Viruses have evolved to create a cellular environment permissive for viral replication in susceptible hosts. Possibly both enabling and resulting from these virus-triggered changes, infected hosts undergo a dramatic transcriptional reprogramming, the analysis of which can shed light on the molecular processes underlying the outcome of virus-host interactions. The study of the transcriptional changes triggered by the plant DNA viruses geminiviruses is potentially hampered by the low representation of infected cells in the total population, a situation that becomes extreme in those cases, like that of Tomato yellow leaf curl virus (TYLCV), in which the virus is restricted to phloem companion cells. In order to gain insight into how different the transcriptional landscapes of TYLCV-infected cells or whole tissues of TYLCV-infected plants might be, here we compare the transcriptional changes in leaf patches infected with TYLCV by agroinfiltration or in systemic leaves of TYLCV-infected plants in Nicotiana benthamiana. Our results show that, in agreement with previous works, infection by TYLCV induces a dramatic transcriptional reprogramming; the detected changes, however, are not equivalent in local and systemic infections, with a much larger number of genes differentially expressed locally, and some genes responding in an opposite manner. Interestingly, a transcriptional repression of the auxin signalling pathway and a transcriptional activation of the ethylene signalling pathway were detected in both local and systemically infected samples. A transcriptional activation of defence was also detectable in both cases. Comparison with the transcriptional changes induced by systemic infection by the geminivirus Tobacco curly shoot virus (TbSV) shows common subsets of up- and down-regulated genes similarly affected by both viral species, unveiling a common transcriptional repression of terpenoid biosynthesis, a process also suppressed by the geminivirus Tomato yellow leaf curl China virus. Taken together, the results presented here not only offer insight into the transcriptional changes derived from the infection by TYLCV in N. benthamiana, but also demonstrate that the resolution provided by local and systemic infection approaches largely differs, highlighting the urge to come up with a better system to gain an accurate view of the molecular and physiological changes caused by the viral invasion.

Viruses reshape the intracellular environment of their hosts, largely through protein-protein interactions, to co-opt processes necessary for viral infection and interference with antiviral defences. Due to genome size constraints and the concomitant limited coding capacity of viruses, viral proteins are generally multifunctional and have evolved to target diverse host proteins. Inference of the virus-host interaction network can be instrumental for understanding how viruses manipulate the host machinery and how re-wiring of specific pathways can contribute to disease. Here, we use affinity purification and mass spectrometry analysis (AP-MS) to define the global landscape of interactions between the geminivirus Tomato yellow leaf curl virus (TYLCV) and its host Nicotiana benthamiana. For this purpose, we expressed tagged versions of each of TYLCV-encoded proteins (C1/Rep, C2/TrAP, C3/REn, C4, V2, and CP) in planta in the presence of the virus. Using a quantitative scoring system, 728 high-confidence plant interactors were identified, and the interaction network of each viral protein was inferred; TYLCV-targeted proteins are more connected than average, and connect with other proteins through shorter paths, which would allow the virus to exert large effects with few interactions. Comparative analyses of divergence patterns between N. benthamiana and potato, a non-host Solanaceae, showed evolutionary constraints on TYLCV-targeted proteins. Our results provide a comprehensive overview of plant proteins targeted by TYLCV during the viral infection, which may contribute to uncovering the underlying molecular mechanisms of plant viral diseases and provide novel potential targets for anti-viral strategies and crop engineering. Interestingly, some of the TYLCV-interacting proteins appear to be convergently targeted by other pathogen effectors, which suggests a central role for these proteins in plant-pathogen interactions, and pinpoints them as potential targets to engineer broad-spectrum resistance to biotic stresses.

Viruses are strict intracellular parasites that rely on the proteins encoded in their genomes for the effective manipulation of the infected cell that ultimately enables a successful infection. Viral proteins have to be produced during the cell invasion and takeover in sufficient amounts and in a timely manner. Silencing suppressor proteins evolved by plant viruses can boost the production of viral proteins; although, additional mechanisms for the regulation of viral protein production likely exist. The strongest silencing suppressor encoded by the geminivirus tomato yellow leaf curl virus (TYLCV) is V2: V2 suppresses both post-transcriptional and transcriptional gene silencing (PTGS and TGS), activities that are associated with its localization in punctate cytoplasmic structures and in the nucleus, respectively. However, V2 has been previously described to largely localize in the endoplasmic reticulum (ER), although the biological relevance of this distribution remains mysterious. Here, we confirm the association of V2 to the ER in Nicotiana benthamiana and assess the silencing suppression activity-independent impact of V2 on protein accumulation. Our results indicate that V2 has no obvious influence on the localization of ER-synthesized receptor-like kinases (RLKs) or ER quality control (ERQC)/ER-associated degradation (ERAD), but dramatically enhances the accumulation of the viral C4 protein, which is co-translationally myristoylated, possibly in proximity to the ER. By using the previously described V2_C84S/86S mutant, in which the silencing suppression activity is abolished, we uncouple RNA silencing from the observed effect. Therefore, this work uncovers a novel function of V2, independent of its capacity to suppress silencing, in the promotion of the accumulation of another crucial viral protein.

SUMMARY: Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon triggering plant defence, interfering with the chloroplast-dependent activation of anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defences following an association with membranes, which is based on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defences from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defence exists in plants, and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an anti-viral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus has the ability to inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homologue, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signalling components have been co-opted to play multiple functions in plants.

Fluorescent timers (FTs), including tandem FTs (tdFTs), enable real-time visualization of precise protein localization and turnover within living cells, an essential property for elucidating protein function. In plants, application of FT has been limited largely because of the mismatch between FT maturation kinetics and the slow growth rate of plant cells. Here, we systematically evaluated multiple FT configurations to identify tdFTs optimized for plant systems. Using Arabidopsis thaliana , we tested a range of fluorescent proteins (FPs) differing in maturation times. Functional tdFT expression was confirmed in diverse organelles. Selected tdFT configurations were developed to track protein biogenesis/turnover in autoimmunity, protein transport in abiotic stress, and polarized protein translocation in determination of cell fate. When the large size of tdFT interfered with protein mobility, we developed a bipartite FT, separately tagging proteins with different FPs. This resource provides a framework for selecting FTs to dissect dynamic protein behaviors in plant cells.