Wheat Dwarf Virus Research Articles

Functional Transcomplementation between Wheat Dwarf Virus Strains in Wheat and Barley.

Wheat dwarf virus, transmitted by the leafhopper Psammotettix alienus in a persistent, non-propagative manner, infects numerous species from the Poaceae family. Data associated with wheat dwarf virus (WDV) suggest that some isolates preferentially infect wheat while other preferentially infect barley. This allowed to define the wheat strain and the barley strain. There are contradictory results in the literature regarding the ability of each of these two strains to infect its non-preferred host. To improve knowledge on the interactions between WDV strains and barley and wheat, transmission experiments were carried out using barcoded P. alienus and an experimental design based on single/sequential acquisitions of WDV strains and on transmissions to wheat and barley. Results showed that (I) WDV strains are transmitted with similar efficiencies by P. alienus males, females and larvae, (II) WDV wheat and barley strains do not infect barley and wheat plants, respectively, and (III) a functional transcomplementation between the wheat and barley strains allows a mixed infection of barley and wheat. The described ability of each WDV strain to infect a non-host plant in the presence of the other viral strain must be considered to analyze data available on WDV host range.

Identification of the wild and cultivated hosts of wheat dwarf virus and oat dwarf virus in Iran.

In the last decade two mastreviruses, Wheat dwarf virus (WDV) and Oat dwarf virus (ODV) have been reported from cereal farms in Iran. In a survey, wild and cultivated hosts of these mastreviruses were studied during 2015 to 2017. Symptomatic small grain cereal samples and weed species were collected and assayed for WDV and/or ODV infection by PCR. While WDV which was detected in 139/284 (49%) of total symptomatic samples, low incidence (2%) was recorded for ODV which was detected only in slender wild oat (Avena barbata Pott ex Link) and red brome (Bromus rubens L.). In agroinfection studies, the clone of ODV infected common oat (A. sativa) and slender wild oat (A. barbata) with the low efficiency and did not infect wheat or barley. ODV was transmitted by the leafhopper Psammotettix alienus, from agroinfected common oat to healthy seedlings. The results show that, in contrast to WDV, ODV has a low incidence and a narrow host range in gramineous plants.

Crystal structure of the Wheat dwarf virus Rep domain.

The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein-ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein-DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24 Å resolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.

Protein Elicitor PeaT1 Efficiently Controlled Barley Yellow Dwarf Virus in Wheat

Barley yellow dwarf virus (BYDV), transmitted by the wheat aphid, generates serious wheat yellow dwarf disease and causes great losses in agriculture. Induced resistance has attracted great attention over recent years as a biological method to control plant pathogens and herbivores. Protein elicitor PeaT1 induces defense response in plants against fungi, viruses, and aphids. In this study, wheat seeds and seedlings were soaked and sprayed with 30 μg/mL PeaT1, respectively. Then seedlings were inoculated with BYDV by viruliferous Schizaphis graminum to detect the control efficiency of PeaT1-induced resistance against BYDV. The control efficiency was over 30% on the 14th and 21st days after the inoculation access period. Quantitative real time polymerase chain reaction (Q-RT-PCR) tests showed that there was less mRNA from the BYDV coat protein in PeaT1-treated wheat seedlings than in the control group. Electrical penetration graph (EPG) tests showed that virus transmission vector S.graminum took a longer time to find probe and feeding sites on PeaT1-treated wheat seedlings. Additionally, PeaT1-treated wheat seedlings gained higher plant height and more chlorophyll a&b. These results showed that PeaT1 efficiently controlled BYDV by inhibiting BYDV proliferation, reducing the virus transmission ability of S. graminum and alleviating the symptoms of dwarfism and yellow colouring caused by BYDV. This study provided a new integrated way to control BYDV biologically.

