The plant rhabdovirus viroporin P9 facilitates insect-mediated virus transmission in barley.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of glycine 14 to threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-GFP (BYGFP) virus, a P9-deleted mutant (BYGFPΔP9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFPΔP9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission.

Barley yellow dwarf virus-GAV 17K protein disrupts thiamine biosynthesis to facilitate viral infection in plants.

Thiamine functions as a crucial activator modulating plant health and broad-spectrum stress tolerances. However, the role of thiamine in regulating plant virus infection is largely unknown. Here, we report that the multifunctional 17K protein encoded by barley yellow dwarf virus-GAV (BYDV-GAV) interacted with barley pyrimidine synthase (HvTHIC), a key enzyme in thiamine biosynthesis. HvTHIC was found to be localized in chloroplast via an N-terminal 74-amino acid domain. However, the 17K-HvTHIC interaction restricted HvTHIC targeting to chloroplasts and triggered autophagy-mediated HvTHIC degradation. Upon BYDV-GAV infection, the expression of the HvTHIC gene was significantly induced, and this was accompanied by accumulation of thiamine and salicylic acid. Silencing of HvTHIC expression promoted BYDV-GAV accumulation. Transcriptomic analysis of HvTHIC silenced and non-silenced barley plants showed that the differentially expressed genes were mainly involved in plant-pathogen interaction, plant hormone signal induction, phenylpropanoid biosynthesis, starch and sucrose metabolism, photosynthesis-antenna protein, and MAPK signaling pathway. Thiamine treatment enhanced barley resistance to BYDV-GAV. Taken together, our findings reveal a molecular mechanism underlying how BYDV impedes thiamine biosynthesis to uphold viral infection in plants.

Bio-control of soil-borne virus infection by seed application of Glycyrrhiza glabra extract and the rhamnolipid Rhapynal.

MAIN CONCLUSION: Seed-application of the natural products protects sugar beet and wheat plants against infection with plasmodiophorid-transmitted viruses and thus may represent an efficient, environmentally friendly, easy and cost effective biocontrol strategy. In times of intensive agriculture, resource shortening and climate change, alternative, more sustainable and eco-friendly plant protection strategies are required. Here, we tested the potential of the natural plant substances Glycyrrhiza glabra leaf extract (GE) and the rhamnolipid Rhapynal (Rha) applied to seeds to protect against infection of sugar beet and wheat with soil-borne plant viruses. The soil-borne Polymyxa betae- and Polymyxa graminis-transmitted viruses cause extensive crop losses in agriculture and efficient control strategies are missing. We show that GE and Rha both efficiently protect plants against infection with soil-borne viruses in sugar beet and wheat when applied to seeds. Moreover, the antiviral protection effect is independent of the cultivar used. No protection against Polymyxa sp. was observed after seed treatment with the bio-substances at our analysis time points. However, when we applied the bio-substances directly to soil a significant anti-Polymyxa graminis effect was obtained in roots of barley plants grown in the soil as well as in the treated soil. Despite germination can be affected by high concentrations of the substances, a range of antiviral protection conditions with no effect on germination were identified. Seed-treatment with the bio-substances did not negatively affect plant growth and development in virus-containing soil, but was rather beneficial for plant growth. We conclude that seed treatment with GE and Rha may represent an efficient, ecologically friendly, non-toxic, easy to apply and cost efficient biocontrol measure against soil-borne virus infection in plants.

The prominent multiplication of Japanese soil-borne wheat mosaic virus co-infected with barley yellow mosaic virus in barley.

Various members of the viral genera Furovirus and Bymovirus are damaging pathogens of a range of crop species. Infection of the soil-borne plasmodiophorid Polymyxa graminis transmits both Japanese soil-borne wheat mosaic virus (JSBWMV) and the barley yellow mosaic virus (BaYMV) to barley, but their interaction during an episode of their co-infection has not been characterized to date. Here, we present an analysis of the titer of JSBWMV and BaYMV in plants of winter barley growing over a five-month period from late fall until mid-spring. Although JSBWMV was detectable in the plants' roots four weeks earlier than BaYMV, the translocation of both viruses from the root to the leaves occurred nearly simultaneously. Both viruses were co-localized in the roots, leaf sheathes, and leaf blades; however, in some stripes of leaf veins where infection by JSBWMV was prominent, BaYMV was not detectable. A substantial titer of both viruses persisted until early spring, after which JSBWMV became more prominent, being in a range of 10 to 100 times abundant of BaYMV. However, JSBWMV was only able to infect a single wheat accession (cv. Norin 61), whereas all of the wheat entries assayed appeared to be immune to BaYMV infection. Overall, our findings highlight the importance of resistance mechanisms against soil-borne viruses in cereal crops, expanding our understanding of plant-virus interactions and potentially informing strategies for crop protection against viral pathogens.

