS) for Functional Characterization of Disease Resistance Genes in Barley Seedlings.

With the recent advances in sequencing technologies, many studies are generating lists of candidate genes associated with specific traits. The major bottleneck in functional genomics is the validation of gene function. This is achieved by analyzing the effect of either gene silencing or overexpression on a specific phenotypic or biochemical trait. This usually requires the generation of stable transgenic plants and this can take considerable time. Therefore any technique that expedites the validation of gene function is of particular benefit in cereals, including barley. One such technique is Virus-Induced Gene Silencing (VIGS), which evokes a natural antiviral defense mechanism in plants. VIGS can be used to downregulate gene expression in a transient manner, but long enough to determine its effects on a specific phenotype. It is particularly useful for screening candidate genes and selecting those with potential for disease control. VIGS based on Barley Stripe Mosaic Virus (BSMV) is a powerful and efficient tool for the analysis of gene function in cereals. Here we present a BSMV VIGS protocol for simple and robust gene silencing in barley and describe it to evaluate the role of the hormone receptor BRI1 (Brassinosteroid Insensitive 1) in barley leaf resistance to Fusarium infection.

Virus-induced gene silencing ofTaERECTAincreases stomatal density in bread wheat

Barley stripe mosaic virus (BSMV)-based virus induced gene silencing (VIGS) is an effective strategy for rapid determination of functional genes in wheat plants. ERECTA genes are reported to regulate stomatal pattern of plants, and manipulation of TaERECTA (a homo-logue of ERECTA in bread wheat) is a potential route for investigating stomatal development. Here, the leucine-rich repeat domains (LRRs) and transmembrane domains of TaERECTA were selected to gain BSMV:ER-LR and BSMV:ER-TM constructs, respectively, targeting TaERECTA for silencing in wheat cultivars ‘Bobwhite’ and ‘Cadenza’, to identify the function of TaERECTA on stomatal patterns. The results showed that reduced expression of TaERECTA caused an increased stomatal and epidermal cell density by average 13.5% and 3.3%, respectively, due to the significantly reduced size of leaf epidermal and stomatal cells, and this led to an increase in stomatal conductance. These suggest that modulation of TaERECTA offers further opportunities in stomatal engineering for the adaptation of photosynthesis in wheat.

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature.

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

HvSRP72 silencing enhanced Blumeria graminis f. sp. hordei growth in compatible interaction with barley

Powdery mildew disease, caused by Blumeria graminis f. sp. hordei (Bgh), is one of the important diseases of barley. Hordeum vulgare Signal Recognition Particle 72 (HvSRP72) was previously identified as differentially induced at 6 h and/or 12 h after infection in powdery mildew disease resistance, suggesting its involvement in pathogen response. Since there is limited information on R gene-mediated signaling cascade, we sought to understand the way which HvSRP72 protein functions in powdery mildew resistance or susceptibility response. HvSRP72 was silenced by barley stripe mosaic virus (BSMV)-induced gene silencing, in order to understand its role in the plant response to pathogen infection. The level of HvSRP72 mRNA in silenced plant samples showed an average 57% reduction with respect to control plants (BSMV:00). The change in resistance or susceptibility to powdery mildew in HvSRP72-silenced plant leaves was determined by measuring the length of hyphal extensions and the germination efficiency upon inoculation. Under susceptible conditions, in HvSRP72-silenced samples spores germinated approximately four times more efficiently and Bgh development was also increased twice with respect to control plants. On the other hand, under the resistant condition, no difference was observed in HvSRP72-silenced samples when compared to the non-silenced ones. Thus, it can be concluded that silencing of HvSRP72 enhances Blumeria graminis f. sp. hordei susceptibility but no change of hyphal growth was observed during resistance response.

Reactions of Triticum urartu accessions to two races of the wheat yellow rust pathogen

Triticum urartu (AA, 2n = 2x = 14), a wild grass endemic to the Fertile Crescent (FC), is the progenitor of the A subgenome in common wheat. It belongs to the primary gene pool for wheat improvement. Here, we evaluated the yellow rust (caused by Puccinia striiformis f. sp. tritici, Pst) reactions of 147 T. urartu accessions collected from different parts of the FC. The reactions varied from susceptibility to strong resistance. In general, there were more accessions with stronger resistance to race CYR33 than to CYR 32. In most cases the main form of defense was a moderate resistance characterized by the presence of necrotic/chlorotic lesions with fewer Pst uredinia on the leaves. Forty two accessions displayed resistance to both races. Histological analysis showed that Pst growth was abundant in the compatible interaction but significantly suppressed by the resistant response. Gene silencing mediated by Barley stripe mosaic virus was effective in two T. urartu accessions with different resistance responses, indicating that this method can expedite future functional analysis of resistance genes. Our data suggest that T. urartu is a valuable source of resistance to yellow rust, and represents a model for studying the genetic, genomic and molecular basis underlying interaction between wheat and Pst.

