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Small RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile membrane-bound compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that Arabidopsis (Arabidopsis thaliana) EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of “tiny RNAs” (10 to 17 nt) that are highly enriched in EVs. This RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses.

Extracellular vesicles (EVs) are small, membrane-enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long-sought-after mechanism for host-induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co-purifying contaminants.

Maintaining high crop yields in an environmentally sustainable manner requires the development of disease-resistant crop varieties. We describe a method to engineer disease resistance in plants by means of an endogenous disease resistance gene from Arabidopsis thaliana named RPS5, which encodes a nucleotide-binding leucine-rich repeat (NLR) protein. RPS5 is normally activated when a second host protein, PBS1, is cleaved by the pathogen-secreted protease AvrPphB. We show that the AvrPphB cleavage site within PBS1 can be substituted with cleavage sites for other pathogen proteases, which then enables RPS5 to be activated by these proteases, thereby conferring resistance to new pathogens. This \"decoy\" approach may be applicable to other NLR proteins and should enable engineering of resistance in plants to diseases for which we currently lack robust genetic resistance.

Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a central role in the innate immune systems of plants and vertebrates. In plants, NLR proteins function as intracellular receptors that detect pathogen effector proteins directly, or indirectly by recognizing effector-induced modifications to other host proteins. NLR activation triggers a suite of defense responses associated with programed cell death (PCD). The molecular mechanisms underlying NLR activation, and how activation is translated into defense responses, have been particularly challenging to elucidate in plants. Recent reports, however, are beginning to shed some light. It is becoming clear that plant NLR proteins are targeted to diverse sub-cellular locations, likely depending on the locations where the effectors are detected. These reports also indicate that some NLRs re-localize following effector detection, while others do not, and such relocalization may reflect differences in signaling pathways. There have also been recent advances in understanding the structure of plant NLR proteins, with crystal structures now available for the N-terminal domains of two well-studied NLRs, a coiled-coil (CC) domain and a Toll-interleukin Receptor (TIR). Significant improvements in molecular modeling have enabled more informed structure-function studies, illuminating roles of intra- and inter-molecular interactions in NLR activation regulation. Several independent studies also suggest that intracellular trafficking is involved in NLR-mediated resistance. Lastly, progress is being made on identifying transcriptional regulatory complexes activated by NLRs. Current models for how plant NLR proteins are activated and how they induce defenses are discussed, with an emphasis on what remains to be determined.

The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Here, we present a structure-function analysis of the CC and LRR domains of RPS5 using transient expression assays in Nicotiana benthamiana. We found that substituting the CC domain of RPS2 for the RPS5 CC domain did not alter RPS5 specificity and only moderately reduced its ability to activate programmed cell death, suggesting that the CC domain does not play a direct role in the recognition of PBS1 cleavage. Analysis of an RPS5-super Yellow Fluorescent Protein fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-4) residues affected RPS5 PM localization, protein stability, and function in an additive manner, indicating that PM localization is essential to RPS5 function. The first 20 amino acids of RPS5 were sufficient for directing super Yellow Fluorescent Protein to the PM. C-terminal truncations of RPS5 revealed that the first four LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for the recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in the autoactivation of RPS5, indicating that the LRR domain must coevolve with the NBS domain. We conclude that the RPS5 LRR domain functions to suppress RPS5 activation in the absence of PBS1 cleavage and promotes RPS5 activation in its presence.

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.

Plant proteins belonging to the nucleotide-binding site–leucine-rich repeat (NBS-LRR) family are used for pathogen detection. Like the mammalian Nod-LRR protein 'sensors' that detect intracellular conserved pathogen-associated molecular patterns, plant NBS-LRR proteins detect pathogen-associated proteins, most often the effector molecules of pathogens responsible for virulence. Many virulence proteins are detected indirectly by plant NBS-LRR proteins from modifications the virulence proteins inflict on host target proteins. However, some NBS-LRR proteins directly bind pathogen proteins. Association with either a modified host protein or a pathogen protein leads to conformational changes in the amino-terminal and LRR domains of plant NBS-LRR proteins. Such conformational alterations are thought to promote the exchange of ADP for ATP by the NBS domain, which activates 'downstream' signaling, by an unknown mechanism, leading to pathogen resistance.

Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to powdery mildew infection, enhanced senescence, and enhanced programmed cell death under both abiotic and biotic stress conditions. All edr1-mediated phenotypes can be suppressed by a specific missense mutation (keg-4) in the KEEP ON GOING (KEG) gene, which encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like (for HECT and RCCl-like) repeats. The molecular and cellular mechanisms underlying this suppression are poorly understood. Using confocal laser scanning microscopy and fluorescent protein fusions, we determined that KEG localizes to trans-Golgi network/early endosóme (TGN/EE) vesicles. Both the keg-4 mutation, which is located in the carboxyl-terminal HERC2-like repeats, and deletion of the entire HERC2-like repeats reduced endosomal localization of KEG and increased localization to the endoplasmic reticulum and cytosol, indicating that the HERC2-like repeats facilitate the TGN/EE targeting of KEG. EDR1 colocalized with KEG to the TGN/EE when coexpressed but localized primarily to the endoplasmic reticulum when expressed alone. Yeast two-hybrid and coimmunoprecipitation analyses revealed that EDR1 and KEG physically interact. Deletion of the HERC2-like repeats abolished the interaction between KEG and EDR1 as well as the KEG-induced TGN/EE localization of EDR1, indicating that the recruitment of EDR1 to the TGN/EE is based on a direct interaction between EDR1 and KEG mediated by the HERC2-like repeats. Collectively, these data suggest that EDR1 and KEG function together to regulate endocytic trafficking and/or the formation of signaling complexes on TGN/vesicles during stress responses.

Nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins mediate pathogen recognition in both mammals and plants. The molecular mechanisms by which pathogen molecules activate NBS-LRR proteins are poorly understood. Here we show that RPS5, a NBS-LRR protein from Arabidopsis, is activated by AvrPphB, a bacterial protease, via an indirect mechanism. When transiently expressed in Nicotiana benthamiana leaves, full-length RPS5 protein triggered programmed cell death, but only when coexpressed with AvrPphB and a second Arabidopsis protein, PBS1, which is a specific substrate of AvrPphB. Using coimmunoprecipitation analysis, we found that PBS1 is in a complex with the N-terminal coiled coil (CC) domain of RPS5 before exposure to AvrPphB. Deletion of the RPS5 LRR domain caused RPS5 to constitutively activate programmed cell death, even in the absence of AvrPphB and PBS1, and this activation depended on both the CC and NBS domains. The LRR and CC domains both coimmunoprecipitate with the NBS domain but not with each other. Thus, the LRR domain appears to function in part to inhibit RPS5 signaling, and cleavage of PBS1 by AvrPphB appears to release RPS5 from this inhibition. An amino acid substitution in the NBS site of RPS5 that is known to inhibit ATP binding in other NBS-LRR proteins blocked activation of RPS5, whereas a substitution thought to inhibit ATP hydrolysis constitutively activated RPS5. Combined, these data suggest that ATP versus ADP binding functions as a molecular switch that is flipped by cleavage of PBS1.