Complete genome sequence of a Moroccan isolate of cereal chlorotic mottle virus.

Cereal chlorotic mottle virus (CCMoV) is a cicadellid-transmitted plant rhabdovirus associated with chlorotic and necrotic streaks on several gramineous hosts and weeds. The virus was initially described in 1979 in Australia, but its genome has never been sequenced. In this study, the complete genome sequence of a Moroccan isolate of CCMoV was generated by high-throughput sequencing from infected oat leaves (Avena sativa). The genome is 13,800 nt long, containing seven open reading frames (ORFs) arranged in the canonical organization of rhabdoviruses: 3'-nucleocapsid (N), phosphoprotein (P), unknown protein (p3), unknown protein (p4), matrix (M), glycoprotein (G), viral polymerase (L)-5'. Pairwise analysis showed that maize fine streak virus (MFSV, genus Gammanucleorhabdovirus) was the closest relative. The amino acid identity values between homologous proteins from CCMoV and MFSV are as follows: 59.27% (N), 36.7% (P), 24% (P3), 62% (P4), 43.70% (M), 49.15% (G), 60.93% (L). Based on its phylogenetic relationship and analogous genome architecture, CCMoV should be assigned as member of the genus Gammanucleorhabdovirus. The low sequence similarity observed between CCMoV and MFSV suggests that CCMoV is a member of a distinct virus species.

Characterization of Blueberry shock virus, an Emerging Ilarvirus in Cranberry.

Blueberry shock virus (BlShV), an Ilarvirus sp. reported only on blueberry, was associated with scarring, disfigurement, and premature reddening of cranberry fruit. BlShV was detected by triple-antibody sandwich enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction, and isometric virions of 25 to 28 nm were observed in cranberry sap. The virus was systemic, although unevenly distributed in plants. The coat protein of BlShV from cranberry shared 90% identity compared with BlShV accessions from blueberry on GenBank. Phylogenetic analysis of isolates of BlShV from cranberry collected from Wisconsin and Massachusetts did not indicate grouping by state. BlShV was detected in cranberry pollen, and seed transmission of up to 91% was observed. Artificial inoculation of cranberry flowers by pollination did not cause virus transmission. In some Nicotiana spp., rub inoculation of leaves with homogenized BlShV-positive cranberry flowers resulted in systemic infection. Cranberry plants recovered from symptoms the year after berry scarring occurred but continued to test positive for BlShV. The virus caused significant reduction in the average number of marketable fruit and average berry weight in symptomatic cranberry plants but recovered plants yielded comparably with healthy plants. Although recovery may limit the immediate economic consequences of BlShV, long-term implications of single- or mixed-virus infection in cranberry is unknown.

Plant Cell Wall Dynamics in Compatible and Incompatible Potato Response to Infection Caused by Potato Virus Y (PVYNTN).

The cell wall provides the structure of the plant, and also acts as a barier against biotic stress. The vein necrosis strain of Potato virus Y (PVYNTN) induces necrotic disease symptoms that affect both plant growth and yield. Virus infection triggers a number of inducible basal defense responses, including defense proteins, especially those involved in cell wall metabolism. This study investigates the comparison of cell wall host dynamics induced in a compatible (potato cv. Irys) and incompatible (potato cv. Sárpo Mira with hypersensitive reaction gene Ny-Smira) PVYNTN-host-plant interaction. Ultrastructural analyses revealed numerous cell wall changes induced by virus infection. Furthermore, the localization of essential defensive wall-associated proteins in susceptible and resistant potato host to PVYNTN infection were investigated. The data revealed a higher level of detection of pathogenesis-related protein 2 (PR-2) in a compatible compared to an incompatible (HR) interaction. Immunofluorescence analyses indicated that hydroxyproline-rich glycoproteins (HRGP) (extensin) synthesis was induced, whereas that of cellulose synthase catalytic subunits (CesA4) decreased as a result of PVYNTN infection. The highest level of extensin localization was found in HR potato plants. Proteins involved in cell wall metabolism play a crucial role in the interaction because they affect the spread of the virus. Analysis of CesA4, PR-2 and HRGP deposition within the apoplast and symplast confirmed the active trafficking of these proteins as a step-in potato cell wall remodeling in response to PVYNTN infection. Therefore, cell wall reorganization may be regarded as an element of "signWALLing"-involving apoplast and symplast activation as a specific response to viruses.

