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Summary It is increasingly clear that chloroplasts play a central role in plant stress responses. Upon activation of immune responses, chloroplasts are the source of multiple defensive signals, including reactive oxygen species (ROS). Intriguingly, it has been described that chloroplasts establish physical contact with the nucleus, through clustering around it and extending stromules, following activation of effector‐triggered immunity (ETI). However, how prevalent this phenomenon is in plant–pathogen interactions, how its induction occurs, and what the underlying biological significance is are important questions that remain unanswered. Here, we describe that the chloroplast perinuclear clustering seems to be a general plant response upon perception of an invasion threat. Indeed, activation of pattern‐triggered immunity, ETI, transient expression of the Rep protein from geminiviruses, or infection with viruses or bacteria all are capable of triggering this response in Nicotiana benthamiana. Interestingly, this response seems non‐cell‐autonomous, and exogenous treatment with H2O2 is sufficient to elicit this relocalization of chloroplasts, which appears to require accumulation of ROS. Taken together, our results indicate that chloroplasts cluster around the nucleus during plant–pathogen interactions, suggesting a fundamental role of this positioning in plant defence, and identify ROS as sufficient and possibly required for the onset of this response.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral 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 can 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 homolog, 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 signaling components have been coopted to play multiple functions in plants.

Summary Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22Rsol) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.

Viruses manipulate the cells they infect in order to replicate and spread. Due to strict size restrictions, viral genomes have reduced genetic space; how the action of the limited number of viral proteins results in the cell reprogramming observed during the infection is a long-standing question. Here, we explore the hypothesis that combinatorial interactions may expand the functional landscape of the viral proteome. We show that the proteins encoded by a plant-infecting DNA virus, the geminivirus tomato yellow leaf curl virus (TYLCV), physically associate with one another in an intricate network, as detected by a number of protein-protein interaction techniques. Importantly, our results indicate that intra-viral protein-protein interactions can modify the subcellular localization of the proteins involved. Using one particular pairwise interaction, that between the virus-encoded C2 and CP proteins, as proof-of-concept, we demonstrate that the combination of viral proteins leads to novel transcriptional effects on the host cell. Taken together, our results underscore the importance of studying viral protein function in the context of the infection. We propose a model in which viral proteins might have evolved to extensively interact with other elements within the viral proteome, enlarging the potential functional landscape available to the pathogen.

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 V2C84S/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.

Viruses are intracellular parasites with a nucleic acid genome and a proteinaceous capsid. Viral capsids are formed of at least one virus-encoded capsid protein (CP), which is often multifunctional, playing additional non-structural roles during the infection cycle. In animal viruses, there are examples of differential localization of CPs associated to the progression of the infection and/or enabled by other viral proteins; these changes in the distribution of CPs may ultimately regulate the involvement of these proteins in different viral functions. In this work, we analyze the subcellular localization of a GFP- or RFP-fused CP from the plant virus (TYLCV; Fam. ) in the presence or absence of the virus upon transient expression in the host plants and tomato. Our findings show that, in agreement with previous reports, when the CP is expressed alone it localizes mainly in the nucleolus and weakly in the nucleoplasm. Interestingly, the presence of the virus causes the sequential re-localization of the CP outside of the nucleolus and into discrete nuclear foci and, eventually, into an uneven distribution in the nucleoplasm. Expression of the viral replication-associated protein, Rep, is sufficient to exclude the CP from the nucleolus, but the localization of the CP in the characteristic patterns induced by the virus cannot be recapitulated by co-expression with any individual viral protein. Our results demonstrate that the subcellular distribution of the CP is a dynamic process, temporally regulated throughout the progression of the infection. The regulation of the localization of the CP is determined by the presence of other viral components or changes in the cellular environment induced by the virus, and is likely to contribute to the multifunctionality of this protein. Bearing in mind these observations, we suggest that viral proteins should be studied in the context of the infection and considering the temporal dimension in order to comprehensively understand their roles and effects in the interaction between virus and host.

