Sorghum mastrevirus-associated alphasatellites: new geminialphasatellites associated with an African streak mastrevirus infecting wild Poaceae plants on Reunion Island.

Nine complete nucleotide sequences of geminialphasatellites (subfamily Geminialphasatellitinae, family Alphasatellitidae) recovered from the wild Poaceae Sorghum arundinaceum collected in Reunion are described and analyzed. While the helper geminivirus was identified as an isolate of maize streak virus (genus Mastrevirus, family Geminiviridae), the geminialphasatellite genomes were most closely related to, and shared ~63% identity with, clecrusatellites. Even though the geminialphasatellite molecules lack an adenine rich-region, they have the typical size of geminialphasatellites, encode a replication-associated protein in the virion sense, and have probable stem-loop structures at their virion-strand origins of replication. According to the proposed geminialphasatellite species and genus demarcation thresholds (88% and 70% nucleotide identity, respectively), the genomes identified here represent a new species (within a new genus) for which we propose the name "Sorghum mastrevirus-associated alphasatellite" (genus "Sorgasalphasatellite").

Lack of transmission of Sugarcane yellow leaf virus in Florida from Columbus grass and sugarcane to sugarcane with aphids or mites.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane (Saccharum spp.), the weed Columbus grass (Sorghum almum) and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18-28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.

Occurrence of putative endornaviruses in non-cultivated plant species in South Louisiana.

Extraction and electrophoretic analysis of viral dsRNA from plants has been used successfully to detect infections by RNA viruses. We used this approach as an initial tool to test non-cultivated plant species for the presence of endornaviruses. Foliar samples were collected from symptomless plants in various locations within East Baton Rouge Parish, Louisiana, USA, and tested for viral dsRNA. After testing 208 plant species belonging to 74 families, five (Geranium carolinianum, Hydrocotyle umbellata, H. prolifera, Sorghum halepense, and Sisyrinchium atlanticum) yielded dsRNAs similar in size to the dsRNAs of members of the family Endornaviridae. The endornavirus nature of the dsRNAs was confirmed by reverse-transcription PCR (RT-PCR) and sequencing the RT-PCR products. Sequence data were used to determine relationships of the putative endornaviruses to members of the family Endornaviridae. The putative endornaviruses were detected in both native and introduced plants species. This is the first survey on the occurrence of endornaviruses in non-cultivated plant species.

Differential molecular response of maize and Johnson grass against maize dwarf mosaic virus and bermuda grass southern mosaic virus.

Maize dwarf mosaic virus (MDMV) and bermuda grass southern mosaic virus (BgSMV) is the most important cereal potyvirus in Iran. Expression of some key genes in maize plants susceptible and tolerant to MDMV or BgSMV and gene expression profile of MDMV and BgSMV compatible or incompatible to Johnson grass plants were studied. Time points of 1, 9, 24 and 72 h after inoculation with both viruses were investigated as well. By analyzing the expression of the genes, it was identified that in maize infected by MDMV and BgSMV, the transcript levels of the peroxiredoxin, GLP, SAM, NPR1 and chlorophyll a-b binding genes were significantly higher in the tolerant than in susceptible plants during the entire experiment. In the BgSMV inoculated Johnson grass plants, some of the genes related to plant defense responses including NPR1, peroxiredoxin and SAM had higher expression level than the Johnson grass plants inoculated by MDMV. Important genes in maize tolerance like NPR1 and MT-LP, were analyzed by trilinear decomposition analysis and genes clustering. The upregulated expression of genes at one-hour post inoculation showed that the plant response to viruses was activated at the early stage of infection. Keywords: MDMV; BgSMV; gene expression; quantitative real-time PCR; trilinear decomposition analysis.

An efficient and improved method for virus-induced gene silencing in sorghum.

BACKGROUND: Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. METHODS: Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. RESULTS: This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. CONCLUSION: In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots.

Genome-wide analysis of alternative splicing landscapes modulated during plant-virus interactions in Brachypodium distachyon.

In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes.

Viruses in maize and Johnsongrass in southern Ohio.

The two major U.S. maize viruses, Maize dwarf mosaic virus (MDMV) and Maize chlorotic dwarf virus (MCDV), emerged in southern Ohio and surrounding regions in the 1960s and caused significant losses. Planting resistant varieties and changing cultural practices has dramatically reduced virus impact in subsequent decades. Current information on the distribution, diversity, and impact of known and potential U.S. maize disease-causing viruses is lacking. To assess the current reservoir of viruses present at the sites of past disease emergence, we used a combination of serological testing and next-generation RNA sequencing approaches. Here, we report enzyme-linked immunosorbent assay and RNA-Seq data from samples collected over 2 years to assess the presence of viruses in cultivated maize and an important weedy reservoir, Johnsongrass (Sorghum halepense). Results revealed a persistent reservoir of MDMV and two strains of MCDV in Ohio Johnsongrass. We identified sequences of several other grass-infecting viruses and confirmed the presence of Wheat mosaic virus in Ohio maize. Together, these results provide important data for managing virus disease in field corn and sweet corn maize crops, and identifying potential future virus threats.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBß, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.

Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells.

Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.

Identification of prevalent potyvirus on maize and johnsongrass in corn fields of Tehran province of Iran and a study on some of its properties.

During a growing season in 2004, 231 leaf samples of virus infected and mosaic and dwarf mosaic symptoms showing maize (Zea mays L.) plants and 258 leaf samples of mosaic showing johnsongrass (Sorghum halepens L.) plants from various corn fields in Tehran province were collected. Serological tests of DAS-ELISA and DIBA were performed on samples using antisera of sugarcane mosaic virus (SCMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and johnsongrasss mosaic virus (JGMV). In both tests performed on leaf samples extractions, all samples reacted strongly with SCMV antiserum and no reaction was seen with other 3 potyviruses antisera. 0.1 M potassium phosphate buffer (pH = 7) containing 2% polyvinyl pyrrolidon (PVP) was used for mechanical inoculation and all isolates were inoculated and propagated on sweet corn cv. Pars 403 and grain sorghum cv. Kimia. In serological tests on the inoculated plants samples also only SCMV was detected. Purification of virus was done using a modified "minipurification" method and the concentration of purified virus was 11.45 mg/ml and ratio of A260/280 = 1.2 was calculated for it. Electron microscopic study using ISEM and decoration method with SCMV antiserum revealed filamentous flexuous particles of SCMV. In SDS-polyachrylamide gel electrophoresis and Western blot test using SCMV antiserum that were performed on infected samples and purified viruses, the molecular weight of the virus coat protein was approximately 37-38 KDa and a difference among the CP weights of various SCMV isolates was not found. Reverse transcription-polymerase chain reaction (RT-PCR) was done using SCMV F3 and SCMV R3 primers and amplified fragments of approximately 900 bp in size were as in expected. The host range study with selected isolates of SCMV showed that the virus isolates were not transmitted by mechanical inoculation on Avena sativa, Panicum miliaceum, Setaria italica, Pennisetum americanum, Hordeum vulgare and Triticum aestivum. The isolates produced red-brown necrotic streaks on sudangrass (Sorghum sudanense) that lately changed in systemic necrosis. In host reaction studies on sorghum (Sorghum bicolor) cultivars, the virus isolates caused severe necrotic and killer reaction on sorghum cultivars Payam, Sepideh and Speed feed, but caused systemic mosaic and non-killer reaction on sorghum cultivars Kimia, KFS2, KFS3 and Jumbo. The present study showed that SCMV is the prevalent potyvirus and the main causal agent of mosaic and dwarf mosaic on maize plants in province. Since the virus is prevalent on johnsongrass plants in marginal areas of corn fields too, it seems that the origin of the virus on corn is from johnsongrass and the virus is a special strain of sugarcane mosaic virus that infects johnsongrass too.

Characterization of a distinct Johnsongrass mosaic virus strain isolated from sorghum in Nigeria.

A virus isolated from sorghum in Nigeria has been partially characterized. It was tested by enzyme-linked immunosorbent assay (ELISA) using antisera to Maize dwarf mosaic virus, Johnsongrass mosaic virus (JGMV), Sugarcane mosaic virus strain-MDB, Sorghum mosaic virus, and Zea mosaic virus. A partial host range, symptom phenotypes for selected sorghum lines, and the mass of the coat protein (CP) subunit was analyzed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid (aa) sequence determined by time-of-flight mass spectrometry (TOFMS). The Nigerian isolate was positive in ELISA to only JGMV antiserum. It infected sorghum and smooth brome but not oat or johnsongrass. It caused necrosis in 12 of 13 tested sorghum lines, while the USA JGMV isolate caused necrosis in only one sorghum line. In SDS-PAGE, the mass of the Nigerian virus CP was 3,000 Da smaller than that of JGMV-MDO. Moreover, TOFMS analyses showed that, while residues 1-7 of the CP aa sequence were identical to those of JGMV (GenBank #A27631), and residues 57-293 were almost identical to residues 67-303 of JGMV, the intermediate region exhibited significant differences, including a 10 aa deletion. These data indicate that the virus should be considered a distinct isolate of JGMV (JGMV-N) and expands the known range of JGMV to Africa.

Sequence diversity in the coat protein coding region of the genome RNA of Johnsongrass mosaic virus in Australia.

Sequence diversity in the coat protein coding region of Australian strains of Johnsongrass mosaic virus (JGMV) was investigated. Field isolates were sampled during a seven year period from Johnsongrass, sorghum and corn across the northern grain growing region. The 23 isolates were found to have greater than 94% nucleotide and amino acid sequence identity. The Australian isolates and two strains from the U.S.A. had about 90% nucleotide sequence identity and were between 19 and 30% different in the N-terminus of the coat protein. Two amino acid residues were found in the core region of the coat protein in isolates obtained from sorghum having the Krish gene for JGMV resistance that differed from those found in isolates from other hosts which did not have this single dominant resistance gene. These amino acid changes may have been responsible for overcoming the resistance conferred by the Krish gene for JGMV resistance in sorghum. The identification of these variable regions was essential for the development of durable pathogen-derived resistance to JGMV in sorghum.