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Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

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.

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.

Barley yellow dwarf virus (BYDV) causes an often-devastating disease of cereals that is most effectively controlled by using plant genotypes that are resistant or tolerant to the virus. New barley lines Vir8:3 and Vir13:8, with pyramided resistance genes against different pathogens and resistance gene Ryd2 against BYDV, are currently being tested. Because microRNAs (miRNAs) are associated with antiviral plant defense, here we compared the miRNA profiles in these lines and in cultivar Wysor (carrying one resistance gene, Ryd2), with and without BYDV infection and after feeding by virus-free aphids, to determine whether the miRNA profile in the resistant variety bear similarities with the newly developed lines. The BYDV titer for each group was also determined and compared to the titer in sensitive cultivar Graciosa. Among 746 miRNAs identified in barley, 66 were known miRNAs, and 680 were novel. The expression of 73 miRNAs differed significantly after BYDV infection, including the strong, specific upregulation of novel miRNA10778 that was conserved across all the barley genotypes. This miRNA belongs to the H box and ACA box (H/ACA) snoR14 family of RNAs (Rf01280) and is associated with pseudourydilation. The expression of 48 miRNAs also differed depending on the barley genotype. The profile of miRNAs expressed in Vir8:3 and Vir13:8 in response to BYDV was similar and differed from that of Wysor. Insights into the expression patterns of miRNAs in response to BYDV in barley provided here will benefit further studies toward understanding the resistance mechanisms and developing novel strategies against virus infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

An isolate of Hydrangea ringspot virus (HdRSV) was obtained in 2008 from infected Hydrangea spp. in the Czech Republic. The isolate (HydCZ) induced chlorotic to mildly necrotic local lesions or chlorotic systemic symptoms, respectively, in Chenopodium quinoa and Nicotiana benthamiana. The genome of HydCZ (6185 nt) was fully sequenced. Phylogenetic analysis revealed that isolate HydCZ shares high nucleotide sequence identity (96.6%) with an isolate (PD109) from the Netherlands. This is the first report of the sequence of an isolate of HdRSV from the Czech Republic and is the second full genome sequence of the virus. [ABSTRACT FROM AUTHOR]

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A biography of associate professor Jaroslav Polák of Crop Research Institute (CRI) in Prague in Czech Republic is presented. He was born on January 25, 1942 and studied and graduated at the Agricultural University in Prague in agronomy. He worked at the Phytopathological Institute in Aschersleben, Germany wherein he acquired more scientific knowledge and technical experience on plant viruses. Polák is a prominent scientist with international reputation in agriculture.