After plants transitioned from water to land around 450 million years ago, they faced novel pathogenic microbes. Their colonization of diverse habitats was driven by anatomical innovations like roots, stomata, and vascular tissue, which became central to plant-microbe interactions. However, the impact of these innovations on plant immunity and pathogen infection strategies remains poorly understood. Here, we explore plant-virus interactions in the bryophyte Marchantia polymorpha to gain insights into the evolution of these relationships. Virome analysis reveals that Marchantia is predominantly associated with RNA viruses. Comparative studies with tobacco mosaic virus (TMV) show that Marchantia shares core defense responses with vascular plants but also exhibits unique features, such as a sustained wound response preventing viral spread. Additionally, general defense responses in Marchantia are equivalent to those restricted to vascular tissues in Nicotiana, suggesting that evolutionary acquisition of developmental innovations results in re-routing of defense responses in vascular plants. Assessment of the evolution of plant-virus interactions suggests rapid reshaping of plant viromes after land colonization, along with rerouting of general defense responses to newly acquired vascular tissues that became focal for viral infections.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and Nucleotide-Binding Leucine Rich Repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence, therefore maintaining that regulatory relationship. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two mutually non-exclusive scenarios: miRNA-dependent regulation of NLR levels might prevent defence mis-activation with negative effects on plant growth and reproduction; or reduction of active miRNA levels in response to pathogen derived molecules (PAMPS and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defence. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defence against the fungal pathogen Plectospharaella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defence and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared to wild-type plants. However, despite MIM472 lines flower at the same time than their wild-type, the end of their reproductive phase is delayed, and they exhibit higher adult biomass, resulting in similar seed yield as the wild-type. Our study highlights how negative consequences of defence activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defence responses.Competing Interest StatementThe authors have declared no competing interest.

After their transition from water to land around 450 million years ago, plants colonized new habitats facing unprecedented pathogenic microbes. That expansion was mostly supported by a growing anatomical complexity based on the acquisition of developmental innovations, such as roots, stomata and vascular tissue. Despite several of those innovations became central for the interaction between plants and their associated microbes, little is known about their impact on plant immune programs and on the diversification of infection strategies of their pathogens. A paradigmatic case is the close relationship between plant vasculature and viruses. Vascular tissues provide a unique cellular environment for viral replication and existence, besides constituting a fast track for viral systemic spread throughout the plant. Since most of our knowledge about plant-virus interactions come from studies in vascular plants, we here present a comparative study to contribute to the understanding of the evolution of plant- virus interactions by molecularly characterizing the interplay between the bryophyte Marchantia polymorpha and viruses. Virome analysis of Marchantia plants shows that they are primarily associated with RNA viruses in natural settings. Additional molecular characterization of the interaction between Marchantia and tobacco mosaic virus (TMV) show conserved basic processes with vascular plants and divergent features. Viral infection triggers an extensive transcriptional reprogramming in Marchantia encompassing broad range defence responses, inhibition of cell cycle and photosynthesis and a sustained wound response that prevents further viral movement. Additionally, infected plants show premature aging and organ maturation. Notably, we found that some core responses that occur in infected areas in Marchantia, were described to be restricted to vascular tissues in Nicotiana, suggesting that evolutionary appearance of developmental innovations that became central in plants-virus interactions resulted in re-routing of defence responses. Finally, we uncover the conserved role of a transcription factor interacting with the TMV silencing suppressor p126 in specifically abrogating TMV infection.Competing Interest StatementThe authors have declared no competing interest.

Key messageAgrobacterium-mediated transient overexpression of E2Fb triggers new cell divisions in pavement cells of Nicotiana benthamiana leaves.Transient transformation in Nicotiana benthamiana enables the study of multiple biological processes in a simple and fast manner. Here, we describe that, upon A. tumefaciens-mediated transient overexpression of the cell cycle regulator E2Fb from either Arabidopsis thaliana or N. benthamiana, cell divisions occur in epidermal pavement cells in N. benthamiana leaves, following a sequence of events that encompasses the nucleus taking a central position and being surrounded by chloroplasts, nuclear division, and formation of a new wall that divides the initial cell in two. Our results indicate that transient expression in N. benthamiana can be used to study cell division in plants, from DNA replication to cell wall formation, in a simple, controlled, and rapid manner.