Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
In sweet cherry (Prunus avium L.), infection by Candidatus Phytoplasma pruni results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet, the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations within eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, that correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we also observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar and location-specific disease outcomes were observed with regards to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.
‘Candidatus Phytoplasma pruni’ infection in cherries causes small, misshapen fruit with poor color and taste, rendering the fruit unmarketable. However, this is a disease with a long development cycle and a scattered, non-uniform symptom distribution in the early stages. To better understand the biology as well as the relationship between pathogen titer and disease expression, we carried out seasonal, spatial, and temporal examinations of ‘Ca. P. pruni’ titer and distribution in infected orchard-grown trees. Sequential sampling of heavily infected trees revealed marked seasonal patterns, with differential accumulation in woody stem and leaf tissues, and most notably within fruit in the early stages of development from bloom to pit hardening. Furthermore, mapping phytoplasma distribution and titer in trees at different stages of infection indicated that infection proceeds through a series of stages. Initially, infection spreads basipetally and accumulates in the roots before populating aerial parts of the trees from the trunk upwards, with infection of specific tissues and limbs followed by an increasing phytoplasma titer. Finally, we observed a correlation between phytoplasma titer and symptom severity, with severe symptom onset associated with 3-4 orders of magnitude more phytoplasma than mild symptoms. Cumulatively, these data aid in accurate sampling and management decision making and furthers our understanding of disease development.
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