First Report of Wheat dwarf virus in Winter Wheat in Estonia

First Report of <i>Wheat dwarf virus</i> in Winter Wheat in Estonia

Virus-Dependent and -Independent Responses of Sitobion avenae (Homoptera: Aphididae) Feeding on Wheat Infected by Transmitted and Nontransmitted Viruses at Transcriptomic Level

Most plant viruses maintain complex interactions with their vector or nonvector insects and can indirectly (via host plants) or directly affect the fitness of insects. However, little is known about the genes involved in the interactions between insects and transmitted or nontransmitted viruses, particularly nontransmitted viruses. Sitobion avenae (Fabricius) is a vector of barley yellow dwarf virus GAV strains (BYDV-GAV), but not a vector of wheat dwarf virus (WDV), which is transmitted by the leafhopper [Psammotettix alienus (Dahlbom)]. In this study, S. avenae was utilized to determine the transcriptomic responses after feeding on wheat infected by each of the two viruses, respectively, using an Illumina Hiseq sequencing platform. The transcriptomic data presented 61,508 genes, of which 854 differentially expressed. Moreover, in addition to sharing 208 genes, the number of differentially expressed genes (DEGs) in S. avenae exposed to BYDV was higher (800) than that when exposed to WDV (262). The DEGs related to the immune system and fitness of S. avenae in response to BYDV-/WDV-infected plants were identified and analyzed using Gene Ontologies (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the number of related DEGs was lower as nonvector than as vector. This study provides the baseline information to further examine molecular mechanisms of how wheat viruses affect S. avenae fitness and immune response either as a vector for BYDV-GAV or as a nonvector for WDV.

ADP ribosylation factor 1 facilitates spread of wheat dwarf virus in its insect vector.

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.

Higher Bacterial Diversity of Gut Microbiota in Different Natural Populations of Leafhopper Vector Does Not Influence WDV Transmission

The bacterial communities in the gut of an insect have important ecological and functional effects on the insect. However, the community composition and diversity of the gut microbiota in insects that vector plant viruses are poorly understood. As an important insect vector, Psammotettix alienus transmits various viruses including wheat dwarf virus (WDV). Here, we used the combination of leafhopper and WDV as model to survey the influence of gut microbiota on virus transmission characteristic of insect vector and vice versa. We have characterized 22 phyla and 249 genera of all gut bacterial communities in the leafhopper populations collected from six geographic regions in China. Community composition and diversity varied across different geographic populations. However, WDV transmission efficiencies of these six field populations were all greater than 80% with no significant difference. Interestingly, the transmission efficiency of WDV by laboratory reared insects with decreased gut bacterial diversity was similar to that of field populations. Furthermore, we found that the composition of the leafhopper gut bacteria was dynamic and could reversibly respond to WDV acquisition. Higher bacterial diversity and abundance of gut microbiota in different leafhopper populations did not influence their WDV transmission efficiency, while the acquisition of WDV changes gut microbiota by a dynamic and reversible manner. This report provides insight into the complex relationship between the gut microbiota, insect vector and virus.

Wheat dwarf virus infectious clones allow to infect wheat and Triticum monococcum plants

We constructed Wheat dwarf virus (WDV) infectious clones in the bacterial plasmids pUC18 and pIPKb002 and tested their ability to inoculate plants using Bio-Rad Helios Gene Gun biolistic inoculation method and Agrobacterium tumefaciens agroinoculation method, and we then compared them with the natural inoculation method via viruliferous P. alienus. Infected plants were generated using both infectious clones, whereas the agroinoculation method was able to produce strong systemic infection in all three tested cultivars of wheat and Triticum monococcum, comparable to plants inoculated by viruliferous P. alienus. Infection was confirmed by DAS-ELISA, and WDV titres were quantified using qPCR. The levels of remaining bacterial plasmid DNA were also confirmed to be zero.

Two mutations in the truncated Rep gene RBR domain delayed the Wheat dwarf virus infection in transgenic barley plants

Wheat dwarf virus (WDV), an important cereal pathogen, is closely related to Maize streak virus (MSV), a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication-associated (RepA) gene (WDVRepA215) and the WDV RepA gene with a mutated retinoblastoma-related protein (RBR) interaction domain (WDVRepA215RBRmut) were cloned into the pIPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers (Psammotettix alienus) fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBRmut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBRmut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.