A new geminialphasatellite associated with wheat dwarf virus identified in winter barley in France.

Using a high-throughput sequencing (HTS) approach, we report the discovery of a new alphasatellite identified in a winter barley plant collected in France in 2022 that was also infected by wheat dwarf virus (WDV). The presence of the satellite and of WDV was confirmed by several independent PCR assays, and the complete genome sequence was determined. The circular satellite genome is 1424 nt long and shows typical hallmarks of members of the subfamily Geminialphasatellitinae, including a replication-associated hairpin with a CAGTATTAC sequence and a Rep-encoding open reading frame (ORF). It also possesses a second ORF, embedded in a different frame within the Rep ORF, which is also observed in clecrusatellites and a few other members of the family Alphasatellitidae. Pairwise sequence comparisons and phylogenetic analysis showed that this satellite represents a novel species. Its closest relatives are in the genus Colecusatellite, but it likely represents a new genus given its divergence from other genera of the subfamily Geminialphasatellitinae. Given that WDV was the only virus observed in coinfection with the satellite, the name "wheat dwarf virus-associated alphasatellite" is proposed for this novel agent.

Yellow Dwarf Virus Resistance in Barley: Phenotyping, Remote Imagery, and Virus-Vector Characterization.

Yellow dwarf viruses (YDVs) spread by aphids are some of the most economically important barley (Hordeum vulgare) virus-vector complexes worldwide. Detection and control of these viruses are critical components in the production of barley, wheat, and numerous other grasses of agricultural importance. Genetic control of plant diseases is often preferable to chemical control to reduce the environmental and economic cost of foliar insecticides. Accordingly, the objectives of this work were to (i) screen a barley population for resistance to YDVs under natural infection using phenotypic assessment of disease symptoms, (ii) implement drone imagery to further assess resistance and test its utility as a disease screening tool, (iii) identify the prevailing virus and vector types in the experimental environment, and (iv) perform a genome-wide association study to identify genomic regions associated with measured traits. Significant genetic differences were found in a population of 192 barley inbred lines regarding their YDV symptom severity, and symptoms were moderately to highly correlated with grain yield. The YDV severity measured with aerial imaging was highly correlated with on-the-ground estimates (r = 0.65). Three aphid species vectoring three YDV species were identified with no apparent genotypic influence on their distribution. A quantitative trait locus impacting YDV resistance was detected on chromosome 2H, albeit undetected using aerial imaging. However, quantitative trait loci for canopy cover and mean normalized difference vegetation index were successfully mapped using the drone. This work provides a framework for utilizing drone imagery in future resistance breeding efforts for YDVs in cereals and grasses, as well as in other virus-vector disease complexes.

Series of Resistance Genes in Barley (Hordeum vulgare) that Control Barley Yellow Mosaic Virus Multiplication and the Root-to-Leaf Systemic Movement.

The infection of young winter barley (Hordeum vulgare L.) root system in winter by barley yellow mosaic virus (BaYMV) can lead to high yield losses. Resistance breeding is critical for managing this virus, but there are only a few reports on resistance genes that describe how the genes control BaYMV propagation and the systemic movement from the roots to the leaves. Here we report a real-time quantitative PCR analysis of the virus in barley roots and leaves carrying BaYMV resistance genes (rym1 to rym15 and an unknown gene) to elucidate the molecular mechanisms underlying the barley response to BaYMV. The resistance mechanism directly targets the virus. Moreover, the resistance genes/cultivars were classified into the following three groups according to their BaYMV titer: (i) immune (BaYMV was undetectable in the roots or leaves), (ii) partially immune (BaYMV was detected in the roots but not in the leaves), and (iii) susceptible (BaYMV was detected in the roots and leaves). Our results clarified the functions of the resistance genes in barley roots and leaves following a BaYMV infection. We anticipate our analysis to be a starting point for more understanding of the correspondence between resistance genes of Triticeae and the soil-borne viruses.

