Targeting of plasmodesmal proteins requires unconventional signals.

Effective cellular signaling relies on precise spatial localization and dynamic interactions among proteins in specific subcellular compartments or niches, such as cell-to-cell contact sites and junctions. In plants, endogenous and pathogenic proteins gained the ability to target plasmodesmata, membrane-lined cytoplasmic connections, through evolution to regulate or exploit cellular signaling across cell wall boundaries. For example, the receptor-like membrane protein PLASMODESMATA-LOCATED PROTEIN 5 (PDLP5), a potent regulator of plasmodesmal permeability, generates feed-forward or feed-back signals important for plant immunity and root development. However, the molecular features that determine the plasmodesmal association of PDLP5 or other proteins remain largely unknown, and no protein motifs have been identified as plasmodesmal targeting signals. Here, we developed an approach combining custom-built machine-learning algorithms and targeted mutagenesis to examine PDLP5 in Arabidopsis thaliana and Nicotiana benthamiana. We report that PDLP5 and its closely related proteins carry unconventional targeting signals consisting of short stretches of amino acids. PDLP5 contains 2 divergent, tandemly arranged signals, either of which is sufficient for localization and biological function in regulating viral movement through plasmodesmata. Notably, plasmodesmal targeting signals exhibit little sequence conservation but are located similarly proximal to the membrane. These features appear to be a common theme in plasmodesmal targeting.

Improving Inter-Helix Contact Prediction With Local 2D Topological Information.

Inter-helix contact prediction is to identify residue contact across different helices in α-helical integral membrane proteins. Despite the progress made by various computational methods, contact prediction remains as a challenging task, and there is no method to our knowledge that directly tap into the contact map in an alignment free manner. We build 2D contact models from an independent dataset to capture the topological patterns in the neighborhood of a residue pair depending it is a contact or not, and apply the models to the state-of-art method's predictions to extract the features reflecting 2D inter-helix contact patterns. A secondary classifier is trained on such features. Realizing that the achievable improvement is intrinsically hinged on the quality of original predictions, we devise a mechanism to deal with the issue by introducing, 1) partial discretization of original prediction scores to more effectively leverage useful information 2) fuzzy score to assess the quality of the original prediction to help with selecting the residue pairs where improvement is more achievable. The cross-validation results show that the prediction from our method outperforms other methods including the state-of-the-art method (DeepHelicon) by a notable degree even without using the refinement selection scheme. By applying the refinement selection scheme, our method outperforms the state-of-the-art method significantly in these selected sequences.

Facilitating viral vector movement enhances heterologous protein production in an established plant system.

Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.

A new algorithm to train hidden Markov models for biological sequences with partial labels.

BACKGROUND: Hidden Markov models (HMM) are a powerful tool for analyzing biological sequences in a wide variety of applications, from profiling functional protein families to identifying functional domains. The standard method used for HMM training is either by maximum likelihood using counting when sequences are labelled or by expectation maximization, such as the Baum-Welch algorithm, when sequences are unlabelled. However, increasingly there are situations where sequences are just partially labelled. In this paper, we designed a new training method based on the Baum-Welch algorithm to train HMMs for situations in which only partial labeling is available for certain biological problems. RESULTS: Compared with a similar method previously reported that is designed for the purpose of active learning in text mining, our method achieves significant improvements in model training, as demonstrated by higher accuracy when the trained models are tested for decoding with both synthetic data and real data. CONCLUSIONS: A novel training method is developed to improve the training of hidden Markov models by utilizing partial labelled data. The method will impact on detecting de novo motifs and signals in biological sequence data. In particular, the method will be deployed in active learning mode to the ongoing research in detecting plasmodesmata targeting signals and assess the performance with validations from wet-lab experiments.

A Tale of Two Domains Pushing Lateral Roots.

Successful plant organ development depends on well-coordinated intercellular communication between the cells of the organ itself, as well as with surrounding cells. Intercellular signals often move via the symplasmic pathway using plasmodesmata. Intriguingly, brief periods of symplasmic isolation may also be necessary to promote organ differentiation and functionality. Recent findings suggest that symplasmic isolation of a subset of parental root cells and newly forming lateral root primordia (LRPs) plays a vital role in modulating lateral root development and emergence. In this opinion article we discuss how two symplasmic domains may be simultaneously established within an LRP and its overlying cells, and the significance of plasmodesmata in this process.

Evaluating molecular movement through plasmodesmata.

Plasmodesmata are membrane-lined cytoplasmic passageways that facilitate the movement of nutrients and various types of molecules between cells in the plant. They are highly dynamic channels, opening or closing in response to physiological and developmental stimuli or environmental challenges such as biotic and abiotic stresses. Accumulating evidence supports the idea that such dynamic controls occur through integrative cellular mechanisms. Currently, a few fluorescence-based methods are available that allow monitoring changes in molecular movement through plasmodesmata. In this chapter, following a brief introduction to those methods, we provide a detailed step-by-step protocol for the Drop-ANd-See (DANS) assay, which is advantageous when it is desirable to measure plasmodesmal permeability non-invasively, in situ and in real-time. We discuss the experimental conditions one should consider to produce reliable and reproducible DANS results along with troubleshooting ideas.