Maize Lethal Necrosis (MLN), an Emerging Threat to Maize-Based Food Security in Sub-Saharan Africa.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.

The Expression of Potato Expansin A3 (StEXPA3) and Extensin4 (StEXT4) Genes with Distribution of StEXPAs and HRGPs-Extensin Changes as an Effect of Cell Wall Rebuilding in Two Types of PVYNTN-Solanum tuberosum Interactions.

The plant cell wall acts not only as a physical barrier, but also as a complex and dynamic structure that actively changes under different biotic and abiotic stress conditions. The question is, how are the different cell wall compounds modified during different interactions with exogenous stimuli such as pathogens? Plants exposed to viral pathogens respond to unfavorable conditions on multiple levels. One challenge that plants face under viral stress is the number of processes required for differential cell wall remodeling. The key players in these conditions are the cell wall genes and proteins, which can be regulated in specific ways during the interactions and have direct influences on the rebuilding of the cell wall structure. The cell wall modifications occurring in plants during viral infection remain poorly described. Therefore, this study focuses on cell wall dynamics as an effect of incompatible interactions between the potato virus Y (PVYNTN) and resistant potatoes (hypersensitive plant), as well as compatible (susceptible plant) interactions. Our analysis describes, for the first time, the expression of the potato expansin A3 (StEXPA3) and potato extensin 4 (StEXT4) genes in PVYNTN-susceptible and -resistant potato plant interactions. The results indicated a statistically significant induction of the StEXPA3 gene during a susceptible response. By contrast, we demonstrated the predominantly gradual activation of the StEXT4 gene during the hypersensitive response to PVYNTN inoculation. Moreover, the in situ distributions of expansins (StEXPAs), which are essential cell wall-associated proteins, and the hydroxyproline-rich glycoprotein (HRGP) extensin were investigated in two types of interactions. Furthermore, cell wall loosening was accompanied by an increase in StEXPA deposition in a PVYNTN-susceptible potato, whereas the HRGP content dynamically increased during the hypersensitive response, when the cell wall was reinforced. Ultrastructural localization and quantification revealed that the HRGP extensin was preferably located in the apoplast, but deposition in the symplast was also observed in resistant plants. Interestingly, during the hypersensitive response, StEXPA proteins were mainly located in the symplast area, in contrast to the susceptible potato where StEXPA proteins were mainly observed in the cell wall. These findings revealed that changes in the intracellular distribution and abundance of StEXPAs and HRGPs can be differentially regulated, depending on different types of PVYNTN-potato plant interactions, and confirmed the involvement of apoplast and symplast activation as a defense response mechanism.

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus.

Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.

Characterization of a Strain of Turnip vein-clearing virus Causing Red Ringspot of Penstemon.

A disease of penstemon (Penstemon digitalis) occurring in commercial nurseries in Minnesota in 2004 to 2006 and characterized by red foliar ringspots, leaf deformation, and plant stunting was found to be caused by a strain of Turnip vein-clearing virus (TVCV) that was named Penstemon ringspot virus (PenRSV). This is the first report of a viral disease of penstemon. The genome organization of PenRSV was similar to that of the crucifer-infecting tobamoviruses. The nucleotide sequence of PenRSV was almost identical (99%) to that of TVCV, but the two viruses differed importantly in host range and symptoms induced. The only sequence difference between PenRSV and TVCV occurred at the 3' end of open reading frame I, where the amino acid sequence FRDSNL in TVCV is replaced by FRGQQL in PenRSV. The experimental host range of PenRSV included species in the families Brassicaceae (Cruciferae), Cactaceae, Cucurbitaceae, Leguminosae, Malvaceae, and Solanaceae. This virus poses a potential threat to commercial nursery and bedding plant production because of its wide host range and because it will escape detection by immunoenzymatic screening procedures for tobamoviruses based on use of antibodies to Tobacco mosaic virus (TMV).

Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).

Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts.

Banana contains a diverse array of endogenous badnaviruses.

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars 'Obino l'Ewai' and 'Klue Tiparot', were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.