Elevated CO2 and virus infection impacts wheat and aphid metabolism

The aphid Rhopalosiphum padi L. is a vector of Barley yellow dwarf virus (BYDV) in wheat and other economically important cereal crops. Increased atmospheric CO2 has been shown to alter plant growth and metabolism, enhancing BYDV disease in wheat. However, the biochemical influences on aphid metabolism are not known. This work aims to determine whether altered host-plant quality, influenced by virus infection and elevated CO2, impacts aphid weight and metabolism. Untargeted 1H NMR metabolomics coupled with multivariate statistics were employed to profile the metabolism of R. padi reared on virus-infected and non-infected (sham-inoculated) wheat grown under ambient CO2 (aCO2, 400µmolmol-1) and future, predicted elevated CO2 (eCO2, 650µmolmol-1) concentrations. Un-colonised wheat was also profiled to observe changes to host-plant quality (i.e., amino acids and sugars). The direct impacts of virus or eCO2 were compared. Virus presence increased aphid weight under aCO2 but decreased weight under eCO2; whilst eCO2 increased non-viruliferous (sham) aphid weight but decreased viruliferous aphid weight. Discriminatory metabolites due to eCO2 were succinate and sucrose (in sham wheat), glucose, choline and betaine (in infected wheat), and threonine, lactate, alanine, GABA, glutamine, glutamate and asparagine (in aphids), irrespective of virus presence. Discriminatory metabolites due to virus presence were alanine, GABA, succinate and betaine (in wheat) and threonine and lactate (in aphids), irrespective of CO2 treatment. This study confirms that virus and eCO2 alter host-plant quality, and these differences are reflected by aphid weight and metabolism.

Temperature affected transmission, symptom development and accumulation of Wheat dwarf virus

One of the biotic agents of yellowing and stunting in wheat and barley cultivations is Wheat dwarf virus (WDV) which is naturally transmitted by the leafhopper Psammotettix alienus (Dahlbom). WDV-Wheat and WDV-Barley isolates of WDV were transmitted to wheat and barley, respectively, using the leafhoppers under four temperature regimes of constant 20, 25, 30, and 35°C. Infection rate, symptom development and virus content of the virus-inoculated plants were determined and the data was statistically analysed. The results showed that the temperature of 25°C was associated with the highest infection rate caused by the viruses. Moreover, P. alienus nymphs were found to be more efficient vectors of WDV than adults, highlighting the importance of nymphs in the epidemiology of wheat dwarf disease. WDV-infected plants incubated at 35°C showed less symptoms than those kept at 20, 25, and 30°C. ELISA results showed that these plants had comparatively low virus content. However, there was no significant difference between the infection rate, symptom development and virus content in plants infected by WDV-Wheat or WDV-Barley.

Could vectors\u2019 fear of predators reduce the spread of plant diseases?

Predators influence the behaviour of prey and by doing so they potentially reduce pathogen transmission by a vector. Arthropod predators have been shown to reduce the consumption of plant biomass by pest herbivores, but their cascading non-consumptive effect on vector insects’ feeding behaviour and subsequent pathogen transmission has not been investigated experimentally before. Here we experimentally examined predator-mediated pathogen transmission mechanisms using the plant pathogen Wheat Dwarf Virus that is transmitted by the leafhopper, Psammotettix alienus. We applied in situ hybridization to localize which leaf tissues were infected with transmitted virus DNA in barley host plants, proving that virus occurrence is restricted to phloem tissues. In the presence of the spider predator, Tibellus oblongus, we recorded the within leaf feeding behaviour of the herbivore using electrical penetration graph. The leafhopper altered its feeding behaviour in response to predation risk. Phloem ingestion, the feeding phase when virus acquisition occurs, was delayed and was less frequent. The phase when pathogen inoculation takes place, via the secretion of virus infected vector saliva, was shorter when predator was present. Our study thus provides experimental evidence that predators can potentially limit the spread of plant pathogens solely through influencing the feeding behaviour of vector organisms.