An evolutionarily conserved motif is required for Plasmodesmata-located protein 5 to regulate cell-to-cell movement.

Numerous cell surface receptors and receptor-like proteins (RLPs) undergo activation or deactivation via a transmembrane domain (TMD). A subset of plant RLPs distinctively localizes to the plasma membrane-lined pores called plasmodesmata. Those RLPs include the Arabidopsis thaliana Plasmodesmata-located protein (PDLP) 5, which is well known for its vital function regulating plasmodesmal gating and molecular movement between cells. In this study, we report that the TMD, although not a determining factor for the plasmodesmal targeting, serves essential roles for the PDLP5 function. In addition to its role for membrane anchoring, the TMD mediates PDLP5 self-interaction and carries an evolutionarily conserved motif that is essential for PDLP5 to regulate cell-to-cell movement. Computational modeling-based analyses suggest that PDLP TMDs have high propensities to dimerize. We discuss how a specific mode(s) of TMD dimerization might serve as a common mechanism for PDLP5 and other PDLP members to regulate cell-to-cell movement.

Auxin-dependent control of a plasmodesmal regulator creates a negative feedback loop modulating lateral root emergence.

Lateral roots originate from initial cells deep within the main root and must emerge through several overlying layers. Lateral root emergence requires the outgrowth of the new primordium (LRP) to coincide with the timely separation of overlying root cells, a developmental program coordinated by the hormone auxin. Here, we report that in Arabidopsis thaliana roots, auxin controls the spatiotemporal expression of the plasmodesmal regulator PDLP5 in cells overlying LRP, creating a negative feedback loop. PDLP5, which functions to restrict the cell-to-cell movement of signals via plasmodesmata, is induced by auxin in cells overlying LRP in a progressive manner. PDLP5 localizes to plasmodesmata in these cells and negatively impacts organ emergence as well as overall root branching. We present a model, incorporating the spatiotemporal expression of PDLP5 in LRP-overlying cells into known auxin-regulated LRP-overlying cell separation pathways, and speculate how PDLP5 may function to negatively regulate the lateral root emergence process.

Publisher Correction: Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle-endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum-plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.

Plasmodesmata at a glance.

Plasmodesmata are cytoplasmic communication channels that are vital for the physiology and development of all plants. They facilitate the intercellular movement of various cargos - ranging from small molecules, such as sugars, ions and other essential nutrients and chemicals, to large complex molecules, such as proteins and different types of RNA species - by bridging neighboring cells across their cell walls. Structurally, an individual channel consists of the cytoplasmic sleeve that is formed between the endoplasmic reticulum and the plasma membrane leaflets. Plasmodesmata are highly versatile channels; they vary in number and structure, and undergo constant adjustments to their permeability in response to many internal and external cues. In this Cell Science at a Glance article and accompanying poster, we provide an overview of plasmodesmata form and function, with highlights on their development and variation, associated components and mobile factors. In addition, we present methodologies that are currently used to study plasmodesmata-mediated intercellular communication.

Plasmodesmata in phloem: different gateways for different cargoes.

The long-distance transport of sugars and nutrients through the phloem is essential for the proper function and growth of vascular plants. However, in addition to essential nutrients and sugars, phloem sap also contains small molecules (e.g. hormones) as well as a diverse population of macromolecules (i.e. proteins small RNAs, and mRNAs), the endogenous functions of which remain largely unknown. Understanding the cellular origins of these mobile macromolecules, their path into and out of the phloem translocation stream, and their fate at their new destination is essential for characterizing their presumptive function. Specialized plasmodesmal connections that regulate phloem entry and exit are central to all of these processes. Here, we highlight new discoveries underscoring plasmodesmal structure and function during unloading of various molecules in the sink, and discuss how these findings shape a new view for the potential function of phloem-mobile macromolecules.

iPTMnet: Integrative Bioinformatics for Studying PTM Networks.

Protein post-translational modification (PTM) is an essential cellular regulatory mechanism, and disruptions in PTM have been implicated in disease. PTMs are an active area of study in many fields, leading to a wealth of PTM information in the scientific literature. There is a need for user-friendly bioinformatics resources that capture PTM information from the literature and support analyses of PTMs and their functional consequences. This chapter describes the use of iPTMnet ( http://proteininformationresource.org/iPTMnet/ ), a resource that integrates PTM information from text mining, curated databases, and ontologies and provides visualization tools for exploring PTM networks, PTM crosstalk, and PTM conservation across species. We present several PTM-related queries and demonstrate how they can be addressed using iPTMnet.

Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of 'funnel plasmodesmata'. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as 'batch unloading'. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots.

Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress.

Plants need to cope with biotic and abiotic stress through well-coordinated cell-to-cell communication to survive as sedentary organisms. Environmental challenges such as wounding, low temperature, oxidative states and pathogen infection are known to affect the symplasmic molecular exchange between plant cells determined by plasmodesmal permeability. However, the signalling pathways and mechanisms by which different environmental stressors affect plasmodesmal permeability are not well understood. Here we show that regulating callose accumulation at plasmodesmal channels is a common strategy to alter plasmodesmal permeability under both pathogen infection and mechanical wounding stress. We have identified Arabidopsis callose synthase 1 (CalS1) and CalS8 as key genes involved in this process, and have integrated these new players into both known and novel signalling pathways that control responses to biotic and abiotic stress. Our studies provide experimental data that indicate the presence of specialized pathways tuned to respond to particular stressors, and new insights into how plants regulate plasmodesmata in response to environmental assaults.

Plasmodesmata Localizing Proteins Regulate Transport and Signaling during Systemic Acquired Immunity in Plants.

Systemic acquired resistance (SAR) in plants is mediated by the signaling molecules azelaic acid (AzA), glycerol-3-phosphate (G3P), and salicylic acid (SA). Here, we show that AzA and G3P transport occurs via the symplastic route, which is regulated by channels known as plasmodesmata (PD). In contrast, SA moves via the extracytosolic apoplast compartment. We found that PD localizing proteins (PDLP) 1 and 5 were required for SAR even though PD permeability in pdlp1 and 5 mutants was comparable to or higher than wild-type plants, respectively. Furthermore, PDLP function was required in the recipient cell, suggesting regulatory function in SAR. Interestingly, overexpression of PDLP5 drastically reduced PD permeability, yet also impaired SAR. PDLP1 interacted with AZI1 (lipid transfer-like protein required for AzA- and G3P-induced SAR) and contributed to its intracellular partitioning. Together, these results reveal the transport routes of SAR chemical signals and highlight the regulatory role of PD-localizing proteins in SAR.

Plasmodesmata: a signaling hub at the cellular boundary.

Effective intercellular communication is crucial for the survival of plants. Because plant cells are encased in rigid cell walls, direct cell-to-cell exchange of cytoplasmic content is only possible through plasmodesmata (PD), membrane-lined nanotubes that connect the cytoplasm of adjacent cells. PD are highly dynamic communication channels that can undergo various structural and functional modifications. Recent findings in the field suggest that defense signaling pathways are tightly linked to the regulation of PD, and the restriction of PD-mediated cell-to-cell communication is an essential innate immune response to microbial pathogens. Moreover, several plasma membrane-bound signaling components, including receptor-like kinases that are known to have non-cell autonomous function or pathogen perception at the cell periphery, are found to also partition to PD. These findings hint at the novel role of PD as a signaling hub for both symplasmic and cross-membrane pathways.

pGenN, a gene normalization tool for plant genes and proteins in scientific literature.

BACKGROUND: Automatically detecting gene/protein names in the literature and connecting them to databases records, also known as gene normalization, provides a means to structure the information buried in free-text literature. Gene normalization is critical for improving the coverage of annotation in the databases, and is an essential component of many text mining systems and database curation pipelines. METHODS: In this manuscript, we describe a gene normalization system specifically tailored for plant species, called pGenN (pivot-based Gene Normalization). The system consists of three steps: dictionary-based gene mention detection, species assignment, and intra species normalization. We have developed new heuristics to improve each of these phases. RESULTS: We evaluated the performance of pGenN on an in-house expertly annotated corpus consisting of 104 plant relevant abstracts. Our system achieved an F-value of 88.9% (Precision 90.9% and Recall 87.2%) on this corpus, outperforming state-of-art systems presented in BioCreative III. We have processed over 440,000 plant-related Medline abstracts using pGenN. The gene normalization results are stored in a local database for direct query from the pGenN web interface (proteininformationresource.org/pgenn/). The annotated literature corpus is also publicly available through the PIR text mining portal (proteininformationresource.org/iprolink/).

Localization of fluorescently tagged protein to plasmodesmata by correlative light and electron microscopy.

Plasmodesmata (PD) are intercellular communication channels that form long, membrane-lined cylinders across cellular junctions. A fluorescent-tagging approach is most commonly used for an initial assessment to address whether a protein of interest may localize or associate with PD domain. However, owing to the dimension of PD being at nanoscale, PD-associated fluorescent signals are detected only as small spots scattered at the cell periphery, hence requiring additional confirmatory evidence. Immunogold labeling provides such information, but suitable antibodies are not always available and morphological preservation is often compromised with this approach. Here we describe an alternative approach using a correlative light and electron microscopy (CLEM) technique, which combines fluorescent imaging and transmission electron microscopy. By employing this method, a clear correlation between fluorescent speckles and the presence of individual or clusters of PD is achieved.