Foxtail mosaic virus: A Viral Vector for Protein Expression in Cereals.

Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function and are particularly useful in plant species for which transformation-based methods are unavailable or are too time and labor demanding, such as wheat (Triticum aestivum) and maize (Zea mays). The virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus and Wheat streak mosaic virus described previously for these species are incapable of expressing free recombinant proteins of more than 150 to 250 amino acids, are not suited for high-throughput screens, and have other limitations. In this study, we report the development of a VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (genus Potexvirus). In this vector, PV101, the gene of interest was inserted downstream of the duplicated subgenomic promoter of the viral coat protein gene, and the corresponding protein was expressed in its free form. The vector allowed the expression of a 239-amino acid-long GFP in both virus-inoculated and upper uninoculated (systemic) leaves of wheat and maize and directed the systemic expression of a larger approximately 600-amino acid protein, GUSPlus, in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as the host-specific toxin ToxA of Parastagonospora nodorum Therefore, this VOX vector opens possibilities for functional genomics studies in two important cereal crops.

Barley stripe mosaic virus γb Protein Subverts Autophagy to Promote Viral Infection by Disrupting the ATG7-ATG8 Interaction.

Autophagy is a conserved defense strategy against viral infection. However, little is known about the counterdefense strategies of plant viruses involving interference with autophagy. Here, we show that γb protein from Barley stripe mosaic virus (BSMV), a positive single-stranded RNA virus, directly interacts with AUTOPHAGY PROTEIN7 (ATG7). BSMV infection suppresses autophagy, and overexpression of γb protein is sufficient to inhibit autophagy. Furthermore, silencing of autophagy-related gene ATG5 and ATG7 in Nicotiana benthamiana plants enhanced BSMV accumulation and viral symptoms, indicating that autophagy plays an antiviral role in BSMV infection. Molecular analyses indicated that γb interferes with the interaction of ATG7 with ATG8 in a competitive manner, whereas a single point mutation in γb, Tyr29Ala (Y29A), made this protein deficient in the interaction with ATG7, which was correlated with the abolishment of autophagy inhibition. Consistently, the mutant BSMVY29A virus showed reduced symptom severity and viral accumulation. Taken together, our findings reveal that BSMV γb protein subverts autophagy-mediated antiviral defense by disrupting the ATG7-ATG8 interaction to promote plant RNA virus infection, and they provide evidence that ATG7 is a target of pathogen effectors that functions in the ongoing arms race of plant defense and viral counterdefense.

Barley stripe mosaic virus (BSMV) as a virus-induced gene silencing vector in maize seedlings.

Barley stripe mosaic virus (BSMV) was the first reported and still widely used virus-induced gene silencing (VIGS) vector for monocotyledons including wheat and barley. Despite BSMV's reported infectivity on maize (Zea mays), the use of the virus as a vector in maize has not been optimized. Here, we assayed infectivity of BSMV in different maize cultivars by vascular puncture inoculation. Through knockdown of the endogenous host phytoene desaturase gene, we demonstrate for the first time that BSMV can be used as a VIGS vector in maize. This adds BSMV to the repertoire of tools available for functional studies in maize.

Barley Stripe Mosaic Virus (Virgaviridae, Hordeivirus) As Etiological Agent of Maize Chlorotic Stripe Disease

The presence of barley stripe mosaic virus (BSMV) (Virgaviridae, Hordeivirus) infection background in maize (Zea mays) crops in the territory of Primorsky krai was demonstrated. Field data and model experiments established that BSMV causes the disease formerly called “maize chlorotic stripe disease” with the following symptoms: general chlorosis of leaves at the early stages of plant development, the emergence of pale yellow, nearly white strikes along the midrib throughout the length of the leaf, and loss of infected plants to the stage of pulling (12–25%; average value of 17.1%) and elongation with the absence of severe death in the later stages. The coefficient of harmfulness for BSMV (37.1%) was determined in the study. We concluded that planned monitoring in relation to BSMV is required for planting maize.

