The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below hostdetrimental levels, and thus to maintain higher long-term replicative capacity. ; This work was funded by grants BIO2010-18541 from the Spanish 'Ministerio de Ciencia e Innovación', and KBBE-204429 from the European Union. FP is financed by a La Caixa PhD fellowship. ; Peer reviewed
Plant RNA (ribonucleic acid) viruses are obligate intracellular parasites with single-stranded (ss) or double-stranded RNA genome(s) generally encapsidated but rarely enveloped. For viruses with ssRNA genomes, the polarity of the infectious RNA (positive or negative) and the presence of one or more genomic RNA segments are the features that mostly determine the molecular mechanisms governing the replication process. RNA viruses cannot penetrate plant cell walls unaided and must enter the cellular cytoplasm through mechanically induced wounds or assisted by a biological vector. After desencapsidation, their genome remains in the cytoplasm where it is translated, replicated and encapsidated in a coupled manner. Replication occurs in large viral replication complexes (VRCs), tethered to modified membranes of cellular organelles and composed by the viral RNA templates and by viral and host proteins. Cis-acting elements located in viral RNA templates regulate the recognition by the virus-encoded RNA-dependent RNA polymerase and possibly contribute to VRC assembly and/or activation. ; Work in the authors' laboratories is supported by grants BFU2012-36095 BIO2013-49053-R and Plant KBBE PCIN-2013-056 from the Ministerio de Economía y Competitividad (MINECO, Spain) and from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 655841. ; Peer Reviewed
[Background] Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons. ; [Results] We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus. ; [Conclusions] Laser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation. ; FP is financed by a La Caixa PhD fellowship and acknowledges support from S. Pasin and L. Lievore. This work was funded by grants BIO2010-18541 and BIO2013-49053-R from the Spanish government. The publication fee was covered partially by the CSIC Open Access Publication Support Initiative through the Unit of Information Resources for Research (URICI). ; Peer reviewed
12 páginas, 8 figuras, 1 tabla. ; [Background]: Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression. [Results]: Nicotiana benthamiana plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus Plum pox virus (PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them started to display sporadic infection foci in leaves approaching maturity. The infection progressed systemically, but at later times the infected plants recovered and returned to an amplicon-inactive state. The failure to detect virus-specific siRNAs in 10.6 plants before amplicon induction and after recovery suggested that a strong amplicon-specific RNA silencing is not established in these plants. However, the coexpression of extra viral silencing suppressors caused some amplicon activation, suggesting that a low level of RNA silencing could be contributing to maintain amplicon repression in the 10.6 plants. The resistance mechanisms that prevent amplicon-derived virus infection were also active against exogenous PPV introduced by mechanical inoculation or grafting, but did not affect other viruses. Amplicon-derived PPV was able to spread into wild type scions grafted in 10.6 rootstocks that did not display signs of amplicon expression, suggesting that resistance has little effect on virus movement. [Conclusions]: Our results suggest that amplicon-derived virus infection is limited in this particular transgenic line by a combination of factors, including the presumed low efficiency of the conversion from the transgene transcript to replicable viral RNA, and also by the activation of RNA silencing and other defensive responses of the plant, which are not completely neutralized by viral suppressors. ; This work was supported by grants BIO2007-67283 from Spanish MEC, CPE03-022-C5-3 from INIA, SAL/ 0185/2006 from Comunidad de Madrid, and KBBE-204429 from European Union. GD was supported by Fundación Carolina. CL was recipient of a fellowship from Comunidad de Madrid. MC was recipient of an I3P fellowship from Consejo Superior de Investigaciones Científicas (CSIC)-Fondo Social Europeo. CSM was supported by a Ramón y Cajal contract. ; Peer reviewed
