RNA viruses have very compact genomes and so provide a unique opportunity to study how evolution works to optimize the use of very limited genomic information. A widespread viral strategy to solve this issue concerning the coding space relies on the expression of proteins with multiple functions. Members of the family Potyviridae, the most abundant group of RNA viruses in plants, offer several attractive examples of viral factors which play roles in diverse infection-related pathways. The Helper Component Proteinase (HCPro) is an essential and well-characterized multitasking protein for which at least three independent functions have been described: (i) viral plant-to-plant transmission; (ii) polyprotein maturation; and (iii) RNA silencing suppression. Moreover, multitudes of host factors have been found to interact with HCPro. Intriguingly, most of these partners have not been ascribed to any of the HCPro roles during the infectious cycle, supporting the idea that this protein might play even more roles than those already established. In this comprehensive review, we attempt to summarize our current knowledge about HCPro and its already attributed and putative novel roles, and to discuss the similarities and differences regarding this factor in members of this important viral family.

Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids. Simultaneous usage of pBBR1- and RK2-based pLX vectors in a two-plasmid/one-Agrobacterium strain strategy allowed multigene delivery to plants. Adoption of pLX vectors will facilitate routine plant transformations and targeted mutagenesis, as well as complex part and circuit characterization.

The helper component proteinase (HCPro) is an indispensable, multifunctional protein of members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by a heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had very reduced infectivity. Making use of different approaches, here, we provide direct evidence of a previously unknown function of HCPro in which the viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is not linked to any of its previously known activities and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, the function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation, and encapsidation, thus contributing to the synchronization of these viral processes, is discussed. IMPORTANCEPotyviruses form one of the most numerous groups of plant viruses and are a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of potyviral progeny. In this case, such a function is performed by the extensively studied viral factor HCPro, which seems to use an unknown mechanism that is not linked to its previously described activities. To our knowledge, this is the first time that a factor different from capsid protein (CP) has been shown to be directly involved in the yield of potyviral particles. Based on the data presented here, we hypothesize that this capacity of HCPro might be involved in the coordination of mutually exclusive activities of the viral genome by controlling correct assembly of CP in stable virions.

Summary Plant defensins are small cysteine‐rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.