Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.
Coat-protein (CP) hybrids between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) were engineered to analyse reported CP-associated differences between these viruses. CP portions delimited by aa 1-59, 60-148 and 149-219 were exchanged in all possible combinations within TAV RNA3. The seven possible chimeras were able to replicate in tobacco protoplasts to similar levels, but only those having residues 1-59 or 60-148 from CMV were infectious to tobacco plants, a common host for CMV and TAV, and formed stable particles. When most of the movement protein (MP) of TAV was substituted for that of CMV, infectivity of CP hybrids did not vary. No hybrid was able to infect cucumber plants, a host for CMV and not for TAV. Need for MP-CP compatibility could explain these results, but shows that MP-CP compatibility conditions the use of CP chimeras to map CP-associated differences between CMV and TAV.The genus Cucumovirus (family Bromoviridae) comprises aphid-transmitted plant viruses with a single-stranded, messenger-sense, tripartite RNA genome encoding five proteins. RNAs 1 and 2 encode proteins 1a and 2a, respectively, which are part of the replicase complex. RNA2 also encodes protein 2b, a post-transcriptional gene-silencing suppressor. RNA3 has two ORFs separated by an intergenic region (IR) and encodes protein 3a and the capsid protein (CP), both of which are necessary for cell-to-cell movement of virus in the host plant (Palukaitis & García-Arenal, 2003). The 3a protein is the movement protein (MP) with RNAbinding and plasmodesmata-gating abilities, and is able to traffic itself and RNA through plasmodesmata (Vaquero et al., 1994;Ding et al., 1995;Li & Palukaitis, 1996). The role of the CP in cell-to-cell movement is unclear. RNA accumulation in protoplasts was diminished considerably for CP deletion mutants and it was proposed that the CP is necessary to protect the (+)-sense viral RNA from degradation in the host cytoplasm (Boccard & Baulcombe, 1993;Suzuki et al., 1991). It could be that CP protection of the viral RNA would be necessary for efficient cell-to-cell movement. Additionally, cell-to-cell movement requires specific compatibility of the MP and CP of different cucumoviruses (Salánki et al., 1997(Salánki et al., , 2004, suggesting that both proteins interact for this function. Both the MP and the CP are required also for long-distance movement through the phloem and the CP has host-specific determinants for access to the phloem (Palukaitis & García-Arenal, 2003).Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) are two cucumoviruses that differ in several properties. The three-dimensional structure of their particles has been resolved, showing high similarity (Smith et al., 2000;Lucas et al., 2002), which agrees with the ability for in vitro assembly of stable particles built of CMV and TAV CP subunits (Chen et al., 1995). Some important differences in the biology of CMV and TAV have been mapped to the CP by using reassortant and chimeric viruses (Palukaitis & García-Arenal, 2003). One ...
Defective RNA molecules (D-RNAs) are being studied in several plant RNA virus groups. In the genus Bromovirus, D-RNAs have been described for Broad bean mottle virus (BBMV), they are formed by a single internal deletion in the RNA2 and have been generated de novo by serial passages. In contrast, in Brome mosaic virus (BMV) D-RNAs are generated by a single or double internal deletion in the RNA3 without serial passages in their hosts. In this work the external effects of the host and the growth temperature in the generation and accumulation of D-RNAs in BBMV, BMV and Cowpea chlorotic mottle virus (CCMV) are studied. The BBMV and BMV D-RNAs were generated and accumulated with or without serial passages of the viruses in different hosts and cultivars. Plants grown at 12, 16, 20 and 24°C in a growth chamber and in a greenhouse (22 ± 5°C) generated D-RNAs after being inoculated with virusfree D-RNAs, with and without passages. The D-RNAs observed in BBMV and BMV presented some common characteristics: both were formed de novo after serial passages or without passages, their deletion borders had short repeated and palindrome sequences that favour recombination. In addition, growing inoculated plants at lower temperatures greatly facilitated the generation and accumulation of D-RNAs. The CCMV did not generate defective molecules in cowpea (Vigna unguiculata) and Nicotiana benthamiana plants after several serial passages or without passages. This is the first time that D-RNAs have been generated in BBMV without passages and BMV with serial passages.Additional key words: BBMV, BMV, CCMV, host effects, temperature conditions. Resumen Análisis de los factores externos que afectan a la formación y acumulación de los D-RNAs en infecciones de BromovirusLos RNAs defectivos (D-RNAs) están siendo estudiados en diversos virus RNA de plantas. En el género Bromovirus se han descrito D-RNAs en el virus del moteado del haba (BBMV), formados mediante una delección interna en el RNA2, que se generan de novo mediante pases seriados del virus sobre un nuevo huésped. En contraste, en el virus del mosaico del bromo (BMV), los D-RNAs se forman por una o dos delecciones en el RNA3 sin necesidad de pases en sus huéspedes. En este trabajo estudiamos algunos factores externos, como los huéspedes, las inoculaciones y las temperaturas de crecimiento en la generación y acumulación de D-RNAs en BBMV, BMV y en el virus del moteado clorótico de la carilla (CCMV). Los D-RNAs de BBMV y BMV fueron generados y acumulados mediante pases seriados y sin pases de los virus en diferentes huéspedes y cultivares. Plantas inoculadas con BBMV y BMV sin D-RNAs, mantenidas a 12, 16, 20 y 24°C en cámaras de crecimiento a temperatura constante o en un invernadero (22 ± 5°C), generaron D-RNAs con o sin pases sobre nuevos huéspedes. Los D-RNAs observados en ambos virus presentan algunas características en común: son formados de novo mediante pases seriados o purificados de hojas infectadas algunas semanas después de la inoculación; los bordes de la delección tie...
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