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. Significance In plants, RNA interference (RNAi) is the main antiviral defense mechanism. RNAi moves cell-to-cell through cytoplasmic channels called plasmodesmata, spreading ahead of the viral infection to immunize tissues before arrival of the virus. How this movement is regulated has been elusive. Here, we identify a plant protein, BAM1, localized at plasmodesmata and promoting RNAi spread; both BAM1 and its closest homolog, BAM2, are required for this process. In agreement with their role in promoting RNAi movement, we found that BAM1 and BAM2 are targeted by a viral effector, which acts as a suppressor of RNAi spread. Our work offers insight into the regulation of cell-to-cell spread of RNAi and provides another example of the arms race between pathogens and hosts.

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 H ₂ O ₂ 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.

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 (csp22^Rsol) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22^Rsol 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 csp22^Rsol 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.

In plants, activation of growth and activation of immunity are opposing processes that define a trade-off. In the past few years, the growth-promoting hormones brassinosteroids (BR) have emerged as negative regulators of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), promoting growth at the expense of defence. The crosstalk between BR and PTI signalling was described as negative and unidirectional, since activation of PTI does not affect several analysed steps in the BR signalling pathway. In this work, we describe that activation of PTI by the bacterial PAMP flg22 results in the reduced expression of BR biosynthetic genes. This effect does not require BR perception or signalling, and occurs within 15 minutes of flg22 treatment. Since the described PTI-induced repression of gene expression may result in a reduction in BR biosynthesis, the crosstalk between PTI and BR could actually be negative and bidirectional, a possibility that should be taken into account when considering the interaction between these two pathways.

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 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 Tomato yellow leaf curl virus (TYLCV; Fam. Geminiviridae) in the presence or absence of the virus upon transient expression in the host plants Nicotiana benthamiana 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.