The translocation of rice yellow mottle virus (RYMV) within tissues of inoculated and systemically infected Oryza sativa L. leaves was characterized by Western immunoblotting, Northern blotting, and electron microscopy of thin sections. In inoculated leaves, RYMV RNA and coat protein first were detected at 3 and 5 days postinoculation, respectively. By 6 days postinoculation, RYMV had spread systemically to leaves, and virus particles were observed in most cell types, including epidermal, mesophyll, bundle sheath, and vascular parenchyma cells. Most of the virions accumulated in large crystalline patches in xylem parenchyma cells and sieve elements. Colocalization of a cell wall marker for cellulosic ␤-(1-4)-D-glucans and anti-RYMV antibodies over vessel pit membranes suggests a pathway for virus migration between vessels. We propose that the partial digestion of pit membranes resulting from programmed cell death may permit virus migration through them, concomitant with autolysis. In addition, displacement of the Ca 2؉ from pit membranes to virus particles may contribute to the disruption of the pit membranes and facilitate systemic virus transport.

Rice yellow mottle sobemovirus (RYMV) is responsible for the yellow mottle disease on rice in Africa. The expression and function of the protein P1 (17.8 kDa) encoded by the first open reading frame (ORF) of RYMV was investigated. Using an antibody raised against purified P1, two proteins with apparent molecular masses of 18 and 19 kDa were identified in in vitro translation reactions of transcripts of the full-length cDNA of RYMV. Likewise, gene products with similar molecular mass were detected in inoculated and systemically infected rice leaves and in infected rice protoplasts. A mutant from which ORF1 nucleotides 88 to 547 were deleted and a frameshift mutant that resulted in truncation of 83 amino acids from the C terminus of P1 were incapable of replicating in protoplasts. In contrast, a mutant that does not express P1 due to a mutation at the initiation codon replicated efficiently in protoplasts but at a reduced level (about 0.5- to 2-fold less) compared to replication of wild-type RNA. None of these mutants caused systemic infection in rice plants. Transgenic rice plants that express P1 complemented the initiation codon mutant, but not the deletion mutants, and produced systemic infection. These experiments demonstrate that P1 of RYMV is dispensible for virus replication, although nucleotide deletions or additions in ORF1 are apparently lethal for virus replication. Furthermore, P1 of RYMV is required for the infection of plants and is important for virus spread.

The haploid ascomycete Diplocarpon rosae is the causal agent of black spot disease on roses, a widespread and devastating disease in the outdoor landscape. In this study, we established a Eurasian collection of 77 monoconidial strains of D. rosae: 50 strains collected on cultivated roses in Europe and Asia, and 27 strains on wild roses in Kazakhstan. To provide tools to describe its biology and to study its genetic diversity, we sequenced two strains of D. rosae using Illumina paired‐end technology. The genome sizes of these two strains were estimated at 31.1 and 35.2 Mb, which are two times smaller than the genome size of the unique strain previously published. A BUSCO analysis confirmed a genome duplication of the strain previously sequenced and partial gene duplication of strains analysed in this study. Using the two genome sequences, 27 polymorphic microsatellite markers were identified. Polymorphism analysis of the 77 strains revealed a strong genetic differentiation between strains from cultivated and wild roses, and a lower diversity within the fungal population from cultivated roses compared to the population from wild roses. Pathogenicity of 10 strains was evaluated on 9 rose cultivars inoculated in the greenhouse. Disease scoring allowed the classification of strains into three groups and the characterization of resistance of rose cultivars. Good correlation observed between resistance scoring in greenhouse conditions and in the field indicates that pathogenicity assays in controlled conditions could be very useful in the near future to rapidly characterize the resistance of new rose varieties to black spot disease.

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