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.
Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.
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In most eukaryotes, RNA silencing is a key element in the regulation of gene expression and defense against pathogens. Plants have developed a defensive barrier against exogenous microorganisms, such as plant-infecting viruses, by specifically targeting and degrading the viral RNAs and thus limiting the negative effects of the diseases caused by them. On the other hand, plant viruses encode for suppressor proteins that repress the host-silencing machinery, hence allowing viral replication and infection establishment. Our current project focuses on the characterization of gene products contributing to the RNA silencing suppressor (RSS) function of Sweet potato virus 2 (SPV2), genus Potyvirus, family Potyviridae. SPV2 infects sweet potatoes (Ipomoea batatas, family Convolvulaceae), one of the most important staple food crops worldwide. Infections by potyvirids result in the high yield losses of sweet potatoes, especially from coinfection with unrelated viruses, and our final goal is to develop efficient control strategies. Our preliminary results analyzing the P1 and HCPro proteases of SPV2, transiently expressed in N. benthamiana together with a reporter GFP construct, revealed that HCPro constitutes a strong RSS. This is a novel finding, and we are currently characterizing the functions of other gene products.
Plant diseases are responsible for considerable economic losses in agriculture worldwide. Recent surveys and metagenomics approaches reveal a higher than expected incidence of complex diseases, like those caused by mixed viral infections. Particularly, frequent cases of mixed infections are co-infections or superinfections of plant viruses belonging to different genera in the families Potyviridae (Ipomovirus or Potyvirus) and Closteroviridae (Crinivirus). The outcome of such multiple infections could modify viral traits, such as host range, titer, tissue and cell tropisms, and even vector preference and transmission rates. Therefore, we believe that understanding the virus–virus, virus–host, and virus–vector interactions would be crucial for developing effective control measures. Since there is still limited knowledge about the molecular mechanisms underlying the different interactions, and how they might contribute to specific diseases in mixed infection, we are analyzing ipomovirus–crinivirus and potyvirus–crinivirus pathosystems, to better understand single and mixed infections in selected susceptible hosts (Cucurbitaceae and Convolvulaceae plants), also incorporating in the study the interactions with insect vectors (whiteflies and aphids). Among other strategies, we are engineering new biotechnological tools, to explore the molecular biology and transmission mechanisms of several viruses implicated in complex diseases, and we are also addressing the possibility to produce virus-like particles (VLPs) through transient expression of the CP of different viruses in Nicotiana benthamiana plants, with the aim to study requirements for virion formation and determinants of transmission. Work supported by project AGL2016-75529-R and grant “Severo-Ochoa” SEV-2015-0533.
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