Development of the differential detection reverse transcription loop-mediated isothermal amplification method for Barley yellow mosaic virus or Japanese soil-borne wheat mosaic virus from barley.

A differential detection reverse transcription loop-mediated isothermal amplification (DD-RT-LAMP) method was developed to detect either Barley yellow mosaic virus (BaYMV) or Japanese soil-borne wheat mosaic virus (JSBWMV) simultaneously. Both primer sets, which recognized either BaYMV or JSBWMV genomic RNA, amplified DNA more efficiently at 65°C using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak annealing temperatures of BaYMV and JSBWMV amplification products using specific primer sets were 86.9°C-87.7°C and 84.5°C-85.0°C, respectively, and were clearly distinguishable during an annealing step following the isothermal amplification, monitored using a fluorescence detection device. In the field samples of barley (Hordeum vulgare L.) tested, BaYMV or JSBWMV were detected by DD-RT-LAMP, and the detection results of DD-RT-LAMP were correspondent with the results of reverse transcription-PCR.

Identification of novel QTL contributing to barley yellow mosaic resistance in wild barley (Hordeum vulgare spp. spontaneum).

BACKGROUND: Barley yellow mosaic disease (BYMD) caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) seriously threatens the production of winter barley. Cultivating and promoting varieties that carry disease-resistant genes is one of the most powerful ways to minimize the disease's effect on yield. However, as the BYMD virus mutates rapidly, resistance conferred by the two cloned R genes to the virus had been overcome by new virus strains. There is an urgent need for novel resistance genes in barley that convey sustainable resistance to newly emerging virus strains causing BYMD. RESULTS: A doubled haploid (DH) population derived from a cross of SRY01 (BYMD resistant wild barley) and Gairdner (BYMD susceptible barley cultivar) was used to explore for QTL of resistance to BYMD in barley. A total of six quantitative trait loci (qRYM-1H, qRYM-2Ha, qRYM-2Hb, qRYM-3H, qRYM-5H, and qRYM-7H) related to BYMD resistance were detected, which were located on chromosomes 1H, 2H, 3H, 5H, and 7H. Both qRYM-1H and qRYM-2Ha were detected in all environments. qRYM-1H was found to be overlapped with rym7, a known R gene to the disease, whereas qRYM-2Ha is a novel QTL on chromosome 2H originated from SRY01, explaining phenotypic variation from 9.8 to 17.8%. The closely linked InDel markers for qRYM-2Ha were developed which could be used for marker-assisted selection in barley breeding. qRYM-2Hb and qRYM-3H were stable QTL for specific resistance to Yancheng and Yangzhou virus strains, respectively. qRYM-5H and qRYM-7H identified in Yangzhou were originated from Gairdner. CONCLUSIONS: Our work is focusing on a virus disease (barley yellow mosaic) of barley. It is the first report on BYMD-resistant QTL from wild barley accessions. One novel major QTL (qRYM-2Ha) for the resistance was detected. The consistently detected new genes will potentially serve as novel sources for achieving pre-breeding barley materials with resistance to BYMD.

Genomic prediction models trained with historical records enable populating the German ex situ genebank bio-digital resource center of barley (Hordeum sp.) with information on resistances to soilborne barley mosaic viruses.

KEY MESSAGE: Genomic prediction with special weight of major genes is a valuable tool to populate bio-digital resource centers. Phenotypic information of crop genetic resources is a prerequisite for an informed selection that aims to broaden the genetic base of the elite breeding pools. We investigated the potential of genomic prediction based on historical screening data of plant responses against the Barley yellow mosaic viruses for populating the bio-digital resource center of barley. Our study includes dense marker data for 3838 accessions of winter barley, and historical screening data of 1751 accessions for Barley yellow mosaic virus (BaYMV) and of 1771 accessions for Barley mild mosaic virus (BaMMV). Linear mixed models were fitted by considering combinations for the effects of genotypes, years, and locations. The best linear unbiased estimations displayed a broad spectrum of plant responses against BaYMV and BaMMV. Prediction abilities, computed as correlations between predictions and observed phenotypes of accessions, were low for the marker-assisted selection approach amounting to 0.42. In contrast, prediction abilities of genomic best linear unbiased predictions were high, with values of 0.62 for BaYMV and 0.64 for BaMMV. Prediction abilities of genomic prediction were improved by up to ~ 5% using W-BLUP, in which more weight is given to markers with significant major effects found by association mapping. Our results outline the utility of historical screening data and W-BLUP model to predict the performance of the non-phenotyped individuals in genebank collections. The presented strategy can be considered as part of the different approaches used in genebank genomics to valorize genetic resources for their usage in disease resistance breeding and research.