Barley stripe mosaic virus infection requires PKA-mediated phosphorylation of γb for suppression of both RNA silencing and the host cell death response.

The Barley stripe mosaic virus (BSMV) γb protein is a viral suppressor of RNA silencing (VSR) and symptom determinant. However, it is unclear how post-translational modification affects the different functions of γb. Here, we demonstrate that γb is phosphorylated at Ser-96 by a PKA-like kinase invivo and invitro. Mutant viruses containing a nonphosphorylatable substitution (BSMVS96A or BSMVS96R ) exhibited reduced viral accumulation in Nicotiana benthamiana due to transient induction of the cell death response that constrained the virus to necrotic areas. By contrast, a BSMVS96D mutant virus that mimics γb phosphorylation spread similarly to the wild-type virus. Furthermore, the S96A mutant had reduced local and systemic γb VSR activity due to having compromised its binding activity to 21-bp dsRNA. However, overexpression of other VSRs in trans or in cis failed to rescue the necrosis induced by BSMVS96A , demonstrating that suppression of cell death by γb phosphorylation is functionally distinct from its RNA silencing suppressor activities. These results provide new insights into the function of γb phosphorylation in regulating RNA silencing and the BSMV-induced host cell death response, and contribute to our understanding of how the virus optimizes the balance between viral replication and virus survival in the host plants during virus infection.

Multi-functional roles of TaSSI2 involved in Fusarium head blight and powdery mildew resistance and drought tolerance

The mutation of the gene encoding a stearoyl-acyl carrier protein fatty acid desaturase (ssi2) has been proved to enhance pathogen resistance in several plants, while it's potential to regulate biotic and abiotic stresses in wheat is still unclear. In this study, we cloned TaSSI2 gene in wheat and provided several evidences of its involvement in multiple biological functions. By using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) in wheat, it was found that TaSSI2 negatively regulated both powdery mildew and Fusarium head blight (FHB) resistance, which was consistent with the phenotype observed in knock-out mutants of Kronos. The expression of TaSSI2 was down-regulated by in vitro treatments of methyl jasmonate (MeJA), but positively regulated by salicylic acid (SA) and abscisic acid (ABA), implying the cross-talk between different hormone signaling pathways involved in wheat to regulate biotic stresses is still to be elucidated. Furthermore, the up-regulated expression of PR4 and PR5 indicated that TaSSI2 probably regulated FHB resistance by depressing the SA signaling pathway in wheat. In addition, the over-expression of TaSSI2 increased the content of linolenic acid (18:3) and subsequently enhanced drought tolerance of transgenic Brachypodium. This phenomenon might be associated with its subcellular localization in the whole cytosol, partly overlapping with Golgi apparatus and the secreted vesicles. As a stearoyl-acyl carrier protein fatty acid desaturase, TaSSI2 was proposed to be involved in cell lipid metabolism and carried targets out of the cell from membrane or wax synthesis, resulting in enhanced drought tolerance in plant.

Molecular analysis of barley stripe mosaic virus isolates differing in their biological properties and the development of reverse transcription loop-mediated isothermal amplification assays for their detection.

Barley stripe mosaic virus (BSMV) is an important seed-transmitted pathogen occurring worldwide. Recently, the occurrence of mild BSMV pathotypes has been observed in barley crops in Poland. In this study, the full-length genome sequences of mild and aggressive Polish and German BSMV isolates was established. Phylogenetic and recombination analysis was performed using Polish and other BSMV isolates described to date. The analysis revealed that Polish isolates differed only in 25 nucleotides, which suggests that point mutations might have had a great impact on the biological properties of the virus. The phylogenetic analysis revealed that the closest relationship was that between European and BSMV-CV42, BSMV-ND18 and BSMV-Type isolates, whereas the highest genetic distance was observed for BSMV-Qasr Ibrim and BSMV-China isolates. A recombination event within the αa protein of BSMV-De-M and BSMV-CV42 isolates was also detected. Moreover, a sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for rapid detection of BSMV isolates. The RT-LAMP assay can be used for routine diagnostics of BSMV in seed and plant material.

