Bioluminescence Production by Turnip Yellows Virus Infectious Clones: A New Way to Monitor Plant Virus Infection.

We used the NanoLuc luciferase bioluminescent reporter system to detect turnip yellows virus (TuYV) in infected plants. For this, TuYV was genetically tagged by replacing the C-terminal part of the RT protein with full-length NanoLuc (TuYV-NL) or with the N-terminal domain of split NanoLuc (TuYV-N65-NL). Wild-type and recombinant viruses were agro-infiltrated in Nicotiana benthamiana, Montia perfoliata, and Arabidopsis thaliana. ELISA confirmed systemic infection and similar accumulation of the recombinant viruses in N. benthamiana and M. perfoliata but reduced systemic infection and lower accumulation in A. thaliana. RT-PCR analysis indicated that the recombinant sequences were stable in N. benthamiana and M. perfoliata but not in A. thaliana. Bioluminescence imaging detected TuYV-NL in inoculated and systemically infected leaves. For the detection of split NanoLuc, we constructed transgenic N. benthamiana plants expressing the C-terminal domain of split NanoLuc. Bioluminescence imaging of these plants after agro-infiltration with TuYV-N65-NL allowed the detection of the virus in systemically infected leaves. Taken together, our results show that NanoLuc luciferase can be used to monitor infection with TuYV.

The N-terminus of the cauliflower mosaic virus aphid transmission protein P2 is involved in transmission body formation and microtubule interaction.

Cauliflower mosaic virus (CaMV) is transmitted by aphids using the non-circulative transmission mode: when the insects feed on infected leaves, virus particles from infected cells attach rapidly to their stylets and are transmitted to a new host when the aphids change plants. Mandatory for CaMV transmission, the viral helper protein P2 mediates as a molecular linker binding of the virus particles to the aphid stylets. P2 is available in infected plant cells in a viral inclusion that is specialized for transmission and named the transmission body (TB). When puncturing an infected leaf cell, the aphid triggers an ultra-rapid viral response, necessary for virus acquisition and called transmission activation: The TB disrupts and P2 is redistributed onto cortical microtubules, together with virus particles that are simultaneously set free from virus factories and join P2 on the microtubules to form the so-called mixed networks (MNs). The MNs are the predominant structure from which CaMV is acquired by aphids. However, the P2 domains involved in microtubule interaction are not known. To identify P2 regions involved in its functions, we generated a set of P2 mutants by alanine scanning and analyzed them in the viral context for their capacity to form a TB, to interact with microtubules and to transmit CaMV. Our results show that contrary to the previously characterized P2-P2 and P2-virion binding sites in its C-terminus, the microtubule binding site is contained in the N-terminal half of P2. Further, this region is important for TB formation since some P2 mutant proteins did not accumulate in TBs but were retained in the viral factories where P2 is translated. Taken together, the N-terminus of P2 is not only involved in vector interaction as previously reported, but also in interaction with microtubules and in formation of TBs.

Turnip Mosaic Virus Is a Second Example of a Virus Using Transmission Activation for Plant-to-Plant Propagation by Aphids.

Cauliflower mosaic virus (CaMV; family Caulimoviridae) responds to the presence of aphid vectors on infected plants by forming specific transmission morphs. This phenomenon, coined transmission activation (TA), controls plant-to-plant propagation of CaMV. A fundamental question is whether other viruses rely on TA. Here, we demonstrate that transmission of the unrelated turnip mosaic virus (TuMV; family Potyviridae) is activated by the reactive oxygen species H2O2 and inhibited by the calcium channel blocker LaCl3 H2O2-triggered TA manifested itself by the induction of intermolecular cysteine bonds between viral helper component protease (HC-Pro) molecules and by the formation of viral transmission complexes, composed of TuMV particles and HC-Pro that mediates vector binding. Consistently, LaCl3 inhibited intermolecular HC-Pro cysteine bonds and HC-Pro interaction with viral particles. These results show that TuMV is a second virus using TA for transmission but using an entirely different mechanism than CaMV. We propose that TuMV TA requires reactive oxygen species (ROS) and calcium signaling and that it is operated by a redox switch.IMPORTANCE Transmission activation, i.e., a viral response to the presence of vectors on infected hosts that regulates virus acquisition and thus transmission, is an only recently described phenomenon. It implies that viruses contribute actively to their transmission, something that has been shown before for many other pathogens but not for viruses. However, transmission activation has been described so far for only one virus, and it was unknown whether other viruses also rely on transmission activation. Here we present evidence that a second virus uses transmission activation, suggesting that it is a general transmission strategy.

Insect transmission of plant viruses: Multilayered interactions optimize viral propagation.

By serving as vectors of transmission, insects play a key role in the infection cycle of many plant viruses. Viruses use sophisticated transmission strategies to overcome the spatial barrier separating plants and the impediment imposed by the plant cell wall. Interactions among insect vectors, viruses, and host plants mediate transmission by integrating all organizational levels, from molecules to populations. Best-examined on the molecular scale are two basic transmission modes wherein virus-vector interactions have been well characterized. Whereas association of virus particles with specific sites in the vector's mouthparts or in alimentary tract regions immediately posterior to them is required for noncirculative transmission, the cycle of particles through the vector body is necessary for circulative transmission. Virus transmission is also determined by interactions that are associated with changes in vector feeding behaviors and with alterations in plant host's morphology and/or metabolism that favor the attraction or deterrence of vectors. A recent concept in virus-host-vector interactions proposes that when vectors land on infected plants, vector elicitors and effectors "inform" the plants of the confluence of interacting entities and trigger signaling pathways and plant defenses. Simultaneously, the plant responses may also influence virus acquisition and inoculation by vectors. Overall, a picture is emerging where transmission depends on multilayered virus-vector-host interactions that define the route of a virus through the vector, and on the manipulation of the host and the vector. These interactions guarantee virus propagation until one or more of the interactants undergo changes through evolution or are halted by environmental interventions.

Identification of host factors potentially involved in RTM-mediated resistance during potyvirus long distance movement.

The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.