Reconceptualizing transcriptional slippage in plant RNA viruses.

RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of them, named transcriptional slippage (TS), is characterized by the appearance of indels in nascent viral RNAs, leading to changes in the open reading frame (ORF). Although members of unrelated viral families express key proteins via TS, the available information about this phenomenon is still limited. In potyvirids (members of the Potyviridae family), TS has been defined by the insertion of an additional A at An motifs (n ≥ 6) in newly synthesized transcripts at a low frequency, modulated by nucleotides flanking the A-rich motif. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases not following this definition. In summary, we observe (i) a high rate of single-nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U8 stretch, and (iii) changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of whole-genome sequences from potyvirids shows a widespread occurrence of species-specific An/Un (n ≥ 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a re-evaluation of TS significance during infections of RNA viruses is required.IMPORTANCETranscriptional slippage (TS) is used by RNA viruses as another strategy to maximize the coding information in their genomes. This phenomenon is based on a peculiar feature of viral replicases: they may produce indels in a small fraction of newly synthesized viral RNAs when transcribing certain motifs and then produce alternative proteins due to a change of the reading frame or truncated products by premature termination. Here, using plant-infecting RNA viruses as models, we discover cases expanding on previously established features of plant virus TS, prompting us to reconsider and redefine this expression strategy. An interesting conclusion from our study is that TS might be more relevant during RNA virus evolution and infection processes than previously assumed.

Specificity of Resistance and Tolerance to Cucumber Vein Yellowing Virus in Melon Accessions and Resistance Breaking with a Single Mutation in VPg.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

The P1 Protein of Watermelon mosaic virus Compromises the Activity as RNA Silencing Suppressor of the P25 Protein of Cucurbit yellow stunting disorder virus.

Mixed viral infections in plants involving a potyvirus and other unrelated virus often result in synergistic effects, with significant increases in accumulation of the non-potyvirus partner, as in the case of melon plants infected by the potyvirus Watermelon mosaic virus (WMV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV). To further explore the synergistic interaction between these two viruses, the activity of RNA silencing suppressors (RSSs) was addressed in transiently co-expressed combinations of heterologous viral products in Nicotiana benthamiana leaves. While the strong RSS activity of WMV Helper Component Proteinase (HCPro) was unaltered, including no evident additive effects observed when co-expressed with the weaker CYSDV P25, an unexpected negative effect of WMV P1 was found on the RSS activity of P25. Analysis of protein expression during the assays showed that the amount of P25 was not reduced when co-expressed with P1. The detrimental action of P1 on the activity of P25 was dose-dependent, and the subcellular localization of fluorescently labeled variants of P1 and P25 when transiently co-expressed showed coincidences both in nucleus and cytoplasm. Also, immunoprecipitation experiments showed interaction of tagged versions of the two proteins. This novel interaction, not previously described in other combinations of potyviruses and criniviruses, might play a role in modulating the complexities of the response to multiple viral infections in susceptible plants.

Assessing the Impact on Virus Transmission and Insect Vector Behavior of a Viral Mixed Infection in Melon.

Mixed viral infections in plants are common, and can result in synergistic or antagonistic interactions. Except in complex diseases with severe symptoms, mixed infections frequently remain unnoticed, and their impact on insect vector transmission is largely unknown. In this study, we considered mixed infections of two unrelated viruses commonly found in melon plants, the crinivirus cucurbit yellow stunting disorder virus (CYSDV) and the potyvirus watermelon mosaic virus (WMV), and evaluated their vector transmission by whiteflies and aphids, respectively. Their dynamics of accumulation was analyzed until 60 days postinoculation (dpi) in mixed-infected plants, documenting reduced titers of WMV and much higher titers of CYSDV compared with single infections. At 24 dpi, corresponding to the peak of CYSDV accumulation, similar whitefly transmission rates were obtained when comparing either individual or mixed-infected plants as CYSDV sources, although its secondary dissemination was slightly biased toward plants previously infected with WMV, regardless of the source plant. However, at later time points, mixed-infected plants partially recovered from the initially severe symptoms, and CYSDV transmission became significantly higher. Interestingly, aphid transmission rates both at early and late time points were unaltered when WMV was acquired from mixed-infected plants despite its reduced accumulation. This lack of correlation between WMV accumulation and transmission could result from compensatory effects observed in the analysis of the aphid feeding behavior by electrical penetration graphs. Thus, our results showed that mixed-infected plants could provide advantages for both viruses, directly favoring CYSDV dissemination while maintaining WMV transmission.