High-resolution mapping of Rym14Hb, a wild relative resistance gene to barley yellow mosaic disease.

KEY MESSAGE: We mapped the Rym14Hb resistance locus to barley yellow mosaic disease in a 2Mbp interval. The co-segregating markers will be instrumental for marker-assisted selection in barley breeding. Barley yellow mosaic disease is caused by Barley yellow mosaic virus and Barley mild mosaic virus and leads to severe yield losses in barley (Hordeum vulgare) in Central Europe and East-Asia. Several resistance loci are used in barley breeding. However, cases of resistance-breaking viral strains are known, raising concerns about the durability of those genes. Rym14Hb is a dominant major resistance gene on chromosome 6HS, originating from barley's secondary genepool wild relative Hordeum bulbosum. As such, the resistance mechanism may represent a case of non-host resistance, which could enhance its durability. A susceptible barley variety and a resistant H. bulbosum introgression line were crossed to produce a large F2 mapping population (n = 7500), to compensate for a ten-fold reduction in recombination rate compared to intraspecific barley crosses. After high-throughput genotyping, the Rym14Hb locus was assigned to a 2Mbp telomeric interval on chromosome 6HS. The co-segregating markers developed in this study can be used for marker-assisted introgression of this locus into barley elite germplasm with a minimum of linkage drag.

Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants.

Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex-reverse transcription-polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

Bymovirus-induced yellow mosaic diseases in barley and wheat: viruses, genetic resistances and functional aspects.

Bymovirus-induced yellow mosaic diseases seriously threaten global production of autumn-sown barley and wheat, which are two of the presently most important crops around the world. Under natural field conditions, the diseases are caused by infection of soil-borne plasmodiophorid Polymyxa graminis-transmitted bymoviruses of the genus Bymovirus of the family Potyviridae. Focusing on barley and wheat, this article summarizes the achievements on taxonomy, geography and host specificity of these disease-conferring viruses, as well as the genetics of resistance in barley, wheat and wild relatives. Moreover, based on recent progress of barley and wheat genomics, germplasm resources and large-scale sequencing, the exploration and isolation of corresponding resistant genes from wheat and barley as well as relatives, no matter what a large and complicated genome is present, are becoming feasible and are discussed. Furthermore, the foreseen advances on cloning of the resistance or susceptibility-encoding genes, which will provide the possibility to explore the functional interaction between host plants and soil-borne viral pathogens, are discussed as well as the benefits for marker-assisted resistance breeding in barley and wheat.

Barley yellow dwarf virus-GAV-derived vsiRNAs are involved in the production of wheat leaf yellowing symptoms by targeting chlorophyll synthase.

BACKGROUND: Wheat yellow dwarf virus disease is infected by barley yellow dwarf virus (BYDV), which causes leaf yellowing and dwarfing symptoms in wheat, thereby posing a serious threat to China's food production. The infection of plant viruses can produce large numbers of vsiRNAs, which can target host transcripts and cause symptom development. However, few studies have been conducted to explore the role played by vsiRNAs in the interaction between BYDV-GAV and host wheat plants. METHODS: In this study, small RNA sequencing was conducted to profile vsiRNAs in BYDV-GAV-infected wheat plants. The putative targets of vsiRNAs were predicted by the bioinformatics software psRNATarget. RT-qPCR and VIGS were employed to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5' RACE was performed to analyze the specific cleavage sites. RESULTS: From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs was mostly 21 and 22 nt, and an A/U bias was observed at the 5' terminus. We also observed that the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis demonstrated that these targets were mostly involved in cell components, catalytic activity and plant-pathogen interactions. The results of RT-qPCR analysis showed that most chloroplast-related genes were downregulated in BYDV-GAV-infected wheat plants. Silencing of a chlorophyll synthase gene caused leaf yellowing that was similar to the symptoms exhibited by BYDV-GAV-inoculated wheat plants. A vsiRNA from an overlapping region of BYDV-GAV MP and CP was observed to target chlorophyll synthase for gene silencing. Next, 5' RACE validated that vsiRNA8856 could cleave the chlorophyll synthase transcript in a sequence-specific manner. CONCLUSIONS: This report is the first to demonstrate that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and the results of this study help to elucidate the molecular mechanisms underlying leaf yellowing after viral infection.