Detection and discrimination of barley- and wheat-specific forms of Wheat dwarf virus in Poland

Wheat dwarf virus (WDV) is one of the most common viruses on cereal crops in Poland. Studies were undertaken aiming at molecular characterization of Polish isolates of the virus. The presence of two main groups, WDV-barley- and WDV-wheat-specific forms, in field samples has been confirmed. Detection and differentiation of WDV isolates was conducted using immuno-capture polymerase chain reactions. The studies were carried out on a set of 68 samples collected from different parts of the country. Obtained results demonstrated that WDV-wheat-specific form can infect all tested cereals: wheat, triticale, rye and barley while WDV-barley-specific form was identified mainly in barley and in rare cases in wheat plants. Comparative analysis of coat protein gene was performed using 16 WDV isolates originated from different hosts revealed high (>98%) nucleotide sequence identity. Moreover, WDV-wheat-specific (Pol-WDV-W) and WDV-barley-specific (WDV-B) isolates were fully sequenced. Based on nucleotide sequence similarity, Pol-WDV-W should be classified as WDV-E and WDV-B as WDV-F strains. This is the first report of the complete sequence of WDV isolates from Poland.

Monoclonal Antibody-Based Serological Detection Methods for Wheat Dwarf Virus.

Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.

TaqMan Multiplex Real-Time qPCR assays for the detection and quantification of Barley yellow dwarf virus, Wheat dwarf virus and Wheat streak mosaic virus and the study of their interactions

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (15°C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at −20°C. By far the most consistent amount of vigorous myceliogenic germination (&gt;75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum.

Detection of barley- and wheat-specific forms of Wheat dwarf virus in their vector Psammotettix alienus by duplex PCR assay

Detection of barley- and wheat-specific forms of Wheat dwarf virus in their vector Psammotettix alienus by duplex PCR assay

Simultaneous detection of wheat dwarf virus, northern cereal mosaic virus, barley yellow striate mosaic virus and rice black-streaked dwarf virus in wheat by multiplex RT-PCR

Wheat dwarf virus (WDV), barley yellow striate mosaic virus (BYSMV), rice black-streaked dwarf virus (RBSDV) and northern cereal mosaic virus (NCMV) are four viruses infecting wheat and causing similar symptoms. In this paper, a multiplex reverse transcription polymerase chain reaction (m-RT-PCR) method has been developed for the simultaneous detection and discrimination of these viruses. The protocol uses specific primer set for each virus and produces four distinct fragments (273, 565, 783 and 1296bp), detecting the presence of RBSDV, BYSMV, WDV and NCMV, respectively. Annealing temperature, concentrations of dNTP, Taq polymerase and Mg2+ were optimized for the m-RT-PCR. The detection limit of the assay was up to 10−2 dilution. The amplification specificity of these primers was tested against a range of field samples from different regions of China, where RBSDV, BYSMV, WDV have been detected. This study fulfills the need for a rapid and specific wheat virus detection that also has the potential for investigating the epidemiology of these new viral diseases.

First Report of the Occurrence of Wheat dwarf virus Infecting Wheat in Slovenia

First Report of the Occurrence of Wheat dwarf virus Infecting Wheat in Slovenia

Viral Vectors for Plant Genome Engineering.

Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. Typically, this is achieved using Agrobacterium mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. The alternate in planta approaches, stably integrating nuclease-encoding cassettes and repair templates into the plant genome, are time consuming, expensive and require extra regulations. More efficient GE reagents delivery methods are clearly needed if GE is to become routine, especially in economically important crops that are difficult to transform. Recently, autonomously replicating virus-based vectors have been demonstrated as efficient means of delivering GE reagents in plants. Both DNA viruses (Bean yellow dwarf virus, Wheat dwarf virus and Cabbage leaf curl virus) and RNA virus (Tobacco rattle virus) have demonstrated efficient gene targeting frequencies in model plants (Nicotiana benthamiana) and crops (potato, tomato, rice, and wheat). Here we discuss the recent advances using viral vectors for plant genome engineering, the current limitations and future directions.