HvGCN2 silencing in barley displays enhanced Blumeria graminis f. sp. hordei susceptibility

Powdery mildew disease, caused by Blumeria graminis f. sp. hordei (Bgh), which belongs to the order Erysiphales, is a major crop disease. The general control nondepressible-2 (GCN2) gene of barley was previously found to be overexpressed during the powdery mildew resistance response. Recently, Arabidopsis thaliana GCN2 (AtGCN2) was shown to be involved in disease resistance against biotrophic and necrotrophic pathogens. In order to understand the function of Hordeum vulgare GCN2 (HvGCN2) in the barley powdery mildew resistance response, this gene was silenced by barley stripe mosaic virus (BSMV), mediated by virus-induced gene silencing (VIGS). This is the first study showing the potential importance of HvGCN2 in powdery mildew disease of barley. Based on our observations, when HvGCN2 was silenced on average by 53.5%, Bgh development was increased by 18.7 to 32.1%, which was determined by primary, secondary and longest hyphae measurements. The number of germinated spores also increased 2.8-fold in HvGCN2 silenced plants compared to control plants (BSMV:00). On the other hand, under the resistant condition, no difference was observed in HvGCN2- silenced plants compared to non-silenced lines although the gene was found to be overexpressed in incompatible interaction.

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell-to-cell movement of Barley stripe mosaic virus.

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA-binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear-cytoplasmic trafficking is required for BSMV cell-to-cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95-104) and a nuclear localization signal (NLS) (amino acids 227-238). NoLS mutations reduced BSMV cell-to-cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine-arginine-rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV-infected cells, Fib2 accumulation increased by about 60%-70% and co-localized with TGB1 in the plasmodesmata. In addition, BSMV cell-to-cell movement in fib2 knockdown transgenic plants was reduced to less than one-third of that of non-transgenic plants. Fib2 also co-localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1-Fib2 interactions play a direct role in cell-to-cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell-to-cell movement.

Virus-Induced Gene Silencing (VIGS) in Aegilops tauschii and Its Use in Functional Analysis of AetDREB2.

Among the availablereverse genetic approaches for studying gene function, virus-induced gene silencing (VIGS) has several advantages. It allows rapid characterization of gene function independent of stable transformation, which is basically difficult to achieve in monocots, and offers the potential to silence individual or multiple genes of a gene family. In order to establish a VIGS system in Aegilops tauschii, modified vectors derived from Barley stripe mosaic virus (BSMV) were used for silencing a phytoene desaturase gene thatprovides a convenient visual reporter for silencing. The results demonstrated a high efficiency of BSMV-VIGS in A. tauschii. Moreover, the BSMV-VIGS system was used to target a 354bp specific region of the Dehydration-responsive element-binding (AetDreb2) gene, resulting in successful silencing of the gene in A. tauschii plants, as verified by real-time qRT-PCR. Indeed, in comparison with plants that were inoculated with an empty vector (BSMV:00), a faster rate of wilting and a lower relative water content were observed in plants inoculated with BSMV:AetDreb2 when they were exposed to drought stress. Therefore, BSMV-VIGS can be efficiently employed as a novel tool for reverse genetics in A. tauschii. It can also be used to study the effects of polyploidization on the gene function by a comparative analysis between bread wheat and its diploid progenitor.

Host-induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust.

SummaryRust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T3 to T4 generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.

Construction of infectious clones of lychnis ringspot virus and evaluation of its relationship with barley stripe mosaic virus by reassortment of genomic RNA segments

Lychnis ringspot virus (LRSV, genus Hordeivirus) was first isolated in 1959, and has been shown to infect several dicot plants in nature. However, due to the lack of infectious cDNA clones, the biological properties and mechanisms underlying LRSV infection are obscure. In this work, we constructed infectious cDNA clones of LRSV and have compiled the complete LRSV genomic (g) RNA sequence. Comparison of nucleotide and amino acid sequences between LRSV and barley stripe mosaic virus (BSMV), the type member of genus Hordeivirus, reveals that despite belonging to the same genus, and replicating in chloroplasts, the viruses are only distantly related. This could be further indicated by the failure of different LRSV/BSMV reassortants to infect N. benthamiana. LRSV infectious cDNA clones provide a useful tool for studies of biological diversity among hordeiviruses, and also may contribute to the understanding of seed transmission in dicot plants.