Development of loop-mediated isothermal amplification assay for rapid detection of genetically different wheat dwarf virus isolates.

Wheat dwarf virus (WDV) is considered as one of the most common viruses on cereal crops. Recently, severe outbreaks of WDV have been observed especially on winter wheat in southwestern part of Poland. Moreover, the presence of genetically different WDV-barley-specific and WDV-wheat-specific forms (WDV-B and WDV-W, respectively) was confirmed. In this study, a loop-mediated isothermal amplification assay (LAMP) was developed for the first time for efficient and rapid detection of WDV-B and WDV-W in infected plants. The reaction was performed using a set of three primer pairs: WDVF3/WDVB3, WDVFIB/WDVBIP and WDVLoopF/WDVLoopB specific for coat protein coding sequence. The amplified products were analyzed by direct staining of DNA, gel electrophoresis and real-time monitoring of the amplification curves. The sensitivity of optimized reaction was tenfold higher in comparison with conventional PCR. LAMP assay developed here is a useful and practical method for the rapid detection of different WDV isolates and can be implemented by phytosanitary services.

Barley yellow dwarf virus Can Be Inoculated During Brief Intracellular Punctures in Phloem Cells Before the Sieve Element Continuous Salivation Phase.

The distinguished intracellular stylet puncture called phloem-pd (potential drop [pd]) produced by Myzus persicae has been associated with the transmission of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus). However, the production of intracellular punctures in phloem cells (phloem-pd) by other aphid species and their role in the transmission of persistently transmitted, phloem-limited viruses are still unknown. Previous studies revealed that inoculation of the persistently transmitted, phloem-limited Barley yellow dwarf virus (BYDV, Luteovirus) is associated mainly with the sieve element continuous salivation phase (E1 waveform). However, the role of brief intracellular punctures that occur before the E1 phase in the inoculation of BYDV by aphids is unknown. We aimed to investigate whether the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae) produced a stereotypical phloem-pd and to study its role in the inoculation of BYDV. The feeding behavior of viruliferous R. padi individuals in barley (Hordeum vulgare) was monitored via the electrical penetration graph (EPG) technique. The feeding process was artificially terminated after the observation of specific EPG waveforms: standard-pds, phloem-pd, and E1. Analysis of the EPG recordings revealed the production of a phloem-pd pattern by R. padi, in addition to a short, distinct E1-like pattern (short-E1), both resulting in successful inoculation of BYDV. Also, the transmission efficiency of BYDV was directly proportional to the time spent by aphids in intracellular salivation in phloem cells. Finally, we discussed the main differences between the inoculation process of semipersistent and persistently transmitted phloem-limited viruses by aphids.

Construction of an Infectious Poa semilatent virus cDNA Clone and Comparisons of Hordeivirus Cytopathology and Pathogenicity.

Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV.

KEY MESSAGE: A novel rare allele of the barley host factor gene eIF4E for BaMMV/BaYMV infection was identified in an Iranian landrace that showed broad resistance to barley yellow mosaic virus disease, and molecular markers facilitating efficient selection were developed. The soil-borne yellow mosaic virus disease caused by different strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) is a major threat to winter barley (Hordeum vulgare) production in Europe and East Asia. However, the exploration of resistant germplasm or casual genes for barley breeding is rather limited in relation to the rapid diversification of viral strains. Here, we identified an Iranian barley landrace 'HOR3298,' which represented complete resistance to BaYMV and BaMMV. In contrast to rym4 and rym5, which act as the predominant source in Europe and East Asia for breeding resistant cultivars over decades and which have been overcome by several virulent isolates, this landrace showed broad-spectrum resistance to multiple isolates of BaYMV/BaMMV in the fields of Germany and China. By employment of bulked segregant RNA sequencing, test for allelism, and haplotype analysis, a recessive resistance gene in 'HOR3298' was genetically mapped coincident with the host factor eukaryotic translation initiation factor 4E (eIF4E, causal gene of rym4 and rym5). The eIF4EHOR3298 allele encoded for a novel haplotype that contained an exclusive nucleotide mutation (G565A) in the coding sequence. The easily handled markers were developed based on the exclusively rare variation, providing precise selection of this allele. Thus, this work provided a novel reliable resistance source and the feasible marker-assisted selection assays that can be used in breeding for barley yellow mosaic virus disease resistance in cultivated barley.